胰腺癌细胞外泌体通过激活线粒体凋亡途径促进β细胞凋亡

发布时间：2018-09-08 09:16

【摘要】：目的:探讨胰腺癌细胞分泌的外泌体(exosome)对胰岛β细胞存活率和功能的影响及作用机制。方法:采用外泌体提取试剂盒提取小鼠胰腺癌细胞Pan02和MPC-83上清液外泌体,经磷钨酸染色后于透射电镜下鉴定形态;外泌体经荧光标记后与小鼠胰岛瘤MIN6细胞共孵育48 h,检测外泌体分泌水平和MIN6细胞的摄取水平;MTT和葡萄糖刺激的胰岛素分泌(GSIS)实验分别检测各组细胞的存活率和胰岛素分泌功能;q PCR检测微小RNA-204(miR-204)和Bcl-2 mRNA的表达;Western blot检测线粒体凋亡信号通路相关蛋白Bcl-2、Bax、caspase-3和细胞色素C(Cyt-C)的表达。结果:透射电镜结果显示2种胰腺癌细胞均能分泌外泌体,且Pan02细胞分泌更多。荧光标记的外泌体与胰岛β细胞共孵育结果显示,β细胞能够大量摄取胰腺癌细胞分泌的外泌体。MTT和GSIS实验结果显示,外泌体处理组的MIN6细胞存活率和高糖刺激的胰岛素分泌量显著低于未处理组(P0.01)。q PCR结果显示胰腺癌细胞分泌的外泌体富含miR-204,且外泌体处理后的MIN6细胞内Bcl-2的mRNA表达显著下调(P0.01)。Western blot结果显示,外泌体处理的MIN6细胞内Bcl-2蛋白表达显著下调(P0.05),Bax、cleaved caspase-3和Cyt-C蛋白表达显著上调(P0.01)。结论:胰腺癌细胞能够分泌外泌体,且该外泌体能够被胰岛β细胞摄取。胰腺癌细胞分泌的外泌体可以降低β细胞存活率和β细胞胰岛素的分泌功能,其机制可能通过外源性上调β细胞内miR-204的表达,进而抑制Bcl-2的mRNA和蛋白表达,最终激活β细胞内线粒体凋亡信号通路。
[Abstract]:Aim: to investigate the effect and mechanism of exocrine (exosome) secreted by pancreatic cancer cells on the survival and function of pancreatic 尾 cells. Methods: the exocrine secretions of mouse pancreatic cancer cells Pan02 and MPC-83 were extracted by exocrine extraction kit, and the morphology was identified by transmission electron microscope after phosphotungstic acid staining. The exocrine was incubated with murine islet tumor MIN6 cells for 48 h after being labeled with fluorescence. The levels of exocrine secretion and the uptake of MIN6 cells were detected. The survival rate and survival rate of each group were detected by (GSIS) assay and glucose-stimulated insulin secretion (GSIS) assay, respectively. The expression of micro RNA-204 (miR-204) and Bcl-2 mRNA was detected by insulin secretory PCR. The expression of Bcl-2,Bax,caspase-3 and Cyt-C were detected by Western blot. Results: transmission electron microscopy showed that both pancreatic cancer cells secreted exocrine bodies and Pan02 cells secreted more. The results of fluorescent labeled exocrine and islet 尾 -cell incubation showed that 尾 -cells could absorb the exocrine secreted by pancreatic cancer cells in large quantities, and the results of GSIS assay showed that 尾 -cells could absorb the exocrine secreted by pancreatic cancer cells. The survival rate of MIN6 cells and insulin secretion stimulated by high glucose in exocrine treatment group were significantly lower than those in untreated group (P0.01) .Q PCR. The results showed that exocrine secreted by pancreatic cancer cells was rich in miR-204, and Bcl-2 mRNA expression in MIN6 cells treated with exosome treatment was significantly lower than that in untreated group (P0.01) .Q PCR. Western blot showed a significant down-regulation (P0.01). The expression of Bcl-2 protein in MIN6 cells treated with exocrine was significantly down-regulated (P0.05). The expression of caspase-3 and Cyt-C protein was significantly up-regulated (P0.01). Conclusion: pancreatic cancer cells can secrete exocrine, and the exocrine can be absorbed by islet 尾 cells. The exocrine secreted by pancreatic cancer cells can reduce the survival rate of 尾 cells and the insulin secretion function of 尾 -cells. The mechanism may be through exogenous up-regulation of miR-204 expression in 尾 cells, and then inhibit the expression of mRNA and protein of Bcl-2. Finally, mitochondrial apoptosis signaling pathway was activated in 尾-cells.
【作者单位】：吉林省肿瘤医院检验科;吉林省疾病预防控制中心理化所;
【基金】：吉林省科技发展计划项目(No.20160101203JC)
【分类号】：R735.9

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