肿瘤干细胞致敏的树突状细胞对脑胶质瘤免疫作用研究
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摘要
脑胶质瘤是颅内最常见的脑肿瘤,与周围组织分界不清,浸润性生长,目前治疗以手术切除为主,术后辅以放疗、化疗,但效果不理想。胶质母细胞瘤手术治疗后,平均生存时间为12-18个月,一般不超过2年,而且近年来治疗效果毫无进展。寻找新的有效治疗方法成为目前研究热点。近年来,随着免疫学及分子生物学的发展,胶质瘤的基因治疗和免疫治疗受到大家的重视,树突状细胞疫苗成为治疗颅内肿瘤的最有希望的免疫治疗方法。
树突状细胞(dendritic cells,DC)是目前已知的人体内功能最强大的、专职抗原呈递细胞,能捕获、加工处理抗原,并将抗原信息呈递给淋巴细胞,从而引起一系列免疫反应。目前已有多种DC疫苗在实验中已取得良好的抗瘤效果。致敏DC的抗原有全肿瘤抗原、特异性抗原肽、肿瘤DNA或RNA等。DC最大的功能是能够处理、呈递肿瘤抗原给T淋巴细胞,促使T淋巴细胞识别肿瘤,产生杀瘤效应。目前尚不清楚胶质瘤的特异性抗原和相关抗原,因此DC呈递的抗原激活T淋巴细胞效率低下。
脑肿瘤干细胞最早由Ignatova:提出,他发现肿瘤组织中只有少数细胞具有不断分裂增殖倾向,它们具有干细胞的生长特性,因此提出了肿瘤干细胞的概念。这些数量极少具有类似干细胞特性的细胞,它们具有自我更新、无限增殖、多潜能分化能力,是胶质瘤产生和复发的根源。脑肿瘤干细胞表达CD133和Nestin,具有高致瘤性,Singh发现接种100个CD133+细胞就能在小鼠颅内形成肿瘤,而接种105个CD133-细胞也未见肿瘤生长,由此可见胶质瘤干细胞具有很强的抗原性。
本实验用C6胶质瘤细胞在无血清培养基中培养,加入细胞因子EGF、bFGF进行诱导,制备胶质瘤干细胞,光镜下观察其形态,免疫荧光检测特异性标志物CD133和Nestin,并进行分化及致瘤性观察。从大鼠骨髓提取单核细胞,加入GM-CSF和IL-4诱导成DC,用胶质瘤干细胞冻融抗原致敏DC制备疫苗,经尾静脉注入荷瘤大鼠体内,能明显延长大鼠的生存时间,病理切片可见较多的CD8+淋巴细胞浸润。
研究目的
胶质瘤是颅内的恶性肿瘤,手术难以切除干净,术后容易复发,术后放疗、化疗效果不理想,因此有必要寻找新的有效的治疗方法。胶质瘤的免疫治疗受到大家的重视。树突状细胞是目前已知的人体内功能最强大的、专职抗原呈递细胞,树突状细胞疫苗已经进行临床试验,证实安全有效。肿瘤干细胞是肿瘤发生发展的根源,具有很强的抗原性。本实验用肿瘤干细胞致敏树突状细胞制备疫苗,研究其对荷瘤大鼠的保护作用,为树突状细胞疫苗的临床应用提供理论基础,为胶质瘤的预防和治疗提供新的途径。
研究方法
1.肿瘤干细胞的培养、鉴定
C6大鼠胶质瘤细胞系快速解冻,用含有10%胎牛血清DMEM培养基培养,置于37℃,5%CO2饱和湿度的恒温培养箱中培养。取对数生长期的C6细胞,接种于无血清培养基中,培养基含2%B27、bFGF 20 ng/ml及EGF 20 ng/ml。免疫荧光检测肿瘤干细胞特异性标志物CD133和Nestin,流式细胞仪检测BTSC表面分子。将10只裸鼠随机分为A、B两组,每组5只,分别于皮下注射肿瘤干细胞及C6细胞,观察肿瘤形成情况,肿瘤切片行HE染色。免疫荧光检测BTSC分化为成熟神经细胞的标志GFAP。
2.DC的分离、培养及疫苗的制备
在无菌条件下冲洗大鼠骨髓腔,获取组织悬液。用淋巴细胞分离液梯度离心,收集界面层单个核细胞。培养箱中孵育2小时,收集非粘附细胞。加入细胞因子GM-CSF(5ng/ml)和IL-4(5ng/ml),第12d天收获成熟的DC。倒置相差显微镜下观察其形态。将分离好的单细胞悬液离心,加入荧光素标记的小鼠抗大鼠OX62单克隆抗体,另一组试管中加入用同样荧光素标记的同型抗体作为对照,流式细胞仪检测OX62表达情况。制备两种DC疫苗。取对数生长期的肿瘤干细胞及C6胶质瘤细胞,分别悬浮成浓度5×106/ml。反复冻融,裂解物离心,肿瘤抗原与DC以3：1比例混合,37℃、5%CO2、饱和湿度培养箱中培养18h(抗原量以冻融前肿瘤细胞的细胞数计算,树突状细胞以细胞数计算),即获得DC-BTSC疫苗及DC-C6疫苗。混合淋巴细胞反应：采用大鼠脾脏来源的淋巴细胞作为反应细胞,DC-BTSC、DC-C6及DC作为刺激细胞。分别设立反应细胞及刺激细胞对照组。于酶标仪上490nm处读取OD值,计算刺激指数(SI)。SI=(混合反应细胞孔OD值-刺激细胞孔OD值)/反应细胞孔OD值
3.DC疫苗对荷瘤大鼠的治疗作用研究
建立C6大鼠胶质瘤模型,将C6细胞注射种植与大鼠右侧尾状核区域。40只成年Wistar大鼠,随机分成A、B、C、D四组,每组10只。7天后A组经尾静脉注射1×107DC-BTSC疫苗(体积100ul)、B组注射1×107DC-C6疫苗、C组为1×107DC,D组为PBS对照,共3次,每次间隔1周。所有荷瘤大鼠死亡后均行病理检查,行HE染色及免疫组化检测CD8+T淋巴细胞浸润情况。生存分析采用Kaplan-Meier方法,生存率比较采用Log-rank检验。
结果
