目的 研究TRAIL基因修饰的骨髓间充质干细胞(MSC)联合吉西他滨对人胰腺癌细胞的杀伤作用.方法 构建重组腺病毒Ad-CMV-TRAIL,体外转染人骨髓间充质干细胞.通过观察绿色荧光的表达强度,确定最佳转染条件.MTT法检测Ad-CMV-TRAIL转染对MSC细胞生长状态的影响.Transwell小室共培养携带TRAIL的MSC和胰腺癌细胞Panc-1,联合应用吉西他滨,AnnexinⅤ-FITC双标法检测凋亡的发生.Western blot法检测Caspase凋亡相关蛋白的表达变化.结果 Ad-CMV-TRAIL以MOI=10体外感染MSC 24 h,显微镜下观察80%以上的MSC表达绿色荧光,转染组细胞高表达TRAIL.Ad-CMV-TRAIL转染之后的MSC仍然可以增殖,活力无明显改变.携带TRAIL的MSC与Panc-1细胞共培养之后,可诱导部分Panc-1细胞发生凋亡,凋亡率达(13.38±3.42)%.而携带TRAIL的MSC与Panc-1细胞共培养之后可以增强吉西他滨的杀伤效果,与单独吉西他滨处理组相比差异显著[(52.10±3.41)% vs.(29.68±1.71)%,P=0.002].Western blot检测结果 显示MSC-TRAIL和吉西他滨共同处理可以诱导凋亡相关的Caspase-8和Caspase-3蛋白活化.结论 MSC可能作为TRAIL的肿瘤靶向载体在胰腺癌的治疗中发挥作用.%Objective To study the killing effect of mesenchymal stem cells(MSCs)modified by TRAIL gene combined with gemcitabine on the pancreatic cancer cells. Methods The adenovirus expressing TRAIL was constructed and infected into the human MSCs in vilro. The intensity of GFP was observed to determine the optimal transfection conditions. MTT assay was used to investigate the influence of Ad-CMV-TRAIL on the growth of MSC. The MSC-TRAIL and Panc-1 cells were co-cultured in Transwell inserts,and gemcitabine was used together to treat the Panc-1 cells. The apoptosis was detected by using An-nexinV-FITC methods. The apoptosis related proteins Caspase 3 and Caspase 8 were detected by using Western blot. Results After being transfected with Ad-CMV-TRAIL(MOI= 10)for 24 h,over 80% MSCs expressed GFP,and the TRAIL was detected in the transected group. The MSCs could still proliferate after Ad-CMV-TRAIL transfection and there were no significant changes about viability. MSCs-TRAIL could induce apoptosis in some Panc-1 cells[(13. 38 + 3. 42)%]. Gemcitabine could significantly enhance the killing effects with MSC-TRAIL,as compared with solely gemcitabine-treated group[(52. 10 + 3. 41)% vs. (29. 68 + 1. 71) % ,P=0. 002]. The apoptosis related proteins Caspase 8 and Caspase 3 were activated in the combined treatment group. Conclusion MSCs can act as an ideal vector for targeted gene therapy of pancreatic cancer.
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