ARHGAP29调控小鼠腭突间充质细胞增殖能力的研究

doi:10.13701/j.cnki.kqyxyj.2022.03.016

口腔医学研究 ›› 2022, Vol. 38 ›› Issue (3): 275-279.DOI: 10.13701/j.cnki.kqyxyj.2022.03.016

ARHGAP29调控小鼠腭突间充质细胞增殖能力的研究

阮文彦^1,2, 侯宇转^1,2, 迟丹丹¹, 于丽丽^1,3, 段小红^2*, 黄永清^1,3*

1.宁夏医科大学口腔医学院宁夏银川 750004;
2.军事口腔医学国家重点实验室,口腔疾病国家临床医学研究中心,陕西省口腔医学重点实验室,第四军医大学口腔医学院口腔生物学教研室陕西西安 710032;
3.宁夏医科大学总医院口腔医院口腔颌面外科宁夏银川 750004

收稿日期:2021-07-26 出版日期:2022-03-28 发布日期:2022-03-25
通讯作者: * 段小红,E-mail: xhduan@fmmu.edu.cn,黄永清,E-mail: yongqinghuang_6510@126.com
作者简介:阮文彦(1995~ ),女,甘肃人,硕士在读,研究方向:唇腭裂。
基金资助:
国家自然科学基金(编号:81960197、81974145、81771052)陕西省重点研发计划重点产业创新链(群)重点项目(编号:2021ZDLSF02-13)宁夏自然科学基金(编号:2021AAC03330)宁夏医科大学总医院公立医院综合改革-重点学科建设及人才培养项目(编号:NYDZY035)宁夏医科大学总医院(自治区临床研究中心)开放课题(编号:2019209)宁夏医科大学校级项目(编号:XM2020186)

ARHGAP29 Regulates Proliferation of Mouse Embryonic Palatal Mesenchymal Cells

RUAN Wenyan^1,2, HOU Yuzhuan^1,2, CHI Dandan¹, YU Lili^1,3, DUAN Xiaohong^2*, HUANG Yongqing^1,3*

1. School of Stomatology, Ningxia Medical University, Yinchuan 750004, China;
2. State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Disease & Shaanxi Key Laboratory of Stomatology, Department of Oral Biology, School of Stomatology, the Fourth Military Medical University, Xi’an 710032, China;
3. Department of Oral and Maxillafacial Surgery, Hospital of Stomatology, the General Hospital of Ningxia Medical University, Yinchuan 750004, China

Received:2021-07-26 Online:2022-03-28 Published:2022-03-25

摘要/Abstract

摘要： 目的: 探讨ARHGAP29对小鼠腭突间充质细胞增殖能力的影响。方法: 免疫组织化学染色确定ARHGAP29在腭突组织中的表达;体外培养GD13.5小鼠腭突间充质细胞,免疫荧光染色鉴定细胞来源;合成3条Arhgap29基因特异性小干扰RNA进行体外沉默实验,利用Real-time PCR和Western blot检测沉默效率;CCK-8法检测Arhgap29基因沉默后腭突间充质细胞增殖能力的改变;实验结果采用GraphPad Prism 8.0.2软件进行统计学分析。结果: 免疫组织化学结果示ARHGAP29在GD13.5小鼠腭突组织中显著表达;免疫荧光染色结果显示Vimentin染色的腭突间充质细胞包浆着色,绿色荧光表达强,Cytokeratin染色的细胞未见有阳性表达;Real-time PCR和Western blot结果显示,相较于阴性对照组和空白对照组,实验组ARHGAP29 mRNA和蛋白表达水平明显降低,其中Arhgap29-siRNA3的沉默效果最好(P<0.001);CCK-8结果显示体外沉默Arhgap29基因48 h后腭突间充质细胞的增殖能力开始上升,转染72 h细胞的增殖能力显著增强(P<0.05)。结论: ARHGAP29可能通过促进腭突间充质细胞的增殖,在腭突发育阶段造成腭突与口腔上皮的黏连,从而影响腭部的正常发育。

关键词: ARHGAP29, 腭突间充质细胞, RNA干扰, 细胞增殖

Abstract: Objective: To investigate the effect of ARHGAP29 on proliferation of cells from mouse embryonic palatal mesenchymal (EPM). Methods: Immunohistochemical technique was used to confirm the expression of ARHGAP29 in mice palate. The mice EPM cells of GD13.5-day were cultured in vitro. Immunofluorescence technique was used to identify the cellular characteristics. The primary cultured EPM cells were transfected with three pair of Arhgap29-siRNA to establish a gene knockdown cell model. The efficiency of gene knockdown was detected by real-time PCR and western blotting methods. The effects of Arhgap29 gene silencing on the proliferation of EMP cells were assessed by CCK-8 assay. Results: The results of immunohistochemistry showed that ARHGAP29 was significantly expressed in the palate of GD13.5 mice. Immunofluorescence detection showed the strong positive green fluorescence staining of Vimentin in cytoplasm and the negative staining of Cytokeratinc. Real-time PCR and western blotting results indicated that the mRNA and protein level of ARHGAP29 were significantly down-regulated in Arhgap29-siRNA group comparing with blank and negative control, and Arhgap29-siRNA3 showed the best silencing effect (P<0.001). The results of CCK8 assay showed that the proliferation ability of EPM cells began to increase after 48 hours Arhgap29-siRNA3 treatment, and the OD value was significantly increased after 72 hours (P<0.05). Conclusion: Our results indicated that Arhgap29 can promote the proliferation of cells from mouse EMP, which may increase the adhesion of palatal shelves and oral epithelium during the embryonic stage, and affect the palate normal development.

Key words: ARHGAP29, palatal mesenchymal cell, RNA interference, cell proliferation

阮文彦, 侯宇转, 迟丹丹, 于丽丽, 段小红, 黄永清. ARHGAP29调控小鼠腭突间充质细胞增殖能力的研究[J]. 口腔医学研究, 2022, 38(3): 275-279.

RUAN Wenyan, HOU Yuzhuan, CHI Dandan, YU Lili, DUAN Xiaohong, HUANG Yongqing. ARHGAP29 Regulates Proliferation of Mouse Embryonic Palatal Mesenchymal Cells[J]. Journal of Oral Science Research, 2022, 38(3): 275-279.

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ARHGAP29调控小鼠腭突间充质细胞增殖能力的研究

ARHGAP29 Regulates Proliferation of Mouse Embryonic Palatal Mesenchymal Cells

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