重组腺相关病毒介导的BMP13和SOX9共转染促进骨髓间充质干细胞向类髓核细胞分化

Recombinant adeno-associated virus-mediated BMP13 and SOX9 co-transfection promotes the differentiation of bone marrow mesenchymal stem cells into nucleus pulposus-like cells

目的观察骨形态发生蛋白13(bone morphogenetic protein 13,BMP13)和性别决定区Y框蛋白9(sex determining region Y box protein 9,SOX9)基因共转染对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向类髓核细胞分化的影响。方法通过腺相关病毒(adeno-associated virus,AAV)将目的基因导入BMSCs中;细胞分为4组:对照组、AAV-SOX9组、AAV-BMP13组和共转染组(AAV-SOX9+AAV-BMP13);应用实时荧光定量PCR技术检测mRNA表达水平;Western blot法检测蛋白表达水平;糖胺聚糖检测试剂盒检测糖胺聚糖的合成水平;MTT法检测细胞增殖。结果共转染组BMP13和SOX9的表达水平明显高于AAV-SOX9组、AAV-BMP13组和对照组(P<0.05);共转染组的糖胺聚糖合成水平、角蛋白19(keratin 19,KRT19)及蛋白聚糖(Aggrecan)和Ⅱ型胶原蛋白(typeⅡcollagen,Col2a1)的表达水平明显高于AAV-SOX9组、AAV-BMP13组和对照组(P<0.05);另外,AAV-SOX9和AAV-BMP13及共转染组的细胞增殖活性明显高于对照组(P<0.05),但AAV-SOX9组和AAV-BMP13组与共转染组的增殖活性相比差异无显著性(P>0.05)。结论 BMP13和SOX9双基因转染的BMSCs向类髓核细胞分化的能力明显高于单基因转染组,为椎间盘突出疾病的细胞治疗提供理论依据和参考。

Purpose To investigate the effect of bone morphogenetic protein 13 （BMP13） and sex determining region Y box protein 9 （SOX9） co-transfection on the differentiation of bone marrow mesenchymal stem cells （BMSCs） into nucleus pulposus-like cells. Methods The target genes were delivered into human BMSCs through recombinant adeno-associated virus （AAV）. Cells were divided into four groups： control group, AAV-BMP13 group, AAV-SOX9 group and co-transfection group （AAV-SOX9 ＋ AAV-BMP13）. The mRNA expression was detected by quantitative polymerase chain reaction analysis. The protein expression was detected by Western blot analysis. The synthesizing level of glycosaminoglycan was measured by a glycosaminoglycan detection kit. Cell proliferation was detected by MTT assay. Results The expression levels of BMP13 and SOX19 were significantly higher in co-transfection group than those in single transfection group and control group （P 〈 0. 05 ）. The synthesizing level of glycosaminoglycan and the expression levels of keratin 19 （KRT19）, aggrecan and type lI collagen （Col2al） were all markedly higher than those in single transfection group and control group （P 〈 0. 05 ）. In addition, cell proliferation activity in AAV-BMP13 group, AAV-SOX9 group and co-transfection group was significantly higher than that in control group （ P 〈 0. 05 ） , whereas no significant difference was observed between co-transfection group and AAV-BMP13 group or AAV-SOX9 group （P 〉 0. 05）. Conclusion These findings suggest that BMP13 and SOX9 double gene- transfected BMSCs have better potential than single gene-transfected BMSCs with respect to differentiation into nucleus pulposus-like cells which provides basic theory and reference for cell therapy of prolapse of intervertebral disc disease.