摘要
目的:建立稳定、成功率高的人子宫肌瘤细胞原代培养方法,探讨不同浓度左炔诺孕酮(levonorgestrel,LNG)对体外培养人子宫肌瘤细胞增殖和凋亡的影响。方法:分别采用酶解法和贴块法原代培养人子宫肌瘤细胞(uterine leiomyoma cell,UtLMC),以α-Actin单抗进行免疫组织化学染色鉴定原代细胞;细胞传代后,加入不同浓度左炔诺孕酮,H-E染色和透射电镜观察细胞的形态学改变,MTT法检测LNG对细胞增殖的调节作用,流式细胞术测定细胞凋亡率,RT-PCR技术分析LNG对人子宫肌瘤细胞凋亡相关基因Bcl-2、IGF-1及Survivin mRNA表达的影响。结果:酶解法和贴块法均成功获得UtLMC,经免疫组化鉴定,纯度达95%以上,并能稳定传12代;5μg/ml左炔诺孕酮对子宫肌瘤细胞无明显作用,当质量浓度达到10μg/ml时可显著抑制细胞增殖,并呈时间、剂量依赖;随着药物浓度的增加,细胞凋亡率亦逐渐升高,与对照组相比有显著性差异(P<0.05);10μg/ml和20μg/ml左炔诺孕酮作用UtLMC后,Bcl-2、IGF-1及Survivin mRNA表达下降。结论:酶解法和贴块法均能获得功能良好的人子宫肌瘤细胞,一定浓度的左炔诺孕酮可抑制其增殖并诱导凋亡,机制与下调Bcl-2、IGF-1及Survivin基因表达有关。
Objective ： To establish a method for stable and efficient culture of human uterine leiomyoma cell（UtLMC） and to explore the effects of levonorgestrel on proliferation and apoptosis of UtLMCs. Methods： Human uterine leiomyoma cells were isolated and cultured by enzymatically dispersed method or explant method. The passaged cells were identified by α-Actin antibody immunohistochemical staining. After exposure to levonorgestrel at different concentrations for 72 h, the morphological changes of UtLMCs were observed by H-E staining and transmission electron microscope 72 h after exposure to levonorgestrel. MTT assay was used to detect the anti-proliferative effect of levonorgestrel. Apoptosis rate of cultured cells was analyzed by flow cytometry. Bcl-2, IGF-1 and Survivin mRNA expressions in UtLMCs were determined by semi-quantitative RT-PCR. Results： Primary cultivated human uterine leiomyoma cells were successfully obtained by the 2 culture methods and with high purity and viability. Lower concentration of levonorgestrel had no inhibitory effect on UtLMC growth ; when the concentration reached 10 μg/ml, levonorgestrel inhibited UtLMC growth in a dose- and time-de pendent manner. Levonorgestrel treatment induced a apoptosis in UtLMCs in a concentration-dependent manner; there was significant difference between the levenorgestrel group and the control group （ P 〈 0.05 ）. The expressions of Bcl-2, IGF-1 and Survivin mRNA were all down-regulated in UtLMC cell after treatment by 10 μg/ml and 20 μg/ml levonorgestrel. Conclusion： Enzymatically dispersed method and explant method both can obtain highly purified and satisfactory UtLMCs. Certain concentration of levonorgestrel can cause decrease in proliferation and increase in apoptosis of UtLMCs. The decreased Bcl-2, IGF-1 and Survivin mRNA expressions may be the molecular mechanisms for the role of levonorgestrel in the inhibition of human uterine leiomyoma growth.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2007年第6期550-556,共7页
Chinese Journal of Cancer Biotherapy
基金
南京市医学科技发展项目(No.YKK06080)
南京医科大学科技发展基金重点项目(No.2005NYDZD21)~~
关键词
左炔诺孕酮
子宫肌瘤细胞
细胞凋亡
基因表达
levonorgestrel
uterine leiomyoma cell
cell apoptosis
gene expression
