【作者】 费宇杰；

【导师】 刘正湘；

【作者基本信息】 华中科技大学 ， 内科学， 2012， 博士

【摘要】 研究目的肿瘤的治疗是一个世界性的难题,攻克癌症术后的复发和转移是当今医学科技攻关的重点与难点。四跨膜蛋白超家族中,CD151是其中重要的分子,其高表达于上皮细胞、内皮细胞、平滑肌和横纹肌细胞、血小板。其参与了多种生物过程,包括细胞粘附、增殖、分化,信号传导,病理性血管的生成和转移等。研究显示,CD151与多种肿瘤相关,其高表达多显示肿瘤病人预后不良。而CD151可以通过胞外大环特异性位点QRD194-196与整合素α3β1或α6β1相连,形成CD151-α3β1/α6β1功能性复合体,从而发挥多种生物学作用。在我们前期的实验研究生发现,CD151对于动物缺血模型有显著的促进血管新生的作用,且这些作用依赖于整合素α3β1/α6β1与CD151形成功能性的复合体。本实验中我们引入前期构建好CD151突变体pAAV-CD151-AAA194-196、体外转染HepG2细胞,观察其对细胞增殖、迁移、侵袭力影响,及其与整合素a3、a6、β1的关系,同时观察观察该突变体Cdc42和Rac1/2/3通路的影响,探讨CD151体外对肿瘤细胞生长的作用及其与整合素的相关性和机制的研究。研究方法1.用去内毒素试剂盒提取pAAV-CD151以及pAAV-CD151-AAA194-196、 pAAV-GFP三种质粒,并基因测序鉴定。2.Attractene Transfection Reagent脂质体转染HepG2细胞,分为四组,pAAV-CD151组、pAAV-CD151-AAA194-196组、pAAV-GFP组和空白对照组。成功转染后提取细胞蛋白,Western blot法检测各组CD151的表达,干预后CCK-8法观察细胞的增殖,细胞划痕实验观察各转染组细胞的迁移力,用Transwell板(孔径8μm),观察各组细胞侵袭力的情况,免疫共沉淀法检测各组整合素a3、a6、β1的表达情况,Western blot去观察各组Cdc42、Rac1/2/3和P-Rac/Cdc42的表达情况。实验结果1.经过基因测序鉴定,成功提取了pAAV-CD151和pAAV-CD151-AAA194-196两种质粒。2.pAAV-CD151-AAA194-196转染组与pAAV-CD151转染组CD151的表达无明显差异。3pAAV-CD151-AAA194-196转染组体外促进HepG2细胞增殖能力较pAAV-CD151转染组减弱。4.pAAV-CD151-AAA194-196转染组体外促进HepG2细胞迁移能力较pAAV-CD151转染组减弱。5.pAAV-CD151-AAA194-196转染组对HepG2细胞侵袭力的作用不及pAAV-CD151转染组,pAAV-CD151转染组能显著增强HepG2细胞侵袭力。6pAAV-CD151转染组P-Rac/Cdc42的表达增强,而突变体转染组未见到此效应。7.免疫共沉淀法检测到pAAV-CD151转染组整合素a3、a6、β1的表达,突变体组未检测到整合素a3、 a6、β1的表达。实验结论1.课题组成功构建了pAAV-CD151-AAA194-196突变体。2.研究表明pAAV-CD151能显著促进HepG2细胞的增值、迁移和侵袭,而突变体组促进HepG2细胞的增值、迁移和侵袭较pAAV-CD151组减弱,pAAV-CD151的此些作用可能通过上调P-Rac/Cdc42的表达来实现。3pAAV-CD151体外促进HepG2细胞的增值、迁移和侵袭依赖于CD151-整合素α3β1/α6β1复合体的形成。更多还原

