HSV-tk、IL-18联合修饰骨髓基质干细胞治疗脑胶质瘤的实验研究
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摘要
胶质瘤作为人类最常见的原发性颅内肿瘤,有很高的发病率和死亡率。约占成人颅内原发肿瘤的30%-50%。迄今的治疗方法都不能从根本上改变这一致命肿瘤的自然转归,因而新疗法的探索成为神经外科的热点课题。近年来随着胶质瘤分子生物学与分子遗传学的发展,同时基因重组、基因转染等技术的成熟,肿瘤基因治疗成为研究的热点,自杀基因治疗是其中最常采用的方案。
理想的脑肿瘤的基因治疗应该是应用最佳的基因转移技术来转移最佳的治疗基因。自杀基因(HSV-tk/GCV)治疗肿瘤的体外及动物实验均取得了惊人的效果,但临床疗效不能令人满意,究其原因是基因治疗的载体无法在体内肿瘤组织中进行广泛分布,从而不能充分发挥其旁观者效应。
胶质瘤患者均表现出了T淋巴细胞抗肿瘤作用的减弱,并且,由于胶质瘤细胞缺乏细胞表面抗原导致的肿瘤抗原呈递作用的减弱,进一步减弱了抗肿瘤免疫。现在的观点认为,IL-18可通过T淋巴细胞、NK细胞发挥抗肿瘤作用,并且通过上调MHC分子的表达、促进CD4+辅助性T细胞向Th1型亚单位的分化增强细胞免疫。
骨髓基质干细胞(BMSCs)是具有迁移能力的细胞,并可稳定的表达外源性转染的某些基因,为胶质瘤基因治疗提供了新的思路。本实验拟用骨髓基质干细胞为载体,观察转染自杀基因的骨髓基质干细胞对脑胶质瘤的治疗作用,再引入IL-18,增强抗肿瘤免疫,观察两者相结合的治疗效果,为以骨髓基质干细胞为载体进行脑胶质瘤的基因治疗提供理论依据。以骨髓基质干细胞为载体,HSV-tk与IL-18联合治疗胶质瘤的研究,在国内外尚无报道。
1.骨髓基质干细胞的培养、鉴定及标记的研究
目的:研究骨髓基质干细胞的培养方法,并对其进行标记,为进一步的体内试验奠定基础。
方法:将出生2周的SD大鼠脱颈处死,消毒后取双侧股骨,自中间剪断后冲出骨髓细胞,加入含20%胎牛血清的完全培养液于37℃、5%CO2孵箱内培养,48小时后半量换液。以后每2-3天半量换液,7-8天传代一次。培养初期细胞较混杂,第6代后细胞形态为均一的纺锤形细胞。MTT法绘制生长曲线。
将培养第6代、已纯化的骨髓基质干细胞消化、计数后应用流式细胞术检测BMSCs CD31、CD34、CD45、CD90、CD44、CD71阳性率。取对数生长期的骨髓基质干细胞进行细胞爬片,待细胞生长至50%时加入BrdU(终浓度10μg/ml),24小时后取出玻片用PBS洗涤,4%多聚甲醛固定15分钟后进行免疫组化及免疫荧光染色,封片观察。将BrdU标记的骨髓基质干细胞注入大鼠颅内,3周后处死大鼠取脑,于穿刺点位置附近作石蜡切片并进行免疫荧光染色。
结果:在培养初期可见大量悬浮细胞,48小时后可见有散在纺锤形贴壁细胞,经多次半量换液后,未贴壁细胞逐渐被去除。细胞初期多以集落形式生长,第6代后细胞呈形态均一的纺锤形,分布均匀。绘制细胞生长曲线,可见细胞呈一种持续的增殖状态,培养2-3天后进入对数生长期。以流式细胞技术检测细胞表面抗原,结果显示为体积均一的细胞群, CD31、CD34、CD45的阳性率分别为5.67%、4.31%、4.42%, CD90、CD44、CD71的阳性率分别为97.17%、98.63%、95.86%。骨髓基质干细胞体外免疫组化显示88.75±4.38%的细胞可被BrdU标记。免疫荧光染色可见胞核染色明显,BrdU标记的骨髓基质干细胞注入体内后3周仍可见明显的BrdU着色,穿刺点位置可见密集的细胞。
结论:通过骨髓全细胞培养的方法可获得高纯度的BMSCs,并可在体外长期培养;采用BrdU标记骨髓基质干细胞具有较高的标记阳性率,并且标记效果稳定。
2.骨髓基质干细胞向脑胶质瘤的趋向性及神经转化潜能
目的:在于研究骨髓基质干细胞向脑胶质瘤的趋向性,及骨髓基质干细胞在体外环境中、在特定细胞因子诱导条件下的细胞转化,以及骨髓基质干细胞在脑胶质瘤环境中的转化情况。
方法:体外培养6~7代的BMSCs接种到预置有多聚赖氨酸包被的盖玻片的培养板内,细胞达到70%融合时,将盖玻片转移至含10%胎牛血清的Neurobasal培养基内培养,加入全反式视黄酸(0.5μmol/L)、脑源性神经生长因子(10ng/mL)。以正常培养的、未加诱导剂的BMSCs作为对照。4d后,取出盖玻片,4%多聚甲醛固定30min,用于免疫荧光检测细胞转化情况。
骨髓基质干细胞趋瘤性研究的体外实验采用空心柱试验及Transwell试验。培养大鼠脑胶质瘤C6细胞,待细胞达到对数生长期时,消化计数,制作大鼠脑胶质瘤模型。模型建立3天后随机抽取20只荷瘤大鼠,在肿瘤对侧大脑立体定向种植BMSCs(8×10~4个悬于2μL PBS)。移植后15d,4%多聚甲醛自大鼠心脏灌注后,取出脑组织制作病理切片。用于转化研究的脑组织制作冰冻切片。
结果:经诱导剂诱导转化3天后,在倒置显微镜下观察,细胞形态改变明显,部分细胞可伸出长的突起,少数突起可与周围的细胞或突起有紧密的接触,另有少部分细胞变圆、脱落;对照组中培养的BMSCs细胞形态未见明显变化。免疫荧光染色显示:在诱导组,GFAP阳性率21.1±6.3%,MAP2阳性率56.4±13.8%,Nestin阳性率8.3±5.2%;在对照组,未发现MAP2、Nestin阳性细胞,GFAP阳性率3.9±2.8%。
空心柱试验显示,随着培养时间的延长,BMSCs很快离开原种植位置,向C6细胞存在位置进行迁移,而后广泛迁移到胶质瘤细胞区域,而3T3细胞仍停留在原位。Transwell试验显示无细胞的培养液(1.1±0.7)和3T3细胞条件培养液(2.4±0.9)的培养孔仅有极个别的BMSCs迁移,两者之间无统计学差异(P>0.05),在C6细胞条件培养液(14.7±4.7)的培养孔有明显的BMSCs迁移,而更大的迁移细胞数是在C6细胞的培养孔(42.7±8.2)。BMSCs在细胞因子条件下的迁移: PDGF诱导的迁移细胞数量最多(32.7±6.3),其次是EGF(26.3±7.4)。b-FGF(7.5±2.8)和VEGF(9.4±3.5)诱导的迁移细胞最少,两者无统计学差异(P>0.05)。在所有诱导因素中,C6细胞诱导了最明显的迁移(P<0.05)。
体内趋瘤性试验显示,有大量的BrdU标记的BMSCs位于正常脑组织与肿瘤的交界位置,而在肿瘤内部只有少许BMSCs。体内转化的免疫荧光染色显示:脑内移植的用Hoechst33258标记的BMSCs部分呈Nestin阳性(8.32±3.41%),部分呈GFAP阳性(32.71±8.66%),部分呈MAP2阳性(11.88±5.16%)。提示移植的部分BMSCs可转化为神经前体细胞,部分可转化为神经元或胶质细胞(Fig7)。对照组2、对照组3中,移植BMSCs的GFAP阳性率分别为31.19±8.76%和29.32±7.75%, Nestin阳性阳性率分别为6.95±3.75%和7.21±3.31%,MAP2阳性率分别为8.76±3.77%和10.59±5.43%,三组间比较无统计学差异(P>0.05)。
结论:骨髓基质干细胞具有明显的向胶质瘤细胞迁移的特性,迁移细胞多聚集于肿瘤边缘,只有少数骨髓基质干细胞可达到肿瘤内部;骨髓基质干细胞在体外特殊诱导环境中及移植到荷瘤大鼠脑内后,可部分转化为神经前体细胞、神经元及胶质细胞。
3.转染HSV-tk的骨髓基质干细胞治疗脑胶质瘤的体内、外实验研究
目的:在于探讨以骨髓基质干细胞为载体,观察转染tk基因的骨髓基质干细胞对脑胶质瘤的杀伤作用。
方法:AdCMV-tk病毒扩增后用于感染骨髓基质干细胞,而后采用RT-PCR方法检测BMSCs/tk对tk基因的转录。而后进一步采用流式细胞术、MTT检测BMSCs/tk细胞表面抗原及生长曲线。Transwell试验检测BMSCs/tk体外趋瘤性;制作大鼠脑胶质瘤模型,将BMSCs/tk移植到肿瘤对侧脑组织,观察其体内趋瘤性。将BMSCs/tk与C6细胞混合培养,采用不同的效靶比及不同的GCV浓度,用MTT法、TUNEL、AnnexinV检测细胞存活率或凋亡率,观察BMSCs/tk的旁观者效应。进一步采用体内试验,观察BMSCs/tk的体内旁观者效应,观察荷瘤大鼠在治疗后的生存期改变。
结果:BMSCs/tk的细胞表面抗原、细胞生长曲线、体内外趋瘤性仍保持着BMSCs的特性。RT-PCR显示,AdCMV-tk腺病毒感染BMSCs后24小时即有明显的tk基因的转录,在48小时达到高峰,而后逐渐降低,在1个月时仍有少量tk基因转录。体外旁观者效应检测显示,随着效应细胞数量的增多,杀伤效果明显增强;随着GCV用量的增多,杀伤效果明显增强。体内试验显示,应用TUNEL法检测BMSCs/tk的体内抗脑胶质瘤作用,对比平均每个视野中肿瘤细胞的凋亡数,BMSCs/βgal组(1.65±1.09)、BMSCs组(1.50±1.43)、PBS组(1.45±1.28)及空白对照组(1.30±1.08)之间无明显差异(P>0.05),但均明显低于BMSCs/HSV-tk组(8.85±3.33) (P<0.05)。
结论:感染AdCMV-tk后, BMSCs可长时间的对tk基因进行转录;BMSCs/tk具有明显的颅内肿瘤趋向性,并保持旺盛的增殖活性;BMSCs/tk在体外及在脑胶质瘤荷瘤大鼠体内可诱导明显的肿瘤细胞凋亡,延长荷瘤大鼠生存期。
4. HSV-tk、IL-18联合修饰骨髓基质干细胞治疗脑胶质瘤的体内、外实验研究
目的:是采用IL-18转染BMSCs,观察BMSCs/IL-18的抗脑胶质瘤效果;进一步采用IL-18及tk联合修饰BMSCs,观察BMSCs/IL-18/tk的体内外抗肿瘤效果。
方法:LXSN/IL-18感染骨髓基质干细胞(BMSCs/IL-18),筛选出阳性克隆后,感染AdCMV/tk病毒(BMSCs/IL-18/tk),而后采用RT-PCR方法检测BMSCs/IL-18、BMSCs/IL-18/tk对IL-18基因、tk基因的转录。而后进一步采用流式细胞术、MTT检测BMSCs/IL-18、BMSCs/IL-18/tk细胞表面抗原及生长曲线。Transwell试验检测BMSCs/IL-18、BMSCs/IL-18/tk体外趋瘤性;制作大鼠脑胶质瘤模型,将BMSCs/IL-18、BMSCs/IL-18/tk移植到肿瘤对侧脑组织,观察其体内趋瘤性。将BMSCs/IL-18、BMSCs/IL-18/tk与C6细胞混合培养,采用不同的效靶比及不同的GCV浓度,用MTT法、TUNEL、AnnexinV检测细胞存活率或凋亡率,观察BMSCs/IL-18、BMSCs/IL-18/tk的旁观者效应。进一步采用体内试验,观察BMSCs/IL-18、BMSCs/IL-18/tk的体内抗脑胶质瘤作用。ELISA法检测BMSCs/IL-18、BMSCs/IL-18/tk对淋巴细胞分泌的影响。免疫组化方法检测BMSCs/IL-18、BMSCs/IL-18/tk治疗后肿瘤组织内部微血管密度、淋巴细胞浸润、细胞凋亡情况。MRI检测治疗后的肿瘤体积改变,观察荷瘤大鼠在治疗后的生存期改变。
结果:BMSCs/IL-18、BMSCs/IL-18/tk的细胞表面抗原、体内外趋瘤性仍保持着BMSCs的特性。细胞生长曲线显示,BMSCs/IL-18、BMSCs/IL-18/tk增殖活性有所下降。RT-PCR显示,BMSCs/IL-18、BMSCs/IL-18/tk可稳定转录IL-18基因, BMSCs/IL-18 /tk可较长时间稳定的转录tk基因,免疫组化显示BMSCs/IL-18、BMSCs/IL-18/tk稳定表达IL-18。体外研究显示,BMSCs/IL-18/tk可产生明显的旁观者效应,且随着效应细胞数量的增多,旁观者效应更加明显,而BMSCs/IL-18未表现出旁观者效应。ELISA法检测显示,BMSCs/IL-18、BMSCs/IL-18/tk可明显增加体外淋巴细胞分泌IFN-γ,并可增加荷瘤大鼠体内血清IFN-γ浓度。体内试验显示,BMSCs/IL-18、BMSCs/IL-18/tk可明显增加荷瘤大鼠脑内胶质瘤肿瘤组织中淋巴细胞浸润数量,减少肿瘤微血管密度,诱导肿瘤细胞凋亡。BMSCs/IL-18/tk可明显延长荷瘤大鼠的生存期。
结论:转染IL-18后,BMSCs/IL-18可稳定转录及表达IL-18基因;感染AdCMV-tk后,BMSCs/IL-18/tk可稳定的转录tk及IL-18; BMSCs/IL-18、BMSCs/IL-18/tk具有明显的颅内肿瘤趋向性,表达BMSCs特异性的表面抗原,保持旺盛的增殖活性;BMSCs/IL-18/tk在体外及在脑胶质瘤荷瘤大鼠体内可诱导明显的肿瘤细胞凋亡,减少肿瘤组织微血管密度,增加CD4~+、CD8~+淋巴细胞的浸润数量,延长荷瘤大鼠生存期。
Malignant glioma, the most common intracranial tumor, accounts for 30-50% of primary brain tumor in adults, with high morbidity, mortality and grim prognosis. Because currently available multiple modalities of treatment have not yet substantially changed the natural history of these lethal neoplasms, new therapeutic approaches are actively being investigated. Over the past few years, with the development in understanding the molecular biology and molecular genetics of glioma, gene therapy of tumor has been the hotspot and suicide gene therapy is one of the most favorite strategy used for malignant gliomas.
