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目的:研究髓母细胞瘤患者中发现的Supressor of Fused(Sufu)的突变位点与Sonic Hedgehog(Shh)信号通路异常激活之间的联系,从而揭示其与肿瘤生长发展之间的关系.方法:构建Sufu突变位点的表达质粒,通过定量PCR、蛋白免疫印迹实验检测Sufu突变体对Sufu-/-细胞内源性Gli1表达水平的影响;通过免疫共沉淀检测Sufu突变体蛋白与Gli1的结合情况;通过MTT实验检测Sufu突变体对人髓母细胞瘤细胞(DAOY)生长增殖的影响.结果:定量PCR和蛋白免疫印迹实验发现,过表达Sufu的错义突变体(突变位点H87R)的Sufu-/-细胞中内源性Gli1表达水平接近于过表达野生型Sufu的Sufu-/-细胞,而过表达3个截短突变体(突变位点R146X、R299X、W430X)Sufu-/-细胞中内源性Gli1表达水平相比于野生型明显增高.正常情况下野生型Sufu可以和Gli1蛋白结合,免疫共沉淀实验发现Sufu的突变体都不同程度地破坏了与Gli1蛋白的结合;蛋白周转速率实验发现Sufu的突变体可以通过蛋白酶体途径降解;MTT实验发现Sufu突变体失去了抑制DAOY细胞生长增殖的能力.结论:髓母细胞瘤患者身上观测到的Sufu突变位点可以驱动肿瘤的形成和生长,并通过细胞功能实验证实了Sufu通过结合Gli蛋白从而抑制其功能.“,”Objective:To investigate the mechanism of aberrant Sonic Hedgehog(Shh)signaling which found in medulloblastom patients' Suppressor of Fused(Sufu)mutations, and to reveal the relationship between Shh pathway and tumor growth or development. Methods:To create Sufu variants expression plasmids, the influence of Sufu mutants on the expression level of Sufu-/-cell endogenous Gli1 were detected by quantitative real-time PCR and Western blot. The combination of Sufu mutants and Gli were detected by CO-IP assay. MTT assay were used to evaluate the effects of Sufu variants on growth and proliferation of human medulloblastoma cells (DAOY). Results:Quantitative PCR and Western blot experiments found that Sufu missense mutant(H87R)expression level of endogenous Gli1 showed wild type inhibition level, three nonsense mutants(R146X, R299X, W430X)of endogenous Gli1 expression level increased obviously;Under normal conditions, WT-Sufu can be combined with Gli1 protein, The CO-IP experiment found that the mutants of Sufu had different degrees of damage to the combination of Gli1 protein. The protein turnover rate experiment showed that Sufu mutants could be degraded by proteasome pathway and MTT assay showed that Sufu mutants lost their ability to inhibit the growth and proliferation of human medulloblastom DAOY cells. Conclusion:Clinically observed mutations in Sufu can drive tumor growth and further elucidates Sufu's role in binding to and suppressing Gli function.