CAJ | 학술논문

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。
배경：연골조절소Ⅰ시일충당단백，주요재연골중표체，이재골수간충질간세포중소량표체。결합과제조적전기연구，추단연골조절소Ⅰ재유도골수간충질간세포향연골세포분화과정중가능회기촉진작용。 　　목적：구건연골조절소Ⅰ표체재체，사기재대서골수간충질간세포중은정상조표체。 　　방법：재대서연골조직중획취연골조절소Ⅰ목적기인，사용 pcDNA3.1(+)질립표체재체구건pcDNA3.1(+)/ChM-I 표체재체。밀도제도리심법화첩벽배양법획득대서골수간충질간세포。용지질체법장구건적pcDNA3.1(+)/ChM-I표체재체전염대서골수간충질간세포，사용G418은정사선전염세포，용RT-PCR 급Western blot 검측연골조절소Ⅰ재세포주내적표체。 　　결과여결론：대양성극륭적쌍매절감정급측서화분석대비，결과현시여연골조절소Ⅰ기인장도상부병차서렬무오，차독마광정학。RT-PCR급western blot검측결과현시，연골조절소Ⅰ적mRNA급단백재골수간충질간세포중은정적고표체。실험성공구건료pcDNA3.1(+)/ChM-I표체재체，병성공장기전염도대서골수간충질간세포중，최종사연골조절소Ⅰ재대서골수간충질간세포중은정상조표체。
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-Ⅰmaybe 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 celllines. 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 successful y 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.