Ideal protocol of brain tumor gene therapy would employ the optimal gene transfer technique with the optical genes transferred. Suicide gene therapy (HSV-tk/GCV) has revealed the encouraging therapeutic effect on glioma in vitro and in vivo animal experiment. However, as far as clinical trials, the results were not satisfactory and the reason might be the poor by-stander effect because of the therapy gene can not distribute in tumor in vivo widely.
Antitumor effect of T lymphocytes of glioma patients were decreased, and because of the glioma antigen present ability was decreased owing to poverty cell special surface antigen, antitumor immunity effect was weakened further. According to present view, IL-18 could play its antitumor ability by activating T lymphocytes and natural killer cells, up-regulating expression of MHC and promoting CD4~+ helper lymphocytes differentiated to Th1 subset to enhance cell mediated immunity.
BMSCs have the ability of immigration and expression of transfected exogenous gene stably which offered the new route of gene therapy against glioma. The experiment planed to employ BMSCs as the therapy gene vector to observe the antitumor effect of BMSCs which infected suicide gene (BMSCs/tk). Furthermore, to enhance antitumor immunity, IL-18 was employed. The study provided a theoretical foundation for the use of BMSCs as vector against glioma. There was no report that BMSCs, as therapy gene vector, were transfected HSV-tk and IL-18 to treat intracranial glioma.
1. The initial research of culturing, characterization and labelling bone marrow stromal stem cells
Objective: The purpose of this part is to research how to culture, identity and label bone marrow stromal stem cells(BMSC), which established foundation for further study.
Methods: Single-cell suspension obtained from femurs of 2-3-week-old SD rats which were sacrificed and then incubated on plastic dishes in Dulbecco’s modified Eagle’s medium(DMEM) containing 20% heat-inactivated-fetal bovine serum(FBS) at 37℃with 5% CO_2 . 48 hours later, shake the culture bottle slowly and nonadherent cells were removed. Adherent cells were further cultured by exchanging half culture medium every 2-3 days to expend. After 7-8 days of incubation, the bone marrow-derived cells were confluent and expended by a 1:2 split. After in vitro culture-expended 6 passages, adherent bone marrow-derived cells were uniformly fibroblast-like in appearance. Cell growth curve was drown by means of MTT.
After subcultured 6 passages and cells were purified, to evaluate the BMSCs characterization, flow cytometry analysis was performed to detect the expression of CD31, CD45, CD90, CD34, CD71, CD44.After subcultured 6 passages, BMSCs, which were at exponential phase of growth, were cultured in flasks which contained several glass coverslips, and after 50% confluent the cells were pulsed for 24 hours with 10μg/ml 5-bromo-2-deoxyuridine (BrdU) (Sigma) in DMEM. Which was followed by fixation with 4% paraformaldehyde for 15 minutes, and then, immunohistochemistry and immunofluorescence were performed and the clips were observed under fluorescence microscope. After being labeled with BrdU, BMSCs were transplanted into rats, brain and the rats were sacrificed 3 weeks later. The brain tissue near the puncture site was made paraffin sections and immunofluorescence was performed.
Results: The results showed that at the initial time, there were lots of suspension cells in the culture dishes.48 hours later, there were several fusiform adherent cells. After several semi-exchange culture medium, non-adherent cells were removed and several cell colonies formed. The BMSCs became comparatively homogeneous in appearance and well-distributed as the cells were 6 passages. The cells were relatively elongated or spindle-shaped. Cell growth curve showed that the cell multiplied actively and were at exponential phase of growth 2-3 later. Surface phenotypic characterization of primary BMSCs detected by flow cytometry showed that the percentage of CD31, CD34, CD45, CD44, CD71, CD90 was 5.67%, 4.31%,4.42%, 97.17%, 98.63%, 95.86% respectively. In vitro immunohistochemistry showed that 88.75±4.38% cells could be labeled with BrdU. Immunofluorescence showed that nucleus were labeled obviously and after being transplanted into rat brain 3 weeks later, lots of BMSCs, which were labeled with BrdU, were observed near the puncture site.
Conclusions: The results showed that purified BMSCs could be harvested through culturing bone marrow cells and could expended culture in vitro in long-term. BMSCs could be labeled with BrdU high efficiency and stably.
2. Tropism for glioma and neural differentiation of bone marrow stromal cells
Objective: The aim was to observe tropism for intracranial glioma of bone marrow stromal cells (BMSCs) and their differentiation in brain of Glioma bearing rats or induced with certain cytokines in vitro.
Methods: After being subcultured in vitro 6-7 passages, BMSCs were seeded into flasks which contained glass coverslips coated with polylysine. After cells reached 70% confluence, the coverslips which contained BMSCs were displaced into Neurobasal medium containing 10% heat-inactivated-fetal bovine serum and alltrans retinoic acid (0.5μmol/L) and brain derived nerve growth factor (10ng/mL). BMSCs were cultured without induced cytokines as control group. 4 days later, the coverslips were taken out and fixation in 4% paraformaldehyde for 30 minutes followed by immunofluorescence to observe cells differentiation.
In vitro experiment of BMSCs tropism for glioma was performed by means of cylinder experiment and Transwell experiment. C6 cells were cultured and, after C6 were at exponential phase of growth, glioma bearing rats were established. 3 days later, 20 glioma bering rats were selected randomly and were transplanted BMSCs(8×10~4cells in 2μL PBS)in the contralateral hemisphere. 15 days later, the rats were fixation with 4% paraformaldehyde perfuse through hearts. And then, the brains were removed and pathological sections were made and frozen section were made for differentiation research.
Results: The results showed that, after being induced 3 days, observed under inverted microscope form, cells form were changed and some of the cells stretch out long branches and some of which got in touch with the other cells or branches. Several cells were rounding and shedding. The form of BMSCs in control group was not changed. Immunofluorescence showed that, in induced group, GFAP positive ratio was 21.1±6.3%,MAP2 positive ratio was 56.4±13.8%,Nestin positive ratio was 8.3±5.2%;in control group,there were no MAP2 and Nestin positive cells could be found,GFAP positive ratio was 3.9±2.8%。
In cylinder experiment, in contrast to 3T3 cells, which remained localized to the area of initial seeding, BMSCs migrated rapidly and interspersed throughout the glioma monolayer, far from the initial site of seeding. Transwell experiment showed that, exposure to cell-free medium (1.1±0.7) or to conditioned medium from 3T3 cells(2.4±0.9) resulting in low levels of migrating BMSCs, there were no statistic difference between the two groups(P>0.05), while exposure to conditioned medium from C6 cells(14.7±4.7) produced significant BMSCs migration, whereas, the largest number of the migration BMSCs was C6 cells(42.7±8.2). Among the four cytokines, PDGF intermediated the maximal migration (32.7±6.3), the 2~(nd) was EGF(26.3±7.4), whereas b-FGF and VEGF had no statistic difference(7.5±2.8, 9.4±3.5). Among all the induced effector, C6 cells induced the obviously immigration (P<0.05).
In vivo tropism for glioma experiment showed that there were lots of BMSCs labeled with BrdU located at the boundary of brain tissue and glioma and there were several BMSCs inside glioma. In vivo differentiation immunofluorescence showed that: some of the BMSCs labeled with Hoechst33258 were Nestin positive (8.32±3.41%), some were GFAP positive(32.71±8.66%) and some were MAP2 positive (11.88±5.16%). This suggested that some of the transplanted BMSCs differentiated into neural precursor cells and some differentiated into neuron and gliocyte. In control group2 and control group3, GFAP positive ratio of transplanted BMSCs was 31.19±8.76% and 29.32±7.75% repectively, Nestin positive ratio of transplanted BMSCs was 6.95±3.75% and 7.21±3.31% repectively and MAP2 positive ratio of transplanted BMSCs was 8.76±3.77% and 10.59±5.43% repectively. There was no statistic difference between the three groups (P>0.05).
Conclusions: BMSCs displayed extensive tropisum for glioma cells and most of immigration BMSCs located on the boundary of glioma and brain, only few immigration BMSCs were in glioma field. BMSCs, in in vitro induce environment or were transplanted into brain of glioma bearing rats, some of the transplanted BMSCs differentiated into neural precursor cells and some differentiated into neuron and gliocyte.
3. In vitro and in vivo study of anti-glioma activity of bone marrow stromal cells transfected with HSV-tk
Objective: The purpose of the research was to study the antitumor effect of BMSCs, as vector of therapy gene, after being transfected with tk gene.
Methods: After being amplification, AdCMV-tk was used to transfected BMSCs (BMSCs/tk). RT-PCR was performed to detect transcription of tk gene by BMSCs/tk. Flow cytometry and MTT were performed to detect BMSCs/tk cells surface antigen and cell growth curve. Transwell experiment was performed to examine BMSCs/tk tropism for glioma in vitro. Glioma bearing rats were established and BMSCs/tk were transplanted into contralateral hemisphere to observe their tropism for glioma in vivo. Co-culture BMSCs/tk and C6, and then, different effector-target ratio and different GCV dose were introduced followed by cell survival ratio and cell apoptosis ratio examination by means of MTT, TUNEL and Annexin V. Experiment were performed to study by-stander effect of BMSCs/tk in vivo and survival time of glioma bearing rats were recorded after being treated with BMSCs/tk-GCV.
Results: The results showed that, the cell surface antigens, cell growth curve and in vitro tropism for glioma of BMSCs were not changed after being infected with AdCMV-tk. RT-PCR results showed that, after being infected with AdCMV-tk 24 hours later, BMSCs/tk could transcribe tk gene obviously and reached its peak at 48 hours. And then, transcription of tk decreased gradually and decreased obviously 1 month later. In vitro by-stander effect experiment showed that, with the increase of effector or GCV dose, kill activity was enhanced obviously. In vivo experiment showed that, by means of TUNEL examination, apoptosis cells number in every view inside glioma was 1.65±1.09 in BMSCs/βgal group, 1.50±1.43 in BMSCs group, 1.45±1.28 in PBS group and 1.30±1.08 in control group and there were no statistic difference between these groups (P>0.05). But the largest apoptosis number was observed in BMSCs/HSV-tk group (8.85±3.33) (P<0.05).
Conclusions: After being transfected with AdCMV-tk, BMSCs could transcript tk gene long term. BMSCs/tk displayed extensive tropisum for intracranial glioma and maintained high multiplication ability. BMSCs/tk induced obviously apoptosis of glioma in vitro and in vivo and prolonged survival time of glima bearing rats. 4. In vivo and in vitro study of therapy effect of bone marrow stromal cells co-transfected with HSV-tk and interleukin 18 on glioma
Objective: The purpose of the study was to observe antitumor effect of BMSCs after being transfected with IL-18. Furthermore, antitumor effect of BMSCs, after being co-transfected with HSV-tk and IL-18, were observed through in vitro and in vivo experiments.
Methods: After being infected with LXSN/IL-18, BMSCs/IL-18 clone was selected followed by infected with AdCMV/tk, thus, BMSCs/IL-18 and BMSCs/IL-18/tk was harvested. RT-PCR were performed to detect the gene transcription of IL-18 and tk by BMSCs/IL-18 and BMSCs/IL-18/tk. Cells surface antigen and cell growth curve were examined of BMSCs/IL-18 and BMSCs/IL-18/tk through flow cytometry and MTT. Transwell experiment was performed to observe in vitro tropism for glioma. Glioma bearing rats were established and BMSCs/IL-18 and BMSCs/IL-18/tk were transplanted into contralateral hemisphere to observed their tropism for glioma in vivo. To observe by-stander effector of BMSCs/IL-18 and BMSCs/IL-18/tk, co-culture BMSCs/IL-18 and C6 or BMSCs/IL-18/tk and C6, and different effector target ratio and different GCV dose were introduced, and then, cell survival ratio and apoptosis ratio was examined by means of MTT, TUNEL and Annexin V. In vivo experiment was performed to detect BMSCs/IL-18 and BMSCs/IL-18/tk antitumor effect. ELISA test was performed to detect the effect of BMSCs/IL-18 and BMSCs/IL-18/tk on lymphocyte secretion of IFN-γ. Immunohistochemistry was introduced to detect microvessel density, lymphocyte infiltration and cell apoptosis inside glioma after being treated with BMSCs/IL-18 and BMSCs/IL-18/tk. MRI examination was performed to observe the volume change of intracranial glioma after treatment and survival time were recorded.
Results: The results showed that the cell surface antigen and tropism for glioma in vitro was not changed after BMSCs were infected with LXSN/IL-18 or LXSN/IL-18 and AdCMV/tk. Cell growth curve showed that BMSCs/IL-18 and BMSCs/IL-18/tk had lower multiply speed than BMSCs. RT-PCR showed that BMSCs/IL-18 and BMSCs/IL-18/tk could transcript IL-18 gene stably and BMSCs/IL-18/tk could transcript tk gene long term. Immunohistochemistry showed that BMSCs/IL-18 and BMSCs/IL-18/tk could express IL-18 stably. In vitro experiment showed that BMSCs/IL-18/tk could display obviously by-stander effect, and with the increase of effectors, by-stander effect was more enhanced. BMSCs/IL-18 did not display by-stander effect. ELISA examination demonstrated that BMSCs/IL-18 and BMSCs/IL-18/tk could increase IFN-γsecretion of lymphocyte and increase IFN-γconcentration of glioma bearing rats serum. In vivo examination showed that BMSCs/IL-18 and BMSCs/IL-18/tk could increase infiltration lymphocytes number and decrease microvessel density and induced glioma cells apoptosis and glioma bearing rats survival time were prolonged obviously after being treated with BMSCs/IL-18/tk.
