摘要
研究背景
我国大肠癌发病率从20年前的十万分之十上升到了今天的十万分之三十,在大城市恶性肿瘤的排名中大肠癌从第六位上升至第二位。大肠癌发病率正以4%的年增长率迅速上升,但治愈率却没有得到根本改变。传统的治疗方法有其自身的局限性,因此寻找新的疗法迫在眉睫。肿瘤自杀基因疗法是近年来肿瘤基因治疗中一个重要的研究策略,尤其是自杀基因的选择与重组,载体系统的优化,基因的靶向性转移表达,以及自杀基因的联合治疗等均有助于提高自杀基因特异性表达,增强杀伤肿瘤细胞的作用。
肿瘤自杀基因治疗是将自杀基因导入肿瘤靶细胞并使之表达,基因编码的酶将无毒的药物前体转化为具有细胞毒性的代谢物,从而诱导靶细胞产生自杀作用,杀伤肿瘤细胞,同时还通过旁观者作用杀伤邻近未转染的肿瘤细胞。目前研究最多的且被美国FDA批准应用于临床试验的自杀基因系统主要有单纯疱疹病毒Ⅰ型胸苷激酶/丙氧鸟苷系统(HSV-TK/GCV)和胞嘧啶脱氨酶/5-氟胞嘧啶系统(CD/5-FC)。此两系统的抗肿瘤效能已在多种肿瘤的体内外实验中得到证实。日本、荷兰、墨西哥等地的学者已开始进行HSV-TK基因治疗前列腺癌的临床试验。目前的临床试验认为,针对人前列腺癌的原位基因治疗安全而有效,具有良好的抗癌生物学活性。鉴于不同类型肿瘤细胞对自杀基因系统的敏感性不同、单一自杀基因系统治疗时部分肿瘤细胞易产生耐药以及CD/5-FC和HSV-TK/GCV系统的作用机理具有很强的互补性,联合应用此两系统不仅可以提高肿瘤细胞杀伤作用,改善治疗效果,而且可以扩大肿瘤治疗谱,减少耐药现象发生。
众所周知,肿瘤的自杀基因治疗虽已成为基因治疗中研究热点之一,但目的基因的靶向性及基因转移的低效率是这一疗法广泛开展的主要障碍。实验研究中大多使用逆转录病毒载体和腺病毒载体进行基因转染治疗,与其它几种常用的病毒载体相比较,逆转录病毒载体病毒滴度低,只能感染分裂期细胞,容纳外源基因的DNA片段长度不超过8 kb。而腺病毒载体感染细胞时,病毒DNA游离在细胞核内,并不整合到染色体上,在体内不能实现稳定的长期表达,且反复应用容易引起免疫反应,因而在应用上这两种病毒载体都受到一定的限制。慢病毒载体最大的特点是可以感染分裂期及非分裂期细胞,容纳外源性目的基因的片段大,可以在体内长期地表达,免疫反应小,安全性较好,可在更多范围的宿主细胞内生成高滴度的病毒,已成为当前基因治疗中载体研究的热点。同时,如能使自杀基因选择性地作用于目的肿瘤细胞而不在正常细胞中表达,将能使该治疗策略更加完善。既往研究利用肿瘤特异性基因调控元件调控自杀基因表达,如以erbB-2、α-人乳白蛋白(hALA)启动子驱动自杀基因进行乳腺癌基因治疗,甲胎蛋白(AFP)基因启动子用于肝癌自杀基因疗法,癌胚抗原(CEA)基因启动子用于肠道肿瘤自杀基因疗法等。上述治疗方案中,每一特异启动子仅能进行针对某一类型肿瘤细胞靶向基因治疗,而绝大多数恶性实体瘤的生物学特性为生长迅速,致使其滋养血管不能及时供应所有肿瘤细胞,该特点使前体药物难以接近所有肿瘤细胞。选择一合适启动子既靶向肿瘤血管内皮细胞,又靶向肿瘤细胞,如此既可杀伤肿瘤细胞,又可靶向破坏肿瘤滋养血管使得其灌流区大量肿瘤细胞缺血性坏死,这样即高效放大了自杀基因的疗效。大量研究证明,KDR在正常人体组织中呈低水平表达,而在绝大多数肿瘤中呈现高表达,且KDR不仅表达于血管内皮细胞,还表达于肿瘤细胞,其表达水平与血管内皮细胞更新速度及肿瘤恶性程度呈正相关。它不仅促进血管内皮细胞分裂、增殖,且诱导肿瘤血管增生,并促使肿瘤细胞生长转移。在肿瘤组织内KDR表达明显高于正常内皮细胞,血管的发生对于肿瘤的生长和转移起着关键的作用。因此,将KDR作为靶点,可为治疗和诊断肿瘤提供了新的理论依据。国内外众多作者报道,KDR在在胃癌、结肠、直肠、卵巢、乳腺癌等多数肿瘤组织中的表达明显增高,并与其生物学行为相关,并伴随肿瘤分级的上升而表达增强。
基于上述研究,为弥补单自杀基因的不足、克服以往启动子仅适用于某一肿瘤细胞的缺点、并利用慢病毒载体的优势,本课题设计思路如下:应用慢病毒载体构建KDR启动子驱动的CD/TK融合基因的重组慢病毒,感染血管内皮细胞ECV304及大肠癌细胞LOVO,观察该治疗系统的体外杀伤作用,为重组慢病毒介导KDR启动子驱动的CD/TK融合基因靶向治疗大肠癌的进一步研究提供依据。
注:本课题来源于:广东省自然科学基金重点项目(013072)。
目的
研究慢病毒介导的KDR启动子驱动的CD/TK双自杀基因系统对大肠肿瘤细胞选择性杀伤作用,并为进一步研究奠定基础。首先利用FUGW慢病毒载体构建携带KDR启动子调控的双自杀基因重组慢病毒,将所构建的重组慢病毒感染LOVO细胞和ECV304细胞,测定感染效率,检测转基因的表达,对转基因细胞施以前药(5-FC和/或GCV),观察转基因细胞对前药的敏感性。最后,以LOVO细胞株建立裸鼠皮下移植瘤动物模型,观察该治疗体系的体内抑瘤效应。
方法
一、重组慢病毒的构建
将FUGW慢病毒载体酶切去掉Ubiqutin启动子,应用PCR扩增出KDR启动子、CD基因、TK基因序列,亚克隆入中间载体pcDNA3,构建pcDNA3-KDRP-CD/TK,然后酶切KDRP-CD/TK片断,插入到FGW中,构建KDR启动子调控下的CD/TK融合基因慢病毒重组质粒FGW-KDRP-CD/TK。重组质粒在2937细胞中包装成病毒,并进一步扩增、纯化,通过荧光显微镜下293T细胞中绿色荧光蛋白(GFP)的表达观察重组病毒的包装与复制,并以PCR方法鉴定重组慢病毒。
二、慢病毒介导的FGW-KDRP-CD/TK融合基因系统对LOVO细胞及ECV304细胞的体外杀伤作用
用重组慢病毒FGW-KDRP-CD/TK感染LOVO细胞、ECV304细胞及LS174T细胞(对照),检测目的基因的表达。在前药5-氟胞嘧啶(5-FC)和/或更昔洛韦(GCV)作用下,测定肿瘤细胞的存活率,观察旁观者效应,并分别通过流式细胞仪、透射电镜观察细胞凋亡与坏死。
三、慢病毒介导FGW-KDRP-CD/TK融合基因系统对大肠癌LOVO细胞裸鼠模型的体内抑瘤效应
采用BALB/C雌性裸鼠,4～6周龄,对数生长期的LOVO细胞接种于裸鼠左侧腋窝皮下,建立大肠癌动物模型,当肿瘤直径达0.5cm时,将荷瘤裸鼠随机分为Ⅰ组:注射重组慢病毒FGW-KDRP-CD/TK与前药5-FC与GCV;Ⅱ组:仅注射前药5-FC与GCV;Ⅲ组:仅注射重组慢病毒FGW-KDRP-CD/TK;Ⅳ组:空白对照,不施加任何处理。每5d测量肿瘤生长状况,估算肿瘤体积,绘制肿瘤生长曲线,25天治疗结束后处死裸鼠,称瘤重,计算肿瘤生长抑制率并观察该治疗体系的体内抑瘤效应,行组织内CD/TK基因表达的检测、肿瘤组织常规病理检查,并观察重要脏器心、肝、脾、肺、肾有无病理变化。
结果
一、重组慢病毒质粒的鉴定
1.重组慢病毒质粒FGW-KDRP-CD/TK的鉴定
在重组慢病毒质粒FGW-KDRP-CD/TK的构建过程中,主要采用酶切和测序的方法进行鉴定。将PCR所得的CD片段、TK片段及KDR分别连于T载体(pMD18-T)送上海申友生物技术有限公司测序,结果正确。FGW-KDRP-CD/TK以HindⅢ和BamHI酶切后电泳出现大小约2.4kb(CDglyTK)片段,结果正确。
2.重组慢病毒质粒在293T细胞的包装及滴度测定
将10μg的转移载体,7.5μg的CMV△R8.2包装质粒,5μg的VSV-G包膜质粒,加入130μL的2mol/L CaCL_2中,磷酸钙法三质粒瞬时共转染293T细胞,24h后通过荧光显微镜即可见GFP荧光表达,72h后收集上清,0.45μm滤器过滤,25000rpm离心90min,沉淀溶于Hanks溶液,-80℃贮存备用。将梯度稀释的病毒液,加入293T细胞中,72h后置于荧光显微镜下观察GFP的表达情况,计数发荧光细胞数目,根据病毒用量和稀释度计算滴度。
