【作者】 邓波；

【导师】 王如文；

【作者基本信息】 第三军医大学 ， 外科学， 2006， 博士

【摘要】 转化生长因子β1(TGF-β1)属于生长因子超家族,其生物学效应非常广泛,主要包括调节细胞增殖和分化、参与胚胎发育调节、促进细胞外基质(ECM)形成和抑制免疫反应等。TGF-β1在包括肺癌在内的多种恶性肿瘤细胞中常呈高表达,并可由癌旁基质细胞产生。目前认为,在肿瘤早期TGF-β1抑制原癌基因c-myc等,阻止细胞生长周期从G1期进入S期;而在肿瘤的中晚期(肿瘤浸润血管逐渐开始形成),TGF-β1可通过促进血管生成、肿瘤细胞迁移、免疫抑制等途径为肿瘤的生长、浸润及转移提供适宜的微环境,使肿瘤细胞表现恶性特征。对非肿瘤细胞的观察发现,TGF-β1对细胞中Fas/FasL蛋白表达具有调控作用。Fas/FasL为重要的细胞凋亡途径,在非肿瘤细胞中TGF-β1通过上调Fas表达,从而激活Fas/FasL凋亡信息链中的凋亡蛋白酶caspase家族诱导细胞发生凋亡。另外,TGF-β1与细胞内TGF-β1受体结合后,亦能通过下游信号smad蛋白激活caspase家族引起细胞凋亡。因此,TGF-β1可看作是一个诱导细胞凋亡的细胞因子,在机体内发挥清除异常细胞的重要作用。但TGF-β1与肿瘤细胞凋亡的关系尚不明确,目前国内外鲜有肿瘤细胞中TGF-β1与Fas/FasL凋亡信号通路之间关系的研究报道。为探讨TGF-β1与肿瘤细胞凋亡的关系,本实验按如下三个部份进行研究。第一部分通过siRNA靶点序列设计软件结合Angela siRNA设计原则,设计、合成TGF-β1特异性siRNA靶点序列;再将靶点序列插入PAVU6+27质粒构建siRNA质粒;通过免疫荧光、免疫印记等方法检测转染siRNA质粒后人A549肺癌细胞株中TGF-β1的表达水平。第二部分将TGF-β1特异性小干扰RNA质粒pOligo1216和无关对照质粒pOligo Control转染肺腺癌A549细胞,检测TGF-β1 siRNA质粒转染后(即TGF-β1被静默后),上述A549细胞中Fas/FasL分子表达与凋亡酶caspase-3、caspase-8的活性;然后通过蛋白转染剂,将TGF-β1蛋白导入A549细胞,检测细胞内TGF-β1蛋白上调后细胞中Fas/FasL分子表达与凋亡酶caspase-3、caspase-8的活性,以了解TGF-β1对人肺腺癌细胞Fas/FasL凋亡通路的影响。第三部分参照Saitoh、Crowley等学者所报告的方法,首先检测人肺腺癌A549细胞与Calu-6细胞的HLA-ABDR等位基因,再构建稳定表达HLA-A2的人肺腺癌细胞系,通过混合淋巴细胞肿瘤细胞培养(MLTC)的方法,对诱导刺激健康人PBL产生肺癌特异性CTL作了初步的研究与探索。更多还原

【Abstract】 TGF-β₁ belongs to super family of growth factor and its biological effect is very common including regulation of cell proliferation and differentiation, regulation of embryonic development, enhancement of ECM formation and suppression of immune- system. TGF-β₁ is highly expressed in many malignant tumor cells including lung cancer cells and it can be expressed in matrix cells around cancer cells. In current opinion, TGF-β1 suppresses proto-oncogene such as c-myc to inhibit cell growth cycle of G1 stage to S stage.But in the middle or later stage of cancer when the tumor infiltrating blood vessels has been forming, TGF-β1 can make tumor cells be of much maliganancy by stimulation of tumor infiltrating blood vessls, by enhancement of cell migration and by immune suppressing.So the microenvironment of growth, infiltration and metastasis for cancer cells is forming. According to the results of observation in normal cells, TGF-β1 can regulate Fas/FasL. Fas/FasL system is a very important apopatosis pathway. In normal cells, TGF-β₁ can upregulat Fas, so that caspases in Fas/FasL pathway are activated to induce cell apoptosis. Moreover, caspases can be activated by combination of Fas with FasL. So TGF-β₁ can be regarded as an important factor that induces apoptosis and plays the role of abnormal cell clearance.But the relationship between TGF-β1 and cancer cell apoptosis is not very clear yet. The experimental report on it is very scarce.In order to study the relationship of TGF-β₁ and cancer apoptosis,the experiment develops according to three steps: step1,siRNA target sequences were analysed and synthesized by siRNA design software plus Angela siRNA principle.Then siRNA vehicle was reformed by the inserting of siRNA target sequences to PAVU6+27. Expressing Level of TGF-β₁ was analysed with indirect immunofluorescence and western blotting after siRNA vehicles were transfected into A549 cells. Step2, the expression level of Fas/FasL and activity of caspase-3/caspase-8 was detected in A549 cells that had been transfected with pOligo1216 or pOligo Control. Then the expression level of Fas/FasL and activity of更多还原