Conclusions: After being transfected with IL-18, BMSCs/IL-18 could transcript and express IL-18 stably. After being transfected with AdCMV-tk, BMSCs/IL-18/tk could transcript tk and IL-18 stably. BMSCs/IL-18 and BMSCs/IL-18/tk displayed extensive tropisum for intracranial glioma and express BMSCs cell surface antigens and maintained high multiplication ability. BMSCs/IL-18/tk could induced obviously apoptosis of glioma in vitro and in vivo, and decrease microvessel density of intracranial glioma, and increase number of infiltration CD4~+ and CD8~+ lymphocyte inside intracranial glioma. BMSCs/IL-18/tk could prolong survival time of glioma bearing rats obviously.
理想的脑肿瘤的基因治疗应该是应用最佳的基因转移技术来转移最佳的治疗基因。自杀基因(HSV-tk/GCV)治疗肿瘤的体外及动物实验均取得了惊人的效果,但临床疗效不能令人满意,究其原因是基因治疗的载体无法在体内肿瘤组织中进行广泛分布,从而不能充分发挥其旁观者效应。
胶质瘤患者均表现出了T淋巴细胞抗肿瘤作用的减弱,并且,由于胶质瘤细胞缺乏细胞表面抗原导致的肿瘤抗原呈递作用的减弱,进一步减弱了抗肿瘤免疫。现在的观点认为,IL-18可通过T淋巴细胞、NK细胞发挥抗肿瘤作用,并且通过上调MHC分子的表达、促进CD4+辅助性T细胞向Th1型亚单位的分化增强细胞免疫。
骨髓基质干细胞(BMSCs)是具有迁移能力的细胞,并可稳定的表达外源性转染的某些基因,为胶质瘤基因治疗提供了新的思路。本实验拟用骨髓基质干细胞为载体,观察转染自杀基因的骨髓基质干细胞对脑胶质瘤的治疗作用,再引入IL-18,增强抗肿瘤免疫,观察两者相结合的治疗效果,为以骨髓基质干细胞为载体进行脑胶质瘤的基因治疗提供理论依据。以骨髓基质干细胞为载体,HSV-tk与IL-18联合治疗胶质瘤的研究,在国内外尚无报道。
1.骨髓基质干细胞的培养、鉴定及标记的研究
目的:研究骨髓基质干细胞的培养方法,并对其进行标记,为进一步的体内试验奠定基础。
方法:将出生2周的SD大鼠脱颈处死,消毒后取双侧股骨,自中间剪断后冲出骨髓细胞,加入含20%胎牛血清的完全培养液于37℃、5%CO2孵箱内培养,48小时后半量换液。以后每2-3天半量换液,7-8天传代一次。培养初期细胞较混杂,第6代后细胞形态为均一的纺锤形细胞。MTT法绘制生长曲线。
将培养第6代、已纯化的骨髓基质干细胞消化、计数后应用流式细胞术检测BMSCs CD31、CD34、CD45、CD90、CD44、CD71阳性率。取对数生长期的骨髓基质干细胞进行细胞爬片,待细胞生长至50%时加入BrdU(终浓度10μg/ml),24小时后取出玻片用PBS洗涤,4%多聚甲醛固定15分钟后进行免疫组化及免疫荧光染色,封片观察。将BrdU标记的骨髓基质干细胞注入大鼠颅内,3周后处死大鼠取脑,于穿刺点位置附近作石蜡切片并进行免疫荧光染色。
结果:在培养初期可见大量悬浮细胞,48小时后可见有散在纺锤形贴壁细胞,经多次半量换液后,未贴壁细胞逐渐被去除。细胞初期多以集落形式生长,第6代后细胞呈形态均一的纺锤形,分布均匀。绘制细胞生长曲线,可见细胞呈一种持续的增殖状态,培养2-3天后进入对数生长期。以流式细胞技术检测细胞表面抗原,结果显示为体积均一的细胞群, CD31、CD34、CD45的阳性率分别为5.67%、4.31%、4.42%, CD90、CD44、CD71的阳性率分别为97.17%、98.63%、95.86%。骨髓基质干细胞体外免疫组化显示88.75±4.38%的细胞可被BrdU标记。免疫荧光染色可见胞核染色明显,BrdU标记的骨髓基质干细胞注入体内后3周仍可见明显的BrdU着色,穿刺点位置可见密集的细胞。
结论:通过骨髓全细胞培养的方法可获得高纯度的BMSCs,并可在体外长期培养;采用BrdU标记骨髓基质干细胞具有较高的标记阳性率,并且标记效果稳定。
2.骨髓基质干细胞向脑胶质瘤的趋向性及神经转化潜能
目的:在于研究骨髓基质干细胞向脑胶质瘤的趋向性,及骨髓基质干细胞在体外环境中、在特定细胞因子诱导条件下的细胞转化,以及骨髓基质干细胞在脑胶质瘤环境中的转化情况。
方法:体外培养6~7代的BMSCs接种到预置有多聚赖氨酸包被的盖玻片的培养板内,细胞达到70%融合时,将盖玻片转移至含10%胎牛血清的Neurobasal培养基内培养,加入全反式视黄酸(0.5μmol/L)、脑源性神经生长因子(10ng/mL)。以正常培养的、未加诱导剂的BMSCs作为对照。4d后,取出盖玻片,4%多聚甲醛固定30min,用于免疫荧光检测细胞转化情况。
骨髓基质干细胞趋瘤性研究的体外实验采用空心柱试验及Transwell试验。培养大鼠脑胶质瘤C6细胞,待细胞达到对数生长期时,消化计数,制作大鼠脑胶质瘤模型。模型建立3天后随机抽取20只荷瘤大鼠,在肿瘤对侧大脑立体定向种植BMSCs(8×10~4个悬于2μL PBS)。移植后15d,4%多聚甲醛自大鼠心脏灌注后,取出脑组织制作病理切片。用于转化研究的脑组织制作冰冻切片。
结果:经诱导剂诱导转化3天后,在倒置显微镜下观察,细胞形态改变明显,部分细胞可伸出长的突起,少数突起可与周围的细胞或突起有紧密的接触,另有少部分细胞变圆、脱落;对照组中培养的BMSCs细胞形态未见明显变化。免疫荧光染色显示:在诱导组,GFAP阳性率21.1±6.3%,MAP2阳性率56.4±13.8%,Nestin阳性率8.3±5.2%;在对照组,未发现MAP2、Nestin阳性细胞,GFAP阳性率3.9±2.8%。
空心柱试验显示,随着培养时间的延长,BMSCs很快离开原种植位置,向C6细胞存在位置进行迁移,而后广泛迁移到胶质瘤细胞区域,而3T3细胞仍停留在原位。Transwell试验显示无细胞的培养液(1.1±0.7)和3T3细胞条件培养液(2.4±0.9)的培养孔仅有极个别的BMSCs迁移,两者之间无统计学差异(P>0.05),在C6细胞条件培养液(14.7±4.7)的培养孔有明显的BMSCs迁移,而更大的迁移细胞数是在C6细胞的培养孔(42.7±8.2)。BMSCs在细胞因子条件下的迁移: PDGF诱导的迁移细胞数量最多(32.7±6.3),其次是EGF(26.3±7.4)。b-FGF(7.5±2.8)和VEGF(9.4±3.5)诱导的迁移细胞最少,两者无统计学差异(P>0.05)。在所有诱导因素中,C6细胞诱导了最明显的迁移(P<0.05)。
体内趋瘤性试验显示,有大量的BrdU标记的BMSCs位于正常脑组织与肿瘤的交界位置,而在肿瘤内部只有少许BMSCs。体内转化的免疫荧光染色显示:脑内移植的用Hoechst33258标记的BMSCs部分呈Nestin阳性(8.32±3.41%),部分呈GFAP阳性(32.71±8.66%),部分呈MAP2阳性(11.88±5.16%)。提示移植的部分BMSCs可转化为神经前体细胞,部分可转化为神经元或胶质细胞(Fig7)。对照组2、对照组3中,移植BMSCs的GFAP阳性率分别为31.19±8.76%和29.32±7.75%, Nestin阳性阳性率分别为6.95±3.75%和7.21±3.31%,MAP2阳性率分别为8.76±3.77%和10.59±5.43%,三组间比较无统计学差异(P>0.05)。
结论:骨髓基质干细胞具有明显的向胶质瘤细胞迁移的特性,迁移细胞多聚集于肿瘤边缘,只有少数骨髓基质干细胞可达到肿瘤内部;骨髓基质干细胞在体外特殊诱导环境中及移植到荷瘤大鼠脑内后,可部分转化为神经前体细胞、神经元及胶质细胞。
3.转染HSV-tk的骨髓基质干细胞治疗脑胶质瘤的体内、外实验研究
目的:在于探讨以骨髓基质干细胞为载体,观察转染tk基因的骨髓基质干细胞对脑胶质瘤的杀伤作用。
方法:AdCMV-tk病毒扩增后用于感染骨髓基质干细胞,而后采用RT-PCR方法检测BMSCs/tk对tk基因的转录。而后进一步采用流式细胞术、MTT检测BMSCs/tk细胞表面抗原及生长曲线。Transwell试验检测BMSCs/tk体外趋瘤性;制作大鼠脑胶质瘤模型,将BMSCs/tk移植到肿瘤对侧脑组织,观察其体内趋瘤性。将BMSCs/tk与C6细胞混合培养,采用不同的效靶比及不同的GCV浓度,用MTT法、TUNEL、AnnexinV检测细胞存活率或凋亡率,观察BMSCs/tk的旁观者效应。进一步采用体内试验,观察BMSCs/tk的体内旁观者效应,观察荷瘤大鼠在治疗后的生存期改变。
结果:BMSCs/tk的细胞表面抗原、细胞生长曲线、体内外趋瘤性仍保持着BMSCs的特性。RT-PCR显示,AdCMV-tk腺病毒感染BMSCs后24小时即有明显的tk基因的转录,在48小时达到高峰,而后逐渐降低,在1个月时仍有少量tk基因转录。体外旁观者效应检测显示,随着效应细胞数量的增多,杀伤效果明显增强;随着GCV用量的增多,杀伤效果明显增强。体内试验显示,应用TUNEL法检测BMSCs/tk的体内抗脑胶质瘤作用,对比平均每个视野中肿瘤细胞的凋亡数,BMSCs/βgal组(1.65±1.09)、BMSCs组(1.50±1.43)、PBS组(1.45±1.28)及空白对照组(1.30±1.08)之间无明显差异(P>0.05),但均明显低于BMSCs/HSV-tk组(8.85±3.33) (P<0.05)。
结论:感染AdCMV-tk后, BMSCs可长时间的对tk基因进行转录;BMSCs/tk具有明显的颅内肿瘤趋向性,并保持旺盛的增殖活性;BMSCs/tk在体外及在脑胶质瘤荷瘤大鼠体内可诱导明显的肿瘤细胞凋亡,延长荷瘤大鼠生存期。
4. HSV-tk、IL-18联合修饰骨髓基质干细胞治疗脑胶质瘤的体内、外实验研究
目的:是采用IL-18转染BMSCs,观察BMSCs/IL-18的抗脑胶质瘤效果;进一步采用IL-18及tk联合修饰BMSCs,观察BMSCs/IL-18/tk的体内外抗肿瘤效果。
方法:LXSN/IL-18感染骨髓基质干细胞(BMSCs/IL-18),筛选出阳性克隆后,感染AdCMV/tk病毒(BMSCs/IL-18/tk),而后采用RT-PCR方法检测BMSCs/IL-18、BMSCs/IL-18/tk对IL-18基因、tk基因的转录。而后进一步采用流式细胞术、MTT检测BMSCs/IL-18、BMSCs/IL-18/tk细胞表面抗原及生长曲线。Transwell试验检测BMSCs/IL-18、BMSCs/IL-18/tk体外趋瘤性;制作大鼠脑胶质瘤模型,将BMSCs/IL-18、BMSCs/IL-18/tk移植到肿瘤对侧脑组织,观察其体内趋瘤性。将BMSCs/IL-18、BMSCs/IL-18/tk与C6细胞混合培养,采用不同的效靶比及不同的GCV浓度,用MTT法、TUNEL、AnnexinV检测细胞存活率或凋亡率,观察BMSCs/IL-18、BMSCs/IL-18/tk的旁观者效应。进一步采用体内试验,观察BMSCs/IL-18、BMSCs/IL-18/tk的体内抗脑胶质瘤作用。ELISA法检测BMSCs/IL-18、BMSCs/IL-18/tk对淋巴细胞分泌的影响。免疫组化方法检测BMSCs/IL-18、BMSCs/IL-18/tk治疗后肿瘤组织内部微血管密度、淋巴细胞浸润、细胞凋亡情况。MRI检测治疗后的肿瘤体积改变,观察荷瘤大鼠在治疗后的生存期改变。
结果:BMSCs/IL-18、BMSCs/IL-18/tk的细胞表面抗原、体内外趋瘤性仍保持着BMSCs的特性。细胞生长曲线显示,BMSCs/IL-18、BMSCs/IL-18/tk增殖活性有所下降。RT-PCR显示,BMSCs/IL-18、BMSCs/IL-18/tk可稳定转录IL-18基因, BMSCs/IL-18 /tk可较长时间稳定的转录tk基因,免疫组化显示BMSCs/IL-18、BMSCs/IL-18/tk稳定表达IL-18。体外研究显示,BMSCs/IL-18/tk可产生明显的旁观者效应,且随着效应细胞数量的增多,旁观者效应更加明显,而BMSCs/IL-18未表现出旁观者效应。ELISA法检测显示,BMSCs/IL-18、BMSCs/IL-18/tk可明显增加体外淋巴细胞分泌IFN-γ,并可增加荷瘤大鼠体内血清IFN-γ浓度。体内试验显示,BMSCs/IL-18、BMSCs/IL-18/tk可明显增加荷瘤大鼠脑内胶质瘤肿瘤组织中淋巴细胞浸润数量,减少肿瘤微血管密度,诱导肿瘤细胞凋亡。BMSCs/IL-18/tk可明显延长荷瘤大鼠的生存期。
结论:转染IL-18后,BMSCs/IL-18可稳定转录及表达IL-18基因;感染AdCMV-tk后,BMSCs/IL-18/tk可稳定的转录tk及IL-18; BMSCs/IL-18、BMSCs/IL-18/tk具有明显的颅内肿瘤趋向性,表达BMSCs特异性的表面抗原,保持旺盛的增殖活性;BMSCs/IL-18/tk在体外及在脑胶质瘤荷瘤大鼠体内可诱导明显的肿瘤细胞凋亡,减少肿瘤组织微血管密度,增加CD4~+、CD8~+淋巴细胞的浸润数量,延长荷瘤大鼠生存期。
Malignant glioma, the most common intracranial tumor, accounts for 30-50% of primary brain tumor in adults, with high morbidity, mortality and grim prognosis. Because currently available multiple modalities of treatment have not yet substantially changed the natural history of these lethal neoplasms, new therapeutic approaches are actively being investigated. Over the past few years, with the development in understanding the molecular biology and molecular genetics of glioma, gene therapy of tumor has been the hotspot and suicide gene therapy is one of the most favorite strategy used for malignant gliomas.