二、慢病毒介导的FGW-KDRP-CD/TK融合基因系统对LOVO细胞及ECV304细胞的体外杀伤作用
1.重组慢病毒体外感染及鉴定
细胞LOVO、ECV304、LS174T分别加入200μl的重组慢病毒悬液(100MOI)培养72小时后,在荧光显微境下观察GFP基因的表达,可见80-90%有绿色荧光存在,三种细胞具有相似的感染率。通过RT-PCR检测发现,除LS174T细胞外,LOVO、ECV304细胞检测有目的基因的表达。
2.重组慢病毒对各细胞的体外抑制效应
将200μl的重组慢病毒悬液(100MOI)感染各细胞株,加入不同浓度的前药培养72h后MTT法检测细胞生长抑制率。结果:感染慢病毒FGW-KDRP-CD/TK的LOVO及ECV304细胞对前药具有较高的敏感性,转基因细胞的存活率随前药浓度的增加而递减。当前药浓度为GCV=100mg/L,5-FC=160mg/L时,LOVO存活率为(5.40±1.85)%,ECV304存活率为(5.26±1.82)%。而感染慢病毒的LS174T细胞对前药不敏感,LS174T存活率仍有(95.02±2.82)%,与其他各组细胞相比差异有显著性意义(F=6567.806,P＜0.001)。感染FGW-KDRP-CD/TK的LOVO、ECV304及LS174T细胞,单用浓度100mg/L的GCV时,生存率分别为(31.96±1.62)%、(29.10±2.86)%、(96.57±1.89)%,单用浓度为160mg/L的5-FC,生存率分别为(29.60±1.91)%、(25.23±2.05)%、(96.20±1.91)%,二者联合时,各细胞生存率分别为(5.40±1.85)%、(5.26±1.82)%、(95.02±2.82)%,提示联合用药比单一用药对LOVO细胞和ECV304细胞均有较强杀伤效应(F=803.123,P＜0.001;F=375.99,P＜0.001;F=1.555,P=0.226)。
3、旁观者效应
将感染慢病毒的细胞与未感染细胞以不同混合培养,观察到该体系明显的旁观者效应,其随转基因细胞所占比例的增加而明显增强。当转基因细胞的比率为40%时,LOVO和ECV304细胞分别有(32.40±1.29)%、(29.95±1.96)%的存活,而LS174T细胞存活率仍在(97.74±1.74)%,三者比较差异均有显著性意义(F=5180.938,P＜0.001)。
4、重组慢病毒对LOVO细胞及ECV304细胞凋亡的影响
转基因LOVO细胞给以前药(GCV:1mg/L;5-FC:40mg/L)处理24h后,流式细胞仪检测发现:治疗组LOVO细胞加药24h后凋亡率为27.09±1.45%,明显高于对照组凋亡率2.96±0.34%(P=0.007)。
转基因ECV304细胞给以前药(GCV:1mg/L;5-FC:40mg/L)处理24h后,流式细胞仪检测发现:治疗组ECV304细胞加药24h后凋亡率为10.85±1.50%,明显高于对照组凋亡率2.25±0.55%(P=0.035)。
5、前药处理前后LOVO细胞的电镜观察
透射电镜下见部分细胞出现细胞体收缩、染色质边聚,有的胞核形态不规则,核发生碎裂,胞浆内可见多个电子密度增强的核碎片,有的可见凋亡小体、(或整个凋亡细胞)被吞噬和降解的现象。部分细胞出现细胞器溶解等坏死征象。
三、慢病毒介导的FGW-KDRP-CD/TK自杀基因系统对大肠癌LOVO细胞裸鼠模型的体内抑瘤效应
1.荷瘤裸鼠肿瘤生长情况
接种肿瘤细胞后10d左右,出现皮下肿瘤结节。裸鼠成瘤达0.5cm后开始治疗,治疗结束时肿瘤标本的称重结果及抑瘤率:Ⅰ组:(21.34±5.72)mg,95.59%:Ⅱ组:(466.61±67.89)mg,3。58%;Ⅲ组:(451.91±48.29)mg,6.61%;Ⅳ组:空白对照组(483.91±51.60)mg。可见,组间瘤重有显著性差异(F=165.805,P＜0.001),Ⅰ组明显缩小,第Ⅱ、Ⅲ、Ⅳ组肿瘤生长情况无统计学意义。
2.肿瘤病理变化及肿瘤组织目的基因表达的检测
前药加慢病毒治疗组肿瘤组织切片中可见片状坏死区,而且这些区域可见大量炎性细胞浸润。RT-PCR检测发现重组慢病毒转基因荷瘤动物肿瘤组织内有CD/TK融合基因表达。
3.治疗后裸鼠重要脏器的组织学观察
为了解该治疗系统对裸鼠有无毒副作用,取治疗组小裸鼠心、肝、脾、肺、肾行常规病理检查未见明显异常。
4.裸鼠可视化大肠癌动物模型
成功的建立了大肠癌裸鼠皮下移植瘤可视化动物模型,LT-9MACIMSYSPULS整体荧光成像系统下,可满意观察到GFP绿色荧光,其优点:①早期发现肿瘤形成;②动态观察肿瘤生长及转移情况等。
结论
1.本实验首先以慢病毒为载体,成功地构建含KDR启动子驱动的CD/TK自杀基因系统的重组慢病毒,重组慢病毒的滴度达6×10~(10)pfu/ml;
2.重组慢病毒对LOVO细胞、ECV304细胞及LS174T细胞均具有较高的转染率,且对三者转染率相似;
3.重组慢病毒能将KDR启动子驱动的CD/TK融合基因转入LOVO细胞及ECV304细胞中并进行有效表达,使该系统对LOVO细胞及ECV304细胞具有满意的靶向杀伤作用,这种杀伤作用具有浓度依赖性,并且联合应用两种前药较单用一种前药更为有效;
4.该治疗体系的作用机制为对靶细胞的直接杀伤作用及旁观者效应,表现为处理后靶细胞的凋亡及坏死;
5.应用LOVO细胞可成功建立大肠癌移植瘤裸鼠普通动物模型及可视化动物模型,并能方便地检测肿瘤的生长、转移;
6.重组慢病毒转基因荷瘤动物肿瘤组织内可表达CD/TK融合基因产物,使该治疗体系具有满意的体内抑瘤效应,表现为肿瘤的生长抑制及肿瘤组织坏死;
7.KDR启动子驱动的CD/TK融合基因系统治疗后,荷瘤裸鼠心、脾、肝、肺、肾等器官无明显组织学变化,提示该系统对荷瘤裸鼠无系统毒副作用。
Background:
In China, the incidence of colon cancer is thrice more than it's twenty years ago as one in million,accompanying with it's rank changed from sixth to second in all cities' cancers.This incidence ascends rapidly at 4% annual growth rate,while no fundamental changes were seen in cure rate.It's urged to find new therapies since the traditional treatments had their limitations.Suicide gene therapy is an important strategy in cancer gene treatment,especially the selection and recombination of suicide gene,the optimization of vector system,the gene targeting transfer expression, and combined suicide gene therapy can increase the specific expression of suicide gene and enhance the effect to kill tumor cells.
