Tca8113-4-1BBL细胞联合CD28单抗在口腔鳞癌免疫中的作用

发布时间：2018-04-10 02:02

本文选题：4-1BBL　切入点：CD28单克隆抗体　出处：《汕头大学》2010年硕士论文

【摘要】：目的在细胞水平,研究口腔癌瘤苗TCV-4-1BBL联合CD28单克隆抗体(CD28 mAb)诱导T淋巴细胞抗肿瘤活性的能力。方法1.建立稳定表达4-1BBL克隆细胞株：通过脂质体法将真核载体pEGFP-4-1BBL转染人口腔鳞癌细胞Tca8113,经G418(400μg/ml)筛选及有限稀释后获得稳定高表达4-1BBL克隆细胞株Tca8113-4-1BBL,分别用RT-PCR和Western blot检测转染细胞中4-1BBL mRNA和蛋白的表达。2.制备瘤苗：将转染与未转染4-1BBL基因的Tca8113细胞用丝裂霉素C (MMC)处理后,制成肿瘤细胞瘤苗,分别命名TCV-4-1BBL、TCV-Tca8113。3. TCV-4-1BBL瘤苗联合CD28 mAb诱导抗肿瘤活性的能力：TCV-4-1BBL联合CD28 mAb与经体外CD3单克隆抗体(CD3 mAb)诱导的人外周血T淋巴细胞共同培养,实验分四组,①Tca8113组：T淋巴细胞+TCV-Tca8113;②Tca8113+CD3 mAb组：T淋巴细胞+CD3 mAb+ TCV-Tca8113;③Tca8113-4-1BBL+CD3 mAb组：T淋巴细胞+CD3 mAb+TCV-4-1BBL;④Tca8113-4-1BBL +CD3 mAb组+CD28 mAb:T淋巴细胞+CD3 mAb+TCV-4-1BBL+CD28 mAb。各实验组培养72小时后,台盼蓝染色计数T细胞、用Cell Counting Kit-8 (CCK-8)测细胞毒性T细胞(CTL)杀伤活性,ELISA法检测细胞因子IL-2、IFN-γ的分泌。结果1.荧光显微镜下观测到转染了pEGFP-4-1BBL的Tca8113细胞(Tca8113-4-1BBL)绿色荧光蛋白的表达,RT-PCR、western blot在mRNA和蛋白水平检测到4-1BBL表达。2.72h后台盼蓝染色计数上述混合培养的T细胞,④组1.78±0.16,高于①组0.917±0.12,②组1.01±0.14及③组1.38±0.15,P0.05,促进T细胞的增殖。3.72h后测CTL的杀伤活性：④组59.2±5.1,高于②组25.7±3.5及③组38.9±3.4,P0.05,促进T细胞的活化。4.72h后各组IL-2的分泌：④组881.2131±78,显著高于①组56.1619±13,②组176.4235±33,③组526.1235±67,P0.05,促进IL-2分泌。5.72h后各组IFN-γ的分泌：④组402.81±34高于其他各组(②组(69.83±13),③组246.87±24,①组37.85±11),促进IFN-γ的分泌。结论pEGFP/neo-4-1BBL真核表达载体在Tca8113细胞中获得稳定表达,口腔癌瘤苗Tca8113-4-lBBL构建成功。在细胞水平显示,TCV-4-1BBL瘤苗联合CD28 mAb,能增强T细胞抗肿瘤免疫功能,促进T细胞增殖、活化,促进IL-2、IFN-γ分泌可能是其机制之一。
[Abstract]:Objective to study the antitumor activity of T lymphocytes induced by oral cancer vaccine (TCV-4-1BBL) combined with CD28 monoclonal antibody (CD28 mAb) at the cell level.Method 1.Expression of 4-1BBL mRNA and protein.Preparation of tumor vaccine: Tca8113 cells transfected and untransfected with 4-1BBL gene were treated with mitomycin C (MMC), and TCV-4-1BBLTCV-TCV-Tca8113.3 respectively.The ability of TCV-4-1BBL tumor Vaccine combined with CD28 mAb to induce Antitumor activity: 1 TCV-4-1BBL combined with CD28 mAb was co-cultured with human peripheral blood T lymphocytes induced by CD3 monoclonal antibody CD3 mAb. the experiment was divided into four groups.T lymphocytes in 1Tca8113 group: TCV-Tca8113Tca8113 CD3 mAb: CD3 mAb TCV-Tca8113TCV-Tca8113-4-1BBL CD3 mAb: CD3 mAb TCV-4-1BBL 4Tca8113-4-1BBL CD3 mAb CD28 mAb:T CD3 mAb TCV-4-1BBL CD28 mAb.After 72 hours of culture, the T cells were counted by trypan blue staining. The cytotoxic T cells were detected by Cell Counting Kit-8 CCK-8 and the secretion of cytokine IL-2 IFN- 纬 was detected by Elisa.Result 1.The expression of green fluorescent protein (RT-PCRwestern blot) was observed in Tca8113 cells transfected with pEGFP-4-1BBL under fluorescence microscope. 4-1BBL expression was detected at the level of mRNA and protein. The expression of 4-1BBL was detected by backstage trypan blue staining for 2.72 hours.Conclusion the eukaryotic expression vector of pEGFP/neo-4-1BBL was stably expressed in Tca8113 cells, and Tca8113-4-lBBL of oral cancer vaccine was successfully constructed.At the cell level, TCV-4-1BBL vaccine combined with CD28 mAbcould enhance the anti-tumor immune function of T cells, promote the proliferation and activation of T cells, and promote the secretion of IL-2 IFN- 纬, which may be one of the mechanisms of TCV-4-1BBL combined with CD28 mAb.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】