Paxillin对骨髓间充质干细胞向类髓核细胞分化的影响

Effect of Paxillin gene on the differentiation of bone marrow mesenchymal stem cells to nucleus pulposus-like cells

中文关键词:  骨髓间充质干细胞  Paxillin  类髓核细胞  真核表达载体

英文关键词:Bone marrow mesenchymal stem cells  Paxillin  Nucleus pulposus-like cells  Eukaryotic expression vector

中文摘要:

  【摘要】 目的：探讨Paxillin对骨髓间充质干细胞（BMSCs）向类髓核细胞分化的影响。方法：取3周龄健康SD大鼠骨髓，采用全骨髓贴壁法培养BMSCs，传至第3代，用流式细胞仪对其表面标志分子进行鉴定。利用pcDNA3.1（-）/Myc-His B质粒构建真核表达载体pcDNA3.1-Paxillin。取第3代BMSCs分为3组：实验组，转染pcDNA3.1-Paxillin；阴性对照组，转染pcDNA3.1（-）/Myc-His B；空白对照组，不转染质粒。分别培养36h后，实验组和阴性对照经G418筛选，取3组BMSCs采用实时荧光定量PCR方法（qRT-PCR）检测各组Paxillin、蛋白多糖（Aggrecan）、Ⅱ型胶原α1（collagen type Ⅱ α1，COL2α1）、性别决定区Y框蛋白9（sex determining region Y box protein 9，SOX9）、角蛋白19（keratin19，KRT19）、配对盒1（paired box 1，PAX1）和叉头蛋白（forkhead box F1，FOXF1）的mRNA表达量；免疫印迹法（Western blot）检测Paxillin、KRT19、PAX1和FOXF1蛋白表达水平。结果：BMSCs原代培养至第3代的细胞形态大体一致，呈长梭形，细胞连接紧密且均匀分布，CD44、CD90呈现高表达（95.6%和96.8%），CD45、CD11则呈现低表达（1.82%和2.16%）。实验组中Paxillin的mRNA和蛋白表达量较阴性对照组和空白对照组升高，差异具有显著性（P＜0.05）；经G418筛选后的BMSCs和髓核细胞形态相似。实验组中COL2α1、SOX9、Aggrecan、KRT19、PAX1、FOXF1的mRNA表达量较阴性对照组和空白对照组显著性升高（P<0.05）；实验组KRT19、PAX1和FOXF1的蛋白表达量显著性高于阴性对照组和空白对照组（P<0.05）。结论：Paxillin可以促进BMSCs向类髓核细胞分化，为椎间盘退变性疾病的细胞移植治疗提供基础。

英文摘要:

  【Abstract】 Objectives: To investigate the effect of Paxillin on the differentiation of bone marrow mesenchymal stem cells(BMSCs) to nucleus pulposus-like cells. Methods: BMSCs from 3-week-old rat bone marrow were cultured by whole bone marrow adherent method until the third generation. Then, BMSCs surface markers were identified by flow cytometry. The eukaryotic expression vector pcDNA3.1-Paxillin was constructed by using pcDNA3.1(-)/Myc-His B vector. The third generation BMSCs were divided into three groups: experimental group, transfected with pcDNA3.1-Paxillin; the negative control group, transfected with pcDNA3.1(-)/Myc-His B; the control group, no transfection. The experimental group and negative control group were screened by using G418 following cultured for 36h. The mRNA expression levels of Paxillin, aggrecan, collagen type Ⅱ α1(COL2α1), sex determining region Y box protein 9(SOX9), keratin19(KRT19), paired box 1(PAX1) and forkhead box F1(FOXF1) in the three BMSCs groups and NP group were detected by quantitative real-time polymerase chain reaction(qRT-PCR). The protein expression levels of Paxillin, KRT19, PAX1 and FOXF1 were measured by Western blot. Results: The 3rd generation of primary culturing BMSCs was fusiform, connected tightly, evenly distributed with high expressions of CD44(95.6%) and CD90(96.8%), and distributed with low expressions of CD11(2.16%) and CD45(1.82%). The mRNA and protein expressions of Paxillin in the experimental group were higher than those in the negative control group and the control group, the difference was significant(P<0.05). The morphologies of the BMSCs selected for 18d by G418 was similar to the nucleus pulposus cells. The mRNA expression levels of COL2α1, SOX9, Aggrecan, KRT19, PAX1 and FOXF1 in the experiemental were significantly up-regulated compared with negative control group and control group(P<0.05). The protein levels of KRT19, PAX1 and FOXF1 were also obviously increased contrasted with negative control group and control group(P<0.05). Conclusions: Paxillin can promote the differentiation of BMSCs to nucleus pulposus-like cells, providing a novel reference for the treatment by using cell transplantation therapy in degenerative disc degeneration disease.