【作者】 赵杰；

【导师】 黄建安；

【作者基本信息】 苏州大学 ， 呼吸内科， 2011， 博士

【摘要】 第一部分瘦素增强外周血γδT淋巴细胞杀伤肺癌细胞的作用及其机制研究目的探讨瘦素在体外对人γδT细胞杀伤作用的影响及可能机制。方法用异戊烯焦磷酸法体外扩增人外周血γδT细胞,用不同浓度的瘦素诱导培养γδT细胞和人肺腺癌细胞A549细胞。MTT检测γδT细胞增殖。用ERK1/2(细胞外信号调节激酶1/2)特异性抑制剂PD98059阻断ERK1/2信号通路后,分别用Western blot法检测ERK1/2,P-ERK1/2(磷酸化细胞外信号调节激酶1/2)蛋白表达水平;用流式细胞术检测瘦素诱导前后的γδT细胞免疫表型TCR-γδ,CD44,NKG2D(自然杀伤细胞活化性受体)的变化;用乳酸脱氢酶法测定瘦素诱导前后γδT细胞的杀伤活性。结果γδT细胞培养10天时从扩增前4.21%增加到70.35%,CD44达94.0%。瘦素能够明显促进γδT细胞增殖,并呈浓度依赖性。瘦素可使ERK1/2信号通路中的靶蛋白发生磷酸化激活,用PD98059阻断ERK1/2信号通路信号通路可显著抑制瘦素促细胞的增殖效应,亦可抑制瘦素对γδT细胞ERK1/2蛋白磷酸化。γδT细胞和肺腺癌细胞A549细胞经不同浓度的瘦素诱导48 h后能显著增强γδT细胞A549细胞的杀伤活性(P<0.05)。瘦素促进γδT细胞上TCR-γδ,NKG2D的表达,但对CD44表达无明显影响。结论瘦素在一定浓度范围内可促进γδT细胞的增殖并增强其杀伤肿瘤细胞作用,瘦素促人γδT细胞增殖效应可能与激活ERK1/2信号通路有关,也可能和其促进细胞高表达NKG2D有关。第二部分人外周血γδT细胞对裸鼠人肺癌移植瘤的治疗作用目的探讨人外周血γδT细胞对已建立的A549肺癌裸鼠的过继免疫治疗作用。方法用人肺癌细胞株A549接种BALB/c裸鼠皮下,建立肺癌裸鼠模型。将已建立肺癌裸鼠模型随机分为两组,对照组用RPMI 1640培养液治疗,治疗组用γδT细胞治疗。从健康人外周血中提取γδT淋巴细胞,体外特异性扩增后浓度调整为5×10~7/ml予0.2ml注入裸鼠腹腔内,观测比较两组种植瘤裸鼠的肿瘤体积、生存时间,并用免疫组化法测定肿瘤组织中c-jun表达。结果裸鼠接种A549人肺癌细胞(5×10~6/ml)后第14天,在接种部位均有肿瘤形成,直径平均为2.7±0.33mm。在首次治疗后49天时测对照组、治疗组平均肿瘤体积分别为0.97±0.32cm~3、0.46±0.25cm~3,治疗组与对照组比较差异有统计学意义(p﹤0.05)。至75天时对照组种植瘤裸鼠全部死亡,治疗组存活6只,治疗组裸鼠最长存活天数为120天,较对照组存活天数延长,两组生存时间有统计学意义差异(p﹤0.05)。γδΤ细胞治疗组肿瘤组织中c-jun表达多呈弱阳性,2例不表达,而对照组均为阳性表达,两组比较差异有统计学意义(P<0.05)。结论人外周血γδT细胞对肺癌裸鼠种植瘤具有显著的抑瘤作用,γδT细胞能下调肿瘤组织中c-jun表达,注射γδT细胞的肺癌裸鼠组有更长的生存时间。更多还原

【Abstract】 PartⅠLeptin Increase the Effect ofγδT Cells Killing on Human Lung Cancer Cell Line A549Objective To explore the effect of leptin on the cytotoxicity ofγδT in vitro and possible mechanisms.Methods Use the isopentenyl pyrophosphate method to amplify human peripheral bloodγδT cells in vitro. After theγδT cells and A549 cells were cultured with different concentrations of leptin, MTT method was used to detect the proliferation of the cells .With ERK1 / 2 inhibitor PD98059 blocked ERK1 / 2 signaling pathway, the ERK1 / 2, P-ERK1 / 2 protein levels were observed by the Western blot, to investigate the changes of CD44, NKG2D on surface ofγδT cells by flow cytometry.Meanwhile, the cytotoxicity ofγδT cells on lung cancer cells were measure by lactate dehydrogenase before and after leptin induction.ResultsγδT cells were amplified from 4.21% to 70.35% in 10 days, CD44 up to 94.0%. Leptin could increase the proliferation ofγδT cells in a dose-dependent manner (P<0.01), but the effect of increasing the proliferation on A549 cells was not obvious.The cytotoxical effects ofγδT Cells on lung cancer cells could be increased afterγδT cells and A549 induced in different concentrations of leptin for 48h.The expression of NKG2D and TCR-γδonγδT cells was up-regulated by treating with leptin but had no effect on CD44 expression.Conclusions Lepint can enhance the proliferationγδT cells and increase its abilities to kill tumor cells . The cytotoxicity ofγδT cells on cancer cells perhaps be mainly mediated through ERK1/2 pathway and by expresion of NKG2D onγδT.PartⅡTherapeutic effect of human peripheralγδT cells on human lung cancer xenograft in nude miceObjective To explore the adoptive immunotherapeutic effect of peripheralγδT cells on nude mice model .Methods Xenografted animal model of human lung cancer was established by innoculating human lung cancer A549 cells into BALB/c nude mice subcutaneously. They were divided into two groups randomly,γδT cells and control group.γδT cells or RPMI-1640 were respectively injected into abdominal cavity of mice. Tumor volume , expression of c-jun and survival days between two groups were compared.Results The nude mice inoculated with 5×10~6/ml lung cancer cells.A tumor was observed in the inoculation site after fourteen days later:the volume of formed tumor in two groups was 0.97±0.32cm~3、0.46±0.25 cm~3 respectively in the 49th day of inoculation. It is obvious that the tumor size in the therapeutic group was large than that of control group. Mice in treatment group survived longer than those in the control group (p﹤0.05). There was a significant difference between two groups in expression of c-jun .(p﹤0.05). ConclusionsγδT cells from human peripheral can inhibit the growth of human lung cancer xenograft in nude mice effectively.γδT cells can down-regulated the c-jun expression of tumor in treatment group. The overall survival between the two nude mice groups was different significantly.更多还原