The Influence Of Cdx2 Treatment On Gastric Cancer Cabiological Behaviors And The Basic Study In Molecule Mechanism

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.