P-gp在人胃癌干细胞中表达的初步研究  

The preliminary study of the expression of P-gp in human gastric cancer stem cells

作　　者：蒋洁敏[1] 乐红红 相芬芬[1] 孔倩倩[1] 詹月萍 许健[2] 吴蓉[1] 康向东[1] 

机构地区：[1]上海中医药大学附属普陀医院检验科,上海200062 [2]上海中医药大学附属普陀医院中心实验室,上海200062

出　　处：《现代肿瘤医学》2016年第15期2347-2351,共5页Journal of Modern Oncology

基　　金：上海市科学技术委员会科研计划项目(编号:134119b1900)

摘　　要：目的:通过无血清法富集人胃癌BGC-823干细胞,并对所获得的BGC-823干细胞生物学特性进行研究。方法:采用无血清悬浮培养方法富集人胃癌BGC-823干细胞球。后续实验分为BGC-823干细胞组和BGC-823细胞组:采用蛋白印迹(Western Blot)法和Hoechst33342染色法对所富集细胞的干性进行鉴定;利用平板克隆实验检测细胞单克隆形成能力;采用CCK-8法检测细胞对紫杉醇的敏感性;RT-PCR法检测P-gp mRNA的表达;细胞免疫荧光双染观察干细胞球CD44、P-gp的表达情况。结果:采用无血清悬浮培养法成功获得干细胞球。CCK-8结果显示,BGC-823干细胞对紫杉醇敏感性较BGC-823细胞降低(P<0.05)。干细胞球单克隆形成能力较人胃癌BGC-823细胞增强(P<0.05)。Western Blot和Hoechst33342染色法结果显示无血清富集的细胞为胃癌干细胞。RT-PCR、细胞免疫组化检测显示BGC-823干细胞CD44、P-gp的表达较BGC-823细胞升高,统计学差异明显(P<0.05)。结论:利用无血清富集法收集的人胃癌BGC-823干细胞球为研究干细胞的理想模型,胃癌干细胞的形成是耐药出现的潜在机制。Objective： To enrich and characterize the human gastric cancer BGC- 823 stem cells（ CSCs） and disscuss their biological characteristics. Methods： By serum- free suspension culturing enrich the human gastric cancer BGC- 823 ball of cells. Identification of stem cells was assessed by Western Blot and Hoechst33342 staining. Drug sensitivity was measured by CCK- 8 assay and P- gp mRNA expression level was tested by RT- PCR. The effects of cell proliferation was measured by clonogenic assay in plate. Expression of P- gp,CD44 protein was determined using immunocytochemistry. Results： Serum- free suspension culture method can be successfully enriched human gastric cancer stem cells,and sensitivity decreased to taxol in BGC- 823 cells（ P〈0. 05）. Colony forming essay indicated that the monoclonal formation capacity of BGC- 8 2 3 CSCs cells were more outstanding than BGC- 823 cells（ P〈0. 05）. The RT- PCR showed that the sphere- forming cells was increased expression of the P- gp mRNA compared to BGC- 823 cells（ P〈0. 05）,and immunofluorescence staining indicated that the expression of CD44,P- gp protein in sphere- forming cells was increased significantly（ P〈0. 05）. Conclusion： Serum- free suspending culture method can enrich the human gastric cancer BGC- 823 cells. The emergence of cancer stem cells may be a potential mechanism for the drug resistance.

关 键 词：肿瘤干细胞 人胃癌BGC-823细胞 无血清 耐药 

分 类 号：R735.32[医药卫生—肿瘤]