Ideal protocol of brain tumor gene therapy would employ the optimal gene transfer technique with the optical genes transferred. Suicide gene therapy (HSV-tk/GCV) has revealed the encouraging therapeutic effect on glioma in vitro and in vivo animal experiment. However, as far as clinical trials, the results were not satisfactory and the reason might be the poor by-stander effect because of the therapy gene can not distribute in tumor in vivo widely.
Antitumor effect of T lymphocytes of glioma patients were decreased, and because of the glioma antigen present ability was decreased owing to poverty cell special surface antigen, antitumor immunity effect was weakened further. According to present view, IL-18 could play its antitumor ability by activating T lymphocytes and natural killer cells, up-regulating expression of MHC and promoting CD4~+ helper lymphocytes differentiated to Th1 subset to enhance cell mediated immunity.
BMSCs have the ability of immigration and expression of transfected exogenous gene stably which offered the new route of gene therapy against glioma. The experiment planed to employ BMSCs as the therapy gene vector to observe the antitumor effect of BMSCs which infected suicide gene (BMSCs/tk). Furthermore, to enhance antitumor immunity, IL-18 was employed. The study provided a theoretical foundation for the use of BMSCs as vector against glioma. There was no report that BMSCs, as therapy gene vector, were transfected HSV-tk and IL-18 to treat intracranial glioma.
1. The initial research of culturing, characterization and labelling bone marrow stromal stem cells
Objective: The purpose of this part is to research how to culture, identity and label bone marrow stromal stem cells(BMSC), which established foundation for further study.
Methods: Single-cell suspension obtained from femurs of 2-3-week-old SD rats which were sacrificed and then incubated on plastic dishes in Dulbecco’s modified Eagle’s medium(DMEM) containing 20% heat-inactivated-fetal bovine serum(FBS) at 37℃with 5% CO_2 . 48 hours later, shake the culture bottle slowly and nonadherent cells were removed. Adherent cells were further cultured by exchanging half culture medium every 2-3 days to expend. After 7-8 days of incubation, the bone marrow-derived cells were confluent and expended by a 1:2 split. After in vitro culture-expended 6 passages, adherent bone marrow-derived cells were uniformly fibroblast-like in appearance. Cell growth curve was drown by means of MTT.
After subcultured 6 passages and cells were purified, to evaluate the BMSCs characterization, flow cytometry analysis was performed to detect the expression of CD31, CD45, CD90, CD34, CD71, CD44.After subcultured 6 passages, BMSCs, which were at exponential phase of growth, were cultured in flasks which contained several glass coverslips, and after 50% confluent the cells were pulsed for 24 hours with 10μg/ml 5-bromo-2-deoxyuridine (BrdU) (Sigma) in DMEM. Which was followed by fixation with 4% paraformaldehyde for 15 minutes, and then, immunohistochemistry and immunofluorescence were performed and the clips were observed under fluorescence microscope. After being labeled with BrdU, BMSCs were transplanted into rats, brain and the rats were sacrificed 3 weeks later. The brain tissue near the puncture site was made paraffin sections and immunofluorescence was performed.
Results: The results showed that at the initial time, there were lots of suspension cells in the culture dishes.48 hours later, there were several fusiform adherent cells. After several semi-exchange culture medium, non-adherent cells were removed and several cell colonies formed. The BMSCs became comparatively homogeneous in appearance and well-distributed as the cells were 6 passages. The cells were relatively elongated or spindle-shaped. Cell growth curve showed that the cell multiplied actively and were at exponential phase of growth 2-3 later. Surface phenotypic characterization of primary BMSCs detected by flow cytometry showed that the percentage of CD31, CD34, CD45, CD44, CD71, CD90 was 5.67%, 4.31%,4.42%, 97.17%, 98.63%, 95.86% respectively. In vitro immunohistochemistry showed that 88.75±4.38% cells could be labeled with BrdU. Immunofluorescence showed that nucleus were labeled obviously and after being transplanted into rat brain 3 weeks later, lots of BMSCs, which were labeled with BrdU, were observed near the puncture site.
Conclusions: The results showed that purified BMSCs could be harvested through culturing bone marrow cells and could expended culture in vitro in long-term. BMSCs could be labeled with BrdU high efficiency and stably.
2. Tropism for glioma and neural differentiation of bone marrow stromal cells
Objective: The aim was to observe tropism for intracranial glioma of bone marrow stromal cells (BMSCs) and their differentiation in brain of Glioma bearing rats or induced with certain cytokines in vitro.
Methods: After being subcultured in vitro 6-7 passages, BMSCs were seeded into flasks which contained glass coverslips coated with polylysine. After cells reached 70% confluence, the coverslips which contained BMSCs were displaced into Neurobasal medium containing 10% heat-inactivated-fetal bovine serum and alltrans retinoic acid (0.5μmol/L) and brain derived nerve growth factor (10ng/mL). BMSCs were cultured without induced cytokines as control group. 4 days later, the coverslips were taken out and fixation in 4% paraformaldehyde for 30 minutes followed by immunofluorescence to observe cells differentiation.
In vitro experiment of BMSCs tropism for glioma was performed by means of cylinder experiment and Transwell experiment. C6 cells were cultured and, after C6 were at exponential phase of growth, glioma bearing rats were established. 3 days later, 20 glioma bering rats were selected randomly and were transplanted BMSCs(8×10~4cells in 2μL PBS)in the contralateral hemisphere. 15 days later, the rats were fixation with 4% paraformaldehyde perfuse through hearts. And then, the brains were removed and pathological sections were made and frozen section were made for differentiation research.
Results: The results showed that, after being induced 3 days, observed under inverted microscope form, cells form were changed and some of the cells stretch out long branches and some of which got in touch with the other cells or branches. Several cells were rounding and shedding. The form of BMSCs in control group was not changed. Immunofluorescence showed that, in induced group, GFAP positive ratio was 21.1±6.3%,MAP2 positive ratio was 56.4±13.8%,Nestin positive ratio was 8.3±5.2%;in control group,there were no MAP2 and Nestin positive cells could be found,GFAP positive ratio was 3.9±2.8%。
In cylinder experiment, in contrast to 3T3 cells, which remained localized to the area of initial seeding, BMSCs migrated rapidly and interspersed throughout the glioma monolayer, far from the initial site of seeding. Transwell experiment showed that, exposure to cell-free medium (1.1±0.7) or to conditioned medium from 3T3 cells(2.4±0.9) resulting in low levels of migrating BMSCs, there were no statistic difference between the two groups(P>0.05), while exposure to conditioned medium from C6 cells(14.7±4.7) produced significant BMSCs migration, whereas, the largest number of the migration BMSCs was C6 cells(42.7±8.2). Among the four cytokines, PDGF intermediated the maximal migration (32.7±6.3), the 2~(nd) was EGF(26.3±7.4), whereas b-FGF and VEGF had no statistic difference(7.5±2.8, 9.4±3.5). Among all the induced effector, C6 cells induced the obviously immigration (P<0.05).
In vivo tropism for glioma experiment showed that there were lots of BMSCs labeled with BrdU located at the boundary of brain tissue and glioma and there were several BMSCs inside glioma. In vivo differentiation immunofluorescence showed that: some of the BMSCs labeled with Hoechst33258 were Nestin positive (8.32±3.41%), some were GFAP positive(32.71±8.66%) and some were MAP2 positive (11.88±5.16%). This suggested that some of the transplanted BMSCs differentiated into neural precursor cells and some differentiated into neuron and gliocyte. In control group2 and control group3, GFAP positive ratio of transplanted BMSCs was 31.19±8.76% and 29.32±7.75% repectively, Nestin positive ratio of transplanted BMSCs was 6.95±3.75% and 7.21±3.31% repectively and MAP2 positive ratio of transplanted BMSCs was 8.76±3.77% and 10.59±5.43% repectively. There was no statistic difference between the three groups (P>0.05).
Conclusions: BMSCs displayed extensive tropisum for glioma cells and most of immigration BMSCs located on the boundary of glioma and brain, only few immigration BMSCs were in glioma field. BMSCs, in in vitro induce environment or were transplanted into brain of glioma bearing rats, some of the transplanted BMSCs differentiated into neural precursor cells and some differentiated into neuron and gliocyte.
3. In vitro and in vivo study of anti-glioma activity of bone marrow stromal cells transfected with HSV-tk
Objective: The purpose of the research was to study the antitumor effect of BMSCs, as vector of therapy gene, after being transfected with tk gene.
Methods: After being amplification, AdCMV-tk was used to transfected BMSCs (BMSCs/tk). RT-PCR was performed to detect transcription of tk gene by BMSCs/tk. Flow cytometry and MTT were performed to detect BMSCs/tk cells surface antigen and cell growth curve. Transwell experiment was performed to examine BMSCs/tk tropism for glioma in vitro. Glioma bearing rats were established and BMSCs/tk were transplanted into contralateral hemisphere to observe their tropism for glioma in vivo. Co-culture BMSCs/tk and C6, and then, different effector-target ratio and different GCV dose were introduced followed by cell survival ratio and cell apoptosis ratio examination by means of MTT, TUNEL and Annexin V. Experiment were performed to study by-stander effect of BMSCs/tk in vivo and survival time of glioma bearing rats were recorded after being treated with BMSCs/tk-GCV.
Results: The results showed that, the cell surface antigens, cell growth curve and in vitro tropism for glioma of BMSCs were not changed after being infected with AdCMV-tk. RT-PCR results showed that, after being infected with AdCMV-tk 24 hours later, BMSCs/tk could transcribe tk gene obviously and reached its peak at 48 hours. And then, transcription of tk decreased gradually and decreased obviously 1 month later. In vitro by-stander effect experiment showed that, with the increase of effector or GCV dose, kill activity was enhanced obviously. In vivo experiment showed that, by means of TUNEL examination, apoptosis cells number in every view inside glioma was 1.65±1.09 in BMSCs/βgal group, 1.50±1.43 in BMSCs group, 1.45±1.28 in PBS group and 1.30±1.08 in control group and there were no statistic difference between these groups (P>0.05). But the largest apoptosis number was observed in BMSCs/HSV-tk group (8.85±3.33) (P<0.05).
Conclusions: After being transfected with AdCMV-tk, BMSCs could transcript tk gene long term. BMSCs/tk displayed extensive tropisum for intracranial glioma and maintained high multiplication ability. BMSCs/tk induced obviously apoptosis of glioma in vitro and in vivo and prolonged survival time of glima bearing rats. 4. In vivo and in vitro study of therapy effect of bone marrow stromal cells co-transfected with HSV-tk and interleukin 18 on glioma
Objective: The purpose of the study was to observe antitumor effect of BMSCs after being transfected with IL-18. Furthermore, antitumor effect of BMSCs, after being co-transfected with HSV-tk and IL-18, were observed through in vitro and in vivo experiments.
Methods: After being infected with LXSN/IL-18, BMSCs/IL-18 clone was selected followed by infected with AdCMV/tk, thus, BMSCs/IL-18 and BMSCs/IL-18/tk was harvested. RT-PCR were performed to detect the gene transcription of IL-18 and tk by BMSCs/IL-18 and BMSCs/IL-18/tk. Cells surface antigen and cell growth curve were examined of BMSCs/IL-18 and BMSCs/IL-18/tk through flow cytometry and MTT. Transwell experiment was performed to observe in vitro tropism for glioma. Glioma bearing rats were established and BMSCs/IL-18 and BMSCs/IL-18/tk were transplanted into contralateral hemisphere to observed their tropism for glioma in vivo. To observe by-stander effector of BMSCs/IL-18 and BMSCs/IL-18/tk, co-culture BMSCs/IL-18 and C6 or BMSCs/IL-18/tk and C6, and different effector target ratio and different GCV dose were introduced, and then, cell survival ratio and apoptosis ratio was examined by means of MTT, TUNEL and Annexin V. In vivo experiment was performed to detect BMSCs/IL-18 and BMSCs/IL-18/tk antitumor effect. ELISA test was performed to detect the effect of BMSCs/IL-18 and BMSCs/IL-18/tk on lymphocyte secretion of IFN-γ. Immunohistochemistry was introduced to detect microvessel density, lymphocyte infiltration and cell apoptosis inside glioma after being treated with BMSCs/IL-18 and BMSCs/IL-18/tk. MRI examination was performed to observe the volume change of intracranial glioma after treatment and survival time were recorded.
Results: The results showed that the cell surface antigen and tropism for glioma in vitro was not changed after BMSCs were infected with LXSN/IL-18 or LXSN/IL-18 and AdCMV/tk. Cell growth curve showed that BMSCs/IL-18 and BMSCs/IL-18/tk had lower multiply speed than BMSCs. RT-PCR showed that BMSCs/IL-18 and BMSCs/IL-18/tk could transcript IL-18 gene stably and BMSCs/IL-18/tk could transcript tk gene long term. Immunohistochemistry showed that BMSCs/IL-18 and BMSCs/IL-18/tk could express IL-18 stably. In vitro experiment showed that BMSCs/IL-18/tk could display obviously by-stander effect, and with the increase of effectors, by-stander effect was more enhanced. BMSCs/IL-18 did not display by-stander effect. ELISA examination demonstrated that BMSCs/IL-18 and BMSCs/IL-18/tk could increase IFN-γsecretion of lymphocyte and increase IFN-γconcentration of glioma bearing rats serum. In vivo examination showed that BMSCs/IL-18 and BMSCs/IL-18/tk could increase infiltration lymphocytes number and decrease microvessel density and induced glioma cells apoptosis and glioma bearing rats survival time were prolonged obviously after being treated with BMSCs/IL-18/tk.
Conclusions: After being transfected with IL-18, BMSCs/IL-18 could transcript and express IL-18 stably. After being transfected with AdCMV-tk, BMSCs/IL-18/tk could transcript tk and IL-18 stably. BMSCs/IL-18 and BMSCs/IL-18/tk displayed extensive tropisum for intracranial glioma and express BMSCs cell surface antigens and maintained high multiplication ability. BMSCs/IL-18/tk could induced obviously apoptosis of glioma in vitro and in vivo, and decrease microvessel density of intracranial glioma, and increase number of infiltration CD4~+ and CD8~+ lymphocyte inside intracranial glioma. BMSCs/IL-18/tk could prolong survival time of glioma bearing rats obviously.