In the suicide gene therapy ,suicide gene is imported into the cancer cells and translated into enzymes which can convert the atoxic prodrug into cytotoxic metabolites,inducing the suicide of targeting cells and the death of cancer cells, meanwhile killing the non-transfected cancer cells through the bystander effect. The most investigated suicide gene system which approved into clinic trials by FDA in US are Herpes simplex virus type 1 thymidine kinase(HSV-TK)/ganciclovir(GCV) and E. coli cytosine deaminase (CD)/5-fluorocytosine (5-FC),the anticancer effect of them have been approved in many tumors in vivo and vitro.The HSV-TK gene therapy has been used in prostate cancer clinical trials in Japan,Holland and Mexico.As shown in recent trials, in situ gene therapy was safe and effective in treating human prostate cancer, with good anticancer biologic activity.HSV-TK/GCV and CD/5-FC systems are most investigated in the suicide gene therapy. Some HSV-TK/GCV and CD/5-FC clinical treatment projects have been validated by FDA.Due to the different sensitivity of cancer cells to the suicide gene therapy,elevating drug resistance treated with only one system, complementarity in the mechanisms of CD/5-FC and HSV-TK/GCV, the combination of these two systems can not only increase the lethal effect of cancer cells, improving the treatment effect, but also enlarge the oncotherapy spectrum, reducing the drug resistance.
It's well known that although the suicide gene therapy of cancer is popular, the low efficiency of purpose gene targeting and gene transfer are the major problems this method meets.The retroviral and adenoviral vectors were wildly used to transfect gene, compared with other viral vectors, the retroviral vector has low virus titer, only infects the division cells, while can contain the size of exogenous gene segment not more than 8kb.When the adenovirus infect cells, their DNA can not combine to chromatosomes but liberate in the nucleus, so it can not reach the long steady expression, and will cause the immune response when being used repeatedly, which limited the usage of these two viral vectors. The features of lentiviral vector such as infecting both division and non-division cells, holding larger segment size of exogenous gene , expressing long in vivo, lower immune response, better safety, producing high titer of virus in host cells at large range have been popular in the current gene therapy research. Meanwhile, this strategy will be more improved when the suicide gene can selectively affected and expressed in the cancer cells not normal cells. The cancer specific regulatory element such as erbB-2,α- hALA promoter were used to promote suicide gene therapy in breast cancer, AFP promoter was used in liver cancer, CEA promoter in intestine cancer and so on. Each specific promoter was used to treat specific type of targeting cancer cells in those treating protocols, while most tumor grows rapidly causing the nourishing vessel cannot supply all the tumor cells which prevents the effect of prodrugs. The suitable promoter can be selected to target the tumor vessel endothelial cells(VECs) and the tumor cells so that it can kill tumor cells and target destroy tumor nourishing vessels which leads to the tumor cells ischemic necrosis in the supplying area, highly amplifying the effect of the suicide gene. Amounts of studies indicated that KDR was expressed at low levels in normal human tissues, while highly expressed in VECs and most tumors cells. The expression of KDR was positively correlated with the VECs renewing rate and tumor malignancy which not only stimulated the division, proliferation of VECs, but also induced blood vessel hyperplasia, spured the growth and metastasis of the tumor cells. The expression of KDR in tumors is obviously higer than the normal endothelial cells, the genesis of vessels plays a key role in the growth and metastasis of tumor .So the application of KDR as a target is a novel theoretical support to diagnosis and treatment of tumor. KDR was reported highly expressed in gastric cancer ,colon cancer, rectal cancer, ovarian cancer, breast cancers and correlated with their biological behaviors, higher when the tumor grading ascends.
Based on these studies, to infect ECV304 and LOVO colon cancer cells by lentiviral KDR promoted CD/TK fusion gene lentivirus and to observe the lethal effects in vitro of this therapy can not only overcome the shortage of using single suicide gene and the limited effect of some promoters which only available on specific tumor cells, but also exert the advantages of lentiviral vector, provide the basis for further study on this gene targeted therapy in colon cancer.
Note: this study was supported by Natural science foundation of Guangdong province (013072).
Objective:
To investigate the selective killing effect of CD/TK double suicide genes drivbed by KDR promoter mediated by lentivirus on colon tumor cells. Firstly, the FUGW lentiviral vector was used to construct the recombinant lentivirus carrying double suicide gene medicated by KDR promoter which were used to infect ECV304 cells and LOVO cells. To determine the efficiency of infection, detect the expression of transgene, treat transgenic cells with prodrug (5-FC and /or GCV) and observe the sensitivity. At last, to build the nude mouse models with transplanted subcutaneous tumor on the base of LOVO cell line and to observe the tumor suppression of this therapy in vivo.
Methods:
1 .The construction of the recombinant lentivirus.
The Ubiqutin promoter in the FUGW lentiviral vector was digested by enzyme, KDR promoter,CD gene sequence,TK gene sequence amplified by PCR were subcloned into the intermedial vector pcDNA3 to construct pcDNA3-KDRP-CD/TK, then the KDRP-CD/TK fragment was digested with restriction enzyme and inserted into the FGW, the lentiviral recombinant plasmid FGW- KDRP-CD/TK with CD/TK fusion gene regulated by the KDR promoter was constructed then.Then recombinant plasmid was packaged in the 293T cells, further amplified and purified, the package and cloning of recombinant virus were observed through the expression of green fluorescent protein(GFP) in 293T cells by fluorescent microscope, the recombinant lentivirus were identified by PCR.
2. The lethal effect of KDRP-CD/TK fusion gene system mediated by lentivirus to LOVO and ECV304 cells in vitro.
The recombinant lentiviral KDRP-CD/TK was used to infect LOVO, ECV304,LS174T(control) cells, the expression of objective gene was observed. The
tumor cell survival rate, the bystander effect were detected under the treatment of prodrug 5-FC/GCV, the cell apoptosis and necrosis were observed through flow cytometry and transmission electron microscope respectively.
3. The anti-tumor effect of FGW-KDRP-CD/TK fusion gene system mediated by lentivirus to the colon cancer LOVO cell athymic mouse model in vivo.
The LOVO cell line in exponential phase of growth was vaccinated subcutaneously into the left axillary area of BALB/C female, 4 to 6 weeks old athymic mouse, to construct the colon cancer model. When the diameter of the tumor reached to 0.5cm, the tumor bearing nude mice were divided into group (ie viruses +5-FC+GCV,viruses only, 5-FC+GCV only,blank)at random. The growth of tumor was measured every five days, the weight of tumor and the tumor inhibitory rate were calculated, the tumor growth state was observed, the anti-tumor effect of the therapy was observed in vivo. After treatment, the CD/TK gene expression in tissue was detected, the routine pathological examination of tumor was made, and major organs such as heart, liver, spleen,lung,kidney were observed to find whether the pathological changes existed.
Results:
1. The identify of the recombinant lentiviral plasmids.
1.1 The identify of the recombinant lentiviral plasmids KDRP-CD/TK.
During the construction of recombinant lentiviral plasmid KDRP-CD/TK, the enzyme cutting and sequencing were made to identify. The CD ,TK fragment and KDR were united to T vector (pMD18-T) respectively and send to Shanghai Shenyou biologic technique LtD for sequencing, results were shown correct. The electrophoresis after enzyme restrictions of FGW-KDRP-CD/TK with Hind III and BamHI shew the product was 2.4kb (CDglyTK), which was correct.
1.2 The package of recombinant lentiviral plasmid in 293T cells and identification of virus titer.
The 10μg transfer vector, 7.5μg CMVAR8.2 packaging plasmid, 5μg VSV-G amicula plasmid were added in 130μL 2mol/L CaCL_2, three plasmids were transiently cotransfected into 293T cells by calcium acid phosphate method, after 24h the GFP fluorescent expression was seen in fluorescent microscope, the supernatant was collected after 72h and filtered by 0.45μm filtrum, then centrifuged at 25000rpm for 90min, precipitate was solved in Hanks solution and stored at - 80℃for use. The gradient diluted virus solution was added into 293T cells, the expression of GFP was detected under fluorescent microscope after 72h, fluorescent cells was counted and the tite was calculated based on the virus dosage and dilution.
2. the lethal effect of KDRP-CD/TK fusion gene system mediated by lentivirus to LOVO and ECV304 cells in vitro .
2.1 The infection and identification of recombinant lentivirus in vitro.
LOVO, ECV304, LS174T cells were added in the 200μl recombinant lentiviral suspension(100MOI) and cultured for 72h, the GFP gene expression was observed by fluorescent microscope, 80-90% cells had the green fluorescence, three types of cells shared similar infection rate. The expression of the objective gene was detected by RT-PCR in the LOVO,ECV304 cells except LS174T cells.
