草酸代谢基因在成人骨髓间充质干细胞中的表达及意义-泌尿外科学专业论文.docx

优秀毕业论文 精品参考文献资料 The Signification and Expression of Oxalate-degredation Genes from Oxalobacter Formigenes in Human Bone Marrow Mesenchymal Stem Cells marrow Mesenchymal Stem Cells Major: surgery Postgraduate: Xu-ming Yang Tutor: Zhang Ze 【Abstract】 Objective：To study cloning oxalate-degredation genes Frc and Oxc of Oxalobacter formigenes into retrovirus vectors pLEGFP-N1 and pBaBE-puro,stably expression in human bone marrow mesenchymal stem cells(hMSCs),obtained the function of oxalate—degradation. Methods：Based on successfully saved genome of Oxalobacter formigenes in our laboratory, according to the gene sequence of Frc (NCBI accession NO.OFU82167) and Oxc (NCBI accession No. M77128), analyzed and designed the primers, the gene frc and oxc were amplified by PCR, then used agarose gel electrophoresis, restriction enzyme digestionrecycling plastic products of the genes Frc and Oxc, retrovirus vectors pLEGFP-N1 and pBaBE-puro, after the products were purified and linked, Transformed into E.coli of DH5α, picked colony PCR, enzyme electrophoresis analysis, verified by sequencing. Retrovirus packaging cells using the methods of calcium phosphate, collected virus fluid from cuture, infectied the normal adult bone marrow mesenchymal stem cells. Used the methods of 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and flow cytometry assay, flow cytometry(FCM) proliferation detected the proliferation of cells,drawing the growth curve. Total RNA were extracted with TRIzol method. cDNA were acquired by RT reaction, then were detected the expression of Frc and Oxc in three groups ( the group of hMSCs, the control group of hMSCs ,the Stable group of hMSCs) from gene level by performing quantitative PCR. Extracted the protein by the protein extraction reagent kit, after gel electrophoresis, transferred to a membrane, closed in night, Joined the primary antibody and second antibody,then incubation, chemiluminescence, developing and other steps.Western blot wer