构建可分解草酸的成人骨髓间充质干细胞系

背景：高草酸尿是结石形成的危险因素之一,利用基因工程和干细胞技术构建具有高代谢草酸能力的细胞系,将成为防治草酸钙结石的有效手段。目的：将产甲酸草酸杆菌的草酸分解基因Frc和Oxc共转染至正常成人骨髓间充质干细胞,构建可分解草酸的成人骨髓间充质干细胞系,使之获得分解草酸的功能。方法：PCR法扩增出Frc和Oxc基因的编码序列,构建真核表达载体pLEGFP-N1-myc-Frc和pBaBE-puro-flag-Oxc,将其共转染至正常成人骨髓间充质干细胞,以转染空载体及未进行转染的正常成人骨髓间充质干细胞作为对照。采用Western blot检测目的基因表达情况;离子色谱法测定转基因后细胞培养液中草酸浓度。结果与结论：酶切鉴定和测序结果显示实验成功扩增了Frc和Oxc基因,并构建了pLEGFP-N1-myc-Frc和pBaBE-puro-flag-Oxc载体。将其转染至正常成人骨髓间充质干细胞后,Western blot检测结果显示其可稳定表达目的蛋白myc-甲酰辅酶A转移酶和flag-草酰辅酶A脱羧酶;离子色谱仪检测结果显示,随着培养时间的延长,转染目的基因的人骨髓间充质干细胞培养液中草酸浓度逐渐下降。而转染空载体及未进行转染的正常成人骨髓间充质干细胞无目的蛋白表达,也无分解草酸能力。说明实验成功构建了可分解草酸的成人骨髓间充质干细胞系,该细胞系可稳定表达草酸分解蛋白Frc和Oxc,并具有草酸分解能力。

Construction of oxalate-degrading adult bone marrow mesenchymal stem cell lines

BACKGROUND: High oxalic acid urine is a risk factor for stone formation. Constructing cell lines with high oxalate metabolic ability using genetic engineering and stem cell technology will become the effective method to prevent and treat calcium oxalate stone. OBJECTIVE:To construct the adult bone marrow mesenchymal stem cell 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 successfully 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 cell culture medium transfected with target gene was decreased gradually. While there was no target protein expression in the non-transfected human bone marrow mesenchymal stem cells as well as the cells transfected with empty vectors. The cells had the ability of oxalate-degradation. The experiment successfully constructs the adult bone marrow mesenchymal stem cell lines that can decompose the oxalic acid, and the cell lines have the ability of oxalate-degradation and can stably express the oxalate decomposition proteins Frc and Oxc.