六味地黄丸、金匮肾气丸及健骨二仙丸含药血清对BMSCs成脂、成骨细胞分化相关基因的影响

Regulation Effects of Liuwei Dihuang Pill， Jingui Shenqi Pill， Jiangu Erxian Pill Containing Serums on Adipogenic and Osteogenic Differentiation related Genes Expressions in the Differentiation Process of Preadipocytes to Osteoblasts

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DOI：

中文关键词: 六味地黄丸金匮肾气丸健骨二仙丸骨髓间充质干细胞前脂肪细胞成骨分化

英文关键词:Liuwei Dihuang Pill Jingui Shenqi Pill Jiangu Erxian Pill mesenchymal stem cell preadipocyte osteogenic differentiation

基金项目:国家自然科学基金资助项目（No.30772817）

作者	单位	E-mail
程志安	广州中医药大学第二临床医学院骨科（广州510120）	chengzhian@126.com
韩凌,危建安,孙静,段晓栋

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中文摘要:

目的研究补肾方药对大鼠骨髓间充质干细胞（BMSCs）来源的前脂肪细胞向成骨分化的影响。方法采用全骨髓贴壁法培养BMSCs，用经典化学方法诱导BMSCs为前脂肪细胞，用六味地黄丸、金匮肾气丸和健骨二仙丸含药血清（含量浓度均为10%，分别代表补肾阴、补肾阳和补肾填精方药），干预前脂肪细胞成骨分化过程；采用反转录－实时荧光定量PCR技术（RT real time qPCR），分别于第6、12和18天，检测成骨分化相关基因RUNX2、ALP、BGP、BMP2、BMP4、SPP1和IGF1 mRNA，及成脂分化相关基因LPL、FABP4和PPARγ mRNA表达水平。结果对于成骨分化相关基因，与对照组比较，第6天各组基因表达无显著变化（2.0>Ratio>0.5）；第12天，金匮肾气丸组IGF1和RUNX2 mRNA表达升高较为显著，相对表达量（Ratio）分别为2.97和1.81；第18天，各组IGF1 mRNA表达均显著上升，六味地黄丸组、金匮肾气丸组和健骨二仙丸组Ratio值分别为3.74、12.60和8.35；各组SPP1 mRNA表达也显著升高，Ratio值分别为2.94、3.18和2.62。对于成脂分化相关基因，第6天，六味地黄丸组和金匮肾气丸组FABP4 mRNA表达显著下降（Ratio分别为0.47和0.40），其他基因表达均下调，但不显著；第12天和第18天，成脂分化基因表达变化均无显著变化（2.0>Ratio>0.5）。结论成骨分化相关基因在补肾方药作用较晚时间出现表达上调的变化，而成脂分化相关基因在补肾方药作用较早时间即出现表达下调的改变，并且提示补肾阳法促进成骨分化、抑制成脂分化作用更为显著。

英文摘要:

ObjectiveTo study the effects of Chinese medical recipes for invigorating Shen on rat bone marrow mesenchymal stem cells （BMSCs） derived preadipocytes′ differentiation to osteoblasts. MethodsThe BMSCs were cultured using whole bone marrow adherence wall method. The BMSCs were induced to preadipocytes by classic chemical method. The osteogenic differentiation process of preadipocytes was intervened by Liuwei Dihuang Pill （LDP）， Jingui Shenqi Pill （JSP）， or Jiangu Erxian Pill （JEP） containing serums （with the concentRation of 10%， on behalf of tonifying Shen yin， tonifying Shen yang， and tonifying Shen essence）. Reverse transcription real time fluorescent quantitative PCR （RT real time qPCR） was used to detect RUNX2， ALP， BGP， BMP2， BMP4， SPP1， and IGF1 mRNA expressions of osteogenic differentiation related genes， mRNA expressions of LPL， FABP4， and PPARγ of adipogenic differentiation related genes on the 6th， the 12th， and the 18th day. ResultsAs for the osteogenic differentiation related gene， when compared with the control group， there was no statistical difference in the gene expression level in the experimental groups on the 6th day （2.0>Ratio>0.5）. On the 12th day， the mRNA expressions of IGF1 and Runx2 increased more significantly in the JSP group， with their relative quantification （Ratio） being 2.97 and 1.81 respectively. On the 18th day the IGF1 mRNA expression significantly increased， being the Ratio value of 3.74， 12.60， and 8.35， respectively， in the LDP group， the JSP group， and the JEP group. The SPP1 mRNA expression also significantly increased， with the Ratio value of 2.94， 3.18， and 2.62， respectively， in the LDP group， the JSP group， and the JEP group. As for adipogenic differentiation related genes， on the 6th day， when compared with the control group， FABP4 mRNA expression significantly decreased in the LDP group and the JSP group （with the Ratio value of 0.47 and 0.40 respectively）. The expression levels of other genes were all down regulated， but not significantly. On the 12th day and 18th day， there was no statistical change in the adipogenic differentiation related genes expressions （2.0>Ratio>0.5）. ConclusionsUp regulation of osteogenic differentiation related genes expression occurred in later time， while down regulation of adipogenic differentiation related genes expression occurred in earlier time after treatment by Chinese medical recipes for invigorating Shen. In general， above data indicated that tonifying Shen yang was more effective in promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs.

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