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目的观察淫羊藿苷促SD大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)骨向分化的作用特点。方法 (1)采用机械分离及全骨髓贴壁法分离培养SD大鼠BMSCs,氨基安替吡啉测酚法检测不同浓度的淫羊藿苷在第3、6、9、12、15、18、21天对细胞上清液中碱性磷酸酶(alkaline phosphatase,ALP)活性的影响,采用茜素红染色法检测各浓度组BMSCs矿化情况,观察不同浓度的淫羊藿苷促BMSCs骨向分化的作用。(2)将BMSCs细胞分为空白对照组、成骨诱导组及淫羊藿苷组(0.5μg/m L),在培养第7、14、21天检测各组细胞上清ALP活性,并于第14及21天分别检测各组细胞ALP阳性染色率与矿化结节数,同时采用实时荧光定量PCR(Real-time PCR)法检测3个不同时间点各组细胞中核心结合蛋白因子(Runt-related transcripition factor-2,Runx2)及骨钙素(Osteocalcin)mRNA的表达水平。结果 (1)0.05~5.0μg/m L淫羊藿苷均可明显提高细胞上清液中ALP活性,其中0.2~2.0μg/m L浓度组细胞结节数亦明显增加(P<0.01)。(2)淫羊藿苷促BMSCs骨向分化时,Runx2 mRNA表达水平及ALP活性均先升后降;Osteocalcin mRNA表达水平则持续上升(P<0.01);与成骨诱导组比较,作用14天后,淫羊藿苷组ALP活性及ALP阳性染色率均明显提高(P<0.05,P<0.01)。结论淫羊藿苷通过上调Runx2基因的表达,促使BMSCs向成骨细胞分化,并通过增加细胞ALP分泌及Osteocalcin基因的表达,促进细胞矿化的发生,从而促使新生成骨细胞的成熟。
Objective To observe the effect of icariin on bone differentiation of bone marrow mesenchymal stem cells (BMSCs) of SD rats. Methods (1) SD rat BMSCs were isolated and cultured by mechanical separation and whole bone marrow adherent method. Aminopigin was used to detect phenylephrine in different concentrations of icarrin at the 3rd, 6th, 9th, 12th, 15th, 21 days on the alkaline phosphatase (ALP) activity in the supernatant of the cells. Alizarin red staining was used to detect the mineralization of BMSCs in different concentration groups. The effects of different concentrations of icariin on the differentiation of BMSCs into bone Role. (2) BMSCs were divided into blank control group, osteogenic induction group and icariin group (0.5μg / mL), ALP activity in each group was detected on the 7th, 14th and 21st day after culture, The numbers of ALP positive staining and mineralized nodules in each group were detected on the 14th and 21st days, respectively. At the same time, real-time PCR was used to detect the expression of core-associated protein (Runt) -related transcripition factor-2, Runx2) and osteocalcin (Osteocalcin) mRNA. Results (1) Icariin 0.05 ~ 5.0μg / m L could obviously improve the ALP activity in the supernatant of the cells, and the number of the cells in the concentration of 0.2 ~ 2.0μg / m L also increased obviously (P <0.01). (2) Icariin promoted the expression of Runx2 mRNA and ALP activity in osteogenic differentiation of BMSCs; Osteocalcin mRNA expression level continued to increase (P <0.01); compared with osteogenic induction group, , Icariin ALP activity and ALP positive staining were significantly increased (P <0.05, P <0.01). Conclusion Icariin can promote the differentiation of BMSCs into osteoblasts by up-regulating the expression of Runx2 gene and promote the mineralization of BMSCs by increasing the secretion of ALP and the expression of Osteocalcin gene, thereby promoting the maturation of newborn osteoblasts.