引文
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1 Arnhold S,Klein H,Klinz FJ,et al.Human bone marrow stroma cells display certain neural characteristics and integrate in the subventricular compartment after injection into the liquor system.Eur J Cell Biol,2006,85 (6):551-565
2 Hamada H, Kobune M, Nakamura K, et al. Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy.Cancer Sci. 2005 ,96(3):149-56
3 Aboody KS,Rainov NG,Rainov NG,et al.Neural stem cells display extensive tropism for pathology in adult brain: Evidence from intracranial gliomas. Proc Natl Acad USA, 2000; 97(23): 12846-12851
4 Lee J, Kuroda S, Shichinohe H, et al. Migration and differentiation of nuclear fluorescence-labeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice.Neuropathology, 2003, 23(3):169-180
5 Buzanska L, Machaj EK, Zablocka B, et al. Human cord blood-derived cells attain neuronal and glial features in vitro. J Cell Sci.,2002,115(10):2131-2138
6 Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res,2002 ,69(6):908-917
7 Pu P, Kang C, Li J, et al. The effects of antisense AKT2 RNA on the inhibition of malignant glioma cell growth in vitro and in vivo. J Neurooncol, 2006,76(1): 1-11
8 Borlongan CV, Lind JG, Dillon-Carter O, et al. Intracerebral xenografts of mouse bone marrow cells in adult rats facilitate restoration of cerebral blood flow and blood-brain barrier. Brain Res, 2004, 1009(1-2):26-33
9 Sato H, Kuwashima N, Sakaida T, et al. Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors. Cancer Gene Ther, 2005, 12(9):757-768 10 Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas.Cancer Res, 2005, 65(8):3307-3318
11 Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 2002, 418 (6893):41-49
12 Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science, 2000, 290 (5497):1779-1782
13 Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood. 2003,102(10):3483-93
14 Lee J, Kuroda S, Shichinohe H, et al. Migration and differentiation of nuclear fluorescence-labeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice.Neuropathology. 2003,23(3):169-80
15 Sanchez-Ramos JR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res. 2002,69(6):880-93
16 Nakano K, Migita M, Mochizuki H, et al. Differentiation of transplantedbone marrow cells in the adult mouse brain. Transplantation. 2001,71(12):1735-40
17 Rismanchi N, Floyd CL, Berman RF, et al. Cell death and long-term maintenance of neuron-like state after differentiation of rat bone marrow stromal cells: a comparison of protocols.Brain Res. 2003,991(1-2):46-55
18 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000,61(4):364-70
19 Hermann A, Gastl R, Liebau S, et al . Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells. J Cell Sci. 2004 ,117(Pt 19):4411-22
20 Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice.Science. 2000,290(5497):1775-9
21 Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow.Science. 2000,290(5497):1779-82
22 Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains.Proc Natl Acad Sci U S A. 1999,96(19):10711-6
23 Nakamura S, Takeda Y, Kanno M, et al. Application of bromodeoxyuridine (BrdU) and anti-BrdU monoclonal antibody for the in vivo analysis of proliferative characteristics of human leukemia cells in bone marrow. Oncology, 1991, 48(4): 285-289
24 Deng W, Obrocka M, Fischer I, et al. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun. 2001, 282(1):148-52
25 Kohyama J, Abe H, Shimazaki T, et al. Brain from bone: efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts toneurons with Noggin or a demethylating agent. Differentiation. 2001,68(4-5):235-44
26 Jiang Y, Henderson D, Blackstad M, et al. Neuroectodermal differentiation from mouse multipotent adult progenitor cells.Proc Natl Acad Sci U S A. 2003, 100 Suppl 1:11854-60
27 Aklyama Y, Radtke C, Kocsis JD.Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J Neurosci. 2002,22(15):6623-30
28 Irons H, Lind JG, Wakade CG, et al. Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immunologic response.Cell Transplant. 2004,13(3):283-94
29 Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood. 2003,102(10):3483-93
30 Sato H, Kuwashima N, Sakaida T, et al. Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors. Cancer Gene Ther. 2005 ,12(9):757-68
31 Borlongan CV, Lind JG, Dillon-Carter O, et al. Intracerebral xenografts of mouse bone marrow cells in adult rats facilitate restoration of cerebral blood flow and blood-brain barrier.Brain Res. 2004,1009(1-2):26-33
32 Chen Q, Long Y, Yuan X, et al. Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors.J Neurosci Res. 2005,80(5):611-9
33 Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res. 2004,94(5):678-85
34 Annabi B, Naud E, Lee YT, et al. Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors.J Cell Biochem. 2004,91(6):1146-58
35 Annabi B,Lee YT, Turcotte S,et al. Hypoxia promotes bone marrow-derived stromal cell migration and tube formation. Stell cells.2003a 21L337-347
36 Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002,99(10):3838-43
37 Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood. 2003,101(9):3722-9
30 Tse WT, Pendleton JD, Beyer WM, et al.Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.Transplantation. 2003,75(3):389-97
38 Nakamura K, Ito Y, Kawano Y, et al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther. 2004 ,1(14):1155-64
39 Lee J, Elkahloun AG, Messina SA, et al. Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells: a potential antiglioma cellular vector.Cancer Res. 2003,63(24):8877-89
40 Abdallah BM, Haack-Sorensen M, Burns JS,et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite extensive proliferation.Biochem Biophys Res Commun,2005, 326(3):527-38
41 Burns JS, Abdallah BM, Guldberg P,et al. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells. Cancer Res, 2005,65(8):3126-35
42 Liu Z, Li Y, Qu R,et al. Axonal sprouting into the denervated spinal cord and synaptic and postsynaptic protein expression in the spinal cord after transplantation of bone marrow stromal cell in stroke rats.Brain Res. 2007Feb 27; [Epub ahead of print]
43 Tseng PY, Chen CJ, Sheu CC, et al. Spontaneous differentiation of adult rat marrow stromal cells in a long-term culture.J Vet Med Sci. 2007,69(2):95-102
44 Lu D, Qu C, Goussev A,et al. Treatment of traumatic brain injury with a combination therapy of marrow stromal cells and atorvastatin in rats.Neurosurgery. 2007,60(3):546-53; discussion 553-4
45 Wright KT, El Masri W, Osman A, et al. Bone marrow stromal cells stimulate neurite outgrowth over neural proteoglycans (CSPG), myelin associated glycoprotein and Nogo-A.Biochem Biophys Res Commun. 2007,354(2):559-66
46 Chen CJ, Ou YC, Liao SL,et al. Transplantation of bone marrow stromal cells for peripheral nerve repair.Exp Neurol. 2007 ,204(1):443-53
47 Yang LY, Huang TH, Ma L. ,et al. Bone marrow stromal cells express neural phenotypes in vitro and migrate in brain after transplantation in vivo.Biomed Environ Sci. 2006 ,19(5):329-35
48 Nandoe RD, Hurtado A, Levi AD, et al. Bone marrow stromal cells for repair of the spinal cord: towards clinical application.Cell Transplant. 2006;15(7):563-77
1 Cho HS, Lee HR, Kim MK.Bystander-mediated regression of murine neuroblastoma via retroviral transfer of the HSV-TK gene. J Korean Med Sci. 2004,19(1):107-12
2 Asklund T, Appelskog IB, Ammerpohl O, et al. Gap junction-mediatedbystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene.Exp Cell Res. 2003,284(2):185-95
3 Ammerpohl O, Thormeyer D, Khan Z,et al.HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells. Biochem Biophys Res Commun. 2004,324(1):8-14
4 Asklund T, Appelskog IB, Ammerpohl O,et al.Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein and connexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells. Eur J Cancer. 2004 ,40(7):1073-81
5 Herrlinger U, Woiciechowski C, Sena-Esteves M, et al.Neural precursor cells for delivery of replication-conditional HSV-1 vectors to intracerebral gliomas. Mol Ther. 2000 ,1(4):347-57
6 Bi X, Zhang JZ. Experimental study of thymidine kinase gene therapy of neuroblastoma in vitro and in vivo. Pediatr Surg Int. 2003,19(5):400-5
7 van Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSV-tk/GCV gene therapy. A review.Curr Gene Ther. 2002,2(3):307-22
8 Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature,2002 ,418(6893):41-49
9 Sandmair AM, Turunen M, Tyynela K, et al.Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: effect of the percentage of thymidine kinase-positive glioma cells on treatment effect, survival time, and tissue reactions.Cancer Gene Ther. 2000,7(3):413-21
10 Rainov NG, Kramm CM, Banning U,et al.Immune response induced by retrovirus-mediated HSV-tk/GCV pharmacogene therapy in patients with glioblastoma multiforme. Gene Ther. 2000,7(21):1853-8
11 Asklund T, Appelskog IB, Ammerpohl O, et al. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene.Exp Cell Res.2003,284(2):185-95
12 Hadaczek P, Mirek H, Berger MS,et al. Limited efficacy of gene transfer in herpes simplex virus-thymidine kinase/ganciclovir gene therapy for brain tumors. J Neurosurg. 2005 ,102(2):328-35
13 Uhl M, Weiler M, Wick W, et al.Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas.Biochem Biophys Res Commun. 2005,328(1):125-9
14 Gentry BG, Im M, Boucher PD, et al.GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity.Gene Ther. 2005,12(13):1033-41
15 Mavria G, Harrington KJ, Marshall CJ, et al. In vivo efficacy of HSV-TK transcriptionally targeted to the tumour vasculature is augmented by combination with cytotoxic chemotherapy.J Gene Med. 2005 ,7(3):263-75
16 Moriuchi S, Glorioso JC, Maruno M, et al.Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkappaBalpha and HSV thymidine kinase.Cancer Gene Ther. 2005,12(5):487-96
17 Kanazawa T, Mizukami H, Okada T, et al. Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. Gene Ther. 2003,10(1):51-8
18 Tsuchiyama T, Kaneko S, Nakamoto Y, et al. Enhanced antitumor effects of a bicistronic adenovirus vector expressing both herpes simplex virus thymidine kinase and monocyte chemoattractant protein-1 against hepatocellular carcinoma. Cancer Gene Ther. 2003 ,10(4):260-9
19 Brockstedt DG, Diagana M, Zhang Y, et al. Development of anti-tumor immunity against a non-immunogenic mammary carcinoma through in vivo somatic GM-CSF, IL-2, and HSVtk combination gene therapy. Mol Ther. 2002,6(5):627-36
20 Chang JW, Lee H, Kim E, et al. Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6glioma. Neurosurgery. 2000 ,47(4):931-8; discussion 938-9
21 Desaknai S, Lumniczky K, Esik O, et al. Local tumour irradiation enhances the anti-tumour effect of a double-suicide gene therapy system in a murine glioma model. J Gene Med. 2003 ,5(5):377-85
22 Boucher PD, Ostruszka LJ, Murphy PJ, et al. Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy. Gene Ther. 2002,9(15):1023-30
1 Cho HS, Lee HR, Kim MK.Bystander-mediated regression of murine neuroblastoma via retroviral transfer of the HSV-TK gene. J Korean Med Sci. 2004,19(1):107-12
2 Asklund T, Appelskog IB, Ammerpohl O, et al. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene. Exp Cell Res. 2003,284(2):185-95
3 Ammerpohl O, Thormeyer D, Khan Z,et al.HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells. Biochem Biophys Res Commun. 2004,324(1):8-14
4 Asklund T, Appelskog IB, Ammerpohl O,et al.Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein andconnexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells. Eur J Cancer. 2004 ,40(7):1073-81
5 Herrlinger U, Woiciechowski C, Sena-Esteves M, et al.Neural precursor cells for delivery of replication-conditional HSV-1 vectors to intracerebral gliomas. Mol Ther. 2000 ,1(4):347-57
6 Bi X, Zhang JZ. Experimental study of thymidine kinase gene therapy of neuroblastoma in vitro and in vivo. Pediatr Surg Int. 2003,19(5):400-5
7 van Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSV-tk/GCV gene therapy. A review.Curr Gene Ther. 2002,2(3):307-22
8 Sandmair AM, Turunen M, Tyynela K, et al.Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: effect of the percentage of thymidine kinase-positive glioma cells on treatment effect, survival time, and tissue reactions.Cancer Gene Ther. 2000,7(3):413-21
9 Rainov NG, Kramm CM, Banning U,et al.Immune response induced by retrovirus-mediated HSV-tk/GCV pharmacogene therapy in patients with glioblastoma multiforme. Gene Ther. 2000,7(21):1853-8
10 Asklund T, Appelskog IB, Ammerpohl O, et al. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene.Exp Cell Res. 2003,284(2):185-95
11 Hadaczek P, Mirek H, Berger MS,et al. Limited efficacy of gene transfer in herpes simplex virus-thymidine kinase/ganciclovir gene therapy for brain tumors. J Neurosurg. 2005 ,102(2):328-35
12 Uhl M, Weiler M, Wick W, et al.Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas.Biochem Biophys Res Commun. 2005,328(1):125-9
13 Gentry BG, Im M, Boucher PD, et al.GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity.Gene Ther. 2005,12(13):1033-41
14 Mavria G, Harrington KJ, Marshall CJ, et al. In vivo efficacy of HSV-TK transcriptionally targeted to the tumour vasculature is augmented by combination with cytotoxic chemotherapy.J Gene Med. 2005 ,7(3):263-75
15 Moriuchi S, Glorioso JC, Maruno M, et al.Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkappaBalpha and HSV thymidine kinase.Cancer Gene Ther. 2005,12(5):487-96
16 Kanazawa T, Mizukami H, Okada T, et al. Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. Gene Ther. 2003,10(1):51-8
17 Tsuchiyama T, Kaneko S, Nakamoto Y, et al. Enhanced antitumor effects of a bicistronic adenovirus vector expressing both herpes simplex virus thymidine kinase and monocyte chemoattractant protein-1 against hepatocellular carcinoma. Cancer Gene Ther. 2003 ,10(4):260-9
18 Brockstedt DG, Diagana M, Zhang Y, et al. Development of anti-tumor immunity against a non-immunogenic mammary carcinoma through in vivo somatic GM-CSF, IL-2, and HSVtk combination gene therapy. Mol Ther. 2002,6(5):627-36
