Genistein、Na3Vo4对椎间盘软骨细胞作用效应的研究

The effect of Genistein and Na3Vo4 on the chondrocyte of intervertebral disc

目的:探讨染料木黄酮(Genistein)、正钒酸钠(Sodium orthovanadate,Na3Vo4)对椎间盘软骨终板软骨细胞的作用效应。方法:使用酶消化法培养大鼠椎间盘软骨终板软骨细胞,将不同浓度Genistein(15、35及55μmol/L)、Na3Vo4(10、20及30μmol/L)加入培养的底3代软骨细胞中,培养7d,MTT检测软骨细胞增殖率,RT-PCR检测Ⅱ型、Ⅸ型胶原和Aggrecan mRNA的表达,定量RT-PCR检测IGFR、PTPn1、PTPn1、IGFR mRNA的表达。结果:干预7d后,15μmol/L Genistein组各项指标与模型组比较变化不明显(P>0.05);35μmol/L Genistein组与模型组比较,软骨细胞增殖率、Ⅱ型胶原及aggrecan mRNA表达显著降低(P<0.01),IGFR mRNA表达较模型组有所降低,但无显著性差异(P>0.05);55μmol/L Genistein组与模型组比较,软骨细胞增殖率、aggrecan基因表达显著下降(P<0.01),Ⅸ型胶原、IGFR mRNA表达较模型组有所降低(P<0.05);各浓度Genistein组PTPn1mRNA表达无显著变化。与模型组比较,10μmol/LNa3Vo4组各项检测指标变化不明显,30μmol/L Na3Vo4组Ⅱ、Ⅸ型胶原mRNA表达明显降低(P<0.01),20及30μmol/L Na3Vo4组软骨细胞增殖率明显下降(P<0.01),Aggrecan以及PTPn1mRNA表达显著降低(P<0.05),IGFRmRNA表达无显著变化(P>0.05)。结论:适当剂量Genistein、Na3Vo4在椎间盘软骨终板软骨细胞增殖、胶原表达以及aggrecan表达等方面起重要作用,Genistein可特异性抑制PTKs中IGFR基因表达,适当剂量Na3Vo4可抑制PTPs中PTPn1的基因表达,降低软骨细胞生物学功能。

Objective:To study the effects of Genistein and Sodium orthovanadate (Na3Vo4) on the chondrocyte in the endplate of intervertebral disc. Methods:The enzyme digestion was used to culture the chondrocyte of the endplate of intervertebral disc. Different kinds of density of Genistein (15、35 or 55μmol/L) and Na3Vo4(10、20 or 30μmol/L)were added in the passage III chondrocyte, which was cultured for 7 days. MTT test was used to examine the growth rate of cartilage cells , RT-PCR test was used to detect the expression of Aggrecan mRNA, type ⅡCollagen and type Ⅸ Collagen , and real time RT-PCR test was used to determine the expression of IGFR , PTPn1 , PTPn1 and IGFR mRNA express. Results:After 7 days, there was no significantly different between the group which was treated with 15μmol/L Genistein and the model group in every test(P>0.05). The growth rate of cartilage cells and the expression of Aggrecan, type Ⅱ Collagen and aggrecan mRNA in the group treated with 35μmol/L Genistein were significantly decreased compared with the model group(P<0.01), while the expression of IGFR mRNA decreased , but not significantly(P>0.05). Compared with model group, the growth rate of cartilage cells and the expression of Aggrecan mRNA decreased significantly(P<0.01), the expression of type Ⅸ Collagen and IGFR mRNA also decreased(P<0.05)in the group treated with 55μmol/L Genistein. There was no significant difference in those three kinds of density group of Genistein. Compared with the model group, all the detection targets did not change obviously in the group treated with 10μmol/L Na3Vo4, while the expression of type Ⅱ Collagen and type Ⅸ Collagen mRNA decreased significantly(P<0.01) in the goup treated with 30μmol/L Na3Vo4 , and in the both of the 20 and 30μmol/L Na3Vo4 groups, the growth rate of cartilage cells decreased significantly significantly(P<0.01), the expression of Aggrecan and PTPn1 mRNA decreased significantly(P<0.05), and the expression of IGFR mRNA did not change remarkably(P>0.05). Conclusion:These results suggested that suitable doses of Genistein and Na3Vo4 play important roles in the growth rate of cartilage cells and the expression of Aggrecan and collagen mRNA. Genistein inhibits the expression of IGFR gene in PTKs specially , while proper dose of Na3Vo4 inhibits the expression of PTPn1 gene in PTPs and depresses the biological function of chondrocyte.