中国骨质疏松杂志

健骨颗粒含药血清调控miR-141对小鼠骨髓间充质干细胞成骨分化的影响

Effect of Jiangu granule drug-containing serum on the osteoblastic differentiation of mouse bone marrow mesenchymal stem cells regulated by miR-141

DOI：10.3969/j.issn.1006.7108.2020.04.006

中文关键词: 中医中药绝经后骨质疏松症健骨颗粒 miR-141 Dlx5/Msx2/Runx2 去卵巢小鼠

英文关键词:Chinese Traditional Medicine postmenopausal osteoporosis Jiangu granules miR-141 Dlx5/Msx2/Runx2 ovariectomized mice

基金项目:国家自然科学基金项目 (81574003)

作者	单位
张楚天1 张文明1,2 林燕萍1* 魏振朴3 杨娟1 张志恒1 孙攀1 王志强3	1.福建中医药大学中西医结合研究院，福建福州350122 2.河南省洛阳正骨医院郑州院区，河南郑州，450016 3.福建中医药大学针灸学院，福建福州350122

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中文摘要:

目的观察健骨颗粒含药血清对去卵巢模型小鼠BMSCs成骨分化的影响，并研究其调控miR-141影响BMSCs分化的可能机制。方法流式细胞计数法鉴定BMSCs；含药血清和空白血清分别干预体外培养的BMSCs；显微镜下观察细胞形态结构，碱性磷酸酶染色、定量检测，茜素红染色；RT-PCR检测miR-141及DLx5、Msx2、Runx2的基因表达情况；Western blot检测DLx5、Msx2及Runx2的蛋白表达情况。结果流式细胞计数法鉴定提取细胞符合实验要求；镜下观察发现两组细胞有差异，含药血清组细胞群落间结晶更为密集；ALP染色及定量、茜素红染色结果含药血清组均优于空白血清组；RT-PCR检测Dlx5、Msx2、Runx2及miR-141的基因表达结果：含药血清组miR-141和Msx2基因表达较空白血清组显著降低（P<0.01），而Dlx5、Runx2的基因表达显著升高（P<0.01）；Western blot检测DLx5、Msx2、Runx2的蛋白表达结果：含药血清组较空白血清组相比，Msx2的蛋白表达略微下降（P<0.05），而Dlx5、Runx2的蛋白表达相对升高（P<0.01）。结论健骨颗粒可以调低miR-141的表达，进而调高Dlx5的表达，削弱Dlx5的同源异形基因 Msx2对 Runx2抑制作用，通过miR-141调控Dlx5/Msx2/Runx2信号通路促进BMSCs成骨分化达到预防和治疗绝经后骨质疏松的目的。

英文摘要:

Objective To investigate whether the drug-containing serum of Jiangu Granule could affect the osteogenic differentiation of mouse bone marrow mesenchymal stem cells (BMSCs) by regulating miR-141. Methods BMSCs were identified by flow cytometry, and BMSCs cultured in vitro were intervened with Jiangu granules-containing serum and blank serum. Cell morphology was observed under microscope, and ALP staining and quantitative detection, and the alizarin red staining were performed. Gene expression of osteogenic-induced factors including miR-141, DLx5, Msx2 and Runx2 was assessed using Quantitative Real-time PCR. Western blot was used to detect the expression of DLx5, Msx2 and Runx2. Results The cells were identified as BMSCs by flow cytometry. Microscopic observation revealed that there were differences between the two groups of cells, and there was more crystallization between the cells of Jiangu granules-containing serum group. The results of ALP staining and quantitative detection and the alizarin red staining showed that the drug-containing serum group was superior to the blank serum group. RT-PCR detection of osteogenic-induced miR-141 and Dlx5, Msx2, Runx2 gene expression results showed that miR-141 and Msx2 of Jiangu granules-containing serum group decreased significantly compared with blank serum group (P <0. 01), while Dlx5 and Runx2 increased significantly (P<0. 01). Western-Blot detection of osteogenic induced protein expression of DLx5, Msx2, Runx2 showed that compared with blank serum group, in Jiangu granules-containing serum group Msx2 was decreased (P<0. 05), and Dlx5 and Runx2 were significantly increased (P<0. 01). Conclusion Jiangu granules-containing serum could inhibit the high expression of osteoblast negative regulator miR-141 in ovariectomized mouse BMSCs. It could regulate the expression of Dlx5/Msx2/Runx2 signal axis of BMP signal pathway and increase the expression of Runx2, the key factor of bone formation, so as to promote osteoblast differentiation and achieve the purpose of preventing and treating osteoporosis.

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