19 Chang JW, Lee H, Kim E, et al. Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6 glioma. Neurosurgery. 2000 ,47(4):931-8; discussion 938-9
20 Desaknai S, Lumniczky K, Esik O, et al. Local tumour irradiation enhances the anti-tumour effect of a double-suicide gene therapy system in a murine glioma model. J Gene Med. 2003 ,5(5):377-85
21 Boucher PD, Ostruszka LJ, Murphy PJ, et al. Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy. Gene Ther. 2002,9(15):1023-30
22 Hwang KS, Cho WK, Yoo J, et al.Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity. Cancer Gene Ther, 2004 ,11(6):397-407
23 Luo Y, Zhou H, Mizutani M,et al.A DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 induces long-lived T-cell memory against tumor recurrence.Cancer Res, 2005 ,65(8):3419-3427
24 Kohyama M, Saijyo K, Hayasida M, et al.Direct activation of human CD8+ cyto- toxic T lymphocytes by interleukin-18. Jpn J Cancer Res,1998 ,89(10): 1041- 1046
25 Kikuchi T, Akasaki Y, Joki T, et al.Antitumor activity of interleukin-18 on mouse glioma cells.J Immunother, 2000 ,23(2):184-189
26 Wataru Hasiimoto, Fumiaki Tanaka, Paul D,et al. Natural killer, but not natural killer T. cells play a necessary role in the promotion of an innate antitumor response induced by IL-18. Int J Cancer. 2003 ,103(4):508-513
27 Tanaka F, Hashimoto W, Robbins PD, et al. Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adeno- viral vector expressing biologically active IL-18.Gene Ther,2002,9(21): 1480- 1486
28 Hashimoto W, Osaki T, Okamura H,et al. Differential antitumor effects of administration of recombinant IL-18 or recombinant IL-12 are mediated primarily by Fas-Fas ligand- and perforin-induced tumor apoptosis, respectively. J Immunol, 1999,163(2):583-589
29 Cao R, Farnebo J, Kurimoto M, et al. Interleukin-18 acts as an angiogenesis and tumor suppressor. FASEB J,1999 ,13(15):2195-2202
30 Coughlin CM, Salhany KE, Wysocka M, et al. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis.J Clin Invest,1998 ,101(6):1441-1452
31 Osaki T, Peron JM, Cai Q, et al. IFN-gamma-inducing factor/IL-18 administration mediates IFN-gamma- and IL-12-independent antitumor effects.J Immunol,1998 ,160(4):1742-1749
32 Baxevanis CN, Gritzapis AD, Papamichail M.In vivo antitumor activity of NKT cells activated by the combination of IL-12 and IL-18. J Immunol,2003 ,171(6):2953-2959
33 Kito T, Kuroda E, Yokota A, et al. Cytotoxicity in glioma cells due to interleukin-12 and interleukin-18-stimulated macrophages mediated by interferon-gamma-regulated nitric oxide.J Neurosurg,2003,98(2):385- 392
34 Yamanaka R, Xanthopoulos KG.Induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis DNA encoding gp100 and IL-18. DNA Cell Biol,2005 ,24 (5):317-324
35 Redlinger RE Jr, Mailliard RB, Lotze MT, et al. Synergistic interleukin-18 and low-dose interleukin-2 promote regression of established murine neuroblastoma in vivo. J Pediatr Surg,2003,38 (3):301 -307
36 Wigginton JM, Lee JK, Wiltrout TA, et al. Synergistic engagement of an ineffec- tive endogenous anti-tumor immune response and induction of IFN- gamma and Fas-ligand - dependent tumor eradication by combined administration of IL-18 and IL-2. J Immunol,2002 ,169(8): 4467-4474
37 Tatsumi T, Huang J, Gooding WE, et al. Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity. Cancer Res,2003,63(19):6378-6386
38 Tanaka F, Hashimoto W, Robbins PD, et al. Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adeno- viral vector expressing biologically active IL-18. Gene Ther,2002 ,9(21):1480- 1486
39 Tanaka F, Hashimoto W,Okamura H,et al. Rapid generation of potent and tumor-specific cytotoxic T lymphocytes by interleukin 18 using dendritic cells and natural killer cells.Cancer Res,2000 ,60(17):4838-4844
40 Chung SW, Cohen EP, Kim TS.Generation of tumor-specific cytotoxic T lympho- cyte and prolongation of the survival of tumor-bearing mice using interleukin-18 -secreting fibroblasts loaded with an epitope peptide. Vaccine, 2004,22(20):2547- 2557
41 Zhang Y, Wang C, Zhang Y,et al.C6 glioma cells retrovirally engineered to express IL-18 and Fas exert FasL-dependent cytotoxicity against glioma formation.Biochem Biophys Res Commun,2004,325 (4) : 1240 -1245
42 Wang Q, Yu H, Zhang L, et al.Vaccination with IL-18 gene-modified, superantigen-coated tumor cells elicits potent antitumor immune response. J Cancer Res Clin Oncol,2001,127(12):718-726
43 Ju DW, Yang Y, Tao Q, et al. Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity.Gene Ther,2000,7(19):1672-1679
44 Xia D,Li F, Xiang J. Engineered fusion hybrid vaccine of IL-18 gene-modified tumor cells and dendritic cells induces enhanced antitumor immunity. Cancer Biother Radiopharm,2004 ,19(3):322-330
45 Golab J. Interleukin 18--interferon gamma inducing factor--a novel player in tumour immunotherapy? Cytokine,2000 ,12(4):332-338
1 Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature,2002 ,418(6893):41-49
2 Deng W, Obrocka M, Fischer I, et al. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commol/Lun,2001,282(1):148-152
3 Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res,2002 ,69(6):908-917
4 Rismanchi N, Floyd CL, Berman RF, et al. Cell death and long-term maintenance of neuron-like state after differentiation of rat bone marrow stromal cells: a comparison of protocols.Brain Res, 2003,991(1-2):46-55
5 Kohyama J, Abe H, Shimazaki T, et al. Brain from bone: efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent.Differentiation,2001,68(4-5):235-244
6 Jiang Y, Henderson D, Blackstad M, et al. Neuroectodermal differentiation from mouse multipotent adult progenitor cells.Proc Natl Acad Sci U S A,2003 ,100 Suppl 1:11854-11860
7 Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science,2000 ,290(5497):1779-1782
8 Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice.Science,2000,290 (5497):1775-1779
9 Lee J, Kuroda S, Shichinohe H, et al. Migration and differentiation of nuclear fluorescence-labeled bone marrow stromal cells aftertransplantation into cerebral infarct and spinal cord injury in mice.Neuropathology,2003 ,23(3):169-180
10 Nakano K, Migita M, Mochizuki H, et al. Differentiation of transplanted bone marrow cells in the adult mouse brain. Transplantation,2001,71(12):1735-1740
11 Aklyama Y, Radtke C, Kocsis JD.Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J Neurosci,2002 ,22(15):6623-6230
12 Irons H, Lind JG, Wakade CG, et al. Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immol/Lunologic response.Cell Transplant,2004,13(3): 283-294
13 Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood, 2003,102(10):3483-3493
14 Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas.Cancer Res, 2005 ,65(8):3307-3318
15 Sato H, Kuwashima N, Sakaida T, et al. Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors. Cancer Gene Ther,2005 ,12(9):757-68
16 Hamada H, Kobune M, Nakamura K, et al. Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy.Cancer Sci,2005 ,96(3):149-156
17 Borlongan CV, Lind JG, Dillon-Carter O, et al. Intracerebral xenografts of mouse bone marrow cells in adult rats facilitate restoration of cerebral blood flow and blood-brain barrier.Brain Res,2004,1009(1-2):26-33
18 Chen Q, Long Y, Yuan X, et al. Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors.J Neurosci Res,2005,80(5):611-619
19 Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cellsexpress genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res,2004 ,94(5):678-685
20 Annabi B, Naud E, Lee YT, et al. Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors.J Cell Biochem,2004 ,91(6):1146-1158
21 Annabi B,Lee YT, Turcotte S,et al. Hypoxia promotes bone marrow-derived stromal cell migration and tube formation. Stell cells.2003a 21L337-347
22 Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood,2002 , 99(10):3838-3843
23 Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood,2003, 101(9):3722-3729
24 Tse WT, Pendleton JD, Beyer WM, et al.Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.Transplantation,2003 ,75(3):389-397
25 Nakamura K, Ito Y, Kawano Y, et al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther,2004,11(14):1155-1164.
26 Lee J, Elkahloun AG, Messina SA, et al. Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells: a potential antiglioma cellular vector.Cancer Res,2003,63(24):8877-8889
27 Abdallah BM, Haack-Sorensen M, Burns JS,et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immol/Lortalized by human telomerase reverse transcriptase gene despite extensive prolif eration.Biochem Biophys Res Commol/Lun,2005 , 326(3):527-538
28 Burns JS, Abdallah BM, Guldberg P,et al. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immol/Lortalized human mesenchymal stem cells. Cancer Res,2005 ,65(8):3126-3135
1 Hwang KS, Cho WK, Yoo J, et al.Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity. Cancer Gene Ther, 2004 ,11(6):397-407
2 Luo Y, Zhou H, Mizutani M,et al.A DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 induces long-lived T-cell memory against tumor recurrence.Cancer Res, 2005 ,65(8):3419-3427
3 Kohyama M, Saijyo K, Hayasida M, et al.Direct activation of human CD8+ cyto- toxic T lymphocytes by interleukin-18. Jpn J Cancer Res,1998 ,89(10): 1041- 1046
4 Kikuchi T, Akasaki Y, Joki T, et al.Antitumor activity of interleukin-18 on mouse glioma cells.J Immunother, 2000 ,23(2):184-189
5 Wataru Hasiimoto, Fumiaki Tanaka, Paul D,et al. Natural killer, but not natural killer T. cells play a necessary role in the promotion of an innate antitumor response induced by IL-18. Int J Cancer. 2003 ,103(4):508-513
6 Tanaka F, Hashimoto W, Robbins PD, et al. Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adeno- viral vector expressing biologically active IL-18.Gene Ther,2002,9(21): 1480- 1486
7 Hashimoto W, Osaki T, Okamura H,et al. Differential antitumor effects of administration of recombinant IL-18 or recombinant IL-12 are mediated primarily by Fas-Fas ligand- and perforin-induced tumor apoptosis, respectively. J Immunol, 1999,163(2):583-589
8 Cao R, Farnebo J, Kurimoto M, et al. Interleukin-18 acts as an angiogenesis and tumor suppressor. FASEB J,1999 ,13(15):2195-2202
9 Coughlin CM, Salhany KE, Wysocka M, et al. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis.J Clin Invest,1998 ,101(6):1441-1452
10 Osaki T, Peron JM, Cai Q, et al. IFN-gamma-inducing factor/IL-18 administration mediates IFN-gamma- and IL-12-independent antitumor effects.J Immunol,1998 ,160(4):1742-1749
11 Baxevanis CN, Gritzapis AD, Papamichail M.In vivo antitumor activity of NKT cells activated by the combination of IL-12 and IL-18. J Immunol,2003 ,171(6):2953-2959
12 Kito T, Kuroda E, Yokota A, et al. Cytotoxicity in glioma cells due to interleukin-12 and interleukin-18-stimulated macrophages mediated by interferon-gamma-regulated nitric oxide.J Neurosurg,2003,98(2):385-392
13 Yamanaka R, Xanthopoulos KG.Induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis DNA encoding gp100 and IL-18. DNA Cell Biol,2005 ,24(5):317-324
14 Redlinger RE Jr, Mailliard RB, Lotze MT, et al. Synergistic interleukin-18 and low-dose interleukin-2 promote regression of established murine neuroblastoma in vivo. J Pediatr Surg,2003,38(3):301-307
15 Wigginton JM, Lee JK, Wiltrout TA, et al. Synergistic engagement of an ineffec- tive endogenous anti-tumor immune response and induction of IFN- gamma and Fas-ligand - dependent tumor eradication by combined administration of IL-18 and IL-2. J Immunol,2002 ,169(8): 4467-4474
16 Tatsumi T, Huang J, Gooding WE, et al. Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity. Cancer Res,2003,63(19):6378-6386
17 Tanaka F, Hashimoto W, Robbins PD, et al. Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adeno- viral vector expressing biologically active IL-18. Gene Ther,2002 ,9(21):1480- 1486
18 Tanaka F, Hashimoto W,Okamura H,et al. Rapid generation of potent and tumor-specific cytotoxic T lymphocytes by interleukin 18 using dendritic cells and natural killer cells.Cancer Res,2000 ,60(17):4838-4844
19 Chung SW, Cohen EP, Kim TS.Generation of tumor-specific cytotoxic T lympho- cyte and prolongation of the survival of tumor-bearing mice using interleukin-18 -secreting fibroblasts loaded with an epitope peptide. Vaccine, 2004,22(20):2547- 2557
20 Zhang Y, Wang C, Zhang Y,et al.C6 glioma cells retrovirally engineered to express IL-18 and Fas exert FasL-dependent cytotoxicity against glioma formation.Biochem Biophys Res Commun,2004 ,325(4):1240-1245
21 Wang Q, Yu H, Zhang L, et al.Vaccination with IL-18 gene-modified, superantigen-coated tumor cells elicits potent antitumor immune response. J Cancer Res Clin Oncol,2001,127(12):718-726
22 Ju DW, Yang Y, Tao Q, et al. Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity.Gene Ther,2000,7(19):1672-1679
23 Xia D,Li F, Xiang J. Engineered fusion hybrid vaccine of IL-18 gene-modified tumor cells and dendritic cells induces enhanced antitumor immunity. Cancer Biother Radiopharm,2004 ,19(3):322-330
24 Golab J. Interleukin 18--interferon gamma inducing factor--a novel player in tumour immunotherapy? Cytokine,2000 ,12(4):332-338
2 Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells.Blood. 2003,102(10):3483-93
3 Lee J, Kuroda S, Shichinohe H, et al. Migration and differentiation of nuclear fluorescence-labeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice.Neuropathology. 2003,23(3):169-80
4 Sanchez-Ramos JR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res. 2002,69(6):880-93
5 Nakano K, Migita M, Mochizuki H, et al. Differentiation of transplanted bone marrow cells in the adult mouse brain. Transplantation. 2001,71(12):1735-40
6 Rismanchi N, Floyd CL, Berman RF, et al. Cell death and long-term maintenance of neuron-like state after differentiation of rat bone marrow stromal cells: a comparison of protocols.Brain Res. 2003,991(1-2):46-55
7 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000,61(4):364-70
8 Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002 ,418(6893):41-9
