Hedgehog-Gli信号通路对胶质瘤细胞增殖、凋亡和侵袭能力的影响

【作者】 王柯；

【导师】 潘力； 车晓明；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 目的最近研究发现脑胶质瘤中存在异常活化的Hedgehog(HH)-Gli信号通路,抑制该通路后肿瘤细胞出现生长缓慢、成瘤能力下降等改变。然而HH-Gli信号通路对胶质瘤细胞周期、凋亡和侵袭能力的影响,以及与临床病理特征的联系目前仍不清楚。方法选取2007-2009年在我科进行手术切除的原发胶质瘤标本110例,使用免疫组化的方法分析细胞核内Gli1表达与肿瘤级别、细胞增殖指数的关系。在体外实验中,我们使用免疫荧光染色和real-time PCR的方法检测胶质瘤细胞系U87、SHG-44、U251和A172中HH-Gli1信号通路的活性状态,然后使用药物环巴胺和小RNA干扰技术在不同水平上影响该信号通路的活性,观察一系列细胞生物学特性的变化,并使用real-time PCR的方法检测与这些细胞生物学特性密切相关的调控基因的改变。结果在各个级别胶质瘤中均存在异常活化的HH-Gli1信号通路,细胞核内Gli1蛋白的表达与肿瘤级别、细胞增殖状态有关。体外培养的四个胶质瘤细胞系具有不同活性的HH-Gli1通路。对于具有该通路活化的胶质瘤细胞U87、SHG-44和U251,环巴胺和siGli1显著地抑制了肿瘤细胞的增殖和侵袭能力,肿瘤细胞停滞于G0／G1期,细胞凋亡率增加。进一步研究结果显示抑制HH-Gli1信号通路后与细胞周期、凋亡和细胞侵袭能力相关的一系列基因的表达水平发生了显著的变化；对于HH-Gli1通路无活性的胶质瘤细胞系A172,针对HH-Gli1信号通路的干预措施未引起明显的细胞生物学行为改变。结论HH-Gli1信号通路可以作为治疗胶质瘤的一个新的靶点,抑制该信号通路的措施通过多种机制发挥治疗作用。更多还原

【Abstract】 Objectives Recent studies have shown aberrant activation of Hedgehog(HH)-Gli signaling pathway exists in a fraction of human gliomas. Blocking the pathway leads to the decrease of tumor cell proliferation and tumorigenicity. However, little is known about the HH-Gli pathway’s impact on glioma cell behavior regarding cell cycle, apoptosis and invasiveness. The relationship between the HH-Gli pathway and the clinicopathological features also remains to be elucidated.Methods 110 primary glioma samples were obtained from those patients who underwent surgical rection in our department from 2007 to 2009. Immunohistochemical staining was performed to analyze the relationship between Glil nuclear staining and pathological grades, and the relationship between Glil nuclear staining and the tumor proliferative index. In in vitro experiments, immunofluorescence staining and real-time PCR were used to detect the HH-Gli 1 activity in glioma cell lines U87, SHG-44, U251 and A172. Then cyclopamine and small RNA interference technique were used to inhibit the pathway activity in these glioma cell lines, and their phenotype changes were recorded. Furthermore, we measured the relative gene expressions by real-time PCR to clarify the molecular mechanisms behind the phenotype changes.Results Aberrant activation of the HH-Gli 1 pathway existed in all four grades of human glioma samples. The nuclear staining of Glil protein was associated with the pathological grades and proliferative index. The four glioma cell lines cultured in vitro harbored different activity of the HH-Gli1 pathway. As for glioma cells with an active pathway like U87, SHG-44 and U251, cyclopamine and siGlil significantly reduced the glioma cell growth and invasion. Cell cycle Go/Gi arrest and enhanced apoptosis were detected after blocking the HH-Gli1 pathway. Further analysis showed that a set of genes related to those phenotype changes saw a remarkable change in their mRNA levels. For A172 cells which have a silent HH-Gli1 pathway, neither cyclopamine nor siGlil produced marked cell behavior changes.Conclusions HH-Gli 1 pathway can serve as a new therapeutic target for human gliomas. Inhibition of the pathway takes its effect through multiple mechanisms.更多还原