Hedgehog信号通路在人角膜缘微环境来源的基质干细胞中表达情况的研究

摘要

AIM: To investigate the expression of Hedgehog signaling pathway in the mesenchymal stem cells derived from human limbal niche ( LNCs) . METHODS:Culture and passage the mesenchymal stem cells derived from LNCs and bone marrow - derived mesenchymal stem cells ( BMMSCs ) in vitro. Using BMMSCs as a positive control, the expression of Hedgehog signaling pathway in LNCs was evaluated by Western blot, immunofluorescence and real-time PCR. Furthermore, different doses ( 1, 5, 10, 15, 20, 25, 30μmol/L) of GANT61 ( the Gli inhibitor ) effect on the proliferation of LNCs and BMMSCs was detected by Cell Count Kit-8. RESULTS: The results of Western blot, immunofluorescence and real-time PCR proved that LNCs expressed Gli - 1, patched and SMO, which are the members of Hedgehog signaling. In addition, GANT61 ( the Gli inhibitor ) inhibited the proliferation of LNCs significantly (P<0. 05). CONCLUSION:Hedgehog signaling pathway plays an important role in the proliferation of LNCs.%目的：探讨Hedgehog信号通路在人角膜缘微环境来源的基质干细胞( LNC)中的表达情况。　　方法：对贴壁生长的人角膜缘来源的基质干细胞( LNC )进行分离培养及传代,对骨髓间充质干细胞系( BMMSC )进行培养传代。通过将BMMSC作为阳性对照,采用Western blot技术,免疫荧光技术, real-time PCR技术从蛋白水平及基因水平验证 Hedgehog 信号通路成员 SHH, patched, SMO,Gli-1在LNC中的表达情况。最后采用Cell Count Kit-8检测不同浓度Gli抑制剂GANT61(空白,1,5,10,15,20,25,30μmol/L)处理LNC和BMMSC48h后的增殖抑制效应反向验证Hedgehog信号通路在LNC中的存在。结果：Western blot技术,免疫荧光技术, real-time PCR技术均证明LNC细胞表达Hedgehog信号通路中的Gli-1, patched, SMO,且Gli蛋白抑制剂GANT61可以显著抑制LNC细胞增殖( P<0.05)。　　结论：Hedgehog信号通路在LNC细胞的增殖过程中发挥重要作用。