9 Hermann A, Gastl R, Liebau S, et al . Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells. J Cell Sci. 2004 ,117(Pt 19):4411-22
10 Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice.Science. 2000,290(5497):1775-9
11 Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow.Science. 2000,290(5497):1779-82
12 Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains.Proc Natl Acad Sci U S A. 1999,96(19):10711-6
13 Nakamura S, Takeda Y, Kanno M, et al. Application of bromodeoxyuridine (BrdU) and anti-BrdU monoclonal antibody for the in vivo analysis of proliferative characteristics of human leukemia cells in bone marrow. Oncology, 1991, 48(4): 285-289
14 Deng W, Obrocka M, Fischer I, et al. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun. 2001, 282(1):148-52
15 Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res. 2002, 69(6):908-17
16 Kohyama J, Abe H, Shimazaki T, et al. Brain from bone: efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent. Differentiation. 2001,68(4-5):235-44
17 Jiang Y, Henderson D, Blackstad M, et al. Neuroectodermal differentiation from mouse multipotent adult progenitor cells.Proc Natl Acad Sci U S A. 2003, 100 Suppl 1:11854-60
18 Aklyama Y, Radtke C, Kocsis JD.Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J Neurosci. 2002,22(15):6623-30
19 Irons H, Lind JG, Wakade CG, et al. Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immunologic response.Cell Transplant. 2004,13(3):283-94
20 Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood. 2003,102(10):3483-93
21 Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas.Cancer Res. 2005,65(8):3307-18
22 Sato H, Kuwashima N, Sakaida T, et al. Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhancedmigratory response and therapeutic potential against murine brain tumors. Cancer Gene Ther. 2005 ,12(9):757-68
23 Borlongan CV, Lind JG, Dillon-Carter O, et al. Intracerebral xenografts of mouse bone marrow cells in adult rats facilitate restoration of cerebral blood flow and blood-brain barrier.Brain Res. 2004,1009(1-2):26-33
24 Chen Q, Long Y, Yuan X, et al. Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors.J Neurosci Res. 2005,80(5):611-9
25 Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res. 2004,94(5):678-85
26 Annabi B, Naud E, Lee YT, et al. Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors.J Cell Biochem. 2004,91(6):1146-58
27 Annabi B,Lee YT, Turcotte S,et al. Hypoxia promotes bone marrow-derived stromal cell migration and tube formation. Stell cells.2003a 21L337-347
28 Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002,99(10):3838-43
29 Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood. 2003,101(9):3722-9
30 Tse WT, Pendleton JD, Beyer WM, et al.Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.Transplantation. 2003,75(3):389-97
31 Nakamura K, Ito Y, Kawano Y, et al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther. 2004 ,1(14):1155-64
32 Lee J, Elkahloun AG, Messina SA, et al. Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells: a potential antiglioma cellular vector.Cancer Res. 2003,63(24):8877-89
33 Abdallah BM, Haack-Sorensen M, Burns JS,et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite extensive proliferation.Biochem Biophys Res Commun,2005, 326(3):527-38
34 Burns JS, Abdallah BM, Guldberg P,et al. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells. Cancer Res, 2005,65(8):3126-35
35 Liu Z, Li Y, Qu R,et al. Axonal sprouting into the denervated spinal cord and synaptic and postsynaptic protein expression in the spinal cord after transplantation of bone marrow stromal cell in stroke rats.Brain Res. 2007 Feb 27; [Epub ahead of print]
36 Tseng PY, Chen CJ, Sheu CC, et al. Spontaneous differentiation of adult rat marrow stromal cells in a long-term culture.J Vet Med Sci. 2007,69(2):95-102
37 Lu D, Qu C, Goussev A,et al. Treatment of traumatic brain injury with a combination therapy of marrow stromal cells and atorvastatin in rats.Neurosurgery. 2007,60(3):546-53; discussion 553-4
38 Wright KT, El Masri W, Osman A, et al. Bone marrow stromal cells stimulate neurite outgrowth over neural proteoglycans (CSPG), myelin associated glycoprotein and Nogo-A.Biochem Biophys Res Commun. 2007,354(2):559-66
39 Chen CJ, Ou YC, Liao SL,et al. Transplantation of bone marrow stromal cells for peripheral nerve repair.Exp Neurol. 2007 ,204(1):443-53
40 Yang LY, Huang TH, Ma L. ,et al. Bone marrow stromal cells express neural phenotypes in vitro and migrate in brain after transplantation invivo.Biomed Environ Sci. 2006 ,19(5):329-35
41 Nandoe RD, Hurtado A, Levi AD, et al. Bone marrow stromal cells for repair of the spinal cord: towards clinical application.Cell Transplant. 2006;15(7):563-77
1 Arnhold S,Klein H,Klinz FJ,et al.Human bone marrow stroma cells display certain neural characteristics and integrate in the subventricular compartment after injection into the liquor system.Eur J Cell Biol,2006,85 (6):551-565
2 Hamada H, Kobune M, Nakamura K, et al. Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy.Cancer Sci. 2005 ,96(3):149-56
3 Aboody KS,Rainov NG,Rainov NG,et al.Neural stem cells display extensive tropism for pathology in adult brain: Evidence from intracranial gliomas. Proc Natl Acad USA, 2000; 97(23): 12846-12851
4 Lee J, Kuroda S, Shichinohe H, et al. Migration and differentiation of nuclear fluorescence-labeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice.Neuropathology, 2003, 23(3):169-180
5 Buzanska L, Machaj EK, Zablocka B, et al. Human cord blood-derived cells attain neuronal and glial features in vitro. J Cell Sci.,2002,115(10):2131-2138
6 Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res,2002 ,69(6):908-917
7 Pu P, Kang C, Li J, et al. The effects of antisense AKT2 RNA on the inhibition of malignant glioma cell growth in vitro and in vivo. J Neurooncol, 2006,76(1): 1-11
8 Borlongan CV, Lind JG, Dillon-Carter O, et al. Intracerebral xenografts of mouse bone marrow cells in adult rats facilitate restoration of cerebral blood flow and blood-brain barrier. Brain Res, 2004, 1009(1-2):26-33
9 Sato H, Kuwashima N, Sakaida T, et al. Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors. Cancer Gene Ther, 2005, 12(9):757-768 10 Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas.Cancer Res, 2005, 65(8):3307-3318
11 Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 2002, 418 (6893):41-49
12 Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science, 2000, 290 (5497):1779-1782
13 Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood. 2003,102(10):3483-93
14 Lee J, Kuroda S, Shichinohe H, et al. Migration and differentiation of nuclear fluorescence-labeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice.Neuropathology. 2003,23(3):169-80
15 Sanchez-Ramos JR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res. 2002,69(6):880-93
16 Nakano K, Migita M, Mochizuki H, et al. Differentiation of transplantedbone marrow cells in the adult mouse brain. Transplantation. 2001,71(12):1735-40
17 Rismanchi N, Floyd CL, Berman RF, et al. Cell death and long-term maintenance of neuron-like state after differentiation of rat bone marrow stromal cells: a comparison of protocols.Brain Res. 2003,991(1-2):46-55
18 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000,61(4):364-70
19 Hermann A, Gastl R, Liebau S, et al . Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells. J Cell Sci. 2004 ,117(Pt 19):4411-22
20 Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice.Science. 2000,290(5497):1775-9
21 Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow.Science. 2000,290(5497):1779-82
22 Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains.Proc Natl Acad Sci U S A. 1999,96(19):10711-6
23 Nakamura S, Takeda Y, Kanno M, et al. Application of bromodeoxyuridine (BrdU) and anti-BrdU monoclonal antibody for the in vivo analysis of proliferative characteristics of human leukemia cells in bone marrow. Oncology, 1991, 48(4): 285-289
24 Deng W, Obrocka M, Fischer I, et al. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun. 2001, 282(1):148-52
25 Kohyama J, Abe H, Shimazaki T, et al. Brain from bone: efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts toneurons with Noggin or a demethylating agent. Differentiation. 2001,68(4-5):235-44
26 Jiang Y, Henderson D, Blackstad M, et al. Neuroectodermal differentiation from mouse multipotent adult progenitor cells.Proc Natl Acad Sci U S A. 2003, 100 Suppl 1:11854-60
27 Aklyama Y, Radtke C, Kocsis JD.Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J Neurosci. 2002,22(15):6623-30
28 Irons H, Lind JG, Wakade CG, et al. Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immunologic response.Cell Transplant. 2004,13(3):283-94
29 Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood. 2003,102(10):3483-93
30 Sato H, Kuwashima N, Sakaida T, et al. Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors. Cancer Gene Ther. 2005 ,12(9):757-68
31 Borlongan CV, Lind JG, Dillon-Carter O, et al. Intracerebral xenografts of mouse bone marrow cells in adult rats facilitate restoration of cerebral blood flow and blood-brain barrier.Brain Res. 2004,1009(1-2):26-33
32 Chen Q, Long Y, Yuan X, et al. Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors.J Neurosci Res. 2005,80(5):611-9
33 Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res. 2004,94(5):678-85
34 Annabi B, Naud E, Lee YT, et al. Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors.J Cell Biochem. 2004,91(6):1146-58
35 Annabi B,Lee YT, Turcotte S,et al. Hypoxia promotes bone marrow-derived stromal cell migration and tube formation. Stell cells.2003a 21L337-347
36 Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002,99(10):3838-43
37 Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood. 2003,101(9):3722-9
30 Tse WT, Pendleton JD, Beyer WM, et al.Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.Transplantation. 2003,75(3):389-97
38 Nakamura K, Ito Y, Kawano Y, et al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther. 2004 ,1(14):1155-64
39 Lee J, Elkahloun AG, Messina SA, et al. Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells: a potential antiglioma cellular vector.Cancer Res. 2003,63(24):8877-89
40 Abdallah BM, Haack-Sorensen M, Burns JS,et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite extensive proliferation.Biochem Biophys Res Commun,2005, 326(3):527-38
41 Burns JS, Abdallah BM, Guldberg P,et al. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells. Cancer Res, 2005,65(8):3126-35
42 Liu Z, Li Y, Qu R,et al. Axonal sprouting into the denervated spinal cord and synaptic and postsynaptic protein expression in the spinal cord after transplantation of bone marrow stromal cell in stroke rats.Brain Res. 2007Feb 27; [Epub ahead of print]
43 Tseng PY, Chen CJ, Sheu CC, et al. Spontaneous differentiation of adult rat marrow stromal cells in a long-term culture.J Vet Med Sci. 2007,69(2):95-102
44 Lu D, Qu C, Goussev A,et al. Treatment of traumatic brain injury with a combination therapy of marrow stromal cells and atorvastatin in rats.Neurosurgery. 2007,60(3):546-53; discussion 553-4
45 Wright KT, El Masri W, Osman A, et al. Bone marrow stromal cells stimulate neurite outgrowth over neural proteoglycans (CSPG), myelin associated glycoprotein and Nogo-A.Biochem Biophys Res Commun. 2007,354(2):559-66
46 Chen CJ, Ou YC, Liao SL,et al. Transplantation of bone marrow stromal cells for peripheral nerve repair.Exp Neurol. 2007 ,204(1):443-53
47 Yang LY, Huang TH, Ma L. ,et al. Bone marrow stromal cells express neural phenotypes in vitro and migrate in brain after transplantation in vivo.Biomed Environ Sci. 2006 ,19(5):329-35
48 Nandoe RD, Hurtado A, Levi AD, et al. Bone marrow stromal cells for repair of the spinal cord: towards clinical application.Cell Transplant. 2006;15(7):563-77
1 Cho HS, Lee HR, Kim MK.Bystander-mediated regression of murine neuroblastoma via retroviral transfer of the HSV-TK gene. J Korean Med Sci. 2004,19(1):107-12
2 Asklund T, Appelskog IB, Ammerpohl O, et al. Gap junction-mediatedbystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene.Exp Cell Res. 2003,284(2):185-95
3 Ammerpohl O, Thormeyer D, Khan Z,et al.HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells. Biochem Biophys Res Commun. 2004,324(1):8-14
4 Asklund T, Appelskog IB, Ammerpohl O,et al.Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein and connexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells. Eur J Cancer. 2004 ,40(7):1073-81
5 Herrlinger U, Woiciechowski C, Sena-Esteves M, et al.Neural precursor cells for delivery of replication-conditional HSV-1 vectors to intracerebral gliomas. Mol Ther. 2000 ,1(4):347-57
6 Bi X, Zhang JZ. Experimental study of thymidine kinase gene therapy of neuroblastoma in vitro and in vivo. Pediatr Surg Int. 2003,19(5):400-5
7 van Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSV-tk/GCV gene therapy. A review.Curr Gene Ther. 2002,2(3):307-22
8 Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature,2002 ,418(6893):41-49
9 Sandmair AM, Turunen M, Tyynela K, et al.Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: effect of the percentage of thymidine kinase-positive glioma cells on treatment effect, survival time, and tissue reactions.Cancer Gene Ther. 2000,7(3):413-21
10 Rainov NG, Kramm CM, Banning U,et al.Immune response induced by retrovirus-mediated HSV-tk/GCV pharmacogene therapy in patients with glioblastoma multiforme. Gene Ther. 2000,7(21):1853-8
11 Asklund T, Appelskog IB, Ammerpohl O, et al. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene.Exp Cell Res.2003,284(2):185-95
12 Hadaczek P, Mirek H, Berger MS,et al. Limited efficacy of gene transfer in herpes simplex virus-thymidine kinase/ganciclovir gene therapy for brain tumors. J Neurosurg. 2005 ,102(2):328-35
13 Uhl M, Weiler M, Wick W, et al.Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas.Biochem Biophys Res Commun. 2005,328(1):125-9
