【作者】 段光军；

【导师】 胡华成；

【作者基本信息】 苏州大学 ， 内科学， 2008， 博士

【摘要】 目的:重组构建人源化ING4腺病毒表达载体,并探究其对非小细胞肺癌NCI-H460细胞株体内外生长抑制作用及其相关分子机制。方法:运用定点突变技术,在已成功克隆mING4(鼠ING4)的基础上,根据hING4(人ING4)氨基酸序列,进行重组构建。设计两对突变引物P1、P2和P3、P4及全长ING4上下游引物P5、P6,通过四轮PCR,将mING4基因序列进行人源化改造,获得了编码hING4氨基酸的基因序列(简称ING4)。将获得酶切目的基因片段,连接到转移载体pAdTrack-CMV上,形成重组转移载体pAdTrack-CMV-ING4。重组转移载体经PmeI酶切后与pAdEasy-1腺病毒载体在BJ5183大肠杆菌中同源重组,得到重组腺病毒载体pAdeasy-1-pAdTrack-CMV-ING4,经PacI酶切后脂质体转染QBI-293A包装细胞,获得重组腺病毒Ad-ING4。通过类似的方法获得了重组空载体病毒Ad-null。将获得同源重组Ad-ING4病毒感染非小细胞肺癌NCI-H460细胞(p53野生型),运用RT-PCR及荧光显微镜检测ING4基因在H460细胞中表达及对H460细胞毒作用(CPE);运用MTT法、细胞集落形成试验及流式细胞仪检测Ad-ING4对肿瘤细胞生长抑制及诱导凋亡作用;运用激光共聚焦显微镜及透射扫描电镜检测H460细胞经ING4基因作用后形态学改变;运用RT-PCR、Western-blotting检测分析Ad-ING4诱导肿瘤细胞凋亡可能分子机制;运用RT-PCR和ELISA方法检测ING4基因对肿瘤主要血管生成相关基因VEGF的作用;在nu/nu裸鼠皮下建立NCI-H460肺癌荷瘤鼠模型,观察Ad-ING4基因对肿瘤生长抑制作用及对血管生成影响。结果:基因序列检测和RT-PCR结果证实获得重组人源化ING4基因,并成功构建重组人源化ING4腺病毒表达载体。RT-PCR和荧光显微镜检测证实ING4基因可以在H460细胞中稳定表达并对H460细胞产生细胞毒作用(CPE);MTT检测和细胞集落形成试验结果表明较对照组(Ad-null组, PBS组)相比,ING4基因可以明显抑制基因治疗组肿瘤细胞生长;流式细胞仪检测证实Ad-ING4可诱导肿瘤细胞凋亡;激光共聚焦显微镜和透射扫描电镜检测显示H460细胞经ING4基因作用后可发生细胞凋亡形态学改变,并出现凋亡小体;RT-PCR、Western-blotting检测结果提示Ad-ING4基因以p53依赖方式诱导肿瘤细胞上调p21和Bax基因表达,下调Bcl-2和Survivin基因表达来诱导肿瘤细胞凋亡;RT-PCR和ELISA检测提示ING4基因可以抑制肿瘤主要血管生成相关基因VEGF表达;体内试验表明Ad-ING4基因可以抑制肿瘤生长并抑制肿瘤体内血管生成。结论:运用定点突变技术对mING4进行了人源化改造,并成功重组构建了人源化ING4腺病毒表达载体;经RT-PCR和荧光显微镜检测证实腺病毒Ad-ING4可以在非小细胞肺癌NCI-H460细胞株中稳定表达;腺病毒Ad-ING4在非小细胞肺癌NCI-H460细胞株中表达可致肺癌细胞产生细胞病变(CPE),并主要通过诱导细胞凋亡和抑制肿瘤血管生成来抑制肿瘤细胞增殖;腺病毒Ad-ING4诱导非小细胞肺癌NCI-H460细胞凋亡的主要分子机制是以p53依赖方式上调促凋亡因子Bax表达,下调Bcl-2、Survivin表达,诱导肿瘤细胞凋亡;腺病毒Ad-ING4同时通过促进p53反应基因p21表达,上调Bax表达,并改变Bax/Bcl-2比例,促进肿瘤细胞凋亡;腺病毒Ad-ING4可以抑制肿瘤主要血管生成相关基因VEGF的表达,并在体内抑制肿瘤血管生成;腺病毒Ad-ING4注射可以抑制H460实体瘤肿瘤生长。更多还原

