目的 基于BMPs-Smad/RUNX2信号通路探讨固本增骨方含药血清对大鼠骨髓间充质干细胞增殖及成骨分化的作用机制。方法 制备固本增骨方含药血清；台盼蓝染色观察细胞生长情况；取P3代大鼠BMSCs分为空白血清组、5%、10%、20%、30%浓度含药血清组及传统诱导组，分别对各组大鼠BMSCs进行培养及成骨诱导，向6组加入相应培养液24 h后幵始测量细胞计数，连续测量4天；成骨诱导2周后，进行碱性磷酸酶染色试剂盒说明书进行染色，光镜下观察各组细胞蓝紫色区域的密集程度；ELISA法检测COL-I、ALP、BGP、OPN蛋白的含量；Real-time PCR检测BMPs信号通路相关基因BMP2、BMP4、Smad1、RUNX2表达。结果 各组固本增骨方含药血清组在培养24h后比空白组及传统成骨诱导组OD值比较差异均具有统计学意义（P < 0.05），并且20%含药血清组较5%、10%及30%含药血清组OD值比较差异具有统计学意义（P < 0.05）；碱性磷酸酶染色后镜下观察在浓度为20%时染色区域最密集；5%、10%、20%含药血清组与其他各组BGP、OPN、ALP、COL-I含量比较差异具有统计学意义（P < 0.05），且20%含药血清组与5%、10%含药血清组比较差异具有统计学意义（P < 0.05）；20%含药血清组与其他各组BMP2、BMP4、Smad1、RUNX2 mRNA表达比较差异均具有统计学意义（P < 0.05）。结论 固本增骨方能促进BMSCs的增殖，并且能促进成骨标志蛋白BGP、OPN、ALP、COL-I蛋白含量的增加，且在20%浓度为最佳，这个机制可能是激活了BMP-Smad/RUNX2信号通路实现的。

Objective To observe the effect of serum containing Guben-Zenggu Prescription(GZP) on proliferation and osteogenic differentiation of rat BMSCs based on bmps-smad signaling pathway and explore the possible mechanism.Methods the serum containing GZP was prepared. Trypan blue staining was used to observe cell growth.BMSCs of P3 generation rats were divided into blank serum group, 5%, 10%, 20%, 30% concentration of drug-containing serum group and traditional induction group.BMSCs of each group were cultured and osteoblastic induction was conducted. The cell count was measured for 4 consecutive days after the corresponding culture medium was added to 6 groups for 24 h. Two weeks after osteogenic induction, the instructions of alkaline phosphatase staining kit were used for staining, and the density of blue and purple areas of cells in each group was observed under light microscope. COL-I, ALP, BGP and OPN proteins were detected by ELISA. Real-time PCR was used to detect the expression of BMPs signaling pathway related genes BMP2, BMP4, Smad1 and RUNX2.Results after 24 hours of cultured values in the serum containing GZP recipe of each group were significantly different from those in the blank serum group and the traditional induction group (P < 0.05), and OD values in the 20% serum containing group were significantly different from those in the 5%, 10% and 30% serum containing group (P < 0.05).The concentration of alkaline phosphatase was the highest when the concentration was 20%.The contents of BGP, OPN, ALP and COL-I in the 5%, 10% and 20% drug-containing serum group were significantly different from those in other groups (P < 0.05), and the differences between the 20% drug-containing serum group and the 5% and 10% drug-containing serum group were statistically significant (P < 0.05). The mRNA expressions of BMP2, BMP4, Smad1 and RUNX2 in the 20% drug-containing serum group were significantly different from those in other groups (P < 0.05).Conclusion BMSCs proliferation can be promoted by bone fixation and bone augmentation, and BGP, OPN, ALP and COL-I protein levels of osteogenic marker proteins can be increased, and the best concentration is at 20%. This mechanism may be realized by activating BMP-Smad/RUNX2 signaling pathway.

R274.9