小檗碱对U251胶质瘤干细胞的诱导分化作用  

Differentiation-inducing effect of berberine on U251 glioma stem cells

作　　者：邹宇辉[1] 王琼[2] 王伟民[1] Zou Yuhui Wang Qiong Wang Weimin(Department of Neurosurgery, Guangzhou General Hospital of Guangzhou Command, Guangzhou, Guangdong 510010, China Information Center, the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China)

机构地区：[1]广州军区广州总医院神经外科,510010 [2]广州医科大学附属第三医院信息科,510150

出　　处：《中国微侵袭神经外科杂志》2016年第12期559-562,共4页Chinese Journal of Minimally Invasive Neurosurgery

基　　金：广州市科技计划项目(编号:201300000150);国家自然科学基金青年科学基金项目(编号:81302201)

摘　　要：目的研究小檗碱对胶质瘤干细胞(GSC)分化的影响。方法用CD133免疫磁珠法由U251胶质瘤细胞中分选培养GSC,并用流式细胞术进行验证;细胞毒性实验评价小檗碱对GSC生长的抑制作用,采用不同浓度小檗碱(0.001、0.01、0.1、1μmol/L)作用于GSC,测量其吸光度值(OD);用RT-PCR、免疫荧光法检测干细胞标记物CD133、Nestin及细胞分化标记物胶质纤维酸性蛋白(GFAP)、微管相关蛋白2(MAP2)的表达水平。结果免疫磁珠法分选后CD133+细胞比例超过98%,无血清培养条件下能形成干细胞球。细胞毒性实验显示:小檗碱对GSC生长的抑制作用随剂量和时间的增加而增强。RT-PCR检测显示:随时间延长,小檗碱可使干细胞标记物CD133、Nestin基因的mRNA表达水平逐步降低,而细胞分化标记物GFAP、MAP2基因的mRNA表达水平逐渐升高。免疫荧光法检测显示:小檗碱能抑制CD133、Nestin蛋白的表达,上调GFAP、MAP2蛋白的表达。结论小檗碱能有效抑制GSC生长,并诱导其分化。Objective To investigate the effect of berberine on glioma stem cell（GSC） differentiation. Methods GSCs were sorted from U251 glioma cells through Magnetic Activated Cell Sorting（MACS） and cultured, the immunomagnetic beads were marked by CD133, and GSCs were identified by flow cytofluorometry. The inhibition of berberine on GSC proliferation was assessed with Cell Counting Kit-8, and GSCs were treated with berberine at different dosages（0.001, 0.01, 0.1 or 1 μmol/L）, then the cell optical density（OD） was detected. The expressions of stem cell markers（CD133, Nestin） and differentiated cell markers（GFAP, MAP2） were detected by real-time PCR（RT-PCR） and immunofluorescence. Results Over 98% sorted cells were CD133 positive cells after MACS, and the sorted cells could form neurospheres in serum-free medium. The Cell Counting Kit-8 test showed that the inhibition of berberine on the GSC growth was enhanced with the increase of berberine dosage and duration. Through RT-PCR test, it was found that berberine gradually decreased the expressions of CD133 and Nestin mRNA, and increased the expressions of GFAP and MAP2 mRNA with the time prolonged. Immunofluorescence assay showed that berberine depressed the expressions of CD133 and Nestin protein, and enhanced the expressions of GFAP and MAP2 protein. Conclusion Berberine can depress GSC proliferation effectively and induce its differentiation.

关 键 词：神经胶质瘤 肿瘤干细胞 小檗碱 细胞毒性实验 

分 类 号：R273[医药卫生—中西医结合]