CD44+CD133+在胃癌干细胞分离与鉴定中的作用研究

The role of CD44+CD133+ in the isolation and identification of gastric cancer stem cells

目的分析验证不同恶性程度、分级、分期的胃癌,其恶性程度与肿瘤干细胞有无关联。方法分离、无血清培养人胃癌原代sphere细胞,通过流式细胞仪检测原代胃癌spheres中CD44+CD133+胃癌干细胞。体外克隆形成实验及RT-PCR检测spheres中干性基因的表达情况鉴定sphere细胞的肿瘤干细胞特性。比较原代胃癌细胞及spheres在裸鼠体内成瘤差异,对移植瘤进行HE染色,免疫组化检测Ki67的表达来比较原代胃癌细胞及sphere在裸鼠体内的增值能力来确定培养的spheres是胃癌干细胞在体外的聚集体。结果经原代无血清培养分选出来的细胞中85.93%的细胞是胃癌干细胞。体外克隆形成实验细胞形态变化不显著。Villin(a)和LGR(b)的表达在胃癌干细胞中均出现了一定程度的上调。结论 CD44和CD133可以作为胃癌干细胞的分选标记,并且胃癌细胞中确实存在胃癌干细胞。

Objective To analyse the relationship between gastric cancer and tumor stem cells in different degrees of malig-nancy,grading and staging. Methods Isolated and serum-free cultured primary human gastric cancer sphere cells,then,detected CD44＋CD133＋gastric cancer stem cells in primary gastric cancer spheres by flow cytometry. Detected the expression of stem gene in sphere cell by in vitro clone formation assay and RT-PCR. Indentified the cancer stem cell characteristics of sphere cells. Com-pared the differences tumor growth in nude mice between primary gastric cancer cell and spheres. HE staining on transplanted tu-mor,used immunohistochemistry detect the expression of Ki67 to compare the proliferation ability of primary gastric cancer cells and sphere in nude mice,then determined the spheres was the aggregate of gastric cancer stem. Results 85.93% primary culture without serum cells were gastric cancer cells. The morphological changes of the cells in vitro clone formation assay were not sig-nificant. The expression of Villin （a） and LGR （b） in gastric cancer stem cells had a certain degree of up regulation. Conclusion CD44＋ and CD133＋ can be used as sorting markers of gastric cancer stem cells,and gastric cancer stem cells do exist in gastric cancer cells.