去分化过程对大鼠骨髓间充质干细胞腱系分化的影响

Effect of dedifferentiation on tendon differentiation of rat bone marrow mesenchymal stem cells

目的探讨大鼠骨髓间充质干细胞在去分化过程中成肌腱细胞分化的潜能。方法采用全骨髓贴壁法分离、培养SD大鼠骨髓间充质干细胞。用含生长分化因子(GDF-5)的培养液诱导骨髓间充质干细胞去分化后,再用含GDF-5的培养基继续诱导其成肌腱分化。3周后,通过反转录-聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)分别检测肌腱相关标记基因如Ⅰ型胶原(ColⅠ)、Ⅲ型胶原(ColⅢ)、肌腱蛋白(TNC)和碱性螺旋环-螺旋转录因子(Scx)和肌腱相关标记蛋白如ColⅠ、TNC和腱调蛋白(Tenomodulin)的表达。结果以均数±标准差表示。多组间数据的比较采用单因素方差分析,两组间比较采用t检验。P<0.05表示差异有统计学意义。结果RT-PCR结果显示:实验组Scx、ColⅠ、ColⅢ、TNC基因表达量为0.635±0.557、0.590±0.020、0.447±0.063、0.613±0.061,显著高于诱导分化组(0.416±0.054、0.406±0.173、0.294±0.013、0.373±0.042,t=17.857,P<0.05;t=12.948,P<0.05;t=7.747,P<0.05;t=-5.212,P<0.05),对照组(0.197±0.019、0.201±0.014、0.216±0.037、0.190±0.014,t=31.077,P<0.05;t=14.419,P<0.05;t=15.054,P<0.05;t=8.013,P<0.05)。Western blot检测结果显示:实验组ColⅠ、TNC和Tenomodulin的蛋白表达分别为1.705±0.159、1.587±0.018、1.616±0.019,显著高于诱导分化组(1.160±0.018、1.164±0.016、1.092±0.022,t=4.825,P<0.05;t=17.395,P<0.05;t=3.961,P<0.05),对照组(0.643±0.047、0.441±0.02l、0.673±0.015,t=13.299,P<0.05;t=37.618,P<0.05;t=9.349,P<0.05)。结论GDF-5能诱导骨髓间充质干细胞成肌腱细胞分化,且去分化过程能提高骨髓间充质干细胞腱系分化的能力。

Objective To investigate the differentiation potential of rat bone marrow mesenchymal stem cells in dedifferentiation process.Methods Bone marrow mesenchymal stem cells(BMSCs)of Sprague Dawley rats were isolated and cultured using a whole bone marrow adherent method.BMSCs were induced to dedifferentiate with culture medium containing growth differentiation factor(GDF-5),and then its tendon differentiation was further induced with culture medium containing GDF-5.After 3 weeks,the expression of typeⅠ(ColⅠ),Collagen typeⅢ(ColⅢ),tenascin-C(TNC),basic Helix-Loop-Helix(scleraxis,SCX)and Tenomodulinwas detected by reverse transcriptase-polymerase chain reaction(RT-PCR)and Western blotting.Results are given as mean±standard deviation.Statistical analysis was performed using Student’s t-test.Multiple comparisons were done using one way ANOVA test.Statistical signicance was set at P<0.05.Results The gene expression of Scx,ColⅠ,ColⅢand TNC in experimental group(0.635±0.557,0.590±0.020,0.447±0.063,0.613±0.061 respectively)was significantly higher than that in induced differentiation group(0.416±0.054,0.406±0.173,0.294±0.013,0.373±0.042,t=17.857,P<0.01;t=12.948,P<0.01;t=7.747,P<0.01;t=-5.212,P<0.05)and control group(0.197±0.019,0.201±0.014,0.216±0.037,0.190±0.014,t=31.077,P<0.01;t=14.419,P<0.01;t=15.054,P<0.01;t=8.013,P<0.05).Western blotting indicated that the expression of ColⅠ,TNC and Tenomodulin in experimental group(1.705±0.159,1.587±0.018,1.616±0.019 respectively)was higher than that in positive control group(1.160±0.018,1.164±0.016,1.092±0.022,t=4.825,P<0.05;t=17.395,P<0.05;t=3.961,P<0.05 respectively)and negative group(0.643±0.047,0.441±0.02l,0.673±0.015 respectively,t=13.299,P<0.05;t=37.618,P<0.05;t=9.349,P<0.05 respectively).Conclusion GDF-5 can induce the differentiation of bone marrow mesenchymal stem cells into tendon cells,and the dedifferentiation process can improve the ability of bone marrow mesenchymal stem cells to differentiate into tendon cells.