左归丸调控miR34a对BMSCs成骨分化能力的影响

Effect of Zuogui Pills on Differentiation Ability of miR34a in BMSCs

目的：探究左归丸调控miR34a对BMSCs成骨分化能力的影响。方法：培养BMSCs,分别用大鼠高、低剂量左归丸含药血清和对照血清干预BMSCs成骨分化过程。采用CCK8检测左归丸对BMSCs的增殖、毒性作用,AKP活性检测BMSCs成骨分化活性,ALP染色检测细胞成骨分化能力,反转录-实时荧光定量技术（RT-q PCR）检测miR34a、Tgif2、Runx2、PPARγmRNA表达;Western-blotting检测Tgif2、RUNX2蛋白表达。结果：CCK8检测发现：随着时间的增加,从第1天到第7天,BMSCs增殖呈上升趋势,各组间增加程度无统计学差异;7~14 d,BMSCs增殖呈下降趋势。AKP活性检测示,干预BMSCs 3 d后,左高组AKP活性显著高于其余两组（P〈0.05）;7 d时,左低、左高组AKP活性均显著高于对照组（P〈0.001）。干预3 d时,ALP染色结果示,左高组ALP染色阳性率高于其余两组;干预7 d时,左高、左低组ALP染色阳性率显著高于对照组。干预3 d时,左高、左低组miR34a mRNA表达较对照组上调,Tgif2、PPARγ2 mRNA表达较对照组下调;干预7 d后,左高组miR34a mRNA表达较对照组显著上调（P〈0.05）;左低、左高组Tgif2 mRNA表达均较对照组显著下调（P〈0.05）;左高组PPARγ2 mRNA表达较对照组显著下调（P〈0.01）;干预3 d后,左高组Tgif2蛋白水平较对照组显著降低（P〈0.01）;而左低组Runx2蛋白水平较对照组显著升高（P〈0.01）;干预7 d后,左低、左高组Tgif2蛋白水平较对照组显著降低（P〈0.001）,而左低、左高组Runx2蛋白水平较对照组显著升高（P〈0.05）。结论：左归丸具有促进BMSCs增殖及成骨分化的作用,其机制可能是通过上调miR34a,抑制其靶基因Tgif2表达,同时下调成脂相关因子PPRAγ和上调成骨核转录因子Runx2。

Objective： To investigate the effect of Zuogui Pills on the differentiation of BMSCs by miR34 a. Methods： BMSCs were cultured and treated with rat blank serum and high and low dose Zuogui pills for osteoblast differentiation. The osteogenic differentiation of BMSCs was detected by CCK8. The osteogenic differentiation of BMSCs was detected by AKP activity. The osteogenic differentiation ability of BMSCs was detected by ALP staining. RT-q PCR was used to detect miR34 a,Tgif2,Runx2 and PPARγ mRNA. The expressions of Tgif2 and Runx2 were detected by Western-blotting. Results： CCK8 showed that the proliferation of BMSCs increased from day 1 to day 7 and there was no significant difference among the three groups. The proliferation of BMSCs between the 7 th and 14 th days were declined. It is showed that after 3 days of intervention in BMSCs,AKP activity in the Zuogui Pills group was significantly higher than that of the other two groups（ P〈0. 05）. At the 7 th day,the AKP activity in the Zuogui Pills low and high dose groups were significantly higher than that in the blank group（ P〈0. 001）. The positive rate of ALP staining in the Zuogui Pills group was higher than that in the other two groups on the 3 rd day after intervention. On the 7 th day,the positive rate of ALP staining in the Zuogui Pills high and low dose groups were significantly higher than that in the blank group. On the 3 rd day,the expressions of miR34 a mRNA in the Zuogui Pills high and low dose groups were higher than that in the blank group. The expressions of Tgif2 and PPARγ2 mRNA were lower than that of the blank group. On the 7 th day,the expression of miR34 a mRNA in the Zuogui Pills high group was significantly higher than that in the blank group（ P〈0. 05）. The expressions of Tgif2 mRNA in the Zuogui Pills high group and low dose groups were lower than that of the blank group（ P〈0. 05）. The expression of PPARγ mRNA in the Zuogui Pills high group was lower than that in the blank group（ P〈0. 05）. On the 3 rd day,the level of Tgif2 protein in the Zuogui Pills high group was significantly lower than that in the blank group（ P〈0. 01）. However,the level of Runx2 protein in the Zuogui Pills low group was higher than that in the blank group（ P〈0. 01）. On the 7 th day,the level of Tgif2 protein in the Zuogui Pills high group was significantly lower than that in the blank group（ P〈0. 001）. The level of Runx2 protein in Zuogui Pills low and high dose groups were significantly higher than that in blank group（ P〈0. 05）. Conclusion：Zuogui Pills can promote the proliferation and osteogenic differentiation of BMSCs. The mechanism may be that the expression of miR34 a is inhibited by inhibiting the expression of Tgif2,and the expression of PPRAγ and the osteogenic transcription factor Runx2 are down-regulated.