【摘要】 背景:软骨调节素Ⅰ是一种糖蛋白,主要在软骨中表达,而在骨髓间充质干细胞中少量表达。结合课题组的前期研究,推断软骨调节素Ⅰ在诱导骨髓间充质干细胞向软骨细胞分化过程中可能会起促进作用。目的:构建软骨调节素Ⅰ表达载体,使其在大鼠骨髓间充质干细胞中稳定上调表达。方法:在大鼠软骨组织中获取软骨调节素Ⅰ目的基因,使用pcDNA3.1(+)质粒表达载体构建pcDNA3.1(+)/ChM-I表达载体。密度梯度离心法和贴壁培养法获得大鼠骨髓间充质干细胞。用脂质体法将构建的pcDNA3.1(+)/ChM-I表达载体转染大鼠骨髓间充质干细胞,使用G418稳定筛选转染细胞,用RT-PCR及Western blot检测软骨调节素Ⅰ在细胞株内的表达。结果与结论:对阳性克隆的双酶切鉴定及测序和分析对比,结果显示与软骨调节素Ⅰ基因长度相符并且序列无误,且读码框正确。RT-PCR及western blot检测结果显示,软骨调节素Ⅰ的mRNA及蛋白在骨髓间充质干细胞中稳定的高表达。实验成功构建了pcDNA3.1(+)/ChM-I表达载体,并成功将其转染到大鼠骨髓间充质干细胞中,最终使软骨调节素Ⅰ在大鼠骨髓间充质干细胞中稳定上调表达。更多还原

【Abstract】 BACKGROUND: Chondromodulin-Ⅰ is expressed mainly in the cartilage, but it is little expressed in mesenchymal stem cells. Combined with the previous study of our group, we concluded that chondromodulin-Ⅰ maybe play an important role in inducing mesenchymal stem cells into chondrocytes accurately. OBJECTIVE: To construct an expression plasmid stably carrying chondromodulin-Ⅰ to up-regulate the expression of chondromodulin-Ⅰ in bone marrow mesenchymal stem cells. METHODS: Specific primers were designed in rat cartilage for chondromodulin-Ⅰ gene, then the pcDNA3.1(+) plasmid expression vector was digested by enzyme and directional connected gene to construct pcDNA3.1(+)/ChM-Ⅰ expression vector. Bone marrow mesenchymal stem cells were obtained from rats using the method of density gradient centrifugation combined with adherent culture. Recombinant plasmid pcDNA3.1(+)/ChM-Ⅰ was transfected into rat bone marrow mesenchymal stem cells with liposome method, and G418 selection was used for stable screen of transfected cells. Reverse transcription-PCR and western blot were used to detect chondromodulin-Ⅰ expression in cell lines.RESULTS AND CONCLUSION: The positive clones were digested by enzyme and were identified and sequenced. The results showed that the reality length and sequence of chondromodulin-Ⅰ gene were consistent with the theoretical values, and reading frame was correct. Reverse transcription-PCR and western blot results showed that the expressions of chondromodulin-ⅠmRNA and protein were markedly up-regulated in bone marrow mesenchymal stem cells. Recombinant plasmid pcDNA3.1(+)/ChM-I was successfully constructed, and transfected into rat bone marrow mesenchymal stem cells. After G418 selection, expression of chondromodulin-Ⅰ was up-regulated stably in rat bone marrow mesenchymal stem cells.更多还原