抑制Hedgehog信号通路对人脑胶质瘤肿瘤干细胞体外增殖的影响

Effect of intervention of Hedgehog signaling pathway on proliferation of human glioma stem cells in vitro

目的探讨抑制Hedgehog信号通路对人脑胶质瘤肿瘤干细胞(GSCs)体外增殖的影响。方法收集我院手术切除的胶质母细胞瘤标本,进行分离、培养和鉴定GSCs,应用环靶明抑制Hedgehog信号通路,采用免疫组化染色检测GSCs Hedgehog信号通路相关蛋白的表达,使用CCK-8试剂盒测定GSCs的增殖活性。结果胶质母细胞瘤标本分离出来的细胞球表达CD133及Nestin;而且表达Hedgehog信号通路关键蛋白Sonic Hedgehog和Smo。环靶明对GSCs增殖的抑制率随浓度的升高显著增高(P<0.05),而且随作用时间延长显著增高(P<0.01)。结论 Hedeghog信号通路的特异性抑制剂环靶明能够显著抑制体外培养GSCs的增殖。

Objective To investigate the expression of Hedgehog signaling pathway in glioma stem cells（GSCs） and the effects ofcyclopamine（a inhibitor of Hedgehog signaling pathway） on the proliferation of GSCs.MethodsThe GSCs were isolated and culturedfrom the glioblastomas removed by neurosurgery in our hospital. Immunohistochemical technique and fluorescence stain were used toidentify GSCs and detect the expression of Hedgehog signaling pathway in GSCs. The effects of cyclopamine on the proliferation of GSCswere analysized by CCK-8.ResultsThe suspended cell masses cultured by serum-free technique expressed CD33 and Nestin cellsurface markers with strong proliferative and self-renewing ability, and was able to differentiated into glioma cells by the induction of10% fetal bovine serum medium. Sonic Hedgehog and Smo, key proteins of Hedgehog signaling pathway, were expressed in GSCs. Theresults of CCK-8 showed that the inhibitory rates of 0.5, 1 and 5 μmol/L cyclopamine to GSCs were（21.93±6.57）%,（32.03±9.13）% and（7.78±16.09）% respectively（P0.01）. The inhibitory rate of cyclopamine to GSCs 4, 12, 24, 36, 48 and 60 hours after the culture in themedia contenting cyclopamine were（25.89±7.11）%,（28.00±10.00）%,（32.98±16.87）%,（35.83±17.44）% and（41.31±16.12）%,（41.58±19.72）% respectively（P0.01）.ConclusionCyclopamine, the inhibitor of Hedgehog signaling pathway, can significantly inhibits theproliferation of GSCs in the concentration- and time-depenent fashions.