1.C6胶质瘤细胞在无血清培养条件下,5～7天左右后形成细胞球,细胞多为透亮圆形,悬浮生长,折光性好。从无血清培养基中分离培养的脑肿瘤干细胞进行荧光染色,可见CD133和Nestin染色阳性；在含血清培养基中,肿瘤干细胞分化后GFAP免疫荧光染色亦呈阳性表达。流式细胞仪显示：诱导后的胶质瘤干细胞MHC-Ⅱ、CD80及CD86表达率为60%、51%、62%,显著高于C6胶质瘤细胞的9%、6%、4%。A组接种肿瘤干细胞小鼠皮下可见肿瘤长出。B组接种干细胞完全培养基中的贴壁细胞小鼠未见肿瘤长出。裸鼠成瘤速度与细胞密度密切相关。浓度越高,肿瘤生长越快。取出移植瘤后经HE染色可见典型胶质瘤组织。将BTSC重新接种于含血清培养基中,细胞球逐渐贴壁,四周伸出的许多细小突起；相互连接成网状,贴壁分化的细胞形态与C6细胞一致。
2.树突状细胞培养的第2天可见培养基中的单个核细胞贴壁生长,第10天可见悬浮细胞明显增多,细胞体积增大,从胞体周围伸出多个长短不一的树突状突起。流式细胞学检查显示OX62表达阳性率为86%。未经诱导的单个核细胞0X62表达率低。MLR结果显示：相同S:R比例下,DC-BTSC刺激淋巴细胞的增殖指数明显高于DC-C6刺激的增殖指数(P<0.01),并且DC与淋巴细胞比例在1：10时刺激能力最强,淋巴细胞增殖最明显,随着两者比例增大或减少刺激能力减弱。
3.立体定向接种胶质瘤后3周,可见大鼠一侧大脑半球上肿瘤组织呈菜花样向外生长,而接种DC-BTSC疫苗组大鼠脑组织未见肿瘤生长。疫苗接种后45天后,取出脑组织行HE染色,A组未见明显肿瘤细胞,其余组可见肿瘤细胞,有不同数量的核分裂相,瘤内坏死。CD8+免疫组化染色见ABC三组均有CD8+T淋巴细胞浸润,其中A组最多,其次为B组,C组散在少量,D组未见CD8+T淋巴细胞浸润。A组中位生存时间为(39±3.12)d,B组为(29±2.01)d,C组为(23±1.42)d,D组为(19±1.02)d。Log-rank检验：4组生存曲线差异总体比较差异有显著统计学意义(P<0.01);A组与B、C、D组分别比较差异均有统计学意义(P<0.05)；B组与C、D组比较差异亦有统计学意义；C、D组比较无统计学意义。
结论
1.无血清培养基中加入细胞因子EGF、bFGF培养C6胶质瘤细胞,可以获得足够数量的肿瘤干细胞。在含血清培养基中分化后可形成与C6一致的肿瘤细胞。
2.诱导后的肿瘤干细胞比诱导前的C6高表达MHC-Ⅱ、CD80、CD86表面分子,抗原性更强。
3.肿瘤干细胞和胶质瘤细胞皮下接种裸鼠,可见肿瘤干细胞致瘤能力更强,并且肿瘤生长速度与接种细胞浓度成正比。
4.从大鼠骨髓获取DC前体细胞,经淋巴细胞分离液分离后,梯度离心,用细胞因子GM-CSF和IL-4联合培养,可以获得足够数量的DC。
5.在同种异体混合淋巴细胞反应(MLR)中,肿瘤干细胞致敏的DC与胶质瘤细胞致敏的DC相比,刺激指数更高,S:R=1:10时,刺激指数最强。
6.胶质瘤干细胞抗原致敏的DC疫苗接种大鼠后,能够诱导机体的免疫反应,抑制C6胶质瘤的生长,延长大鼠的生存时间。
7.胶质瘤干细胞抗原致敏的DC疫苗对大鼠具有良好的免疫保护作用,其效果优于胶质瘤细胞致敏的DC疫苗及未经抗原致敏的DC,为胶质瘤的免疫治疗提供了新的途径。
Glioma is the most common intracranial tumor.It present as diffuse toumors with invasion into normal brain tissue.After surgery,radiation or chemotherapy the glioma often recur or progress. The clinical therapeutic effect to glioma is disappointing. Despite the improvement of local therapies, such as surgery and radiation, the mean survival time for patients carrying a diagnosis of glioblastoma multiforme remains virtually unchanged. On average, patients survive 12 to 18 months, with few patients surviving beyond 2 years. Therefore it is urgent to find effective therapies of treating glioma.Recently,with the development of immunology and molecular biology, gene therapy and immunotherapy are rapidly gaining momentum. One of the most promising immunotherapeutic approaches for the treatment of glioma is the vaccination of cancer patients with dendritic cells (DC) pulsed with tumor antigens.
