【作者】 高爽；

【导师】 韩昱晨；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的Rho家族蛋白是Ras超家族中小分子量G蛋白的成员，RhoC是Rho亚族蛋白之一，通过其下游靶分子促进肌动蛋白微丝骨架的聚合，对细胞运动及肿瘤侵袭和转移发挥重要作用，关于RhoC与肺癌关系尚不清楚。RhoGDI为RhoC的调节因子，曾认为其抑制Rho蛋白的活性。近年来的研究显示RhoGDI对Rho蛋白还可能有激活作用，其在肿瘤的侵袭及进展中办可能发挥双重作用。有研究结果显示RhoGDIβ在不同类型肿瘤的侵袭和转移中发挥两种相反的作用，而RhoGDIα与肿瘤侵袭和转移的关系尚无报道。本实验中，我们通过检测不同侵袭能力肺癌细胞的RhoC、RhoGDIα的mRNA及蛋白表达水平，初步探讨RhoC、RhoGDIα与肺癌转移的关系。材料与方法1、材料与试剂人肺癌细胞系BE1，LH7，A549，A2均由北京医学院惠赠，人支气管上皮细胞HBE购自中南大学湘雅中心实验室，兔抗人RhoC、羊抗人RhoGDIα多克隆抗体购自Santa Cruz生物技术公司，Trizol总RNA提取试剂购自天根(Tiangen)公司，逆转录试剂盒、PCR试剂盒购自大连宝生物(TaKaRa)公司。2、实验方法(1) Westernblot：采用考马斯亮蓝法进行蛋白定量。SDS—聚丙烯酰胺凝胶电泳，转印(恒压50V，2h)，一抗4℃孵育过夜，辣根过氧化物酶标记二抗37℃孵育2h，DAB显色。经凝胶成像系统采集，灰度值测定。(2) RT—PCR：TRNzol试剂提取细胞总RNA后，逆转录获得cDNA，均按说明书操作。PCR扩增产物使用1.5％琼脂糖，120V电泳30min，溴化乙啶(EB)染色，凝胶成像系统下观察。(3) Transwell小室体外侵袭实验：微孔滤膜上平铺Matrigel胶，小室下室加含血清的培养液，上室加细胞悬液，常规培养12h，70％L甲醇固定45min，苏木素染色10min，高倍镜下计数膜背面上的穿膜细胞。(4)免疫荧光：细胞爬片，Triton-100打孔，一抗4℃过夜，避光滴加荧光二抗，Hoechst染核，50％甘油封片，荧光显微镜下观察、照像。3、统计学处理应用SPSS for Windowsl 1.5统计分析软件进行数据处理，采用单因素方差分析．P＜0.05为有统计学意义实验结果1、RhoC、RhoGDIα蛋白在肺癌细胞中的表达4种肺癌细胞中RhoC、RhoGDIα蛋白的表达量明显高于与正常人支气管上皮细胞；高侵袭能力肺巨细胞癌细胞BE1的RhoC、RhoGDIα蛋白表达量明显高于低侵袭能力肺巨细胞癌细胞LH7。在BE1、LH7细胞胞浆内均可检测到RhoC及RhoGDIα蛋白的表达。2、RhoC、RhoGDIαmRNA在肺癌细胞中的表达高侵袭能力肺巨细胞癌细胞BE1的RhoC、RhoGDIα的mRNA表达量高于低侵袭能力肺巨细胞癌细胞LH7。3．Transwell小室对癌细胞侵袭力的测定BE1细胞的体外侵袭力明显高于LH7细胞。结论1、RhoC、RhoGDIα在肺癌细胞中表达高于正常人支气管上皮细胞。2、RhoC、RhoGDIα的表达与肺癌细胞的体外侵袭能力相关。更多还原

【Abstract】 INTRODUCTIONRho protein family is one member of Small GTP-binding proteins of Rassuperthmily. RhoC is one member of Rho subtribe, which take a pivotal role in theprocess of tumor invasion and metastasis. However, there is no studies about RhoCexpression in lung carcinoma and its correlation with metastasis of lung cancer.RhoGDIs have been regarded as regulators to inactivate Rho proteins, however,evidence is accumulating that the classical function of rhoGDIs as universal chaperonesfor GDP-bound proteins may need to be revisited. Several studies have investigated thelevel of expression of rhoGDIs in various cancer cells compared with normal cells,revealing opposite patterns depending on the tumor cell considered. Likewise, rhoGDIβmRNA levels were increased in ovarian adenocarcinoma, and this upregulationcorrelated with the malignancy of tumors. In addition, the induction of motility ofcancer cells by the autocrine motility factor cytokine appeared to induce anupregulation of rhoGDIβmRNA and protein in vitro. However, less studies reportedthe correlation of Rho-GDIαexpression and metastasis of cancer. In the present study,We investigate the expression of RhoC and Rho-GDIαmRNA and protein in four lungcarcinoma cell (BE1, LH7, A2, A549) and Homo sapien Bronchus Epithelial cell(HBE)and their correlation with metastasis of lung cancer. METERAL AND METHODS1. Material and ReagentsHuman lung cancer cells BE1, LHT, A549, A2 were obtained from Beijing MedicalUniversity. Homo sapien bronchusepithelial cell was purchased from ZhongnanUniversity Xiangya laboratory. RPMI 1640 were purchased from Gibco. The firstantibodies were rabbit polyclonal antibody to RhoC and goat polyclonal antibody toRho-GDIαwas purchased from Santa Cruz. Trizol was purchased from Tiangen.2. MethodsThe expressions of RhoC, Rho-GDIαprotein were detected with western blot.The expressions of RhoC, Rho-GDIαRNA were detected with reverse-transcription polymerase chain reaction (RT-PCR).The expressions and locations of RhoC, Rho-GDIαwere detected with immuno-fluorescence.The invasion ability of BE1 and LH7 in vitro was detected with BioCoat MatrigelInvasion Chambers with 8.0 micron pore transwells coated with extracellular matrixproteins.3. Statistical analysisThe SPSS 11.5 software was employed to ayalyze the data. P＜0.05 wasconsidered as statistical significance.RESULTSThe expression level of RhoC and Rho-GDIαprotein in various lung carcinomacell was significantly higher than that in HBE. The expression of RhoC and Rho-GDIαprotein in BE1(the cell with high metastasis capability) was significantly higher thanthat in LH7(the cell with low metastasis capability). The expression of RhoC andRho-GDIαmRNA in BE1(the cell with high metastasis capability) was significantlyhigher than that in LH7(the cell with low metastasis capability). The invasion ability ofBE1 in vitro was significantly higher than LH7. The expression of RhoC and Rho-GDIαin BE1 and LH7 was all located in endochylema.CONCLUSIONThe expression level of RhoC and Rho-GDIαin lung carcinoma cell was higherthan in HBE.The expression of RhoC and Rho-GDIαcorrelates with metastasis of lung cancer.更多还原