摘要
目的观察二氟甲基鸟氨酸(DFMO)对人宫颈永生化上皮细胞细胞周期的影响,并探讨其细胞周期调控机制。方法宫颈永生化上皮细胞系(H8细胞)单层培养,应用磺酰罗丹明B(SRB)法,绘制细胞生长曲线,并获得抑制50%细胞生长所需药物浓度(IC50)。应用流式细胞仪分析细胞周期。应用免疫荧光分析检测细胞周期调控因子cyclinD1、CDK4、p21的蛋白表达。应用RT-PCR半定量检测cyclinD1、CDK4、p21的mRNA表达。结果DFMO对宫颈永生化上皮细胞(H8)有生长抑制作用,DFMO对H8细胞作用第5天的IC50为1mM。DFMO作用后使H8细胞阻滞于G1期。DFMO作用后降调节H8细胞cyclinD1蛋白(P<0.01)和mRNA(P<0.05)的表达。DFMO对H8细胞的CDK4蛋白表达无明显影响(P>0.05),但使CDK4 mRNA的水平降低(P<0.05)。DFMO对H8细胞p21蛋白和mRNA表达均无明显影响(P>0.05)。结论DFMO使宫颈永生化上皮细胞细胞周期阻滞于G1期,其作用机制之一主要是通过降调节cyclinD1 mRNA和蛋白的表达。DFMO可能作为一种细胞周期调控剂来治疗宫颈癌前病变。
Objective To investigate the effects of difluoromethylornithine （DFMO） on the cell cycles of immortalized human cervical epithelial cells, and its regulation mechanisms. Methods Immortalized human cervical epithelial cell line （H8） was used in this study. The effects of DFMO on proliferation were tested by growth curves of monolayer culture, and concentrations required for a 50% growth inhibition （IC50） with a 5 -day treatment were determined by the sulforhodamine B （SRB） assay. Cellular DNA content was measured and analyzed using flow cytometry. The protein expressions of cyclin D1/CDK4/p21 were measured by immunological fluorescence assay. The mRNA expressions of cyclin D1/CDK4/p21 were determined by RT- PCR semiquantitatively. Results DFMO suppressed the proliferation of the immortalized cervical epithelial cells （H8）. The concentration required for a 50% growth inhibition （IC50） of H8 cells with a 5 -day DFMO treatment was 1mM. H8 cells treated with DFMO were arrested at G1 phase, and both cyclin D1 protein （P〈0.01） and cyclin D1 mRNA （P〈0. 05） were decreased. No significant change of CDK4 was found in H8 cells after treated with DFMO （P〉0.05）, but the mRNA expression was decreased （P〈0.05）. No changes of p21 or its mRNA was observed after DFMO treatment （P〉0.05）. Conclusions DFMO arrests the immortalized cervical epithelial cells at G1 phase via down - regulation of cyclin D1 and its mRNA. DFMO may be used as a cell cycle regulatory agent for treatment of cervical pre - cancerous lesions.
出处
《中国妇产科临床杂志》
2007年第3期202-205,240,共5页
Chinese Journal of Clinical Obstetrics and Gynecology
关键词
二氟甲基鸟氨酸
宫颈
永生化
肿瘤
细胞周期
difluoromethylornithine （DFMO）
cervix
immortalization
neoplasms
cell cycle