2.2 The inhibition effect of recombinant lentivirus to cells in vitro.
The 200μl recombinant lentiviral suspension(100MOI) was used to infect each cell lines, the different concentration of prodrugs were added and cultured for 72h, then the MTT was used to detect the inhibitory rate of cell growth.Results: the LOVO and ECV304 cells infected by lentiviral FGW-KDRP-CD/TK had higher sensitivity to the prodrug, the survival rate of the transgenic cells decreased when the concentration of prodrug increased. When the concentration of GCV and 5-FC were 100mg/L and 160mg/L respectively, the survival rate of LOVO cells was (5.40±1.85)%, the ECV304 was (5.26±1.82)%. While the LS174T cells infected by lentivirus were not sensitive to prodrug, the survival rate was (95.02±2.82)%, compared with other groups the significant difference existed (F=6567.806, P< 0.001) . The survival rate of FGW-KDRP-CD/TK infected LOVO,ECV304 and LS174T cells treated with 100mg/L GCV were (31.96±1.62) % ,(29.10±2.86) %,(96.57±1.89)% respectively; while treated with 160mg/L 5-FC the rates were (29.60±l .91)%、(25.23±2.05)%、(96.20±1.91)% respectively; and the combination of both drugs were (5.40±1.85)%、(5.26±1.82)%、(95.02±2.82)% respectively. This suggested that the combined use of two drugs had more lethal effect than either one in LOVO and ECV304 cells (F=803.123, P<0.001; F= 375.99, P<0.001; F=1.555, P=0.226) .
2.3 Bystander effect.
The obvious bystander effect could be observed in the different proportion mixed culture of the lentivirus infected cells and uninfected cells, the effect was obviously enhanced when the transgenic cells ratio increased. when this ratio was 40%, there were (32.40±1 .29)% LOVO cells and(29.95±1.96) % ECV304 cells existed respectively, while the survival ratio of LS174T cells was (97.74±1.74)%,the significant statistical difference was seen among them (F=5180.938,P<0.001) .
2.4 The effect of recombinant lentivirus to the apoptosis in LOVO and ECV304 cells.
The transgenic LOVO cells was dealt with prodrug for 24h( GCV: 1mg/L; 5-FC: 40mg/L) and then observed with flow cytometry: the apoptosis rate of LOVO cells treated group was 27.09±1.45%, significantly higher than the ratio of 2.96±0.34% in control group( P=0.007). The same procedure was done in the ECV304 transgenic cells , the apoptosis rate of ECV304 cells treated group was 10.85±1.50%, significantly higher than the ratio of 2.25±0.55% in control group( P=0.035).
2.5 The transmission electron microscopy observation of prodrug dealt LOVO cells
Cell body construction,chromatin fringe, irregular nucleus shape, nuclear fragmentation, several enhanced electron desity nuclear fragments in cytoplasm, apoptosis body, or the phagocytosis and degradation of entire apoptosis could be found in some cells under the transmission electron microscopy. Some cells may exhibit the necrosis signs like organelles dissolve.
3. The anti-tumor effect of FGW-KDRP-CD/TK fusion gene system mediated by lentivirus to the colon cancer LOVO cell athymic mouse model in vivo .
3.1 The growth of cancer in tumor bearing nude mice.
The subcutaneous tumor node was found in the 10d after tumor cells injection.
When the diameter of node reached 0.5cm, the nude mouse received treatment and the weight of tumor sample and tumor inhibited rate were recorded as follows: group I (21.34±5.72)mg,95.59%; group II :(466.61±67.89)mg, 3.58%; groupIII (451.91±48.29)mg, 6.61%; group IV: (483.91±51.60) mg;the significant difference was seen between four groups (F =165.805,P<0.001) ,weight in groups I decreased obviously,tumor weights hadn't difference between between II group,III group, IV group.
3.2 Pathological changes and the detection of objective gene expression in tumor.
Focal necrosis areas were seen infiltrated by amounts of inflammatory cells in the tumor tissue sections in group treated with prodrug and lentivirus. The expression of CD/TK fusion gene at mRNA levels was detected by RT-PCR in the recombinant lentivirus transgenic tumor bearing animal's tumor tissue.
3.3 The histological observation in the nude mouse's major organs after treatment.
In order to understand if the treatment system had toxic side effects to the nude mouse, the routine pathologic tests of heart, liver, spleen, lung,kidney were done in the treated group with no obvious abnormal findings.
3.4 The visible colon cancer nude mice model.
The visible subcutaneous transplanted colon cancer nude mice model was successfully constructed. The GFP green fluorescence could be observed under the LT-9MACIMSYSPULS general fluorescence imaging system. The features included:①Find the tumor as early as possible.②Observe the tumor growth and metastasis dynamically.
Conclusions:
1 .The KDRP promoted CD/TK suicide gene system of recombinant lentivirus was successfully constructed based on the lentiviral vector, the virus tite was 6×10~(10) pfu/ml
2.The recombinant lentivirus could be highly transfected to LOVO cells, ECV304 cells and LS174T cells, the transfection efficiency was almost the same;
3.The recombinant lentivirus could transfect the the KDRP promoted CD/TK fusion gene to LOVO cells and ECV304 cells and express effectively, with the satisfied targeting lethal effect which was concentration dependent. The combination of both prodrugs were more effective than either one;
4.The mechanism of this therapy were direct tumoricidal activity and bystander effect to the targeting cells, causing the apoptosis and necrosis of target cells;
5.The common and visible animal model of colon cancer transplanted nude mice could be constructed using LOVO cells, it's convenient to detect the growth, transfer;
6.CD/TK fusion gene product could be expressed in the tumor tissues of recombinant lentivirus transgenic tumor bearing animals, made the satisfied anti-tumor effect in vivo, such as tumor growth inhibiting and tissue necrosis;
7.After the KDRP promoted CD/TK fusion gene therapy, no obvious histological changes were seen in heart, liver, kidney, lung,spleen of the tumor bearing nude mice, suggested that this system had no toxic side effects to the tumor bearing nude mice.
引文
[1]Uckert W,Kammertons T,Haack K,et al.Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase)but not single gene transfer allows reliable elimination of tumor cells in vivo.Hum Gene Ther,1998,9(6):855-865.
[2]Aghi M,Kramm CM,Chou TC,et al.Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine deaminase gene therapies.J Natl Cancer Inst,1998,90(5):370-380.
[3]Rogulski KR,Kim JH,Kin SH,et al.Glioma cells transduced with an Escherichia coli CD/HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitiveity.Hum Gene Ther,1997,8(1):73-85.
[4]Brummelkamp TR,Bernards R,Agami R.Stable suppression of tumorigenicity by virus-mediated RNA interference.Cancer Cell 2002;2:243-247.
[5]李振宇,徐开林,潘秀英.慢病毒载体构建及结构优化.国外医学:分子生物医学分册.2002;24:310-313.
[6]Rubinson DA,Dillon CP,Kwiatkowski AV,et al.A lentivirus-based system to functionally silence genes in primary mammalian cells,stem cells and transgenic mice by RNA interference.Nat Genet.2003;33:401-406.
[7]Guse]]a GL,Fedorova E,Hanss B,et al.Lentiviral gene transduction of kidney.Hum Gene Ther.2002;13:407-14.
[8]Follenzi A,Sabatino G,Lombardo A,et al.Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors.Hum Gene Ther.2002;13:243-60.
[9] Zhang Z , Shirakawa T , Hinta N , et al. Combination with CD/5-Fc gene therapy enhances killing of human bladder cancer cells by radiation. J Gene Med .2003:5:860.
[10] Asklund T , Appelskog I B , Ammerpohl O , et al. Gap junction mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSV-TK gene. Exp Cell Res. 2003:284(2): 183.
[11] Suhardja A .Hoffman H. Role of growth factors and their receptors in proliferation of microvascular endothelial cell.Microsc Res Tech .2003:60:70-75.
[12] Brenner RM, Nayak NR ,Slayden OD et al .Premenstrual and menstrual changes in the macaque and human endometrium: relevance to endometriosis. Ann N Y Acad Sci. 2002:955:60-74 .
[13] Harada Y , Ogata Y, Shirouzu K .Expression of vascular endothelial growth factor and its receptor KDR/FLK-1(FETAL LIVER KINASE-l)as prognostic factors in human colorectal cancer. Int J Clin Oncol .2001:6 :221-228.
[14] Ryden L .linderholm B , Niesen NH et al .Tumor specific VEGF-A and VEGFR2/KDR protein are co-expressed in breast cancer. Breast Cancer Res Treat .2003:82(3):147-54
[15] Fine BA ,Valente PT ,Dey T et al .VEGF , flt-1 , and KDR/flk-1 as prognostic indication in endothelial carcinomaJ. Gynecol Oncol. 2000:76(1): 33-39.
[16] Cha MS ,Lee MJ , Je GH et al .Endogenous production of nitric oxide by vascular endothelial growth factor down regulates prolife of chorio-carcinoma cell.Biochem Biophys Res Commun. 2000;282(4): 1061-1066.
[17] Kamiyama M, Ichikawa Y,Ishikawa T et al .VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric and peritoneal dissemination of human gastric cancer in nudemice. Cancer Gene Ther. 2002;90(13):197.
