miR-181a-5p对人颌骨骨髓间充质干细胞成骨分化的影响

doi:10.3969/j.issn.1005-4979.2022.05.002

摘要/Abstract

摘要：

目的：探讨骨折后骨改建中miR-181a-5p对人骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）成骨分化的作用。方法：提取正颌手术患者废弃骨块中的BMSCs，将其转染后分为对照组、miR-181a-5p过表达组、miR-181a-5p沉默组。成骨诱导14 d后，分析各组矿化结节形成、碱性磷酸酶（alkaline phosphatase, ALP）表达。成骨诱导21 d后，实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）分析各组成骨相关基因的表达。结果：miR-181a-5p过表达组相比对照组，其ALP表达更高，成骨相关基因Ⅰ型胶原（collagen type 1,COL1）、Runt相关转录因子2（runt related transcription factor 2, Runx2）表达更高，矿化结节形成更多；miR-181a-5p沉默组相比对照组，其ALP表达更低，成骨相关基因COL1、Runx2表达更低，矿化结节形成更少。结论：miR-181a-5p对BMSCs成骨分化具有促进作用。

关键词: miR-181a-5p, 骨髓间充质干细胞, 成骨分化

Abstract:

Objective: To investigate the effect of miR-181a-5p on osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) during bone remodeling after bone fracture. Methods: BMSCs were extracted from the discarded bone of patients who received orthognathic surgery and were divided into control group, miR-181a-5p overexpression group and miR-181a-5p silencing group after transfection. After 14-day osteogenic induction, the formation of mineralized nodules and the expression of alkaline phosphatase (ALP) were analyzed. And the expression of osteogenic genes were analyzed by real-time quantitative polymerase chain reaction(RT-qPCR) after 21 days. Results: Compared with the control group, the miR-181a-5p overexpression group had higher expression level of ALP, higher expression level of osteogenic genes such as collagen type 1 (COL1), runt related transcription factor 2 (Runx2), and more formation of mineralized nodules. The miR-181a-5p silencing group had lower expression level of ALP, osteogenic genes COL1, Runx2, and less formation of mineralized nodules than that of the control group. Conclusion: miR-181a-5p can promote the osteogenic differentiation of BMSCs.

Key words: miR-181a-5p, bone marrow mesenchymal stem cells, osteogenic differentiation

中图分类号:

R782

夏煜星, 廖崇珊, 康非吾. miR-181a-5p对人颌骨骨髓间充质干细胞成骨分化的影响[J]. 《口腔颌面外科杂志》, 2022, 32(5): 272-277.

XIA Yuxing, LIAO Chongshan, KANG Feiwu. Effects of miR-181a-5p on osteogenic differentiation of human bone marrow mesenchymal stem cells of jaw[J]. 《Journal of Oral and Maxillofacial Surgery》, 2022, 32(5): 272-277.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2022/V32/I5/272

图/表 5

表1 引物序列

Table 1 Primer sequences

基因名称	引物序列5′-3′	引物序列3′-5′
U6	GCTTCGGCAGCACATATACTAAAAT	CGCTTCACGAATTTGCGTGTCAT
miR-181a	TGGTGAAGACGCCAGTGGA	TGGTGAAGACGCCAGTGGA
GAPDH	GCACCGTCAAGGCTGAGAAC	TGGTGAAGACGCCAGTGGA
COL1	GAGGGCCAAGACGAAGACATC	CAGATCACGTCATCGCACAAC
Runx2	TGGTTACTGTCATGGCGGGTA	TCTCAGATCGTTGAACCTTGCTA
OCN	GGCGCTACCTGTATCAATGG	GTGGTCAGCCAACTCGTCA

图1 人BMSCs的形态学观察、鉴定及转染 A. 人BMSCs P0代1 d（×20）；B. 人BMSCs P2代3 d（×10）；C. miR-181a-5p转染率，**代表P<0.01，与NC组比较。D—G. 人BMSCs表面标志物CD90、CD73、CD44、CD105表达情况。

Figure 1 Morphological observation, identification and transfection of human BMSCs

图2 ALP染色和ALP活性检测 A. ALP活性检测，***表示P<0.001，与control组比较；B. 各组光镜下ALP染色图（×10）；C. 各组直观下ALP染色图。

Figure 2 ALP staining and detection of ALP activity

图3 ALP染色和茜素红染色 A. 光镜下各组ALP染色图（×10）；B. 直观下各组ALP染色图；C. 光镜下各组茜素红染色图（×10）；D. 直观下各组茜素红染色图。

Figure 3 ALP staining and alizarin red staining

图4 RT-qPCR检测成骨相关因子COL1、Runx2、OCN A. 人BMSCs中COL1的mRNA表达情况；B. 人BMSCs中Runx2的mRNA表达情况；C. 人BMSCs中OCN的mRNA表达情况。*代表P<0.05，**代表P<0.01，***代表P<0.001，与control组比较。

Figure 4 Detection of osteogenic related factors COL1, Runx2 and OCN by RT-qPCR

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