【作者】 李雷；

【导师】 肖强；

【作者基本信息】 广西医科大学 ， 外科学， 2009， 博士

【摘要】 胃癌(Gastric cancer)是我国最常见的消化道肿瘤,其年患病率和死亡率均为世界平均水平的2倍以上。在广西壮族自治区,胃癌是仅次于肝癌的最常见的恶性肿瘤,其死亡率位居第二位。近二十年来,胃癌的发病率和死亡率在广西有明显上升趋势,已成为危害广西居民健康和生命的主要疾病之一。目前常见胃癌中由胃黏膜腺体細胞生成的腺癌占绝大多数,约90%。慢性胃炎-胃黏膜萎缩-肠化生-不典型增生-胃癌这一演进模式已为国内外多数学者赞同,不完全性肠化与肠型胃癌关系最为密切。而在多细胞生物体的发生发展过程中,同源基因(Homeobox Gene)编码决定位置信息的遗传因子,是生物体中一类专门调控生物形体的基因。其中Cdx家族对消化道特别是结肠和小肠上皮的发育起着关键的作用,特别是Cdx2基因与胃癌前期改变-胃黏膜肠化生之间有着紧密的联系。尽管国内外对Cdx2基因在胃癌组织中表达情况的研究已经积累了很多资料,但是对Cdx2在胃癌中的功能研究却很少。本课题在体外-体内实验的研究基础上,利用细胞和现代分子生物学等技术,通过构建真核表达载体研究过表达Cdx2基因在体外环境中对胃癌细胞的生物学影响;通过建立裸鼠人胃癌原位移植瘤的动物模型观察其对移植瘤生长、发育和转移的作用;通过构建Cdx2基因的逆转录病毒载体对裸鼠胃癌皮下瘤进行感染试验,为基因治疗胃癌提供新的实验和理论依据,指导进一步的临床应用;最终通过基因芯片扫描技术研究Cdx2过表达引起的基因表达谱改变情况,探讨其在胃癌细胞中产生作用的分子机制。实验内容包括以下四部分:第一部分Cdx2真核表达载体的构建及其对胃癌细胞MGC-803生物学行为的影响目的:通过构建人同源基因Cdx2真核表达载体,转染胃癌细胞MGC-803,观察过表达Cdx2基因对胃癌细胞生物学性状的影响。方法:从人胃癌组织中扩增出Cdx2基因的ORF全长,构建pCMV-Cdx2-HA真核表达载体;脂质体介导法瞬时转染pCMV-HA和pCMV-Cdx2-HA至人胃癌细胞MGC-803 ,两组细胞命名为MGC-803/EV,MGC-803/Cdx2。应用RT-PCR检测各组Cdx2基因的mRNA表达情况,Western blot检测各组Cdx2蛋白的表达情况。MTT法观测细胞增殖,透射电镜观察转染细胞形态学的改变,流式细胞仪分析各组细胞周期和凋亡。结果:成功构建pCMV-Cdx2-HA真核表达载体,在瞬时转染48h的MGC-803/Cdx2组细胞中检测到较高水平Cdx2基因的mRNA和蛋白表达。与未转染组、MGC-803/EV组比较:MGC-803/Cdx2组的胃癌细胞在转染72h时生长受到明显抑制,细胞周期G0 /G1期比例上升,S期比例下降,胃癌细胞的凋亡率也明显升高(P<0.05)。透射电镜显示MGC-803/Cdx2组的胃癌细胞出现明显的凋亡表现。结论:过表达Cdx2基因明显抑制胃癌细胞的生长,提示Cdx2参与胃癌的生长调控。第二部分Cdx2过表达胃癌细胞稳定株对裸鼠人胃癌移植瘤体内增殖和转移的影响目的:筛选稳定过表达Cdx2基因的胃癌细胞株,检测细胞功能的改变;构建裸鼠的人胃癌移植瘤模型,观察过表达Cdx2基因对胃癌体内成瘤的影响。方法:将pCMV-HA和pCMV-Cdx2-HA转染进人胃癌细胞MGC-803,通过G418筛选出稳定胃癌细胞株,经过RT-PCR、Western-blot鉴定后命名为空载体组(MGC-803/EV)和转染组(MGC-803/Cdx2)。对其进行细胞划痕和生长曲线等细胞功能试验,使用流式细胞仪检测其细胞周期和凋亡改变。建立裸鼠人胃癌移植瘤模型,观察移植瘤的生长情况;应用RT-PCR及Western-blot分析各组标本中Cdx2基因的mRNA和蛋白表达水平;以HE染色观察各组标本中肿瘤转移情况。结果:成功筛选出稳定胃癌细胞株MGC-803/Cdx2和MGC-803/EV。细胞划痕试验中MGC-803/Cdx2组愈合率明显低于MGC-803/EV和MGC-803组(P<0.05);生长曲线中MGC-803/Cdx2组生长速度明显低于其他两组(P<0.05)。同时MGC-803/Cdx2组细胞侵袭和克隆能力也明显下降,而细胞周期也在G0 /G1期出现阻滞、细胞凋亡明显。裸鼠人胃癌移植瘤模型解剖发现:裸鼠胃壁成瘤率MGC-803/EV组为90%, MGC-803/Cdx2组为73.33%,胃壁移植瘤的体积MGC-803/Cdx2组(13.42±2.34)mm3小于MGC-803/EV组(17.59±2.80) mm3,两者存在显著差异(P<0.05),但MGC-803/Cdx2组裸鼠的肿瘤转移率尚不能认为与MGC-803/EV组不同。结论:成功构建裸鼠人胃癌转移模型,真实模拟人胃癌生长过程,证实过表Cdx2基因在体内-体外均可抑制胃癌生长。第三部分Cdx2逆转录病毒感染对裸鼠人胃癌皮下瘤生长的影响目的:研究肿瘤的基因疗法,建立含有Cdx2基因的逆转录病毒包装细胞PA317/Cdx2,观察逆转录病毒感染后裸鼠胃癌皮下瘤生长情况。方法:应用基因重组技术将Cdx2基因克隆到逆转录病毒载体pLXSN中构建pLXSN-Cdx2载体。将逆转录病毒载体pLXSN和Cdx2重组体分别转染进包装细胞PA317中,经G418筛选出稳定包装细胞株分别命名为PA317/PLX和PA317/Cdx2,使用RT-PCR检测其转染效果。收集两组稳定细胞株培养上清,通过感染NIH3T3细胞测定病毒滴度。建立裸鼠人胃癌皮下瘤模型,观察Cdx2重组逆转录病毒感染后对肿瘤成瘤体积的影响,探索基因治疗效果。结果:重组逆转录病毒载体pLXSN-Cdx2经酶切鉴定证实与目的片断一致,表明重组质粒构建成功。经G418筛选后获得多个抗性克隆,重组体组经过RT-PCR检测均证实Cdx2基因正确克隆进PLXSN。pLXSN和pLXSN-Cdx2经NIH3T3细胞测定病毒滴度分别为3.0×105 CFU/ml和2.4×105 CFU/ml。裸鼠人胃癌皮下瘤模型显示pLXSN-Cdx2感染裸鼠皮下瘤后使得肿瘤生长减缓。结论:成功建立起含有Cdx2基因的逆转录病毒包装细胞PA317/Cdx2,证实Cdx2在胃癌具有肿瘤基因治疗的潜力。第四部分Cdx2过表达对胃癌细胞MGC-803作用机制的研究目的:研究过表达Cdx2基因引起胃癌细胞MGC-803基因表达谱改变。方法:提取稳转pCMV-HA(MGC-803/EV)和pCMV-HA/Cdx2(MGC-803/Cdx2)细胞的总RNA,应用基因芯片技术对差异基因进行芯片分析,对结果进行统计分析。结果:基因芯片分析发现了两组细胞MGC-803/EV和MGC-803/Cdx2间的差异表达基因(以表达差异≥2.0或≤0.5倍为限)共551条,其中上调基因249条,下调基因302条。经过分析,这些差异基因同细胞的增生、分化和细胞的信号传导密切相关。结论:应用基因芯片技术成功筛选出可能与Cdx2过表达后抑制胃癌细胞MGC-803生长的相关基因,为进一步研究Cdx2基因在胃癌中的作用机制提供新的依据。更多还原