[18] Hunt S. Technology evaluation : IMC - 1C , IMclone systems. Curr Opin Mol Ther . 2001;3(4):418.
[19] Isayeva T, Kumar S, Ponnazhagan S. Antiangiogenic gene therapy for cancer. Int J Oncol. 2004:25:335-343.
[20] Lin P, Buxton JA, Acheson A, et al. Antiangiogenic gene therapy targetingothe endothelium-specific receptor tyrosine kinase Tie2. Proc Natl Acad Sci U S A. 1998;95:8825-8834.
[21] Bergelson JM, Cunningham JA, Droguett G, et al. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science. 1997:275:1320-1323.
[22] Wickham TJ, Mathias P, Cheresh DA, Nemerow GR. Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment.Cell. 1993:73:309-319.
[23] Weitzman MD, Kyostio SR, Kotin RM, Owens RA. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA. Proc Natl Acad Sci U S A. 1994:91:5808-5812.
[24] Miller AD. Retroviral vectors. Curr Top Microbiol Immunol. 1992; 158:1-24.
[25] Li Z, Dullmann J, SchiedlmeierB, et al. Jurine leukemia induced by retroviral gene marking. Science. 2002:296:497.
[26] Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMOS-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science.2003:302:415-419.
[27] Deisseroth A. Normal and pathological functions of mammalian retroelements. Proc Natl Acad Sci U S A. 2005:102:12292-12293.
[28] Grote D, Russell SJ, Cornu TI, et al. Live attenuated measles virus induces regression of human lymphoma xenografts in immunodef icient mice. Blood.2001;97:3746-3754.
[29] Nussbaum 0, Broder CC, Moss B, et al. Functional and structural interactions between measles virus hemagglutinin and CD46. J Virol. 1995:69:3341-3349.
[30] Dorig RE, Marcil A, Richardson CD. CD46, a primate-specific receptor for measles virus.Trends Microbiol. 1994:2:312-318.
[31] Tatsuo H, Ono N, Tanaka K, Yanagi Y. SLAM(CDwl50) is a cellular receptor for measles virus. Nature. 2000;406:893-897.
[32] Valyi-Nagy T, Sheth V, Clement C, et al. Herpes simplex virus entry receptor nectin-1 is widely expressed in the murine eye. Curr Eye Res. 2004;29:303-309.
[33] Kaner RJ, Baird A, Mansukhani A, et al. Fibroblast growth factor receptor is a portal of cellular entry for herpes simplex virus type 1. Science. 1990:248:1410-1413.
[34] Wood MJ, Byrnes AP, Pfaff DW, et al. Inflammatory effects of genetransfer into the CNS with defective HSV-1 vectors. Gene Ther. 1994:1:283-291.
[35] Kucharczuk JC, Randazzo B, Chang MY, et al.Use of a replicationr- estricted herper virus to treat experimental human malignant mesothelioma. Cancer Res.1997;57:466-471.
[36] Barker E, Planelles V. Vectors derived from the human immunodeficiency virus, HIV-1.Front Biosci. 2003;8:491-510.
[37] Shichinohe T, Bochner BH, Mizutani K, et al. Development of lentiviral vectors for antiangiogenic gene delivery. Cancer Gene Ther. 2001:8:879-889.
[38] Pfeifer A, Kessler T, Silletti S, et al. Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2. Proc Natl Acad Sci U S A. 2000:97:12227-12232.
[39] De Palma M, Venneri MA, Naldini L. In vivo targeting of tumor endothelial cells by systemic deliverydelivery of lentiviral vectors. Hum Gene Ther.2003:14:1193-1206.
[40] Cefai D, Simeoni E, Ludunge KM, et al. Multiply attenuated, self-inactivating lentiviral vectors efficiently transduce human coronary artery cells in vitro and rat arteries in vivo.J Mol Cell Cardiol. 2005:38:333-344.
[41] EscarpeP, et al. Development of a sensitive assay for detection of replication-competent recombinant lentivirus in largescale HIV-based vector preparations. Mol Ther. 2003;8:332 - 341.
[42] Lu X. Antisense-mediated inhibition of human immunodeficiency virus (HIV) replication by use of an HIV type 1-based vector results in severely attenuated mutants incapable of developing resistance. J Virol. 2004:78:7079-7088.
[43] Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1-8.
[44]Yanzheng Liu,Albert Deisseroth.Tumor vascular targeting therapy with viral vectors.Blood.2006;107:3027-3033.
[45]Folkman J.What is the evidence that tumors are angiogenesis dependent?.J Natl Cancer Inst.1990;82(1):4-6.
[46]傅建新,王玮,白霞,等.肿瘤细胞系血管内皮生长因子及其受体共表达的研究.癌症.2002;21(11):1217-1221.
[47]Suhardja A,Hoffman H.Role of growth factors and their receptors in proliferation of microvascular endothelial cells.Microsc Res Tech.2003;60(1):70-75.
[48]Qi L,Robinson WA,Brady BM,et al.Migration and invasion of human prostate cancer cells is related to expression of VEGF and its receptors.Anticancer Res.2003;23(5A):3917-3922.
[49]Beierle EA,Dai W,Langham MR Jr,et al.Expression of VEGF receptors in cocultured neuroblastoma cells.J Sure Res,.2004;119(1):56-65.
[50]Chabner BA,Roberts TG,Jr.Timeline:Chemotherapy and the war on cancer.Nat Rev Cancer.2005;5(1),65-72.
[51]Chatelut E,Delord JP,Canal P.Toxicity patterns of cytotoxic drugs.Invest.New Drugs.2003;21(2),141-8.
[52]Scripture CD,Figg WD.Drug interactions in cancer therapy.Nat Rev Cancer.2006;6(7),546-58.
[53]Jain RK.Delivery of molecular and cellular medicine to solid tumors.Adv Drug Deliv Rev.2001;46(1-3),149-68.
[54]Jain RK.Normalization of tumor vasculature:an emerging concept in antiangiogenic therapy.Science.2005;307(5706),58-62.
[55]Munn LL.Aberrant vascular architecture in tumors and its importance in drug-based therapies.Drug Discov Today.2003;8(9),396-403.
[56] Daniel Portsmouth, Juraj Hlavaty .Matthias Renner. Suicide genes for cancer therapy . Molecular Aspects of Medicine, In Press, Corrected Proof, Available online 10 January 2007.
[57] Seth P. Vector-mediated cancer gene therapy: an overview. Cancer Biol Ther. 2005:4(5):512-7.
[58] Gunzburg WH, Salmons B. Use of cell therapy as a means of targeting chemotherapy to inoperable pancreatic cancer. Acta Biochim. Pol. 2005:52(3):601-7.
[59] Pereboeva L, Curiel DT. Cellular vehicles for cancer gene therapy: current status and future potential. BioDrugs . 2004;18(6):361-85.
[60] Agarwal S, Nikolai B, Yamaguchi T, et al. Construction and use of retroviral vectors encoding the toxic gene barnase. Mol Ther. 2006;14(4): 555-63.
[61] Johannes L, Decaudin D. Protein toxins: intracellular trafficking for targeted therapy. Gene Ther. 2005:12(18):1360-8.
[62] Tubiana M. Tumor cell proliferation kinetics and tumor growth rate. Acta Oncol. 1989;28(1):113-21.
[63] McGeoch DJ, Dalrymple MA, Davison AJ, et al. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol. 1988;69(7):1531-74.
[64] Chen SH, Pearson A, Coen DM. Failure of thymidine kinase-negative herpes simplex virus to reactivate from latency following efficient establishment. J Virol. 2004:78(1):520-3.
[65] Moolten FL. Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res. 1986:46(10):5276-81.
[66] Klatzmann D, Cherin P, Bensimon G, et al. A phase I/II dose-escalation study of herpes simplex virus type 1 thymidine kinase "suicide" gene therapy for metastatic melanoma. Study Group on Gene Therapy of Metastatic Melanoma. Hum Gene Ther. 1998a;9(17):2585.
[67] Klatzmann D, Valery CA, Bensimon G., et al. A phase I/II study of herpes simplex virus type 1 thymidine kinase"suicide" gene therapy for recurrent glioblastoma.Study Group on Gene Therapy for Glioblastoma.Hum Gene Ther. 1998b;9(17):2595-604.
[68] Rainov NG. A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum Gene Ther.2000;11 (17):2389-401.
[69] Fillat C, Carrio M, Cascante A. et al. Suicide gene therapy mediated by the Herpes Simplex virus thymidine kinase gene/Ganciclo-vir system:fifteen years of application. Curr Gene Ther. 2003;3 (1): 13-26.
