BMP9通过Orai1途径促进小鼠骨髓间充质干细胞骨向分化

基础医学与临床 ›› 2021, Vol. 41 ›› Issue (10): 1470-1475.

BMP9通过Orai1途径促进小鼠骨髓间充质干细胞骨向分化

周家旺¹, 陆国海², 李晓林^2*

1.南通大学附属吴江医院,苏州市第九人民医院骨科,江苏苏州 215200;
2.南京医科大学附属苏州医院,苏州市立医院(北区) 骨科, 江苏苏州 215008

收稿日期:2020-11-06 修回日期:2021-07-05 发布日期:2021-09-29
通讯作者: *lxl65506@163.com
基金资助:
苏州市科技局项目(SYS2019101)

BMP9 promotes bone differentiation of bone marrow mesenchymal stem cells via Orai1 pathway

ZHOU Jia-wang¹, LU Guo-hai², LI Xiao-lin^2*

1. Department of Orthopaedics, the Ninth People's Hospital of Suzhou, Wu Jiang Hospital Affiliated to Nantong University, Suzhou 215200;
2. Department of Orthopaedics, the North District of Suzhou Municipal Hospital, the Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou 215008, China

Received:2020-11-06 Revised:2021-07-05 Published:2021-09-29
Contact: *lxl65506@163.com

摘要/Abstract

摘要： 目的探索钙释放激活钙通道蛋白1(Orai1)在骨形态发生蛋白9(BMP9)诱导骨髓间充质干细胞(BMSCs)中的作用。方法贴壁法分离BMSCs,取第3代BMSCs加入10^－5mol/L BMP9在不同时间点观察Orai1 mRNA表达;Orai1基因敲减后利用RT-qPCR和Western blot检测转染效果;利用茜素红染色、碱性磷酸酶(ALP)染色观察BMP9诱导BMSCs骨向分化效果,RT-qPCR检测骨性标志物RUNX2、骨桥蛋白(OPN)、骨钙素(OCN)基因表达。将细胞复合在骨组织工程支架材料羟基磷灰石/聚乳酸/壳聚糖(hydroxyapatite/PLA/chitosan)植入裸鼠皮下,4周后通过HE染色和Masson染色观察体内成骨效果。结果 BMP9诱导第5天开始Orai1 mRNA表达逐渐升高(P＜0.05);基因敲减后Orai1基因和蛋白表达显著降低(P＜0.05);Orai1基因敲减后成骨效果显著下降(P＜0.05),并且骨性标志物RUNX2、OPN和OCN基因表达显著下降(P＜0.05);过表达BMP9能显著提高体内BMSCs异位成骨能力,Orai1敲减后显著抑制了这一效果(P＜0.05)。结论 BMP9促进BMSCs骨向分化,可能是通过调控Orai1发挥效应。

关键词: 骨形态发生蛋白9(BMP9), Orai1, 骨髓间充质干细胞(BMSCs), 骨向分化

Abstract: Objective To explore the role of calcium release activated calcium channel protein 1 (Orai1) in bone marrow mesenchymal stem cells (BMSCs) osteogenesis induced by bone morphogenetic protein 9 (BMP9). Methods BMSCs were isolated by adherent method, and the third generation BMSCs were treated with 10^－5mol/L of BMP9, and the mRNA expression of Orai1 was measured at different time points. RT-qPCR and Western blot were used to value the transfection efficiency after Orai1 knockdown. The osteogenic markers of Runx2, OPN and OCN were detected by RT-qPCR. Hydroxyapatite/PLA/chitosan, a scaffold material for bone tissue engineering, was implanted into the subcutaneous of nude mice. After 4 weeks, the osteogenic effect was examined by microscopy. Results The mRNA expression of Orai1 increased gradually at the 5th day after BMP9 induction (P＜0.05). The gene and protein expression of Orai1 decreased significantly after Orai1 knockdown (P＜0.05). Besides, the osteogenic effect of Orai1 knockdown decreased significantly, and the expression of Runx2, OPN and OCN gene decreased significantly (P＜0.05). Moreover, BMP9 could significantly improve the ectopic osteogenic ability of BMSCs, and Orai1 knockdown significantly inhibited this effect (P＜0.05). Conclusions BMP9 plays an important role in osteogenic differentiation of BMSCs potentially by regulating Orai1.

Key words: bone morphogenetic protein 9 (BMP9), Orai1, bone marrow mesenchymal stem cells (BMSCs), osteogenic differentiation

中图分类号:

周家旺, 陆国海, 李晓林. BMP9通过Orai1途径促进小鼠骨髓间充质干细胞骨向分化[J]. 基础医学与临床, 2021, 41(10): 1470-1475.

ZHOU Jia-wang, LU Guo-hai, LI Xiao-lin. BMP9 promotes bone differentiation of bone marrow mesenchymal stem cells via Orai1 pathway[J]. Basic & Clinical Medicine, 2021, 41(10): 1470-1475.

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