【Abstract】 Gastric cancer are the most common gastrointestinal cancers in China, annual morbidity and mortality are 2 times higher than the world’s average rate.In our Guangxi, gastric cancer are the most common malignant tumor after liver cancer,and and its mortality ranked second place. During the past two decades, there is clear upward trend of gastric cancer morbidity and mortality in Guangxi, gastric cancer has become one of the major diseases hazard to health and the lives of the residents.At present, the common gastric cancer, including adenocarcinoma, lymphoma, sarcoma and so on, the vast majority is gastric glands of the adenocarcinoma, about 90%.Now, chronic gastritis - gastric mucosal atrophy - intestinal metaplasia - dysplasia - gastric cancer model are agreeded by the majority of scholars all over th world,especially in incomplete intestinal metaplasia and intestinal-type gastric cancer are closely related.In the development of the organism, homeobox gene decide the location of the information encoded genetic factor, are in a class of specialized physical biology of gene regulation.Cdx family plays a key role in development of the digestive tract, especially colon and small intestinal epithelium.Its protein through the helix - loop - helix combination in the DNA corresponding region in the form of transcription factors regulating the expression of DNA.In particular, Cdx2 gene is close linkage with early changes of gastric cancer - gastric intestinal metaplasia. Our research studied the biological effects in gastric cancer cells overexpressed Cdx2 gene in vitro - in vivo by used cellular and molecular biology techniques.Observed gastric cancer cells changes of growth, development and metastasis in nude mice by orthotopic transplantation of gastric cancer,to explore the mechanism of Cdx2 and provide a new experimental and theoretical basis gene therapy for gastric cancer to guide further clinical application. Include the following four parts:Part 1 Construction of eukaryotic expression plasmid of Cdx2 and its expression in human gastric carcinoma cell line MGC 803 cellsObjective To construct an eukaryotic expression vector with human caudal-related homeobox 2(Cdx2) gene,and to investigate the transfection and expression of pCMV-Cdx2-HA eukaryotic expression plasmid in MGC-803 cells.Methods pCMV-Cdx2-HA eukaryotic expression plasmid was constructed by DNA recombinant method. pCMV-Cdx2-HA was transfecting into MGC-803 cells with effectene reagent. Cdx2 gene was detected by Reverse transcription polymerase chain reaction(RT-PCR) and western blot techniques. The effects of Cdx2 overexpression on the growth of human gastric carcinoma MGC-803 cells in vitro was assessed by measuring MTT, flow cytometric and transmission electron microscopy analysis.Results The eukaryotic expression vector pCMV-Cdx2-HA has been successfully constructed.The levels of Cdx2 mRNA and protein was higher in MGC-803 transfecting with pCMV-Cdx2-HA.In MGC-803 cells transfecting with pCMV-Cdx2-HA, cell proliferation was significantly inhibited, the ultrastructural changes of the cells was destroyed, cell cycle progression was blocked in G0/G1 and apoptosis rate was higher (P<0.05) in comparison with nontransfected cells or cells transfected with empty vector.Conclusions These results demonstrate that Cdx2 gene plays an inhibition role in gastric carcinoma and may save as a target for the development of novel therapeutic interventions to treat this disease.Part 2 The effects of gastric cancer cells stably overexpressing Cdx2 on growth of transplanted human gastric cancer in nude miceObjective: To construct a gastric cancer cell line MGC-803 stably