[70] Floeth FW, Shand N, Bojar H , et al. Local inflammation and devascularization—-in vivo mechanisms of the "bystander effect" in VPC-mediated HSV-Tk/GCV gene therapy for human malignant glioma. Cancer Gene Ther. 2001;8(11):843-51.
[71] Prados MD, McDermott M, Chang SM, et al. Treatment of progressive or recurrent glioblastoma multiforme in adults with herpes simplex virus thymidine kinase gene vectorproducer cells followed by intravenous ganciclovir administration: a phase I/II multi- institutional trial. J Neurooncol. 2003;65(3):269-78.
[72] Germano IM, Fable J, Gultekin SH, et al. Adenovirus/herpes simplex- thymidine kinase/ganciclovir complex: preliminary results of a phase I trial in patients with recurrent malignant gliomas. J Neurooncol. 2003;65(3):279-89.
[73] Immonen A, Vapalahti M, Tyynela K, et al. AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study. Mol Ther. 2004;10(5):967-72.
[74] Ayala G, Satoh T, Li R, et al. Biological response determinants in HSV-tk+ ganciclovir gene therapy for prostate cancer. Mol Ther. 2006:13(4): 716-28.
[75] Freytag SO, Khil M, Stricker H, et al. Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer Res. 2002;62(17):4968-76.
[76] Fujita T, The BS, Timme T L, et al. Sustained long-term immune responses after in situ gene therapy combined with radiotherapy and hormonal therapy in prostate cancer patients. Int J Radiat Oncol Biol Phys. 2006;65(1):84-90.
[77] Kubo H, Gardner TA, Wada Y, et al. Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther. 2003:14(3): 227-41.
[78] Miles B J, Shalev M, Aguilar-Cordova E, et al. Prostate-specific antigen response and systemic T cell activation after in situ gene therapy in prostate cancer patients failing radiotherapy. Hum Gene Ther.2001;12 (16):1955-67.
[79] Satoh T, The BS, Timme TL, et al. Enhanced systemic T-cell activation after in situ gene therapy with radiotherapy in prostate cancer patients. Int J Radiat Oncol Biol Phys. 2004;59(2):562-71.
[80] Teh BS, Ayala G., Aguilar L, et al. Phase I—II trial evaluating combined intensity-modulated radiotherapy and in situ gene therapy with or without hormonal therapy in treatment of prostate cancer-interim report on PSA response and biopsy data. Int J Radiat Oncol Biol Phys;2004;58(5):1520-9.
[81] Freytag SO, Khil M, Stricker H, et al. Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer Res. 2002;62(17):4968-76.
[82] Sterman DH, Recio A, Vachani A, et al. Long-term follow-up of patients with malignant pleural mesothelioma receiving high-dose adenovirus herpes simplex thymidine kinase/ganciclovir suicide gene therapy. Clin Cancer Res. 2005;11(20):7444-53.
[83] Chevez-Barrios P, Chintagumpala M, Mieler W, et al. Response of retinoblastoma with vitreous tumor seeding to adenovirus-mediated delivery of thymidine kinase followed by ganciclovir. J Clin Oncol. 2005;23(31):7927-35.
[84] Hasenburg A , Fischer D C, Tong XW, et al. Histologic and immunohistochemical analysis of tissue response to adenovirus- mediated herpes simplex thymidine kinase gene therapy of ovarian cancer. Int J Gynecol Cancer. 2002;12(1):66-73.
[85] Hasenburg A,Tong XW, Fischer DC, et al. Adenovirus-mediated thymidine kinase gene therapy in combination with topotecan for patients with recurrent ovarian cancer: 2. 5-year follow-up. Gynecol Oncol. 2001;83(3):549-54.
[86] Thompson J. Protocol #0207-544: a phase study to evaluate the safety and pharmacokinetics of Pro-1, a liposome-encapsulated thymidine kinase gene formulation, in patients with stage IV metastatic melanoma. Hum Gene Ther.2003;14(3):310-2.
[87] Smitt PS, Driesse M, Wolbers J. et al. Treatment of relapsed malignant glioma with an adenoviral vector containing the herpes simplex thymidine kinase gene followed by ganciclovir. Mol Ther. 2003; 7(6): 851-8.
[88] Immonen A, Vapalahti M, Tyynela K, et al. AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study. Mol Ther. 2004;10(5):967-72.
[89] Danielsen S, Kilstrup M, Barilla K, et al. Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase. Mol. Microbiol. 1992;6(10):1335-44.
[90] Erbs P, Exinger F, Jund R. Characterization of the Saccharomyces cerevisiae FCYl gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans.Curr Genet.1997;31(1):1-6.
[91] Andersen L, Kilstrup M, Neuhard J. Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K 12. Involvement of the glnLG and purR genes in the regulation of codA expression. Arch Microbiol. 1989;152 (2):115-8.
[92] Austin EA, Huber B E. A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. Mol Pharmacol. 1993; 43(3): 380-7.
[93] Mullen CA, Coale MM, Lowe R, et al. Tumors expressing the cytosine deaminase suicide gene can be eliminated in vivo with 5-fluorocytosine and induce protective immunity to wild type tumor. Cancer Res.1994;54(6):1503-6.
[94] Anderson LM, Krotz S, Weitzman SA. Breast cancer-specific expression of the Candida albicans cytosine deaminase gene using a transcriptional targeting approach. Cancer Gene Ther. 2000;7(6): 845-52.
[95] Stackhouse MA, Pederson LC, Grizzle WE, et al. Fractionated radiation therapy in combination with adenoviral delivery of the cytosine deaminase gene and 5-fluorocytosine enhances cytotoxic and antitumor effects in human colorectal and cholangiocarcinoma models. Gene Ther.2000;7(12):1019-26.
[96] Tai CK, Wang WJ, Chen TC, et al. Single-shot, multicycle suicide gene therapy by replication-competent retrovirus vectors achieves long-term survival benefit in experimental glioma. Mol Ther. 2005; 12 (5):842-51.
[97] Zhang JH, Wan MX, Pan BR, et al. Cytotoxicity of HSVtk and hrTNF-alpha fusion genes with IRES in treatment of gastric cancer. Cancer Lett. 2006a;235(2):191-201.
[98] Fuerer C, Iggo R. 5-Fluorocytosine increases the toxicity of Wnt-targeting replicating adenoviruses that express cytosine deaminase as a late gene. Gene Ther. 2004;11(2):14251.
[99]Conrad C, Miller CR, Ji Y, Gomez-Manzano C, et al. Delta24-hyCD adenovirus suppresses glioma growth in vivo by combining oncolysis and chemosensitization. Cancer Gene Ther. 2005;12(3):284-94.
[100]Barton KN, Paielli D, Zhang Y, et al. Second-generation replication-competent oncolytic adenovirus armed with improved suicide genes and ADP gene demonstrates greater efficacy without increased toxicity.Mol Ther. 2006;13(2):347-56.
[101]Freytag SO, Stricker H, Pegg J, et al. Phase I study of replication-competent adenovirus-mediated double-suicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate-to high-risk prostate cancer. Cancer Res. 2003; 63 (21): 7497-506.
[102]Fujimori M. Genetically engineered bifidobacterium as a drug delivery system for systemic therapy of metastatic breast cancer patients. Breast Cancer. 2006;13(1):27-31.
[103]Liu SC, Minton NP, Giaccia AJ, et al. Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis. Gene Ther. 2002;9(4):291-6.
[104]Yi C, Huang Y, Guo ZY, Wang SR. Antitumor effect of cytosine deaminase/5fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma. Acta Pharmacol Sin. 2005;26 (5): 629-34.
[105]Nemunaitis J, Cunningham C, Senzer N. et al. Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients. Cancer Gene Ther. 2003;10(10):737-44.
[106]Thust R, Tomicic MT, Grabner R, et al. Cytogenetic detection of a trans-species bystander effect: induction of sister chromatid exchanges in raurine 3T3 cells by ganciclovir metabolized in HSV thymidine kinase gene-transfected Chinese hamster ovary cells. Mutagenesis. 2004;19(1):27-33.
[107]Tanaka T , Kanai F , Okabe S , et al. Adenovirus2mediated prodruggene therapy for carcinoembryonic antigen2producing human gastric carcinoma cells i n vit ro. Cancer Research. 1996;56(15): 1341-1345.
[108]Sandalon Z , Fusenig N E , McCutcheon J , et al. Suicide gene ther2 apy for premalignant disease : a new strategy for the treatment of in2 traepithelial neoplasia. Gene Therapy. 2001;8(3):232-238.
[109]Burrows F J , Gore M , Smiley W R , et al. Purified herpes simplex virus thymidine kinase retroviral particies : III. Characterization of bystander killing mechanism in transfected tumor cells. Cancer Gene Therapy. 2002;9(1):87-95.