【Abstract】 ObjectiveThe treatment of tumors is a worldwide problem, the fight against cancer recurrence and metastasis after surgery is important and difficult of today’s medical technology research.CD151is the member of the tetraspanin family to be associated as a promoter of tumor metastasis of malignant cells. Tetraspanins form membrane complexes with integrins. Here we constructed CD151-AAA mutant to assess the roles of Rac, Cdc42and P-Rac/Cdc42, and the effects of CD151-integrin complexes on proliferation, migration and invasion of HepG2cells. pAAV-CD151and CD151-AAA mutant were constructed and used to transiently transfect HepG2mediated with Qiagen Attractene Transfection Reagent. After transfection, the expression of CD151was measured by Western blotting. Cell proliferation assay was evaluated using the Cell Counting Kit-8(CCK-8) method, Cell migration assay was assessed by cell-wounding healing assay and cell invasion assay was evaluated in Microchemotaxis Chambers using FBS as the chemotactic stimulus. The potential involvement of various signaling path-ways was explored using relevant antibodies. The association between CD151and integrins was evaluated by immunoblotting analysis. We found that CD151promotes cell proliferation migration and chemotaxis. In addition, CD151increases phospho-Rac/cdc42activity. Further, CD151-AAA mutant decreased cellular proliferation, migration, and invasion. Moreover, CD151-AAA mutant abrogated the association between CD151and integrins. These data suggest that CD151form complexes by interacting with integrins, particularly α3β1and α6β1, to exert its function on HepG2cells. The mechanism is possibly related to the Rac, Cdc42and P-Rac/Cdc42signalling pathway. Methods1. extract plasmid pAAV-CD151, pAAV-CD151-AAA194-196、pAAV-GFP.2. Using Attractene Transfection Reagent liposomal to transfect HepG2cells, divided into four groups, pAAV-CD151group, pAAV-CD151-AAA194-196group, pAAV-GFP group and blank control group.Extracting each groups cell protein to detect the expression of CD151by Western blot,the proliferation of the cells was observed by CCK-8assay, Cell migration assay was assessed by cell-wounding healing assay and cell invasion assay was evaluated in Transwell plate (pore size8μm). observe the expression of integrin α3、α6and (31by co-immunoprecipitation assay, detect the expression of Cdc42、Rac1/2/3and P-Rac/Cdc42by Western blot.Results1. Successfully extract pAAV-CD151-AAA194"196mutant.2. The expression of CD151in the cells transfected with pAAV-CD151significantly increased as compared with the control and pAAV-GFP groups48h after transfection. In addition, pAAV-CD151-AAA transfection showed no significant difference of protein expression compared with CD151group.3. HepG2transfected with pAAV-CD151significantly enhanced HepG2proliferation as compared with the groups transfected with pAAV-GFP and the control. pAAV-CD151-AAA transfection significantly inhibited the proliferative effects as compared with the groups transfected with pAAV-GFP and the control group.4. In the wound healing assay cells transfected with pAAV-CD151significantly enhanced wound healing, The ability of pAAV-CD151-AAA mutant cells to wound healing was markedly delayed compared with that of pAAV-CD151transfectant cells.5. pAAV-CD151significantly promoted HepG2invasion compared with the control and pAAV-GFP groups. pAAV-CD151-AAA could not promote cell invasion.6. transfection with pAAV-CD151increased expression of phosphorylated phospho-Rac/cdc42compared with the control group and the GFP group, whereas transfection with pAAV-CD151-AAA had no change on the expression of phospho-Rac/cdc42.7. pAAV-CD151group could detect the expression of a3, a6and (31by Immunoblotting analysis,while pAAV-CD151-AAA group could not.Conclusion1. Successfully extract pAAV-CD151-AAA194-196mutant..2. Studies show that the pAAV-CD151promote HepG2cells proliferation, migration and invasion, while the function of mutant group is weakened, the mechanism may be through the increase the expression of P-Rac/Cdc42.3. PAAV-CD151promoting HepG2cells proliferation migration and invasion in vitro depends on the formation of CD151-integrin α3β1/α6β1complex.更多还原