【Abstract】 Objective: To construct a recombinant hING4 adenovirus vector and to explore the potential mechanisms of ING4 gene in suppressing non-small cell lung cancer NCI-H460 cells growth in vitro and in vivo.Methods: Using site-specific mutagenesis technique, mING4 gene was changed into hING4 gene after fourth cycle PCR. hING4 cDNA was introduced into the shuttle plasmid pAdTrack-CMV, the recombinant shuttle plasmid (pAdTrack-CMV-ING4) and the backbone plasmid(pAdEasy-1) were linealized with PmeI digestion and then under co-transformation in bacteria E. coil BJ5183.Then, the newly recombinant plasmid pAdEasy-1-pAdTrack-CMV-ING4 was linearized with Pac I and was transferred into QBI-293A cells to form Ad-ING4.The control virus Ad-null with GFP was constructed in the same manner.The recombinant adenoviruses were amplified in QBI-293A cells, and viruses were purified from these cells to obtain viral stock.Non-small cell lung cancer NCI-H460 cells were transfected with Ad-ING4, RT-PCR and fluorescent microscopy were used to detect ING4 expression in H460 cells, Cytotoxicity assay and colony formation assay were used to evaluate the role of ING4 on cell growth, Flow cytometry was used to evaluate the role of ING4 in inducing apoptosis, Laser scan confocal microscope(LSCM) and transmission electron microscope were used to observe the morphology change of H460 cell. RT-PCR and Western-blotting were used to explore the potential mechanism of ING4 gene in inducing apoptosis. RT-PCR and ELISA were used to evaluate the role of ING4 on angiogenesis related gene vascular endothelial growth factor (VEGF), the effect and mechanism of Ad-ING4 in vivo was observed through the H460 subcutaneous model in nu/nu mice.Results: Sequencing and RT-PCR results proved that recombinant hING4 gene was constructed successfully. ING4 gene was also proved expression in Ad-ING4 transfected H460 cells by RT-PCR and fluorescent microscopy. Cytopathic effect (CPE) was found in Ad-ING4 transfected H460 cells. Cytotoxicity assay and colony formation assay results show that the viability and growth of Ad-ING4 transfected H460 cells were significantly inhibited compared with Ad-null and PBS control groups. RT-PCR and Western-blotting results proved that apoptosis related gene Bax was up-regulted expression whereas Bcl-2 and Survivin were down-regulated expression in Ad-ING4 transfected H460 cells. p53-responsive gene p21 was also up-regulated in Ad-ING4 transfected H460 cells. RT-PCR and ELISA result show that ING4 gene can reduce the expression of VEGF in Ad-ING4 transfected H460 cells. Tumor growth and tumor vessel formation were repressed in H460 subcutaneous model.Conclusion: The recombinant adenovirus hING4 vector was constructed successfully, and Ad-ING4 can express in H460 cells which cause obviously CPE in ING4 transfected cells compared with control group cells. Ad-ING4 can repress H460 cells growth in vitro and vivo mainly through inducing apoptosis and inhibiting angiogenesis. Ad-ING4 induced H460 cells apoptosis via up-regulation Bax expression and down-regulation Bcl-2 and Survivin expression in a p53 dependent manner, accompanied by p53-responsive gene p21 up-regulation which might promote the expression of Bax, subsequently change the ratio of Bax/Bcl-2,leading to the apoptosis of H460 cells. RT-PCR and ELISA results proved that the expression of VEGF was significantly reduced in Ad-ING4 transfected H460 cells compared with Ad-null and PBS groups. In mice bearing tumors, intratumoral injections of Ad-ING4 significantly inhibited tumor growth.更多还原