[110]Lee KC, Hamstra DA, Bullarayasamudram S, et al. Fusion of the HSV-1 tegument protein vp22 to cytosine deaminase confers enhanced bystander effect and increased therapeutic benefit. Gene Ther. 2006; 13 (2): 127-37.
[111]Pierrefite C V , Baque P , Gavelli A , et al. Subcutaneous or intra2 hepatic injection of suicide gene modified tumor cells induces a sys2 temic antitumor response in a metastatic model of colon carcinoma in rats. Gut;2002 :50(3):387-391.
[112]Huber BE, Austin EA, Good SS, et al. In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer Res, 1993, 53(19):4619-4626.
[113]于波,李世拥,安萍,等.双启动子引导自杀基因靶向杀伤5-FU耐药肿瘤细胞的研究.中华外科杂志,2002,40(11):811-813.
[114]Rygaard J,Povlsen CO.Heterotransplantation of a human malignant tumour to "Nude" mice.Acta Pathol Microbiol Scand.1969;77(4):758-60.
[115]Nilsson K,Giovanella BC,Stehlin JS,Klein G.Iumorigenicity of human hematopoietic cell lines in athymic nude mice.Int J Cancer.1977;19(3):337-44.
[116]时彦胜,耿志贤,战大伟等.SCID小鼠和BALB/c裸鼠对两株人体肿瘤细胞系移植敏感性的研究.中国实验动物学杂志.2000;10:526.
[117]Tanaka E,Yamashita JI,Hayashi.A pulmonary metastatic model of human non-small cell lung carcinoma cells t hat produce a neut rophil elastase-like molecule in severe combined immunodeficiency mice.Chest.2003;123:1248-1253.
[118]王允,周清华,覃扬.外源性FHIT基因表达对肺腺癌细胞株A549恶性表型的逆转作用.Chin J Med Genet.2002:19:225-229.
[1]Naldini.L,Blomer U,Gallay P,et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J].Science,1996;272:263-7.
[2]Poeschla E,Gilbert J,Li X et al.Identification of a human immunodeficiency virus type 2(HIV-2)encapsidation determinant and transduction of nondividing human cells by HIV-2-based lentivirus vectors[J].J Virol,1998;72:6527-36.
[3]Mangeot PE,Negre D,Dubois B,et al.Development of minimal lentivirus vectors derived from simian immunodeficiency virus(SIVmac251)and their use for gene transfer into human Dendritic cells[J].J Virol,2000;74:8307-15.
[4] Poeschla EM, Wong-Staal F, Looney DJ. Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors[J].Nat Med, 1998;4:354-7.
[5] Berkowitz R, lives H, Lin WY, et al. Construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus[J].J Virol, 2001;75:3371-82.
[6] Mselli-Lakhal L, Favier C, Da Silva, et al. Defective RNA packaging is responsible for low transduction efficiency of CAEV-based vectors[J]. Arch Virol, 1998;143:681-95.
[7] Olsen JC. Gene transfer vectors derived from equine infectious anemia virus[J].Gene Ther, 1998;5:1481-7.
[8] Mitrophanous K, Yoon S, Rohll J, et al. Stable gene transfer to the nervous system using a non-primate lentiviral vector. Gene Ther, 1999;6: 1808-18.
[9] Luigi Naldini, Ulrike Blomer, Philippe Gallay, et al. In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector[J]. Science, 1996:272:263.
[10] T Kafri, U Blomer, DA Peterson et al.Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors[J]. Nat Genet,1997;17(3):314-7.
[11] T Iwakuma, Y Cui, LJ Chang. Self-inactivating lentiviral vectors with U3 and U5 modifications[J]. Virology, 1999;261(1):120-32.
[12] C Delenda. Lentiviral vectors: optimization of packaging, Transd- Uction and gene expression[J]. J Gene Med,2004;6 Suppl l:S125-38.
[13] Chinnasamy D, Chinnasamy N, Enriquez MJ et al. Lentiviral-mediated gene transfer intohuman lymphocytes: role of HIV—1 accessory proteins[J]. Blood, 2000;96:1309-16.
[14] Costello E, Munoz M, Buetti E et al. Gene transfer into stimulated and unstimulated T lymphocytes by HIV-1-derived lentiviral vectors[J]. Gene Ther, 2000;7:596-604.
[15] Rossi GR, Mautino MR, Morgan RA. High-efficiency lentiviral vector- mediated gene transfer into murine macrophages and activated splenic B lymphocytes[J].Hum Gene Ther,2003:14:385-91.
[16] Negre D, Mangeot PE, Duisit G, et al. Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cel1s[J]. Gene Ther, 2000;7:1613-23.
[17] Mangeot PE, Duperrier K, Negre D, et al. High levels of transduction of human dendritic cells with optimized SIV vectors[J].Mol Ther, 2002;5: 283-90.
[18] Johnston JC, Gasmi M, Lim LE, et al. Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors[J].J Virol, 1999;73:4991-5000.
[19] Molina RP, Matukonis M, Paszkiet B et al. Mapping of the bovine immunodeficiency virus Packaging signal and RRE and incorporation into a minimal gene transfer vector[J]. Virology,2002;304:10 - 23.
[20] Curran MA, Kaiser SM, Achacoso PL, et al. Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors[J]. Mol Ther,2000;1:31 - 38.
[21] Mautino MR, Keiser N, Morgan RA. Improved titers of HIVbased lentiviral vectors using the SRV-1 constitutive transportelement. Gene Ther,2000;7:1421-24.
[22] Nappi F, Schneider R, Zolotukhin A, et al. Identification of a novel posttranscriptional regulatory element by using a rev- and RRE-mutated human immunodeficiency virus type 1 DNA proviral clone as a molecular trap[J].J Virol, 2001;75:4558-69.
[23] Kotsopoulou E, KimVN, Kingsman AJ et al. A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codonoptimized HIV-1 gag-pol gene[J].J Virol, 2000;74:4839-52.
[24] Wagner R, Graf M, Bieler K, et al. Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus:implications for the safety of lentiviral vectors in process citation] [J].Hum Gene Ther, 2000;11: 2403-2413.
[25] Kuate S, Wagner R, Uberla K. Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectors[J].J Gene Med, 2002;4:347 - 355.
[26] Pandya S, Boris-Lawrie K, Leung NJ et al. Development of an Rev-independent, minimal simian immunodeficiency virus-derived vector system[J]. Hum Gene Ther, 2001;12:847 - 857.
[27] Roberts TM, Boris-Lawrie K. The 5_ RNA terminus of spleen necrosis virus stimulates translation of nonviral mRNA[J].J Virol, 2000; 74:8111-18.
[28] Wu X, Wakefield JK, Liu H, et al. Development of a novel translentiviral vector that affords predictable safety[J].Mol Ther, 2000;2:47-55.
[29] Cherepanov P, Pluymers W, Claeys A et al. High-level expression of active HIV-1 integrase from a synthetic gene in human cells [J]. FASEB J, 2000:14:1389-99.
[30] Towers GJ, Hatziioannou T, Cowan S, et al. Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors[J].Nat Med, 2003:9:1138-43.
[31] Kootstra NA, Munk C, Tonnu N et al. Abrogation of postentry restriction of HIV-1 based lentiviral vector transduction in simian cells[J].Proc Natl Acad Sci U S A, 2003:100:1298-1303.
[32] Stitz J, Muhlebach MD, Blomer U, et al. A novel lentivirus vector derived from apathogenic simian immunodeficiencyvirus[J]. Virology, 2001:291:191-7.
[33] Goujon C, Jarrosson-Wuilleme L, Bernaud J et al. Heterologous human immunodeficiency virus type 1 lentiviral vectors packaging a simian immunodeficiency virusderived genome display a specific postentry transduction defect in dendritic cells[J].J Virol, 2003:77: 9295-304.
[34] Negre D, Mangeot PE, Duisit G, et al. Characterization of novel safe lentiviral vectors derived from simian immunodeficiencyvirus (SIVmac251) that efficiently transduce mature human dendritic cells[J]. Gene Ther, 2000:7:1613-23.
[35] D' CostaJ, Harvey-WhiteJ, QasbaP, et al. HIV-2 derived lentiviral vectors:gene transfer in Parkinson' s and Fabry disease models in vitro[J].J Med Virol, 2003:71:173-82.
[36] Browning MT, Schmidt RD, Lew KA, et al. Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions[J]. J Virol, 2001;75: 5129-40.
[37] Reiser J, Lai Z, Zhang XY, et al. Development of multigene and regulated lentivirus vectors[J].J Virol, 2000; 74:10589-99.
