猪TGF-β_1基因重组慢病毒载体的构建及其在BMSCs中的表达  

CONSTRUCTION OF RECOMBINANT PORCINE TRANSFORMING GROWTH FACTOR β_1 GENE LENTIVIRAL VECTOR AND ITS EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS

作　　者：赖建明[1] 林建华[2] 林文平[3] 吴朝阳[2] 

机构地区：[1]福建医科大学附属龙岩市第一医院骨科,福建龙岩364000 [2]福建医科大学附属第一医院骨科 [3]福建医科大学附属第二医院骨科

出　　处：《中国修复重建外科杂志》2012年第7期849-854,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：福建省科技计划重点项目(2010Y0023)~~

摘　　要：目的构建猪TGF-β1重组慢病毒表达载体,并转染BMSCs,为构建组织工程骨软骨提供TGF-β1修饰的BMSCs,作为持续、高效的种子细胞。方法将已获取的目的基因TGF-β1cDNA包装至慢病毒载体中,通过PCR及基因测序对阳性克隆进行鉴定,并测定病毒滴度。取2月龄巴马香猪(体重约15 kg)骨髓制备BMSCs,取第2～3代用于实验。用TGF-β1重组慢病毒载体以感染复数(multiplicity of infection,MOI)为10、50、70、100、150分别转染BMSCs,通过激光共聚焦显微镜观察,并以Western blot检测不同MOI值的转染效果,确定最佳MOI值。用TGF-β1重组慢病毒载体以最佳MOI值感染BMSCs作为实验组,以空载体转染的BMSCs(空载体组)及未转染的BMSCs(空白组)作为对照,通过RT-PCR、免疫细胞化学染色、ELISA等方法检测TGF-β1基因及蛋白在BMSCs中的表达情况,并检测Ⅱ型胶原表达情况。结果经PCR及基因测序鉴定TGF-β1重组慢病毒表达载体构建成功,并成功转染BMSCs,激光共聚焦显微镜下可观察到强绿色荧光;Western blot示MOI为70时转染效果最佳;RT-PCR示实验组TGF-β1基因的表达量明显高于空载体组及空白组,差异有统计学意义(P<0.05);免疫细胞化学染色示实验组TGF-β1蛋白及Ⅱ型胶原呈阳性表达,而空载体组及空白组呈弱阳性或阴性表达;ELISA示实验组TGF-β1蛋白至转染后21 d仍有较高表达。结论 TGF-β1重组慢病毒表达载体可成功转染BMSCs,TGF-β1蛋白可长期、稳定表达,促使BMSCs向成软骨细胞方向分化。Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 （TGF-β1） gene and transfect bone marrow mesenchymal stem cells （BMSCs） so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated from bone marrow of the 2-month-old Bama miniature pigs （weighing 15 kg）, and the 2nd and 3rd generations of BMSCs were harvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors （TGF-β1 vector group） respectively at multiplicity of infection （MOI） of 10, 50, 70, 100, and 150; then the effects of transfection were detected by laser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector （empty vector group） and non-transfected BMSCs （non-transfection group） were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type Ⅱ. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group （P 〈 0.05）. Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type Ⅱ in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-�

关 键 词：组织工程 BMSCS TGF-β1 Ⅱ型胶原 关节软骨 基因治疗 慢病毒载体 猪 

分 类 号：R346[医药卫生—基础医学]