【作者】 付国斌；

【导师】 程书钧； 高燕宁； 林东昕；

【作者基本信息】 中国协和医科大学 ， 肿瘤学， 2009， 博士

【摘要】 世界范围内肺癌发病率和病死率居各恶性肿瘤首位,严重危害人们健康,目前临床上缺乏有效的诊疗措施,肺癌患者的五年生存率不足15%。鉴定和研究相关癌基因和抑癌基因的功能有望为肺癌防治提供新的靶标,因而一直是肺癌研究领域内的热点。我们实验室的前期工作从中国人肺鳞癌差异表达cDNA文库中发现、克隆并初步鉴定得到两条新基因,一为候选抑癌基因DENND2D,一为候选癌基因OLC1。本课题主要围绕候选抑癌基因DENND2D展开深入研究,探讨其功能、作用机制和可能的应用前景。为明确DENND2D基因在生理状态和肿瘤中的差异表达谱信息,我们首先利用Northern blot方法在肺、睾丸和胎盘组织中检测到了与预测大小一致的该基因天然转录本,而在心脏、脑和肌肉组织中则未检出DENND2D的表达;利用Real Time PCR方法我们在16例肺鳞癌患者的肿瘤与正常配对组织中检测了DENND2D的差异表达情况,以2倍差异作为接受标准,发现它在50%的肿瘤组织中低表达。为深入研究基因功能,我们首先建立了DENND2D的四环素诱导表达腺病毒系统,实现了基因的可控表达。针对在人肺癌细胞系H1299的研究发现,DENND2D的诱导表达可以直接引发细胞凋亡,并且凋亡发生率呈时间一剂量依赖性上升。进一步研究发现诱导表达DENND2D后,H1299细胞线粒体膜电位下降,Caspase 9和Caspase 3的蛋白水平活性增高,应用广谱Caspase抑制剂和针对Caspase 9的抑制剂后,DENND2D的凋亡诱导作用明显下调,初步证实了DENND2D可以直接诱导Caspase依赖的线粒体途径凋亡。我们利用融合标签蛋白示踪了DENND2D的蛋白定位提示为DENND2D定位于胞浆。同时我们在蛋白水平证明了DENND2D虽然激活JNK通路,但是DENND2D的凋亡诱导作用并非JNK依赖,应用JNK显性负突变体和特异JNK抑制剂SP600125均未对DENND2D的凋亡诱导作用产生影响。在肺鳞癌细胞系H520和肺腺癌细胞系A549中的研究结果提示,DENND2D的凋亡诱导作用不受DENND2D本底表达情况的影响。最后,我们采用裸鼠移植瘤模型从体内水平证实了DENND2D的抑瘤活性并利用Tunel方法原位检测瘤内凋亡情况,发现DENND2D肿瘤抑制作用可能也是通过诱导细胞凋亡实现。针对候选癌基因OLC1,为进行后续上皮细胞转化实验,我们构建了OLC1的诱导表达腺病毒系统。为了实现外周血血浆中OLC1蛋白的检测分析,我们与中国医学科学院基础医学研究所陈实平教授课题组合作,已经制备并鉴定了10株OLC1蛋白的单克隆抗体。为明确OLC1基因的生理学和病理学功能,我们与中国医学科学院医学实验动物研究所张连峰教授课题组合作,分别制备得到了全身广泛表达和肺组织特异表达OLC1的转基因小鼠:组织学研究发现二者都有明显的Ⅱ型肺泡上皮细胞增生,提示了OLC1的生理学功能线索。目前OLC1相关实验仍在进行之中。同时作为附加研究内容,我们还对来自肺癌差异表达cDNA文库的候选癌基因RAP2B的功能作了初步探讨。研究发现RAP2B在约67%(18/27)肺鳞癌肿瘤组织中高表达,在体外具有恶性转化Rat1细胞能力,并且可能通过激活NF-kappaB信号通路在肺癌发生发展中发挥作用。更多还原

【Abstract】 Lung cancer is the leading cause of cancer death worldwide.There are still no effective methods for early detection and therapy of the malignant disease,and the 5 year survival rate of lung cancer patients is below 15%,due to poor understanding of the mechanisms involved in lung cancer development and progerssion. Identification and research on the oncogenes and the tumor suppressor genes will provide new targets of prevention and treatment to lung cancer.Two lung cancer-related novel genes had been obtained from the different expression cDNA libraries previously established in this lab,one is the candidate oncogene OLC1,the other is candidate tumor suppressor DENND2D.This project was mainly focused on the functional study for the two genes,especially on DENND2D.To understand expression status of DENND2D gene in human normal and pathological tissues,using Northern blotting analysis we detected the natural transcript of DENND2D with the predicted molecular size in lung,testis and placenta tissues,but there was no detectable expression of this gene in heart,brain and muscle tissues.Then we tested the expression levels of DENND2D with Real-Time PCR,in 16 pairs of tumor and normal lung tissue samples derived from patients of lung squamous cell carcinoma.There was 50%of the tumor samples showing decreased expression of DENND2D,setting 2-fold as a cutoff ratio.To explore the gene function,we construct a Tet-on inducible expression system with adenovirus as the vector,and successfully get the gene expression under control. Induced expression of DENND2D can directly induce apoptosis in lung cancer cells H1299,and the apoptosis incidence presented in manner of the time-dose dependent of DENND2D expression level.With induced DENND2D expression,the loss of mitochondria membrane potential,and the activity of Caspase 3 and Caspase 9 which is key regulator of mitochondria-apoptosis pathway were increased;whereas when treated the cells with general Caspase inhibitor and specific Caspase 9 inhibitor,the DENND2D induced apoptosis was significantly down-regulated.It suggests that DENND2D could induce Caspase dependent mitochondrial apoptosis.However, there was no experimental evidence support the DENND2D as a mitochondria protein, using fusion tagged protein to show this protein location.Moreover,we found that DENND2D could activate the JNK pathway,but the DENND2D induced apoptosis is in JNK independent manner because it was not affected by the JNK dominant negative mutant and the JNK inhibitor SP600125.Combine the results in lung cancer cell line A549 and H520,we could summary up that the DENND2D induced apoptosis was not affected by the endogenous DENND2D expression.With xenograft of H1299 cells in nude mice,we confirmed the tumor suppressor activity of DENND2D in vivo,and it also seemed that DENND2D could induce apoptosis in vivo.For study on OLC1 gene,an inducible adenovirus expression system of OLC1 was also successfully constructed.Several monoclonal antibodies against OLC1 protein were prepared in hope of measuring OLC1 protein levels in the circulating plasma.To identify the OLC1 gene fuction,the gene engineering mouse model were established,and both the general expression transgenic and the lung specific expression transgenic mouse showed the hyperplasia of typeⅡalveolar cells,giving the physiology function clue to this gene.Further investigations are still undertaking.更多还原