As the most impotant functional antigen presenting cell, DC can capture、process and present antigen to lymphocyte.Recently, much research of different kinds of DC vaccine has got satisfactory antitumor effect. DC can present kinds of antigen such as the whole tumor antigen specific antigen、neoplasm DNA or RNA. The most impotant function of DC is processing and presenting antigen to lymphocyte,which can evoke anti-tomour effect.It is unknown about the specific and related antigen of glioma,so the anti-tumor effect is limited.
BTSC were first reported by Ignatova.He observed small part of glioma cell posseses the abilities of self-regenerate, which caused the proliferation of glioma,namely BTSC. Similar to the NSCs, BTSC posses the abilities of self-regenerate and multipotential differentiation,which were connected to originate of glioma.BTSC expressed CD133+ and Nestin.Singh discovered only 100 CD133+cells could generate glioma in rat brain,but 105 CD133-cells could not do it. The result showed BTSC had strong antigenicity.
In our study,C6 cell line was cultured in serum-free medium with EGF, bFGF to obtain tumor cell spheres. The morphous of BTSC was observed under microscope before and after differentiation.Specific markers such as CD 133 and nestin, were employed to recognize BTSC by immunofluorescence cytochemistry techniques. The monocytes from rat bone were cultured in RPMI-1640 containing GM-CSF and IL-4 to obtain DC. Dendritic cells pulsed with brain tumor stem cells can provide good protect for tumor-bearing rat. Pathological section of the tumor have the most inflammation and CD8+T lymphocytes.
Objective
Glioma is the malignant intracranial tumor. Bescause it is very hard to be cured with the conventional therapy including surgery,radiation and chemotherapy,the recurrence rate of glioma is much high.The immunotherapy of glioma has become hot spot. Dendritic cells are the professional antigen presenting cells in vivo.DC vaccine has been applied in clinical trials,which has been safely and effectively. BTSC are the reasons of glioma occuring and recurring.They have good antigenicity of glioma. The aim of our study is to investigate the effect of dendritic cells pulsed with brain tumor stem cells. It will provide the experimental foundation for clinical immunotherapy of glioma.
