生长分化因子5转染对大鼠骨髓间充质干细胞增殖分化的影响

Effect of Growth Differentiation Factor 5 Gene Transfer on the Proliferation and Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

目的:探讨生长分化因子5(GDF5)基因转染对大鼠骨髓间充质干细胞(BMSCs)增殖分化的影响。方法:采用密度梯度离心法分离培养大鼠BMSCs,pc DNA3.1(+)/h GDF5重组质粒转染大鼠BMSCs后,利用免疫荧光法、聚合酶链式反应(PCR)法检测GDF5蛋白及mRNA的表达,利用3H-TdR检测细胞DNA合成,采用流式细胞仪检测细胞生长周期比例;利用甲苯胺蓝染色、阿辛蓝染色及PCR法检测Ⅱ型胶原的表达。结果:pc DNA3.1(+)/h GDF5重组质粒转染大鼠BMSCs后可以稳定表达,转染pc DNA3.1(+)/h GDF5重组质粒的BMSCs cpm值明显高于未转染BMSCs组(P<0.05);转染pc DNA3.1(+)/h GDF5重组质粒的BMSCs的G1期和G2期细胞的比例下降,S期细胞的比例上升;转染pc DNA3.1(+)/h GDF5重组质粒的BMSCs的Ⅱ型胶原的表达水平增高。结论:pc DNA3.1(+)/h GDF5重组质粒可以成功转染大鼠BMSCs,促进BMSCs增殖并诱导其向软骨细胞分化。

Objective： To explore the effect of gene transfection of growth differentiation factor 5（ GDF5） on the proliferation and differentiation of rat bone marrow mesenchymal stem cells（ BMSCs）.Methods： Rat BMSCs were isolated by density-gradient centrifugation. The protein and mRNA expression of GDF5 were detected by immunofluorescence and polymerase chain reaction（ PCR） methods after pc DNA3. 1（ ＋） / h GDF5 recombinant plasmid transfection into BMSCs. The DNA synthesis count and cell growth cycle were measured by 3H-TdR and flow cytometry. The expression of collagen Ⅱ was detected by toluidine blue staining,alcian blue staining and PCR method. Results： GDF5 gene was stably expressed after pc DNA3. 1（ ＋） / h GDF5 recombinant plasmid transfection into BMSCs. The value of cpm was significantly higher in the transfection group than in the non-transfection group（ P〈 0. 05）. The proportion of G1 and G2 phase cells decreased and the proportion of S cells increased in the BMSCs with pc DNA3. 1（ ＋） / h GDF5 recombinant plasmid transfection. The expression of collagen II increased in the BMSCs with pc DNA3. 1（ ＋） / h GDF5 recombinant plasmid transfection. Conclusion： pc DNA3. 1（ ＋） / h GDF5 recombinant plasmid was successfully transfected into rat BMSCs. The gene could promote proliferation and induce chondrogenesis of the BMSCs.