左归丸和右归丸对大鼠脂肪干细胞增殖及成骨分化影响的比较

Comparison of the effect of Zuogui pill and Yougui pill on the proliferation and osteogenic differentiation of rat adipose-derived stem cells

目的比较左归丸、右归丸对大鼠脂肪干细胞(ASCs)增殖和成骨性分化的影响。方法成年大鼠15只,随机分为3组,分别以左归丸(ZGW组)、右归丸(YGW组)和生理盐水(CON组)连续灌胃后制备含药血清。胶原酶消化法分离大鼠脂肪干细胞,成骨诱导21 d后,茜素红染色对细胞进行鉴定。分别采用CCK-8法、PNPP法测定加药诱导后ASCs的增殖能力及碱性磷酸酶表达差异;比色法检测细胞外钙离子浓度。Western Blot法测定加药诱导7 d后的Runx2蛋白表达量。结果 ASCs细胞呈长梭形生长,增殖旺盛;茜素红染色显示为阳性。CCK-8法检测结果显示,加药24 h及48 h时,YGW组OD值显著高于ZGW组及CON组(P<0.05);加药72 h时,ZGW组、YGW组OD值均显著高于CON组(P<0.05),而两个加药组间并无显著性差异(P>0.05)。PNPP检测结果显示,ZGW组、YGW组OD值均显著高于CON组(P<0.05);其中YGW组OD值显著高于ZGW组(P<0.05)。YGW组细胞外钙离子浓度显著升高(P<0.05)。Western Blot结果显示,YGW组Runx2蛋白表达显著上调(P<0.05)。结论与滋肾阴法相比,温肾阳法在促进ASCs增殖及成骨分化方面均表现出明显优势。

Objective To compare the effect of Zuogui pill and Yougui pill on the proliferation and osteogenic differentiation of rat adipose-derived stem ceils （ASCs）. Methods Fifteen adult rats were randomly divided into 3 groups： Zuogui pill group （ZGW group）, Yougui pill group （YGW group）, and normal saline group （CON group）. Drug containing-serum of rats was acquired after continuous lavage with decoction of Zuogui pill and Yougui pill, and physiological saline, respectively. Rat ASCs were isolated using collagenase digestion method. After 21-day ostegenie induction, the cells were identified using Alizarin red staining. The proliferation of ASCs and the expression of alkaline phosphatase after drug intervention were detected using CCK-8 and PNPP methods. Extracellular Ca2＋ concentration was detected using colorimetric method. The expression of Runx2 protein after 7-day drug induction was detected using Western blotting. Results The ASCs were mainly spindle-shaped and proliferated quickly. The result of Alizarin red staining was positive. The results of CCK-8 method showed that the OD value was significantly higher in YGW group than that in both ZGW group and CON group after 24-hour and 48-hour culture （ P 〈 0. 05 ）. And after 72-hour culture, the OD value in both YGW group and ZGW group was higher than that in CON group （ P 〈 0.05 ） , while no significant difference between the two drug-intervened groups was observed （P 〉0.05）. The result of PNPP detection showed that the OD value in both YGW group and ZGW group was significantly higher than tilat in CON group （P 〈 0.05 ）. Compared with that in ZGW group, the OD value in YGW group was higher （P 〈 0.05 ）. The extracellular Ca2 ＋ concentration was higher in YGW group （ P 〈 0.05）. The result of Western blotting showed that the expression of Runx2 protein increased significantly in YGW group （ P 〈 0.05 ）. Conclusion Compared with the method of nourishing the kidney- yin, the method of warming the kidney-yang has obvious advantage in promoting both the proliferation and the osteogenic differentiation of ASCs.