Methods 1. The culture and identification of BTSC
C6 glioma cells were firstly cultured in DMEM containing 10% FCS. C6 cells which were in exponential phase of growth were transplanted to serum-free medium containing B27 supplement, basic fibroblast growth factor(20ng/ml) and recombinant epidermal growth factor(20ng/ml). The expression of special markers CD 133 and Nestin was detected by immunofluorescence. Method of flow cytometry were used to determine cell surface molecule of BTSC. Divided 10 nude mouse into two groups randomly, each group had 5 rats.One group accept BTSC by subcutaneous injections,the other accept C6 cells by the same way. we applied immunofluorescence to detect the expression of special markers of mature nerve cells such as GFAP.
2. The isolation and culture of DC.
Rat bone marrow monocytes isolated from rat femurs and tibias were cultured in RPMI1640 containing 10% FCS, granulocyte macrophage-colony stimulating factor(GM-CSF,5ng/ml)and interleukin-4(IL-4,5ng/ml). The mature DC were harvested after 12 days.The mature DC were analyzed by morphology with phase microscope; The expressions of specific marker OX62 was analyzed by flow cytometry. C6 glioma cells of logarithm growth period were collected through digestion and calculated.We prepare BTSC and C6 cells antigen by freezing-melt method. The antigen and DC were cocultured by the ratio 3:1.The function of two kinds of vaccines were examined by mixed lymphocyte rection (MLR).
3. The treatment of glioma by DC vaccine
To established rat C6 brain-glioma model. C6 tumor cells were cultured in vitro and then the C6 cells at logarithmic growth phase were injected intracranially under the stereotactic monitor into the right caudate nucleus.40 Wistar rats were divided into group A、B、C and D randomly, each group had 10 rats. Group A was injected with DC-BTSC vaccines of 1 X 107 cells through tail veins once a week for 3 weeks. Group B was treated with DC-C6 vaccines. The same number of DC and the same volume PBS were applied to group C and group D. Tomor samples were examined with HE staining and immunohistochemistry. The survival time of rats was analyzed by Log-rank survival analysis.
Results
1. C6 glioma cells were cultured in serum-free medium. After five to seven days,they presented as typical tumor spheres,which appeared globular and floating in medium. The expression of special markers CD 133 and Nestin marked by immunofluorescence was positive. When cultured in serum-supplemented medium,BTSC began to differentiate and express mature neurocyte antigens such as GFAP. Flow cytometry results showed:the average positive rate of MHC-Ⅱ、CD80 and CD86 was 60%、51% and 62% respectively,which was significantly higher than that of C6 cells(9%、6%、4%). Tumor spheres were transplanted to rats in group A and tumor nodules were seen after several weeks.But no tumors were formed in group B injected with attached cells in serum-free medium.The size of formed tumor in nude mouse was related to cell density injected.The pathological section of transplanted tumor with HE staining showed typical characterization of C6 cells.When the cultural condition was changed into serum-supplemented medium,BTSC attached and appeared to reticulate.The characterization of differentiated cell was the same as C6 cells.
2. When cultured with cytokines,after 10 days, PBMCs attached developed to free-floating DC, which had typical morphological dendrites. The positive rate of special surface marker OX62 on DC was 86% by flow cytometry analysis,which was low on PBMCs uninduced with cytokine.MLR showed:the stimulation index of DC-BTSC was significantly higher than that of DC-C6 at the same ratio (P< 0.01),which was the highest at stimulator to responder ratio of 1:10.
3. After 3 weeks implanted C6 tumor cells by stereotaxis, the cerebral hemisphere of rats was observed the cauliflower-like growth of tumor. But no tumors were formed in the group protected by DC-BTSC vaccine. The pathological sections stained with HE showed the growth of tumor cells and intratumoral necrosis in group B、C and D.In contrast,they were not shown in group A. Immunohistology staining demonstrated the infiltration with CD8+T lymphocyte in group A、B and C.The MST of rats were as fllowing:(39±3.12)days of group A、(29±2.01)days of group B、(23±1.42)days of group C and (19±1.02)days of group D. The survival analysis showed group A was statistically significant in constrast to the other groups(P<0.05).
Conclusion
1. We could get enough BTSC by culturing C6 glioma cells in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The characterization of differentiated cell was the same as C6 cells.
2. The expression of MHC-Ⅱ、CD80 and CD86 on BTSC was higher than that of C6 glioma cells.
3. The growth of tumor in nude mouse injected with BTSC was faster than that of nude mouse injected with C6 glioma cells.
4. We got enough DC by culturing rat bone marrow cells in RPMI1640 containing GM-CSF and IL-4.
5. MLR showed:the stimulation index of DC-BTSC was significantly higher than that of DC-C6 at the same ratio,which was the highest at stimulator to responder ratio of 1:10.
