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miR-431-5p靶向Smad4影响人牙髓干细胞增殖、分化的实验研究
引用本文:张建,吴建坤,刘丽丽,林秀雅,刘学丽. miR-431-5p靶向Smad4影响人牙髓干细胞增殖、分化的实验研究[J]. 口腔生物医学, 2022, 0(4): 229-233
作者姓名:张建  吴建坤  刘丽丽  林秀雅  刘学丽
作者单位:1. 沧州市中心医院2. 沧州市中心医院口腔科
基金项目:河北省沧州市重点研发计划自筹项目
摘    要:
目的:探讨miR-431-5p对人牙髓干细胞(DPSCs)增殖、分化的调控作用及机制。方法:分别将miR-431-5p mimic(miR-431-5p mimic组)、Smad4过表达载体(pc-Smad4组)及二者共转染(pc-Smad4+miR-431-5p mimic组)DPSCs细胞。采用双荧光素酶报告基因实验验证miR-431-5p与Smad4靶向关系;qRT-PCR检测转染效率;CCK-8实验检测细胞增殖能力;茜素红染色观察钙化结节;碱性磷酸酶(ALP)染色试剂盒和ALP活性检测ALP表达及活性;Western blot检测成牙分化标志物骨钙蛋白(OCN)及Smad4蛋白表达。结果:共转染miR-431-5p mimic和野生型Smad4报告基因载体后,DPSCs细胞的荧光素酶活性显著降低(P<0.05)。与mimic-NC组比较,miR-431-5p mimic组miR-431-5p表达上调,Smad4表达下调,细胞增殖能力增加,钙化结节数目减少,ALP活性降低,OCN蛋白表达下调(均P<0.05);pc-Smad4组细胞增殖能力降低(P<0.05),钙化结节数目增加,ALP活性增加,OCN蛋白表达上调(均P<0.05)。与pc-Smad4组比较,pc-Smad4+miR-431-5p mimic组细胞增殖能力显著增加,钙化结节数目减少,ALP活性降低,OCN蛋白表达下调(均P<0.05)。结论:miR-431-5p可通过靶向抑制Smad4蛋白表达抑制DPSCs成牙分化并促进其增殖。

关 键 词:牙髓干细胞  miR-431-5p  Smad4  增殖  成骨分化  
收稿时间:2022-07-01

Experimental study about the effects of miR-431-5p on the proliferation and differentiation of human dental pulp stem cells by targeting Smad4
Abstract:
Objective:To explore the regulation effect and mechanism of miR-431-5p on the proliferation and differentiation of human dental pulp stem cells (DPSC). Methods:The third molars of healthy people were collected to isolate and culture DPSC. In the experiments, mimic-NC group, miR-431-5p mimic group, pc-Smad4 group and pc-Smad4+miR-431-5p mimic group were set up. DPSC were transfected with lipid plastid transfection. The targeted relationship between miR-431-5p and Smad4 was verified by double luciferase reporter gene assay. The cells proliferation was detected by CCK-8. The calcified nodules were observed by alizarin red staining. The expression and activity of alkaline phosphatase (ALP) were detected by ALP staining kits and ALP activity test. The expressions of osteogenic differentiation marker osteocalcin (OCN) and Smad4 were detected by Western blot. Results:After co-transfection of miR-431-5p mimic and wild-type Smad4 reporter gene vector, luciferase activity of DPSC was significantly reduced (P<0.01), while which was not significantly changed after co-transfection of miR-431-5p mimic and mutant Smad4 reporter gene vector (P>0.05). Compared with mimic-NC group, expression of miR-431-5p was up-regulated, expression of Smad4 was down-regulated, ability of cells proliferation was increased, staining of calcified nodules was light, ALP activity was reduced, and expression of OCN protein was down-regulated in miR-431-5p mimic group (P<0.05). Compared with mimic-NC group, ability of cells proliferation was decreased, staining of calcified nodules was deepen, ALP activity was increased, and the expression of OCN was up-regulated in pc-Smad4 group (P<0.05). Compared with pc-Smad4 group, ability of cells proliferation was significantly increased, staining of calcified nodules was light, ALP activity was decreased, and the expression of OCN protein was down-regulated in pc-Smad4+miR-431-5p mimic group (P<0.05). Conclusions:The miR-431-5p can inhibit osteogenic differentiation of DPSC and promote their proliferation by inhibiting the expression of Smad4 protein.
Keywords:dental pulp stem cell   miR-431-5p   Smad4  proliferation  osteogenic differentiation  
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