【作者】 王颖；

【导师】 邹多宏；

【作者基本信息】 安徽医科大学 ， 口腔临床医学， 2017， 硕士

【摘要】 目的通过慢病毒载体基因转染技术,体外研究miR-21诱导拉布拉多犬的骨髓间充质干细胞(Bone marrow mesenchymal stem cell,BMSCs)成骨向分化的作用,并应用多种方法进行检测,为将来的体内骨缺损修复提供实验数据支持。方法使用全骨髓贴壁培养法在体外培养拉布拉多犬BMSCs;构建慢病毒载体Lenti-miR-21-Luciferase与Lenti-Lac Z-Luciferase,分别转染犬BMSCs,将研究分为miR-21实验组与Lac Z对照组;转染后4 d,倒置显微镜下观察细胞,通过生物发光成像法观察转染后两组细胞的示踪报告基因Luciferase的荧光发光强度检测慢病毒转染效率;MTT比色法检测慢病毒转染后BMSCs的增殖情况;利用细胞划痕实验比较转染后BMSCs的迁移能力;慢病毒转染后的第14天,通过茜素红S染色与碱性磷酸酶(ALP)染色观察BMSCs的体外骨形成情况;在转染后的第0、1、4、7、14 d,分别提取miR-21实验组与Lac Z对照组BMSCs的m RNAs和蛋白;通过Real time-PCR技术和Western blot检测成骨关键因子骨钙素(OCN)和骨桥蛋白(OPN)的表达。结果显微镜下观察BMSCs形态较为均一,多呈纺锤形或长梭形,细胞排列呈旋涡状或放射状融合,生长状态良好;MOI值为10时,BMSCs的转染效率最高,Luciferase的荧光发光强度检测结果表明示踪基因Luciferase在BMSCs中连接成功;miR-21实验组划痕宽度减少平均值为372.33μm,而Lac Z对照组仅为161.33μm,P﹤0.001(F=0.10),差异具有统计学意义,提示miR-21实验组的BMSCs细胞爬行迁移能力显著增强;钙结节染色与ALP染色结果提示,相较于Lac Z对照组,miR-21实验组中钙结节与ALP的表达均显著提高;Real time-PCR定量结果显示,miR-21实验组的BMSCs在慢病毒转染后7 d即有OCN基因和OPN基因的显著高表达,而在转染后的14 d,两基因仍维持较高表达水平,结果提示miR-21能够显著增强BMSCs中关键成骨相关因子的表达(P﹤0.05);Western blot检测结果显示,miR-21可以显著上调BMSCs成骨分化中OCN和OPN的蛋白表达(P﹤0.05),且表达趋势与m RNA的表达趋势相近。结论以慢病毒作为载体的miR-21能够成功转染BMSCs,并在细胞中能够持续高效的增强成骨相关因子的表达。miR-21可以显著促进BMSCs的骨向分化,为进一步体的内实验奠定基础。更多还原

【Abstract】 Objective To explore the effect of miR-21 on the osteogenic differentiation of Labrador dog bone marrow mesenchymal stem cells(BMSCs)in vitro.Methods Labrador dog BMSCs were cultured in vitro using whole bone marrow adherent method,Lenti-miR-21-Luciferase and Lenti-Lac Z-Luciferase lentiviral vector were constructed and then were transfected into canine BMSCs.The study was divided into miR-21 experimental group and Lac Z control group.After 4 days of transfection,observe the cells under inverted microscope.Detect the transfection efficiency of lentivirus by observing fluorescence intensity of tracer reporter Luciferase through bioluminescence imaging methods.MTT colorimetric assay was used to detect the proliferation of BMSCs after lentivirus transfection.The cell scratch test was used to compare the migration ability of BMSCs after lentiviral vector transfection.The m RNA and protein of BMSCs were extracted on 0,1,4,7,and 14 days.The expression of osteocalcin(OCN)and osteopontin(OPN),the key factors of osteogenic differentiation of BMSCs,were detected by Real time-PCR and Western blot.Results Microscopic observation shows the shape of cell is uniform,mostly spindle-shaped or long spindle and the cells were arranged in a spiral or radial fusion,also grow well.The transfection efficiency of BMSCs was the highest and the result of Luciferase’s fluorescence luminescence intensity test suggest that the tracer gene Luciferase is successfully linked in BMSCs when the MOI value is 10.The mean reduction of scribe width in miR-21 group was 372.33 μm while that of Lac Z control group was only 161.33 μm,P<0.001(F = 0.10),differences were statistically significant.The result suggests the crawling ability of BMSCs cell in miR-21 experimental group increased significantly.Alizarin Red S Calcium nodule staining and ALP staining results suggest the expression of calcium nodules and ALP in BMSCs was significantly increased in miR-21 group comparing with Lac Z group.Real time-PCR quantitative results show BMSCs in the miR-21 experimental group were significantly expressed in OCN and OPN genes at 7 days after lentivirus transfection.At the 14 th day after transfection,the two genes were still highly expressed.The results showed that miR-21 could significantly enhanced the expression of osteogenic factors in BMSCs(P<0.05).Western blot analysis shows that miR-21 can significantly increase the expression of OCN and OPN protein in osteogenic differentiation of BMSCs(P<0.05),and the expression trend is close to that of m RNA.Conclusion miR-21 played as a carrier with lentivirus,can successfully transfected BMSCs and efficiently enhance the expression of osteogenesis-related factors in cells.The miR-21 could significantly promote the osteogenic differentiation of BMSCs,laying the foundation for further in vivo experiments.更多还原