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

摘要

Objective To compare the effect of Zuogui pill and Yougui pill on the proliferation and osteogenic differentiation of rat adipose-derived stem cells ( 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 ostegenic 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 that 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.%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。