环孢素 A 治疗后牙龈中视网膜母细胞瘤蛋白磷酸化的上调：体内和体外研究。

Up-regulation of retinoblastoma protein phosphorylation in gingiva after cyclosporine A treatment: an in vivo and in vitro study.

机构信息

Department of Periodontology, School of Dentistry, National Defense Medical Center and Tri-Service General Hospital, Taipei, Taiwan.

出版信息

J Periodontal Res. 2011 Apr;46(2):158-63. doi: 10.1111/j.1600-0765.2010.01312.x. Epub 2010 Dec 28.

DOI:10.1111/j.1600-0765.2010.01312.x

PMID:21198643

Abstract

BACKGROUND AND OBJECTIVE

Cyclosporine A can induce gingival cell proliferation; however, the precise molecular regulation of the proliferation is uncertain. Therefore, this study was carried out to examine, in vivo and in vitro, the expression of genes and proteins associated with gingival cell proliferation after treatment with cyclosporine A.

MATERIAL AND METHODS

Forty Sprague Dawley rats with right maxillary posterior edentulous gingivae were assigned to a cyclosporine A group (30 mg/kg daily of cyclosporine A, administered orally) or a control group (administered mineral oil only). The animals were killed 4 wk after treatment. The edentulous gingivae were dissected out and analyzed for the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (Rb1) mRNA and/or protein, and phosphorylated Rb1 (pRb1), by real-time RT-PCR or immunohistochemistry. In human gingival fibroblast (HGF) cultures, the expression of PCNA, CDK4, cyclin D1 and Rb1 proteins and Rb1 phosphorylation were determined by western blotting after cyclosporine A treatment (0-10(4) ng/mL).

RESULTS

Proliferating cell nuclear antigen and cyclin D1 mRNAs (Pcna and Ccnd1, respectively) were expressed more strongly in the gingivae of cyclosporine A-treated animals than in the gingivae of the controls. Immunohistochemical analyses showed that a greater number of gingival cells stained positive for cyclin D1, CDK4 and pRb1 in the cyclosporine A group than in the control group. Increased expression of cyclin D1, CDK4 and PCNA proteins was observed in HGFs after cyclosporine A treatment. The phosphorylation of Rb1 was enhanced in HGFs after treatment with cyclosporine A at concentrations of 10(2)-10(3) ng/mL.

CONCLUSION

The increases in cyclin D1, PCNA and CDK4, together with the enhanced phosphorylation of Rb1, suggest that cyclosporine A promotes cell-cycle progression through the G(1)/S transition in the gingiva.

摘要

背景与目的

环孢素 A 可诱导牙龈细胞增殖；然而，增殖的确切分子调控尚不清楚。因此，本研究旨在体内和体外研究环孢素 A 处理后与牙龈细胞增殖相关的基因和蛋白的表达。

材料与方法

40 只右侧上颌后牙缺失的 Sprague Dawley 大鼠被分为环孢素 A 组（每天 30mg/kg 环孢素 A，口服）或对照组（仅给予矿物油）。治疗 4 周后处死动物。取出无牙牙龈并通过实时 RT-PCR 或免疫组织化学分析增殖细胞核抗原（PCNA）、周期蛋白 D1、细胞周期蛋白依赖性激酶 4（CDK4）和视网膜母细胞瘤蛋白（Rb1）mRNA 和/或蛋白以及磷酸化 Rb1（pRb1）的表达。在人牙龈成纤维细胞（HGF）培养物中，通过 Western 印迹法检测环孢素 A 处理（0-10（4）ng/mL）后 PCNA、CDK4、cyclin D1 和 Rb1 蛋白和 Rb1 磷酸化的表达。

结果

与对照组相比，环孢素 A 处理动物的牙龈中 PCNA 和 cyclin D1 mRNA（分别为 Pcna 和 Ccnd1）的表达更强。免疫组织化学分析显示，环孢素 A 组中更多的牙龈细胞 cyclin D1、CDK4 和 pRb1 染色阳性。环孢素 A 处理后 HGFs 中 cyclin D1、CDK4 和 PCNA 蛋白表达增加。环孢素 A 以 10（2）-10（3）ng/mL 的浓度处理 HGFs 后，Rb1 磷酸化增强。