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[目的]探讨胃癌干细胞中差异表达microRNA及其对胃癌干细胞生物学特性(自我更新、侵袭、耐药能力)的调控作用。[方法]采用无血清悬浮培养法和干细胞标志物流式分选术分离人胃癌干细胞;分别采用胃癌细胞球形成实验、体外侵袭、耐药实验鉴定胃癌干细胞的生物学特性;microRNA表达谱芯片检测人胃癌干细胞中差异表达microRNA;采用定量逆转录PCR(qRT-PCR)技术检测相关差异表达microRNA的表达情况,验证microRNA芯片结果;相关microRNA抑制剂干扰后,分别进行胃癌细胞球形成实验、体外侵袭与耐药实验检测microRNA对人胃癌干细胞自我更新、侵袭、耐药能力的影响。[结果 ]成功分离得到人胃癌干细胞CD44(+)亚群,其具有典型的肿瘤干细胞生物学特性:较强的自我更新能力、侵袭和耐药能力。与CD44(-)细胞亚群相比,人胃癌干细胞CD44(+)细胞亚群高表达miRNAs共有17个;低表达miRNAs共有33个。采用q PCR技术验证microRNA表达谱芯片相关结果,得到:CD44(+)亚群中miR-196a-5p、miR-155-5p、miR-30a、miR-30b、miR-30c表达上调,miR-1246、miR-195-5b表达下调。抑制相关高表达microRNA(miR-196a-5p、miR-155-5p、miR-30a、miR-30b、miR-30c)表达后,肿瘤干细胞的自我更新能力、侵袭和耐药能力均下降。[结论 ]胃癌干细胞中具有多种差异表达microRNA,microRNA对胃癌干细胞生物学特性具有调控作用。
[Objective] To investigate the differential expression of microRNA in gastric cancer stem cells and its regulation on the biological characteristics of gastric cancer stem cells (self-renewal, invasion, drug resistance). [Methods] Human gastric cancer stem cells were isolated by serum-free suspension culture and flow cytometry (FCM). The biological characteristics of gastric cancer stem cells were identified by cell formation of gastric cancer cells, invasion and drug resistance in vitro respectively. The microRNA expression microarray Gastric cancer stem cells differentially expressed microRNA; quantitative reverse transcription PCR (qRT-PCR) technology to detect the expression of differentially expressed microRNA to verify microRNA chip results; relevant microRNA inhibitor interference, respectively, gastric cancer cell formation experiments, in vitro invasion and Drug resistance test to detect microRNA on human gastric cancer stem cells self-renewal, invasion, drug resistance. [Result] The CD44 (+) subpopulation of human gastric cancer stem cells was successfully isolated, which has the typical biological characteristics of cancer stem cells: strong ability of self-renewal, invasion and drug resistance. Compared with the CD44 (-) cell subpopulation, there were 17 miRNAs highly expressed in CD44 (+) cell subpopulation of human gastric cancer stem cells, and 33 miRNAs were low expressed. QPCR was used to validate the microRNA expression profile. The results showed that the expression of miR-196a-5p, miR-155-5p, miR-30a, miR-30b and miR-30c in CD44 + , MiR-195-5b downregulation. After inhibiting the expression of related high expression microRNAs (miR-196a-5p, miR-155-5p, miR-30a, miR-30b and miR-30c), the self-renewal ability, invasion and drug resistance of tumor stem cells decreased. [Conclusion] There are many differentially expressed microRNAs in gastric cancer stem cells, which regulate the biological characteristics of gastric cancer stem cells.