【摘要】 目的:构建人骨形态发生蛋白2反转录病毒表达载体,探讨以转人骨形态发生蛋白2基因骨髓基质细胞为种子细胞的组织工程骨修复骨缺损的可行性。方法:实验于2001-05/2003-01在北京大学医学部完成。①采用DNA重组技术,将骨形态发生蛋白2cDNA克隆到pLXSN反转录病毒质粒,经酶切及测序鉴定正确后,包装成为pLXSN/BMP2重组反转录病毒。取15d龄BALB/c系小鼠5只用于骨髓基质细胞的提取。将重组病毒感染小鼠骨髓基质细胞,经筛选后,聚合酶链反应鉴定人骨形态发生蛋白2cDNA在转基因骨髓基质细胞中的整合情况。②转基因骨髓基质细胞的骨形态发生蛋白2表达情况采用免疫印渍杂交和核糖核酸印渍杂交技术检测。转基因骨髓基质细胞碱性磷酸酶的表达采用钙-钴法染色检测,用以评定表达骨形态发生蛋白2的生物活性。③取6周龄BALB/c小鼠6只,将转基因骨髓基质细胞与羟基磷灰石构建人工骨植于小鼠骨骼肌内,体内成骨情况予组织学观察评估。结果:①pLXSN/BMP2质粒的鉴定结果:经酶切及DNA测序证实重组pLXSN/BMP2序列正确。②重组病毒的滴度测定结果:包装的病毒滴度为(6～8)×105cfu/mL。③基因整合及表达的分析:聚合酶链反应证实转基因骨髓基质细胞基因组中有骨形态发生蛋白2cDNA整合;核糖核酸印渍杂交和蛋白免疫印渍杂交证实转基因骨髓基质细胞稳定表达人骨形态发生蛋白2;钙-钴法染色证实转基因骨髓基质细胞碱性磷酸酶阳性。④小鼠体内成骨情况:甲苯胺蓝染色的组织切片镜下观察显示转骨形态发生蛋白2基因骨髓基质细胞构成人工骨可见少量软骨细胞及软骨基质存在,软骨细胞呈巢状分布。结论:成功地构建了pLXSN-BMP2重组反转录病毒载体,该载体不仅有效地表达骨形态发生蛋白2蛋白,表达的骨形态发生蛋白2能够诱导未分化的骨髓基质细胞向成骨细胞方向分化,且在体内肌肉环境下能促进成骨。更多还原

【Abstract】 AIM: To construct retroviral vector encoding human bone morphogenetic protein-2(BMP2) cDNA for evaluating the possibility of repairing the bone defects with tissue-engineered bone taking bone marrow-derived stromal cells containing BMP2 cDNA as seed cells. METHODS: The experiment was finished in Peking University Health Science Center from May 2001 to January 2003. ①Human BMP2 cDNA from the plasmid pSP65/BMP2 was cloned into retroviral plasmid pLXSN by DNA recombinant technique, then identified correct by restriction endonuclease analysis and DNA sequence analysis to construct pLXSN/BMP2 plasmid. Stromal cells were derived from bone marrow of 5 BALB/c mice at the age of 15 days, and then were infected by recombinant retrovirus. The integration of human BMP2 cDNA into transduced bone marrow-derived cell genomic DNA was detected by polymerase chain reaction(PCR). ②The expression of BMP2 in tranduced bone marrow-derived cells was detected by Northern and Western blotting. The activity of alkaline phosphatase(ALP) was detected by calcium-cobalt staining in the transduced bone marrow-derived cells so as to assess the bioactivity of BMP2. ③The artificial bone constructed by transduced bone marrow-derived stromal cells and hydroxyapatite was implanted into the skeletal muscles of 6 BALB/c mice at age of 6 weeks for the histological observation of in vivo osteogenesis. RESULTS: ①The recombinant pLXSN/BMP2 plasmid was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. ②The titer of recombinant retrovirus pLXSN/BMP2 was (6 to 8)×105 cfu/mL. ③It was shown by PCR that the transduced bone marrow-derived stromal cells were integrated by BMP2 cDNA; The expression of human BMP2 in the tranduced bone marrow-derived stromal cells was confirmed by Northern and Western blotting; There was positive activity of ALP in the transduced bone marrow-derived stromal cells by means of calcium-cobalt staining. ④From tissue sections stained by toluidine blue, a small quantity of chondrocytes distributing in nest-like shape and cartilage matrix were observed in the artificial bone constructed by bone marrow-derived stromal cells containing BMP2 cDNA. CONCLUSION: The recombinant retroviral vector encoding BMP2 was successfully constructed. BMP2 protein was not only expressed effectively in the transduced bone marrow-derived stromal cells, but also could induce the undifferentiated bone marrow-derived stromal cells to differentiate into osteoblasts and enhance bone formation in muscles in vivo.更多还原