摘要
目的:研究罗格列酮对人宫颈癌Hela细胞生长及凋亡影响,并初步探讨其作用机制。
方法:体外培养Hela细胞, MTT比色法检测罗格列酮对Hela细胞活力的相对抑制率; AO/EB染色荧光显微镜观察罗格列酮诱导Hela细胞凋亡形态学改变; DNA凝胶电泳确证罗格列酮诱导Hela细胞凋亡作用; PI染色流式细胞仪检测罗格列酮对宫颈癌Hela细胞Sub-G1细胞百分率的影响;FITC荧光标记免疫细胞化学法检测罗格列酮对人宫颈癌Hela细胞COX-2蛋白表达的影响。
结果:在MTT比色法的预实验中,罗格列酮(1.0、10.0、100.0、200.0、500.0μmol/L)分别处理宫颈癌Hela细胞24、48、72小时;结果显示,其抑制细胞活力的作用时间为48小时作用显著;200.0、500.0μmol/L的罗格列酮与100.0μmol/L的罗格列酮药效相当,无统计学意义。在其后正式实验中,取罗格列酮的浓度梯度为1.0、10.0、100.0μmol/L,作用48小时;结果表明,其细胞活力相对抑制率分别为(15.42±2.23)%、(54.34±2.78)%、(82.31±4.87)%,呈剂量依赖性抑制宫颈癌Hela细胞活力,IC50值为9.66μmol/L。AO/EB染色荧光显微镜观察罗格列酮处理后,部分宫颈癌Hela细胞呈现典型凋亡细胞形态特征。罗格列酮(100.0μmol/L)处理宫颈癌Hela细胞48h,琼脂糖凝胶电泳呈现“梯形”DNA条带。PI染色FCM分析发现1.0、10.0、100.0μmol/L的罗格列酮作用于宫颈癌Hela细胞48h后,Sub-G1细胞百分率分别是(5.15±0.48) %、(17.7±0.81)%、(38.9±0.98)%,呈浓度依赖性。FITC荧光标记免疫细胞化学法显示1.0、10.0、100.0μmol/L的罗格列酮作用于宫颈癌Hela细胞48h后,细胞内COX-2蛋白阳性表达率分别为(39.59±0.32)%、(30.36±0.81)%、(20.12±0.46)%,呈浓度依赖性下调。
结论: 1)罗格列酮具有抑制人宫颈癌Hela细胞活力作用,呈浓度依赖性。
2)罗格列酮具有诱导人宫颈癌Hela细胞凋亡作用,呈浓度依赖性。
3)罗格列酮抑制人宫颈癌Hela细胞活力和诱导细胞凋亡作用可能与降低细胞内COX-2蛋白表达相关。
Objective: To investigate the rosiglitazone on induction of growth inhibition and apoptosis of human cervical cancer Hela cells line, and its mechanism in vitro.
Methods: Human cervical cancer Hela cells were cultured in vitro. MTT assay was used to determine the effect of rosiglitazone on the viability of Hela cells. Fluorescence after AO/EB staining was used to observe the morphologic changes of apoptosis induced by rosiglitazone in Hela cells line. DNA agarose gel electrophoresis was used to test apotosis induced by rosiglitazone in Hela cells line. Flow cytometry (FCM) using PI staining was used to analyse the Sub-G1 cell population treated with rosiglitazone. COX-2 was determined using cell immunofluorescence.
Results: In preliminary experiment, Treatment of Hela cells with various concentrations of 1.0, 10.0, 100.0, 200.0, 500.0μmol/L rosiglitazone for 24, 48, 72 hours, respectively. MTT assay showed that the best time is 48 hours, and efficacy of both 200.0, 500.0μmol/L of rosiglitazone for inhibition of viability in Hela cells was nearly equal, there was no significant. In subsequent experiments, we used the experimental concentration gradient 1.0, 10.0, 100.0μmol/L and incubated for 48 hours. The results shown that the inhibitory rateof relative ecell viability was (15.42±2.2)%, (54.34±2.78)%,(82.31±4.87)%, respectively, and its IC50 was 9.66μmol/L. Typical morphologic changes of apoptosis in Hela cells could be observed after treatment with rosiglitazone by fluorescence microscope using AO/EB fluorescence staining. DNA agarose gel electrophoresis shown that DNA ladder bands could appear after treatment with rosiglitazone at 100.0μmol / L for 48h. Flow cytometry(FCM) analysis after PI stainning indicated that the percentage of sub-G1 cell population in Hela cells was( 5.15±0.48 )%,(17.7±0.81)%,(38.9±0.98)% respectively after treatment with rosiglitazone at the concentrations of 1.0, 10.0, 100.0μmol/L for 48h. FITC fluorescence labeling immunocytochemistry showed treatment with 1.0, 10.0, 100.0μmol/L of rosiglitazone reduced positive cells for COX-2 protein expression to(39.59±0.32)%, (30.36±0.81)% , (20.12±0.46)%, in a concentration dependent manner.
Conclusion:
1) Rosiglitazone posses the inhibitory effect of the viability of Hela cells, in a dose-dependent manner.
2) Rosiglitazone can significantly induce apoptosis of Hela cells, in a dose-dependent manner.
3) Inhibition of cell viability and induction of apoptosis by rosiglitazone in human cervical cancer Hela cells may be associated with downregulation of intracellular COX-2 protein expression.
引文
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