人骨髓间充质干细胞外泌体对胰腺癌细胞的影响及作用机制

Molecular mechanisms of pancreatic cancer cells regulation by exosome derived from human bone marrow mesenchymal stem cells in vitro

目的探讨人骨髓间充质干细胞(BMSCs)来源的外泌体调控胰腺癌细胞的分子机制。方法提取BMSCs分泌的外泌体并对其加以鉴定。随后将人胰腺癌细胞株(PANC-1和AsPC-1)各自分为3组:对照组、外泌体组和抑制剂组,抑制剂组加入磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)信号通路靶向抑制剂LY294002和外泌体。采用蛋白免疫印迹法检测PI3K、AKT及其磷酸化蛋白p-PI3K、p-AKT的表达,ELISA法检测胰腺癌细胞标志物B7-H4、糖类抗原19-9的表达水平。细胞计数实验检测细胞增殖能力,流式细胞术检测细胞凋亡,Transwell实验检测细胞侵袭和迁移能力。结果成功提取BMSCs分泌的外泌体。与对照组相比,外泌体组PANC-1和AsPC-1的细胞标志物B7-H4、糖类抗原19-9表达、细胞增殖率、细胞侵袭力及迁移能力均下降,而凋亡率增加,差异均有统计学意义(均P<0.001),抑制剂组上述指标与对照组相比差异无统计学意义(均P>0.05);对照组PANC-1细胞p-PI3K、p-AKT的蛋白表达水平分别为(5.81±1.87)、(5.30±1.21),加入外泌体后分别上调为(12.74±3.28)、(11.22±2.35)(均P<0.001),而AsPC-1细胞p-PI3K、p-AKT的蛋白表达水平分别为(4.75±0.87)、(4.15±1.32),加入外泌体后分别上调为(10.83±3.19)、(9.28±2.33)(均P<0.001),差异均有统计学意义。抑制剂组p-PI3K以及p-AKT水平与对照组相比差异无统计学意义(均P>0.05)。结论BMSCs分泌的外泌体可通过激活PI3K/AKT信号通路抑制胰腺癌细胞的增殖、侵袭和迁移能力,起到抑癌的作用。

Objective To investigate the molecular mechanism by which exosomes secreted from human bone marrow mesenchymal stem cells(BMSCs)regulate pancreatic cancer(PC)cells.Methods The exosomes secreted by BMSCs were extracted and identified.Human PC cell lines(PANC-1 and AsPC-1)were then divided into 3 groups:simple cell group,exosome group and inhibitor group.For inhibitor group,PI3K/AKT Signaling Pathway inhibitor LY294002 was added,followed by exosomes.Subsequently,the protein expression of phosphatidylinositol 3-kinase(PI3K)protein kinase B(AKT)and their phosphorylated types was detected by Western-blot,and the contents of PC cell markers B7-H4 and CA199 were detected by ELISA kit.The CCK-8 experiment was mainly conducted to detect the proliferation ability of cells,the flow cytometry to detect apoptosis,and the Transwell experiment to detect the migration and invasion ability of cells.Results The exosomes secreted by BMSCs were extracted successfully.The expression of PC cell markers B7-H4 and CA199,the proliferation rate,and the invasion and migration ability of cancer cells were all decreased for both PANC-1 and AsPC-1,while the apoptosis rate increased,all of which had statistical significance(P<0.001).There were no significant differences for the above parameter in inhibitor group compared with the simple cell group(P>0.05).The p-PI3K and p-Akt of PANC-1 cells in simple cell group were(5.81±1.87),(5.30±1.21),while up-regulated to(12.74±3.28),(11.22±2.35)with statistically significant differences(P<0.001)after exosomes were added.Meanwhile,the above 2 kinds of protein expression of AsPC-1 cells in simple cell group were(4.75±0.87),(4.15±1.32),while increased to(10.83±3.19),(9.28±2.33)in exosome group with statistically significant differences(P<0.001).On the other hand,there was no significant differences for the protein expression of P-PI3K and P-AKT in inhibitor group(P>0.05)compared with the simple cell group.Conclusion Exosomes secreted by BMSCS can inhibit the proliferation of PC cells and its migration and invasion ability by activating PI3K/AKT signaling pathway,thus playing a role in antitumor effect.