[摘要] 目的:探讨人脐带来源间充质干细胞（umbilical cord-derived mesenchymal stem cell, UC-MSC）经PI3K/AKT信号通路对人肺腺癌A549 细胞凋亡和增殖的影响及其作用机制。方法:采用酶消化法从人脐带组织中分离UC-MSC并进行体外培养，用流式细胞术鉴定所得到的UC-MSC的免疫表型。收集UC-MSC培养上清，建立UC-MSC条件培养基与肺腺癌A549 细胞株的体外间接共培养体系。采用CCK-8 法检测A549 细胞增殖率，通过Annexin V/PI 染色流式细胞术检测A549 细胞的凋亡率，通过PI染色法判断肿瘤细胞的细胞周期分布；qPCR实验检测CyclinD1、BAX、Bcl-2 等PI3K/AKT通路下游凋亡与增殖相关基因的转录水平；Wb测定PI3K/AKT通路相关蛋白质表达水平。结果:人脐带组织分离培养3 周后，可见纤维状细胞铺满培养瓶，平行排列或呈旋涡状生长。流式细胞术检测发现所得细胞表达MSC标志分子CD73、CD90、CD105，不表达CD45 和HLA-DR。UC-MSC条件培养基与肺腺癌A549 细胞株在体外间接共培养后，与对照组比较，A549 细胞的增殖率明显降低、凋亡率明显升高，其细胞周期明显停滞在G1期（均P<0.01）。PI3K/AKT信号通路相关分子CyclinD1 和Bcl-2 转录下调、BAX的转录上调（均P<0.01）；UCMSC条件培养基培养的A549 细胞总AKT水平不变，p-AKT蛋白表达呈剂量性依赖下降（P<0.01）。结论:UC-MSC明显影响肺腺癌A549 细胞的增殖和凋亡能力，细胞周期停滞在G1 期，其主要作用机制是UC-MSC抑制A549 细胞PI3K/AKT信号通路，为探索UC-MSC临床应用的安全性和有效性提供实验依据。

[Abstract] Objective: To investigate the effects of umbilical cord-derived mesenchymal stem cells (UC-MSCs) on apoptosis and proliferation of human lung adenocarcinoma A549 cells via PI3K/AKT signaling pathway, and to explore the mechanism. Methods: UCMSCs were isolated from human umbilical cord tissues by enzyme digestion method and cultured in vitro. The immunophenotypes of the obtained MSCs were identified by flow cytometry. The culture supernatant of UC-MSCs was collected to establish an indirect in vitro co-culture system of UC-MSCs conditioned medium and lung adenocarcinoma A549 cell line. Proliferation of A549 cells was detected by CCK-8 assay; apoptosis of A549 cells was determined by Annexin V/PI double staining, and cell cycle distribution of tumor cells was determined by PI staining. The transcription levels of apoptosis and proliferation associated downstream genes in the PI3K/AKT pathway, such as CyclinD1, BAX and Bcl-2, were detected by quantitative polymerase chain reaction (qPCR). Moreover, Wb was utilized to detect the expression levels of PI3K/AKT pathway-related proteins. Results: The culture flask was filled with fibroblast-like cells, arranged in parallel, with spiral growth after three weeks of isolation and culture of human umbilical cord tissues. The flow cytometry results revealed that the MSC markers CD73, CD90 and CD105, but not CD45 and HLA-DR, were expressed on obtained cells. After indirect in vitro co-culture of UC-MSCs conditioned medium and lung adenocarcinoma A549 cells, the proliferation rate of A549 cells was significantly decreased; the apoptosis rate was significantly increased, and the cell cycle was obviously arrested at the G1 phase as compared with the control group (all P<0.01). The transcription levels of PI3K/AKT signaling pathway-related factors, CyclinD1 and Bcl-2 were down-regulated, and the transcription level of BAX was up-regulated (all P<0.01). The total AKT was not changed, but p-AKT protein expression was decreased in a dose-dependent manner in A549 cells cultured in UC-MSCs conditioned medium (P<0.01). Conclusion: UC-MSCs can affect the proliferation and the apoptosis of A549 cells, and arrest cells in G1 phase. The main mechanism is that UC-MSCs can inhibit the PI3K/AKT signaling pathway in A549 cells, providing an experimental basis for exploring the safety and effectiveness of clinical application of UC-MSCs.

国家自然科学基金资助项目（No.81270430）