14 Gentry BG, Im M, Boucher PD, et al.GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity.Gene Ther. 2005,12(13):1033-41
15 Mavria G, Harrington KJ, Marshall CJ, et al. In vivo efficacy of HSV-TK transcriptionally targeted to the tumour vasculature is augmented by combination with cytotoxic chemotherapy.J Gene Med. 2005 ,7(3):263-75
16 Moriuchi S, Glorioso JC, Maruno M, et al.Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkappaBalpha and HSV thymidine kinase.Cancer Gene Ther. 2005,12(5):487-96
17 Kanazawa T, Mizukami H, Okada T, et al. Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. Gene Ther. 2003,10(1):51-8
18 Tsuchiyama T, Kaneko S, Nakamoto Y, et al. Enhanced antitumor effects of a bicistronic adenovirus vector expressing both herpes simplex virus thymidine kinase and monocyte chemoattractant protein-1 against hepatocellular carcinoma. Cancer Gene Ther. 2003 ,10(4):260-9
19 Brockstedt DG, Diagana M, Zhang Y, et al. Development of anti-tumor immunity against a non-immunogenic mammary carcinoma through in vivo somatic GM-CSF, IL-2, and HSVtk combination gene therapy. Mol Ther. 2002,6(5):627-36
20 Chang JW, Lee H, Kim E, et al. Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6glioma. Neurosurgery. 2000 ,47(4):931-8; discussion 938-9
21 Desaknai S, Lumniczky K, Esik O, et al. Local tumour irradiation enhances the anti-tumour effect of a double-suicide gene therapy system in a murine glioma model. J Gene Med. 2003 ,5(5):377-85
22 Boucher PD, Ostruszka LJ, Murphy PJ, et al. Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy. Gene Ther. 2002,9(15):1023-30
1 Cho HS, Lee HR, Kim MK.Bystander-mediated regression of murine neuroblastoma via retroviral transfer of the HSV-TK gene. J Korean Med Sci. 2004,19(1):107-12
2 Asklund T, Appelskog IB, Ammerpohl O, et al. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene. Exp Cell Res. 2003,284(2):185-95
3 Ammerpohl O, Thormeyer D, Khan Z,et al.HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells. Biochem Biophys Res Commun. 2004,324(1):8-14
4 Asklund T, Appelskog IB, Ammerpohl O,et al.Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein andconnexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells. Eur J Cancer. 2004 ,40(7):1073-81
5 Herrlinger U, Woiciechowski C, Sena-Esteves M, et al.Neural precursor cells for delivery of replication-conditional HSV-1 vectors to intracerebral gliomas. Mol Ther. 2000 ,1(4):347-57
6 Bi X, Zhang JZ. Experimental study of thymidine kinase gene therapy of neuroblastoma in vitro and in vivo. Pediatr Surg Int. 2003,19(5):400-5
7 van Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSV-tk/GCV gene therapy. A review.Curr Gene Ther. 2002,2(3):307-22
8 Sandmair AM, Turunen M, Tyynela K, et al.Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: effect of the percentage of thymidine kinase-positive glioma cells on treatment effect, survival time, and tissue reactions.Cancer Gene Ther. 2000,7(3):413-21
9 Rainov NG, Kramm CM, Banning U,et al.Immune response induced by retrovirus-mediated HSV-tk/GCV pharmacogene therapy in patients with glioblastoma multiforme. Gene Ther. 2000,7(21):1853-8
10 Asklund T, Appelskog IB, Ammerpohl O, et al. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene.Exp Cell Res. 2003,284(2):185-95
11 Hadaczek P, Mirek H, Berger MS,et al. Limited efficacy of gene transfer in herpes simplex virus-thymidine kinase/ganciclovir gene therapy for brain tumors. J Neurosurg. 2005 ,102(2):328-35
12 Uhl M, Weiler M, Wick W, et al.Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas.Biochem Biophys Res Commun. 2005,328(1):125-9
13 Gentry BG, Im M, Boucher PD, et al.GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity.Gene Ther. 2005,12(13):1033-41
14 Mavria G, Harrington KJ, Marshall CJ, et al. In vivo efficacy of HSV-TK transcriptionally targeted to the tumour vasculature is augmented by combination with cytotoxic chemotherapy.J Gene Med. 2005 ,7(3):263-75
15 Moriuchi S, Glorioso JC, Maruno M, et al.Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkappaBalpha and HSV thymidine kinase.Cancer Gene Ther. 2005,12(5):487-96
16 Kanazawa T, Mizukami H, Okada T, et al. Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. Gene Ther. 2003,10(1):51-8
17 Tsuchiyama T, Kaneko S, Nakamoto Y, et al. Enhanced antitumor effects of a bicistronic adenovirus vector expressing both herpes simplex virus thymidine kinase and monocyte chemoattractant protein-1 against hepatocellular carcinoma. Cancer Gene Ther. 2003 ,10(4):260-9
18 Brockstedt DG, Diagana M, Zhang Y, et al. Development of anti-tumor immunity against a non-immunogenic mammary carcinoma through in vivo somatic GM-CSF, IL-2, and HSVtk combination gene therapy. Mol Ther. 2002,6(5):627-36
19 Chang JW, Lee H, Kim E, et al. Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6 glioma. Neurosurgery. 2000 ,47(4):931-8; discussion 938-9
20 Desaknai S, Lumniczky K, Esik O, et al. Local tumour irradiation enhances the anti-tumour effect of a double-suicide gene therapy system in a murine glioma model. J Gene Med. 2003 ,5(5):377-85
21 Boucher PD, Ostruszka LJ, Murphy PJ, et al. Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy. Gene Ther. 2002,9(15):1023-30
22 Hwang KS, Cho WK, Yoo J, et al.Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity. Cancer Gene Ther, 2004 ,11(6):397-407
23 Luo Y, Zhou H, Mizutani M,et al.A DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 induces long-lived T-cell memory against tumor recurrence.Cancer Res, 2005 ,65(8):3419-3427
24 Kohyama M, Saijyo K, Hayasida M, et al.Direct activation of human CD8+ cyto- toxic T lymphocytes by interleukin-18. Jpn J Cancer Res,1998 ,89(10): 1041- 1046
25 Kikuchi T, Akasaki Y, Joki T, et al.Antitumor activity of interleukin-18 on mouse glioma cells.J Immunother, 2000 ,23(2):184-189
26 Wataru Hasiimoto, Fumiaki Tanaka, Paul D,et al. Natural killer, but not natural killer T. cells play a necessary role in the promotion of an innate antitumor response induced by IL-18. Int J Cancer. 2003 ,103(4):508-513
27 Tanaka F, Hashimoto W, Robbins PD, et al. Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adeno- viral vector expressing biologically active IL-18.Gene Ther,2002,9(21): 1480- 1486
28 Hashimoto W, Osaki T, Okamura H,et al. Differential antitumor effects of administration of recombinant IL-18 or recombinant IL-12 are mediated primarily by Fas-Fas ligand- and perforin-induced tumor apoptosis, respectively. J Immunol, 1999,163(2):583-589
29 Cao R, Farnebo J, Kurimoto M, et al. Interleukin-18 acts as an angiogenesis and tumor suppressor. FASEB J,1999 ,13(15):2195-2202
30 Coughlin CM, Salhany KE, Wysocka M, et al. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis.J Clin Invest,1998 ,101(6):1441-1452
31 Osaki T, Peron JM, Cai Q, et al. IFN-gamma-inducing factor/IL-18 administration mediates IFN-gamma- and IL-12-independent antitumor effects.J Immunol,1998 ,160(4):1742-1749
32 Baxevanis CN, Gritzapis AD, Papamichail M.In vivo antitumor activity of NKT cells activated by the combination of IL-12 and IL-18. J Immunol,2003 ,171(6):2953-2959
33 Kito T, Kuroda E, Yokota A, et al. Cytotoxicity in glioma cells due to interleukin-12 and interleukin-18-stimulated macrophages mediated by interferon-gamma-regulated nitric oxide.J Neurosurg,2003,98(2):385- 392
34 Yamanaka R, Xanthopoulos KG.Induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis DNA encoding gp100 and IL-18. DNA Cell Biol,2005 ,24 (5):317-324
35 Redlinger RE Jr, Mailliard RB, Lotze MT, et al. Synergistic interleukin-18 and low-dose interleukin-2 promote regression of established murine neuroblastoma in vivo. J Pediatr Surg,2003,38 (3):301 -307
36 Wigginton JM, Lee JK, Wiltrout TA, et al. Synergistic engagement of an ineffec- tive endogenous anti-tumor immune response and induction of IFN- gamma and Fas-ligand - dependent tumor eradication by combined administration of IL-18 and IL-2. J Immunol,2002 ,169(8): 4467-4474
37 Tatsumi T, Huang J, Gooding WE, et al. Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity. Cancer Res,2003,63(19):6378-6386
38 Tanaka F, Hashimoto W, Robbins PD, et al. Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adeno- viral vector expressing biologically active IL-18. Gene Ther,2002 ,9(21):1480- 1486
39 Tanaka F, Hashimoto W,Okamura H,et al. Rapid generation of potent and tumor-specific cytotoxic T lymphocytes by interleukin 18 using dendritic cells and natural killer cells.Cancer Res,2000 ,60(17):4838-4844
40 Chung SW, Cohen EP, Kim TS.Generation of tumor-specific cytotoxic T lympho- cyte and prolongation of the survival of tumor-bearing mice using interleukin-18 -secreting fibroblasts loaded with an epitope peptide. Vaccine, 2004,22(20):2547- 2557
41 Zhang Y, Wang C, Zhang Y,et al.C6 glioma cells retrovirally engineered to express IL-18 and Fas exert FasL-dependent cytotoxicity against glioma formation.Biochem Biophys Res Commun,2004,325 (4) : 1240 -1245
42 Wang Q, Yu H, Zhang L, et al.Vaccination with IL-18 gene-modified, superantigen-coated tumor cells elicits potent antitumor immune response. J Cancer Res Clin Oncol,2001,127(12):718-726
43 Ju DW, Yang Y, Tao Q, et al. Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity.Gene Ther,2000,7(19):1672-1679
44 Xia D,Li F, Xiang J. Engineered fusion hybrid vaccine of IL-18 gene-modified tumor cells and dendritic cells induces enhanced antitumor immunity. Cancer Biother Radiopharm,2004 ,19(3):322-330
45 Golab J. Interleukin 18--interferon gamma inducing factor--a novel player in tumour immunotherapy? Cytokine,2000 ,12(4):332-338
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1 Hwang KS, Cho WK, Yoo J, et al.Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity. Cancer Gene Ther, 2004 ,11(6):397-407
2 Luo Y, Zhou H, Mizutani M,et al.A DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 induces long-lived T-cell memory against tumor recurrence.Cancer Res, 2005 ,65(8):3419-3427
3 Kohyama M, Saijyo K, Hayasida M, et al.Direct activation of human CD8+ cyto- toxic T lymphocytes by interleukin-18. Jpn J Cancer Res,1998 ,89(10): 1041- 1046
4 Kikuchi T, Akasaki Y, Joki T, et al.Antitumor activity of interleukin-18 on mouse glioma cells.J Immunother, 2000 ,23(2):184-189
5 Wataru Hasiimoto, Fumiaki Tanaka, Paul D,et al. Natural killer, but not natural killer T. cells play a necessary role in the promotion of an innate antitumor response induced by IL-18. Int J Cancer. 2003 ,103(4):508-513
6 Tanaka F, Hashimoto W, Robbins PD, et al. Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adeno- viral vector expressing biologically active IL-18.Gene Ther,2002,9(21): 1480- 1486
7 Hashimoto W, Osaki T, Okamura H,et al. Differential antitumor effects of administration of recombinant IL-18 or recombinant IL-12 are mediated primarily by Fas-Fas ligand- and perforin-induced tumor apoptosis, respectively. J Immunol, 1999,163(2):583-589
8 Cao R, Farnebo J, Kurimoto M, et al. Interleukin-18 acts as an angiogenesis and tumor suppressor. FASEB J,1999 ,13(15):2195-2202
9 Coughlin CM, Salhany KE, Wysocka M, et al. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis.J Clin Invest,1998 ,101(6):1441-1452
10 Osaki T, Peron JM, Cai Q, et al. IFN-gamma-inducing factor/IL-18 administration mediates IFN-gamma- and IL-12-independent antitumor effects.J Immunol,1998 ,160(4):1742-1749
11 Baxevanis CN, Gritzapis AD, Papamichail M.In vivo antitumor activity of NKT cells activated by the combination of IL-12 and IL-18. J Immunol,2003 ,171(6):2953-2959
12 Kito T, Kuroda E, Yokota A, et al. Cytotoxicity in glioma cells due to interleukin-12 and interleukin-18-stimulated macrophages mediated by interferon-gamma-regulated nitric oxide.J Neurosurg,2003,98(2):385-392
13 Yamanaka R, Xanthopoulos KG.Induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis DNA encoding gp100 and IL-18. DNA Cell Biol,2005 ,24(5):317-324
14 Redlinger RE Jr, Mailliard RB, Lotze MT, et al. Synergistic interleukin-18 and low-dose interleukin-2 promote regression of established murine neuroblastoma in vivo. J Pediatr Surg,2003,38(3):301-307
15 Wigginton JM, Lee JK, Wiltrout TA, et al. Synergistic engagement of an ineffec- tive endogenous anti-tumor immune response and induction of IFN- gamma and Fas-ligand - dependent tumor eradication by combined administration of IL-18 and IL-2. J Immunol,2002 ,169(8): 4467-4474
16 Tatsumi T, Huang J, Gooding WE, et al. Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity. Cancer Res,2003,63(19):6378-6386
17 Tanaka F, Hashimoto W, Robbins PD, et al. Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adeno- viral vector expressing biologically active IL-18. Gene Ther,2002 ,9(21):1480- 1486
18 Tanaka F, Hashimoto W,Okamura H,et al. Rapid generation of potent and tumor-specific cytotoxic T lymphocytes by interleukin 18 using dendritic cells and natural killer cells.Cancer Res,2000 ,60(17):4838-4844
19 Chung SW, Cohen EP, Kim TS.Generation of tumor-specific cytotoxic T lympho- cyte and prolongation of the survival of tumor-bearing mice using interleukin-18 -secreting fibroblasts loaded with an epitope peptide. Vaccine, 2004,22(20):2547- 2557
20 Zhang Y, Wang C, Zhang Y,et al.C6 glioma cells retrovirally engineered to express IL-18 and Fas exert FasL-dependent cytotoxicity against glioma formation.Biochem Biophys Res Commun,2004 ,325(4):1240-1245
21 Wang Q, Yu H, Zhang L, et al.Vaccination with IL-18 gene-modified, superantigen-coated tumor cells elicits potent antitumor immune response. J Cancer Res Clin Oncol,2001,127(12):718-726
22 Ju DW, Yang Y, Tao Q, et al. Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity.Gene Ther,2000,7(19):1672-1679
23 Xia D,Li F, Xiang J. Engineered fusion hybrid vaccine of IL-18 gene-modified tumor cells and dendritic cells induces enhanced antitumor immunity. Cancer Biother Radiopharm,2004 ,19(3):322-330
24 Golab J. Interleukin 18--interferon gamma inducing factor--a novel player in tumour immunotherapy? Cytokine,2000 ,12(4):332-338