6. DC loading with BTSC lysates can provide immunity protect for the rats loaded with C6 glioma cells,which could prolong those live time.
7. DC loading with BTSC lysates can provide a higher level of immunity protect against gliomas than those loading with C6 cells,which provide a new method in the immuntherapy of brain glioma.
树突状细胞(dendritic cells,DC)是目前已知的人体内功能最强大的、专职抗原呈递细胞,能捕获、加工处理抗原,并将抗原信息呈递给淋巴细胞,从而引起一系列免疫反应。目前已有多种DC疫苗在实验中已取得良好的抗瘤效果。致敏DC的抗原有全肿瘤抗原、特异性抗原肽、肿瘤DNA或RNA等。DC最大的功能是能够处理、呈递肿瘤抗原给T淋巴细胞,促使T淋巴细胞识别肿瘤,产生杀瘤效应。目前尚不清楚胶质瘤的特异性抗原和相关抗原,因此DC呈递的抗原激活T淋巴细胞效率低下。
脑肿瘤干细胞最早由Ignatova:提出,他发现肿瘤组织中只有少数细胞具有不断分裂增殖倾向,它们具有干细胞的生长特性,因此提出了肿瘤干细胞的概念。这些数量极少具有类似干细胞特性的细胞,它们具有自我更新、无限增殖、多潜能分化能力,是胶质瘤产生和复发的根源。脑肿瘤干细胞表达CD133和Nestin,具有高致瘤性,Singh发现接种100个CD133+细胞就能在小鼠颅内形成肿瘤,而接种105个CD133-细胞也未见肿瘤生长,由此可见胶质瘤干细胞具有很强的抗原性。
本实验用C6胶质瘤细胞在无血清培养基中培养,加入细胞因子EGF、bFGF进行诱导,制备胶质瘤干细胞,光镜下观察其形态,免疫荧光检测特异性标志物CD133和Nestin,并进行分化及致瘤性观察。从大鼠骨髓提取单核细胞,加入GM-CSF和IL-4诱导成DC,用胶质瘤干细胞冻融抗原致敏DC制备疫苗,经尾静脉注入荷瘤大鼠体内,能明显延长大鼠的生存时间,病理切片可见较多的CD8+淋巴细胞浸润。
研究目的
胶质瘤是颅内的恶性肿瘤,手术难以切除干净,术后容易复发,术后放疗、化疗效果不理想,因此有必要寻找新的有效的治疗方法。胶质瘤的免疫治疗受到大家的重视。树突状细胞是目前已知的人体内功能最强大的、专职抗原呈递细胞,树突状细胞疫苗已经进行临床试验,证实安全有效。肿瘤干细胞是肿瘤发生发展的根源,具有很强的抗原性。本实验用肿瘤干细胞致敏树突状细胞制备疫苗,研究其对荷瘤大鼠的保护作用,为树突状细胞疫苗的临床应用提供理论基础,为胶质瘤的预防和治疗提供新的途径。
研究方法
1.肿瘤干细胞的培养、鉴定
C6大鼠胶质瘤细胞系快速解冻,用含有10%胎牛血清DMEM培养基培养,置于37℃,5%CO2饱和湿度的恒温培养箱中培养。取对数生长期的C6细胞,接种于无血清培养基中,培养基含2%B27、bFGF 20 ng/ml及EGF 20 ng/ml。免疫荧光检测肿瘤干细胞特异性标志物CD133和Nestin,流式细胞仪检测BTSC表面分子。将10只裸鼠随机分为A、B两组,每组5只,分别于皮下注射肿瘤干细胞及C6细胞,观察肿瘤形成情况,肿瘤切片行HE染色。免疫荧光检测BTSC分化为成熟神经细胞的标志GFAP。
2.DC的分离、培养及疫苗的制备
在无菌条件下冲洗大鼠骨髓腔,获取组织悬液。用淋巴细胞分离液梯度离心,收集界面层单个核细胞。培养箱中孵育2小时,收集非粘附细胞。加入细胞因子GM-CSF(5ng/ml)和IL-4(5ng/ml),第12d天收获成熟的DC。倒置相差显微镜下观察其形态。将分离好的单细胞悬液离心,加入荧光素标记的小鼠抗大鼠OX62单克隆抗体,另一组试管中加入用同样荧光素标记的同型抗体作为对照,流式细胞仪检测OX62表达情况。制备两种DC疫苗。取对数生长期的肿瘤干细胞及C6胶质瘤细胞,分别悬浮成浓度5×106/ml。反复冻融,裂解物离心,肿瘤抗原与DC以3：1比例混合,37℃、5%CO2、饱和湿度培养箱中培养18h(抗原量以冻融前肿瘤细胞的细胞数计算,树突状细胞以细胞数计算),即获得DC-BTSC疫苗及DC-C6疫苗。混合淋巴细胞反应：采用大鼠脾脏来源的淋巴细胞作为反应细胞,DC-BTSC、DC-C6及DC作为刺激细胞。分别设立反应细胞及刺激细胞对照组。于酶标仪上490nm处读取OD值,计算刺激指数(SI)。SI=(混合反应细胞孔OD值-刺激细胞孔OD值)/反应细胞孔OD值
3.DC疫苗对荷瘤大鼠的治疗作用研究
建立C6大鼠胶质瘤模型,将C6细胞注射种植与大鼠右侧尾状核区域。40只成年Wistar大鼠,随机分成A、B、C、D四组,每组10只。7天后A组经尾静脉注射1×107DC-BTSC疫苗(体积100ul)、B组注射1×107DC-C6疫苗、C组为1×107DC,D组为PBS对照,共3次,每次间隔1周。所有荷瘤大鼠死亡后均行病理检查,行HE染色及免疫组化检测CD8+T淋巴细胞浸润情况。生存分析采用Kaplan-Meier方法,生存率比较采用Log-rank检验。
结果
1.C6胶质瘤细胞在无血清培养条件下,5～7天左右后形成细胞球,细胞多为透亮圆形,悬浮生长,折光性好。从无血清培养基中分离培养的脑肿瘤干细胞进行荧光染色,可见CD133和Nestin染色阳性；在含血清培养基中,肿瘤干细胞分化后GFAP免疫荧光染色亦呈阳性表达。流式细胞仪显示：诱导后的胶质瘤干细胞MHC-Ⅱ、CD80及CD86表达率为60%、51%、62%,显著高于C6胶质瘤细胞的9%、6%、4%。A组接种肿瘤干细胞小鼠皮下可见肿瘤长出。B组接种干细胞完全培养基中的贴壁细胞小鼠未见肿瘤长出。裸鼠成瘤速度与细胞密度密切相关。浓度越高,肿瘤生长越快。取出移植瘤后经HE染色可见典型胶质瘤组织。将BTSC重新接种于含血清培养基中,细胞球逐渐贴壁,四周伸出的许多细小突起；相互连接成网状,贴壁分化的细胞形态与C6细胞一致。