[38] Kafri T, van PraagH, Gage FH, Verma IM. Lentiviral vectors:regulated gene expression[J]. Mo1 Ther, 2000;1:516-21.
[39] Ogueta SB, Yao F, Marasco WA. Design and in vitro characterization of a single regulatory module for efficient control of gene expression in both plasmid DNA and a selfinactivating lentiviral vector[J].Mol Med, 2001:7:569-79.
[40] Vigna E, Cavalieri S, Ailles L, et al. Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors[J].Mol Ther, 2002:5:252-61.
[41] Regulier E, Pereira de Almeida L, Sommer B et al. Dose-dependent neuroprotective effect of ciliary neurotrophic factor delivered via tetracycline-regulated lentiviral vectors in the quinolinic acid rat model of Huntington' s disease[J]. Hum Gene Ther, 2002; 13:1981-90.
[42] Johansen J, Rosenblad C, Andsberg K, et al. Evaluation of Teton system to avoid transgene down-regulation in ex vivo gene transfer to the CNS[J]. Gene Ther, 2002;9:1291-301.
[43] Koponen JK, Kankkonen H, Kannasto J, et al. Doxycyclineregulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo[J]. Gene Ther, 2003;10:459-66.
[44] Salmon P, Oberholzer J, Occhiodoro T et al. Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes[Jl.Mol Ther, 2000:2:404-14.
[45] Mitta B, Rimann M, Ehrengruber MU, et al. Advanced modular self- inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction[J]. Nucleic Acids Res,2002;30:113.
[46] Chang LJ, Zaiss AK. Self-inactivating lentiviral vectors and a sensitive Cre-loxP reporter system[J]. Methods Mol Med, 2003:76:367-82.
[47] Karreman S, Hauser H, Karreman C. On the use of double FLP recognition targets (FRTs) in the LTR of retroviruses for the construction of high producer cell lines[J].Nucleic Acids Res,1996:24:1616-24.
[48] Chantret I, Dupre T, Delenda C, et al. Congenital disorders of glycosylation type Ig is defined by a deficiency in dolichyl-P-mannose:Man7GlcNAc2-PP-dolichyl mannosyltransferase [J].J Biol Chem,2002;277:25 815-22.
[49] Chantret I, Dancourt J, Dupre T, et al. A deficiency in dolichyl- P-glucose:GlclMan9GlcNAc2-PP-dolichyl alpha3-glucosyl transferase defines a new subtype of congenital disorders of glycosylation[J]. J Biol Chem,2003:278:9962-71.
[50] Richard E, Geronimi F, Lalanne M, et al.A bicistronic SINlentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines[J].J Gene Med, 2003;5: 737-47.
[51] Azzouz M, Martin-Rendon E, Barber RD, et al.Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson' s disease[J].J Neurosci, 2002;22:10302-12.
[52] Zhu Y, Feuer G, Day SL, et al. Multigene lentiviral vectors based on differential splicing and translational control[J]. Mol Ther,2001;4: 375-82.
[53] Zhu Y, Planelles V. A multigene lentiviral vector system based on differential splicing[J]. Methods Mol Med, 2003:76:433-48.
[54] Murthy RC, McFarland TJ, Yoken J, et al. Corneal transduction to inhibit angiogenesis and graft failure[J]. Invest Ophthalmol Vis Sci,2003:44:1837-42.
[55] M Wiznerowicz , Trono. arnessing HIV for therapy, basic research and biotechnology[J]. Trends Biotechnol, 2005;23(1):42-7.
[56] Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector [J]. Science 1996; 272:263-7
[57] Kordower, J. H. et al. () Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson' s disease[J]. Science, 2000;290:767-73
[58] Mazarakis, N. D. et al. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery[J]. Hum. Mol.Genet, 2001;10:2109-21.
[59] Azzouz,M et al.VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature, 2004;429: 413-17.
[60] Lo Bianco, C. et al. a-Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson' s disease[J]. Proc Natl Acad Sci USA, 2002:99:10813-8.
[61] Regulier, E. et al. Early and reversible neuropathology induced by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum[J]. Hum Mol Genet, 2003:12:2827-36.
[62] de Almeida, L. P. et al. Lentiviral-mediated delivery of mutant huntingtin in the striatum of rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein length[J].J Neurosci, 2002:22:3473-83.
[63] Moreau-Gaudry, F. et al. High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors[J]. Blood, 2001:98:2664-72.
[64] Chen, W. et al. Lentiviral vector transduction of hematopoietic stem cells that mediate long-term reconstitution of lethally irradiated mice[J]. Stem Cells, 2000:18:352-9.
[65] Hanazono, Y. et al. Gene transfer into nonhuman primate hematopoietic stem cells: implications for gene therapy[J]. Stem Cells, 2001;19:12-23.
[66] Horn, P. A. et al. Lentivirus-mediated gene transfer into hematopoietic repopulating cells in baboons[J]. Gene Ther, 2002; 9:1464-71.
[67] Woods, N. B. et al. Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells[J].Blood,2000;96:3725-33.
[68] Follenzi, A, et al. Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice[J]. Blood, 2004;103:3700-9.
[69] Nguyen, T. H. et al. Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes [J].Mol Ther, 2002;6:199 - 209.
[70] Kang, Y. et al. In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River virus glycoproteins[J].J Virol, 2002; 76:9378-88.
[71] VandenDriessche, T. et al. Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen- presenting cells in vivo[J]. Blood, 2002:100:813-22.
[72] Pfeifer, A. et al. Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging[J].Mol Ther, 2001;3:319-22.
[73] Park, F. et al.The effect of age on hepatic gene transfer with self-inactivating lentiviral vectors in vivo[J].Mol Ther, 2003:8:314-23.
[74] Park F, et al. Efficient lentiviral transduction of liver requires cell cycling in vivo[J].Nat Genet,2000;24:49 - 52.
[75] Ohashi K , et al. Role of hepatocyte direct hyperplasia in lentivirus-mediated liver transduction in vivo[J].Hum Gene Ther 2002; 13:653-63.
[76] Elbashir S M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature, 2001 ;411:494—8.
[77] Brummelkamp T R, et al. A system for stable expression of short interfering RNAs in mammalian cells[J]. Science, 2002;296:550-3.
[78] Abbas-Terki T, et al. Lentiviral-mediated RNA interference[J]. Hum Gene Ther, 2002;13:2197-201.
[79] Wiznerowicz M, Trono, D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference [J].J Virol, 2003:77:8957-61.
[80] Rubinson, D.A. et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference[J]. Nat Genet, 2003;33:401-6.
[81] Gupta S, et al. Inducible, reversible and stableRNAinterference in mammalian cells[J]. Proc Natl Acad Sci USA, 2004;101:1927-32.
[82] Matsukura S, et al. Establishment of conditional vectors for hairpin siRNA knockdowns[J].Nucleic Acids Res, 2003;31:e77.
[83] Van dewatering M, et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector[J]. EMBO Rep, 2003:4:609-15.
[84] Wang J, et al. Stable and controllable RNA interference: investigating the physiological function of glutathionylated actin [J]. Proc Natl Acad Sci USA, 2003:100:5103-6.
[85] Cherry S R, et al. Retroviral expression in embryonic stem cells and hematopoietic stem cells[J].Mol Cell Biol, 2000:20:7419-26.
[86] Lois C, et al, Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors[J]. Science, 2002;295:868-72.
[87] Pfeifer A, et al. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos[J]. Proc Natl Acad Sci USA, 2002;99:2140-45.
[88] Hofmann A, et al. Efficient transgenesis in farm animals by lentiviral vectors[J]. EMBO Rep, 2003:4:1054-60.
[89] Hofmann A, et al. Generation of transgenic cattle by lentiviral gene transfer into oocytes[J].Biol Reprod, 2004:71:405-9.
[90] Wolfgang M J, et al. Rhesus monkey placental transgene expression after lentiviral gene transfer into preimplantation embryos [J]. Proc Natl Acad Sci USA, 2001:98:10728-32.
[91] Hamra FK, et al. Production of transgenic rats by lentiviral transduction of male germ-line stem cells[J].Proc Natl Acad Sci USA,2002; 99:14931-36.
[92] EscarpeP, et al. Development of a sensitive assay for detection of replication-competent recombinant lentivirus in largescale HIV-based vector preparations[J].Mol Ther, 2003:8:332-41.
[93] Lu X, et al. Anti sense-mediated inhibition of human immunodeficiency virus (HIV) replication by use of an HIV type 1-based vector results in severely attenuated mutants incapable of developing resistance[J]. J Virol, 2004:78:7079-88.
[94] Bushman F D. Integration site selection by lentiviruses:biology and possible control[J].Curr Top Microbiol Immunol, 2002;261:165-77.