Construction of oxalate-degrading adult bone marrow mesenchymal stem cell lines / 中国组织工程研究

ABSTRACT

BACKGROUND: High oxalic acid urine is a risk factor for stone formation. Constructing cel lines with high oxalate metabolic ability using genetic engineering and stem cel technology wil become the effective method to prevent and treat calcium oxalate stone. OBJECTIVE: To construct the adult bone marrow mesenchymal stem cel lines that can decompose the oxalic acid, through co-transfecting the oxalic acid degradation genes Frc and Oxc of oxalobacter formigenes into the normal adult bone marrow mesenchymal stem cells. METHODS: Frc and Oxc were amplified by PCR, and the eukaryotic expression vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed, then co-transfected into the normal adult bone marrow mesenchymal stem cells. The non-transfected bone marrow mesenchymal stem cells and the cells transfected with empty vectors were as control. Western blot was performed to detect the expression of the objective genes;the concentration of oxalate in the culture medium after transgenic was determined by ion chromatography. RESULTS AND CONCLUSION: Restriction enzyme digestion and sequencing results showed that the Frc and Oxc genes were successful y amplified, and the vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed. After tranfected into the bone marrow mesenchymal stem cells, the Western blot results showed that transfected bone marrow mesenchymal stem cells could stably express the target protein myc-formyl coenzyme A transferase enzyme and the flag-oxalyl coenzyme A decarboxylase; ion chromatography test results showed with the prolonging of the culture time, the concentration of oxalic acid in the human bone marrow mesenchymal stem cel culture medium transfected with target gene was decreased gradual y. While there was no target protein expression in the non-transfected human bone marrow mesenchymal stem cells as wel as the cells transfected with empty vectors. The cells had the ability of oxalate-degradation. The experiment successful y constructs the adult bone marrow mesenchymal stem cel lines that can decompose the oxalic acid, and the cel lines have the ability of oxalate-degradation and can stably express the oxalate decomposition proteins Frc and Oxc.