2.树突状细胞培养的第2天可见培养基中的单个核细胞贴壁生长,第10天可见悬浮细胞明显增多,细胞体积增大,从胞体周围伸出多个长短不一的树突状突起。流式细胞学检查显示OX62表达阳性率为86%。未经诱导的单个核细胞0X62表达率低。MLR结果显示：相同S:R比例下,DC-BTSC刺激淋巴细胞的增殖指数明显高于DC-C6刺激的增殖指数(P<0.01),并且DC与淋巴细胞比例在1：10时刺激能力最强,淋巴细胞增殖最明显,随着两者比例增大或减少刺激能力减弱。
3.立体定向接种胶质瘤后3周,可见大鼠一侧大脑半球上肿瘤组织呈菜花样向外生长,而接种DC-BTSC疫苗组大鼠脑组织未见肿瘤生长。疫苗接种后45天后,取出脑组织行HE染色,A组未见明显肿瘤细胞,其余组可见肿瘤细胞,有不同数量的核分裂相,瘤内坏死。CD8+免疫组化染色见ABC三组均有CD8+T淋巴细胞浸润,其中A组最多,其次为B组,C组散在少量,D组未见CD8+T淋巴细胞浸润。A组中位生存时间为(39±3.12)d,B组为(29±2.01)d,C组为(23±1.42)d,D组为(19±1.02)d。Log-rank检验：4组生存曲线差异总体比较差异有显著统计学意义(P<0.01);A组与B、C、D组分别比较差异均有统计学意义(P<0.05)；B组与C、D组比较差异亦有统计学意义；C、D组比较无统计学意义。
结论
1.无血清培养基中加入细胞因子EGF、bFGF培养C6胶质瘤细胞,可以获得足够数量的肿瘤干细胞。在含血清培养基中分化后可形成与C6一致的肿瘤细胞。
2.诱导后的肿瘤干细胞比诱导前的C6高表达MHC-Ⅱ、CD80、CD86表面分子,抗原性更强。
3.肿瘤干细胞和胶质瘤细胞皮下接种裸鼠,可见肿瘤干细胞致瘤能力更强,并且肿瘤生长速度与接种细胞浓度成正比。
4.从大鼠骨髓获取DC前体细胞,经淋巴细胞分离液分离后,梯度离心,用细胞因子GM-CSF和IL-4联合培养,可以获得足够数量的DC。
5.在同种异体混合淋巴细胞反应(MLR)中,肿瘤干细胞致敏的DC与胶质瘤细胞致敏的DC相比,刺激指数更高,S:R=1:10时,刺激指数最强。
6.胶质瘤干细胞抗原致敏的DC疫苗接种大鼠后,能够诱导机体的免疫反应,抑制C6胶质瘤的生长,延长大鼠的生存时间。
7.胶质瘤干细胞抗原致敏的DC疫苗对大鼠具有良好的免疫保护作用,其效果优于胶质瘤细胞致敏的DC疫苗及未经抗原致敏的DC,为胶质瘤的免疫治疗提供了新的途径。
Glioma is the most common intracranial tumor.It present as diffuse toumors with invasion into normal brain tissue.After surgery,radiation or chemotherapy the glioma often recur or progress. The clinical therapeutic effect to glioma is disappointing. Despite the improvement of local therapies, such as surgery and radiation, the mean survival time for patients carrying a diagnosis of glioblastoma multiforme remains virtually unchanged. On average, patients survive 12 to 18 months, with few patients surviving beyond 2 years. Therefore it is urgent to find effective therapies of treating glioma.Recently,with the development of immunology and molecular biology, gene therapy and immunotherapy are rapidly gaining momentum. One of the most promising immunotherapeutic approaches for the treatment of glioma is the vaccination of cancer patients with dendritic cells (DC) pulsed with tumor antigens.
As the most impotant functional antigen presenting cell, DC can capture、process and present antigen to lymphocyte.Recently, much research of different kinds of DC vaccine has got satisfactory antitumor effect. DC can present kinds of antigen such as the whole tumor antigen specific antigen、neoplasm DNA or RNA. The most impotant function of DC is processing and presenting antigen to lymphocyte,which can evoke anti-tomour effect.It is unknown about the specific and related antigen of glioma,so the anti-tumor effect is limited.
