肝癌干细胞源性外泌体对肝癌细胞增殖耐药性的影响

Effect of hepatoma stem cell-derived exosomes on proliferation and drug resistance of hepatocellular carcinoma cells

目的观察肝癌干细胞外泌体对肝癌细胞增殖和耐药性的影响。方法流荧光细胞技术筛选人肝癌细胞株MHCC97干细胞,超速离心提取干细胞外泌体。MTT和软琼脂克隆形成实验检测细胞增殖情况;MTT法检测细胞对化疗药物的敏感程度,实时荧光定量RT-PCR和Western Blot检测细胞增殖和耐药相关基因和蛋白表达水平。结果实验肝癌细胞各时间点OD值要明显高于对照组和普通组(P<0.05)。实验组肝癌细胞对5-氟尿嘧啶和阿霉素的半数抑制浓度明显高于对照组和普通组(P<0.05)。RT-PCR和Western blot结果显示:实验组肝癌干细胞外泌体中survivin、c-myc、ras、ABCG2、MDR-1、MRP-1基因mRNA和蛋白的表达均较对照组的普通肝癌细胞明显增强,而P53和PTEN的表达则较对照组明显降低,差异有统计学意义(P<0.05)。结论肝癌干细胞外泌体能促进肝癌细胞的增殖,诱导肝癌细胞对化疗药物的耐药,其机制可能通过调控细胞增殖和耐药相关基因蛋白表达来实现。

Objective To observe the effect of exosomes secreted by hepatoma stem cells on the proliferation and drug resistance of hepatoma cells.Methods Human hepatoma cell line MHCC97 was screened by flow cytometry,and the exosomes were extracted by ultracentrifugation.MTT and soft agar colony forming assay were used to detect the cell proliferation.MTT method was used to detect the sensitivity of cells to chemotherapeutic drugs.Real time fluorescent quantitative RT-PCR and Western blot were used to detect the expression levels of genes and proteins related to cell proliferation and drug resistance.Results The OD value of experimental hepatoma cells at each time point was significantly higher than that of control group and common group(P<0.05).The median inhibitory concentration of 5-FU and adriamycin in experimental group was significantly higher than that in control group and common group(P<0.05).The results of RT-PCR and Western blot showed that the mRNA and protein expressions of survivin,c-myc,RAS,ABCG2,MDR-1,MRP-1 genes in the exosomes of HCC stem cells in the experimental group were significantly higher than those in the control group,while the expressions of p53 and PTEN were significantly lower than those in the control group(P<0.05).Conclusion The exocrine of HCC stem cells can promote the proliferation of HCC cells and induce the drug resistance of HCC cells.The mechanism may be achieved by regulating cell proliferation and drug resistance related gene protein expression.