龟鹿二仙胶含药血清介导大鼠骨髓间充质干细胞的成骨分化.

Guilu Erxian Gum medicated serum mediated osteogenic differentiation of rat bone marrow mesenchymal stem cells.

BACKGROUND: On the basis of studying the proliferation of bone marrow mesenchymal stem cells, revealing their differentiation mechanism is one of the hot topics in the study of prevention and treatment of osteoporosis. OBJECTIVE: To explore the relationship between the molecular mechanism of Guilu Erxian Gum medicated serum that induces osteogenic differentiation of bone marrow mesenchymal stem cells to prevent osteoporosis and the activation of extracellular signal-regulated kinase 1/2/E26 transcription factor 1 signaling pathway factors. METHODS: Passage 3 commercial SD rat bone marrow mesenchymal stem cells were purchased, and divided into fetal bovine serum group, blank serum group, Guilu Erxian Gum medicated serum group, classical induction group, pathway inhibitor group, and Guilu Erxian Gum+pathway inhibitor group. Osteogenic differentiation was conducted according to group conditions, and the fluid was altered every 3 days. At 7 days after intervention, alkaline phosphatase activity detection kit was used to determine the osteogenic differentiation level of bone marrow mesenchymal stem cells in the first four groups. At 14 days after intervention, alizarin red staining was used to verify the level of bone formation and mineralization of bone marrow mesenchymal stem cells in the first four groups. Real-time fluorescent quantitative PCR was used to detect the gene expression levels of alkaline phosphatase, core binding protein 2, peroxisome proliferator activated receptor γ, and E26 transcription factor 1 in bone marrow mesenchymal stem cells of the first four groups. Western blot assay was utilized to determine protein expression of type I collagen, alkaline phosphatase, core binding protein 2, peroxisome proliferator activated receptor γ, E26 transcription factor 1, extracellular signal-regulated kinase 1/2 and phosphorylated extracellular signal-regulated kinase 1/2 in bone marrow mesenchymal stem cells of the six groups. RESULTS AND CONCLUSION: (1) Compared with the blank serum group, the mRNA expression levels of alkaline phosphatase, core binding protein 2, and E26 transcription factor 1 were significantly higher in the Guilu Erxian Gum medicated serum group, while the mRNA expression levels of the adipogenic differentiation specific index peroxisome proliferator activated receptor γ were inhibited. (2) Compared with the fetal calf serum group and the blank serum group, the expression levels of osteogenic differentiation-related proteins, such as type I collagen, alkaline phosphatase, core binding protein 2, E26 transcription factor 1, and phosphorylated extracellular signal-regulated kinase 1/2 were significantly increased; and the expression level of peroxisome proliferator activated receptor γ protein was also suppressed. The use of MAPK signaling pathway inhibitor PD98059 could down-regulate the expression levels of osteogenic differentiation-related proteins and up-regulate the expression levels of peroxisome proliferator activated receptor γ protein. (3) Compared with the fetal bovine serum group and the blank serum group, the alkaline phosphatase activity (P < 0.05) and calcification capacity (P < 0.05) of bone marrow mesenchymal stem cells were significantly improved in the Guilu Erxian Gum medicated serum group. (4) The results show that Guilu Erxian Gum medicated serum may mediate the osteogenic differentiation of bone marrow mesenchymal stem cells through extracellular signal-regulated kinase 1/2/E26 transcription factor 1 signaling pathway factors, which may be one of its important mechanisms for preventing and treating osteoporosis. [ABSTRACT FROM AUTHOR]

背景：在研究骨髓间充质干细胞增殖的基础上揭示其分化机制是防治骨质疏松症研究的热门课题之一。 目的：探讨龟鹿二仙胶含药血清诱导骨髓间充质干细胞成骨分化以防治骨质疏松症的分子机制与激活细胞外信号调节激酶 1/2/E26 转录因 子 1 信号通路因子之间的相关性。 方法：购买商品化第 3 代 SD 大鼠骨髓间充质干细胞，根据加入不同成分培养基分为胎牛血清组、空白血清组、龟鹿二仙胶含药血清组、经 典诱导组以及通路抑制剂组、龟鹿二仙胶含药血清+通路抑制剂组，根据分组条件进行成骨诱导分化，每 3 d 换液1次，干预7 d时采用碱性 磷酸酶活性检测试剂盒测定前4组骨髓间充质干细胞的成骨分化水平；干预 14 d 时采用茜素红染色观察前4组骨髓间充质干细胞的成骨矿化 水平，实时荧光定量 PCR 检测前 4 组骨髓间充质干细胞中碱性磷酸酶、Runt 家族相关转录因子 2、过氧化物酶体增殖物激活受体γ和E26转录 因子 1 的基因表达水平，Western blot 法检测6组骨髓间充质干细胞中Ⅰ型胶原、碱性磷酸酶、Runt 家族相关转录因子 2、过氧化物酶体增殖 物激活受体γ、E26 转录因子1、细胞外信号调节激酶 1/2 和磷酸化细胞外信号调节激酶 1/2 蛋白表达。 结果与结论：①与空白血清组比较，龟鹿二仙胶含药血清组碱性磷酸酶、Runt家族相关转录因子 2、E26 转录因子 1 mRNA 的表达水平均明 显升高，成脂分化特异性指标过氧化物酶体增殖物激活受体γ mRNA 的表达水平则受到抑制；②与胎牛血清组和空白血清组比较，Ⅰ型胶 原、碱性磷酸酶、Runt 家族相关转录因子 2、E26 转录因子1、磷酸化细胞外信号调节激酶 1/2 等成骨分化相关蛋白表达水平明显升高，过氧 化物酶体增殖物激活受体γ蛋白表达水平同样受到抑制，使用 MAPK 信号通路抑制剂 PD98059 可下调上述成骨分化相关蛋白表达水平，上调 过氧化物酶体增殖物激活受体γ蛋白表达水平；③与胎牛血清组和空白血清组比较，龟鹿二仙胶含药血清组骨髓间充质干细胞的碱性磷酸 酶活性 (P < 0.05) 和钙化能力 (P < 0.05) 明显提高；④结果表明，龟鹿二仙胶含药血清可能通过调节细胞外信号调节激酶 1/2/E26 转录因子1信 号通路因子介导骨髓间充质干细胞成骨分化，这可能是其防治骨质疏松症的重要机制之一。 [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu is the property of Chinese Journal of Tissue Engineering Research and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)