BTSC were first reported by Ignatova.He observed small part of glioma cell posseses the abilities of self-regenerate, which caused the proliferation of glioma,namely BTSC. Similar to the NSCs, BTSC posses the abilities of self-regenerate and multipotential differentiation,which were connected to originate of glioma.BTSC expressed CD133+ and Nestin.Singh discovered only 100 CD133+cells could generate glioma in rat brain,but 105 CD133-cells could not do it. The result showed BTSC had strong antigenicity.
In our study,C6 cell line was cultured in serum-free medium with EGF, bFGF to obtain tumor cell spheres. The morphous of BTSC was observed under microscope before and after differentiation.Specific markers such as CD 133 and nestin, were employed to recognize BTSC by immunofluorescence cytochemistry techniques. The monocytes from rat bone were cultured in RPMI-1640 containing GM-CSF and IL-4 to obtain DC. Dendritic cells pulsed with brain tumor stem cells can provide good protect for tumor-bearing rat. Pathological section of the tumor have the most inflammation and CD8+T lymphocytes.
Objective
Glioma is the malignant intracranial tumor. Bescause it is very hard to be cured with the conventional therapy including surgery,radiation and chemotherapy,the recurrence rate of glioma is much high.The immunotherapy of glioma has become hot spot. Dendritic cells are the professional antigen presenting cells in vivo.DC vaccine has been applied in clinical trials,which has been safely and effectively. BTSC are the reasons of glioma occuring and recurring.They have good antigenicity of glioma. The aim of our study is to investigate the effect of dendritic cells pulsed with brain tumor stem cells. It will provide the experimental foundation for clinical immunotherapy of glioma.
Methods 1. The culture and identification of BTSC
C6 glioma cells were firstly cultured in DMEM containing 10% FCS. C6 cells which were in exponential phase of growth were transplanted to serum-free medium containing B27 supplement, basic fibroblast growth factor(20ng/ml) and recombinant epidermal growth factor(20ng/ml). The expression of special markers CD 133 and Nestin was detected by immunofluorescence. Method of flow cytometry were used to determine cell surface molecule of BTSC. Divided 10 nude mouse into two groups randomly, each group had 5 rats.One group accept BTSC by subcutaneous injections,the other accept C6 cells by the same way. we applied immunofluorescence to detect the expression of special markers of mature nerve cells such as GFAP.