overexpressing Cdx2 gene and study its’effects on tumor volume of human gastric cancer transplanted in nude mice.Methods: The MGC-803 cells were transfected pCMV-HA and pCMV-Cdx2-HA by using the lipofectamin transfegtion respectively. Use G418 to selected stable cell line,Cdx2 was detected by RT-PCR and western blot.The effects of Cdx2 on the growth of human gaxtric carcinoma cell MGC-803 in vitro was assessed by scratching assay and measuring MTT.The cells stably expression of Cdx2 (MGC-803/Cdx2) and control (MGC-803/EV) were inoculated into nude mice subcutis.Then,the histologically intact xenograft tissues were transplanted to stomach wall.The xenografts were observed by tumor formation rate and volume in 30 days.Results:The stably cell line MGC-803/Cdx2 and MGC-803/EV were screened successfully. All assay showed that the migration and cell proliferation of MGC-803/Cdx2 were significantly inhibited in comparison with MGC-803/EV and MGC-803 cells(P<0.05). The transplantation formation rate of MGC-803/EV group was 90% and tumor volume was ( 17.59±2.80 ) mm3.In contrast,the formation rate of MGC-803/Cdx2 group was 73.33% and tumor volume was(13.42±2.34)mm3,were less than MGC-803/EV group(P<0.05).Conclusions: Transplanted human gastric cancer in nude mice closelt correlates with the course of the tumor in clinical patients.Overexpression of Cdx2 can inhibit tumor growth of transplanted human gastric cancer in nude micePart 3 Retrovirus-mediated Cdx2 gene therapy for human gastric cancer xenogeneic graft in nude mouse subcutaneousObjective: Study of tumor gene therapy, establish retrovira cell line PA317/Cdx2 containing Cdx2 gene. Observation affectes of retroviral infection on human gastric cancer transplantated in nude mouse subcutaneous.Method:Construction of the package cells PA317/Cdx2 with recombinant retroviral vector containing Cdx2 gene.Retroviral vectors were then titrated,injected at xenograft tumors.The volumes of tumors in nude mouse subcutaneous were observed.Result:The retroviral vector pLXSN-Cdx2 has been successfully constructed.The titer of retroviral was 2.4×105 .The recombinant retroviral vector containing Cdx2 gene restrained the growth of xenograft tumors by normal group and control group(P<0.05).Conclusion:Cdx2 have a curative effect on gastric cancer xenogeneic graft in nude mouse subcutaneous. Part 4 Screening of genes differentially expressed in MGC-803 cells transfected with Cdx2 gene using Gene microarrayObjective: To investing the effects of cdx2 gene on the expression profile of gastric cancer cell line MGC-803.Method:The MGC-803 cells were transfected by pcmv-cdx2-ha and pcmv-ha by using the lipofectamin transfegtion respectively.The RNA was isolated. High-throughout gene microarray was used to screen the effects of cdx2 gene on the expression profile of gastric cancer cell line MGC-803.Result:Using an extrance limit of≥2.0 or≤0.5 ,there were 249 up-regulated and 302 down-regulated genes identified by microarray which were commonly differentially espressed.The expression of these related genes belonged to many functional categories.Conclusion:With gene microarrary technology,the influence of cdx2 gene on the espression profile of gastric cancer cell line MFC-803 is extrnsive.The altered genes may be useful to clarify the mechanism of gastric cancer genesis.更多还原