2. The isolation and culture of DC.
Rat bone marrow monocytes isolated from rat femurs and tibias were cultured in RPMI1640 containing 10% FCS, granulocyte macrophage-colony stimulating factor(GM-CSF,5ng/ml)and interleukin-4(IL-4,5ng/ml). The mature DC were harvested after 12 days.The mature DC were analyzed by morphology with phase microscope; The expressions of specific marker OX62 was analyzed by flow cytometry. C6 glioma cells of logarithm growth period were collected through digestion and calculated.We prepare BTSC and C6 cells antigen by freezing-melt method. The antigen and DC were cocultured by the ratio 3:1.The function of two kinds of vaccines were examined by mixed lymphocyte rection (MLR).
3. The treatment of glioma by DC vaccine
To established rat C6 brain-glioma model. C6 tumor cells were cultured in vitro and then the C6 cells at logarithmic growth phase were injected intracranially under the stereotactic monitor into the right caudate nucleus.40 Wistar rats were divided into group A、B、C and D randomly, each group had 10 rats. Group A was injected with DC-BTSC vaccines of 1 X 107 cells through tail veins once a week for 3 weeks. Group B was treated with DC-C6 vaccines. The same number of DC and the same volume PBS were applied to group C and group D. Tomor samples were examined with HE staining and immunohistochemistry. The survival time of rats was analyzed by Log-rank survival analysis.
Results
1. C6 glioma cells were cultured in serum-free medium. After five to seven days,they presented as typical tumor spheres,which appeared globular and floating in medium. The expression of special markers CD 133 and Nestin marked by immunofluorescence was positive. When cultured in serum-supplemented medium,BTSC began to differentiate and express mature neurocyte antigens such as GFAP. Flow cytometry results showed:the average positive rate of MHC-Ⅱ、CD80 and CD86 was 60%、51% and 62% respectively,which was significantly higher than that of C6 cells(9%、6%、4%). Tumor spheres were transplanted to rats in group A and tumor nodules were seen after several weeks.But no tumors were formed in group B injected with attached cells in serum-free medium.The size of formed tumor in nude mouse was related to cell density injected.The pathological section of transplanted tumor with HE staining showed typical characterization of C6 cells.When the cultural condition was changed into serum-supplemented medium,BTSC attached and appeared to reticulate.The characterization of differentiated cell was the same as C6 cells.
2. When cultured with cytokines,after 10 days, PBMCs attached developed to free-floating DC, which had typical morphological dendrites. The positive rate of special surface marker OX62 on DC was 86% by flow cytometry analysis,which was low on PBMCs uninduced with cytokine.MLR showed:the stimulation index of DC-BTSC was significantly higher than that of DC-C6 at the same ratio (P< 0.01),which was the highest at stimulator to responder ratio of 1:10.
3. After 3 weeks implanted C6 tumor cells by stereotaxis, the cerebral hemisphere of rats was observed the cauliflower-like growth of tumor. But no tumors were formed in the group protected by DC-BTSC vaccine. The pathological sections stained with HE showed the growth of tumor cells and intratumoral necrosis in group B、C and D.In contrast,they were not shown in group A. Immunohistology staining demonstrated the infiltration with CD8+T lymphocyte in group A、B and C.The MST of rats were as fllowing:(39±3.12)days of group A、(29±2.01)days of group B、(23±1.42)days of group C and (19±1.02)days of group D. The survival analysis showed group A was statistically significant in constrast to the other groups(P<0.05).
Conclusion
1. We could get enough BTSC by culturing C6 glioma cells in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The characterization of differentiated cell was the same as C6 cells.
2. The expression of MHC-Ⅱ、CD80 and CD86 on BTSC was higher than that of C6 glioma cells.
3. The growth of tumor in nude mouse injected with BTSC was faster than that of nude mouse injected with C6 glioma cells.
4. We got enough DC by culturing rat bone marrow cells in RPMI1640 containing GM-CSF and IL-4.
5. MLR showed:the stimulation index of DC-BTSC was significantly higher than that of DC-C6 at the same ratio,which was the highest at stimulator to responder ratio of 1:10.
6. DC loading with BTSC lysates can provide immunity protect for the rats loaded with C6 glioma cells,which could prolong those live time.
7. DC loading with BTSC lysates can provide a higher level of immunity protect against gliomas than those loading with C6 cells,which provide a new method in the immuntherapy of brain glioma.
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