二甲双胍对子宫内膜异位症在位内膜细胞增殖与凋亡的影响

Effects of Metformin on the Cultured Eutopic Endometrial Cells From Endometriosis Patients

目的研究二甲双胍(metformin)对体外培养子宫内膜异位症(EMS)在位内膜细胞增殖与凋亡的影响。方法选择2012年1月至7月于河北医科大学附属邢台市人民医院妇科就诊的50例EMS患者的在位子宫内膜为研究对象,并纳入研究组(均经腹腔镜或开腹手术确诊)。选择同期在本院因宫颈病变、子宫纵隔、卵巢畸胎瘤、单纯卵巢囊肿行手术治疗的88例患者的子宫内膜为对照组(均排除EMS)。两组患者的年龄等一般临床资料比较,差异均无统计学意义(P>0.05)。建立细胞模型,以不同浓度(1μmol/L、10μmol/L、100μmol/L、1 000μmol/L)二甲双胍干预24h、48h、72h,通过3-(4,5二甲基噻唑-2)-2,5二苯基四氮唑溴盐(MTT)法检测不同浓度二甲双胍对EMS在位内膜细胞增殖的影响,并应用流式细胞术(FCM)检测细胞周期改变及细胞凋亡情况,应用酶联免疫吸附(ELISA)法测定检测其对Bcl-2、Bax表达水平的影响。本研究遵循的程序符合河北医科大学附属邢台市人民医院人体试验委员会制定的伦理学标准,得到该委员会批准,并与受试对象签署临床研究知情同意书。结果不同浓度二甲双胍对对照组患者的正常在位内膜细胞增殖无明显抑制作用。二甲双胍干预48h、72h后,EMS患者的在位内膜细胞增殖明显受到抑制(P<0.05),以二甲双胍浓度为1 000μmol/L时最为明显(P<0.01),并呈时间-剂量依赖性。二甲双胍干预后EMS患者在位内膜细胞G1期比例明显高于对照组(P<0.05)。二甲双胍浓度为10μmol/L、100μmol/L、1 000μmol/L时,EMS在位内膜细胞的Bcl-2表达水平较对照组降低,Bax表达水平较对照组升高,差异均有统计学意义(P<0.05)。结论二甲双胍可显著抑制EMS在位内膜细胞增殖,促进其凋亡,阻滞细胞周期进程。

Objective To study effects of metformin on the cultured eutopic endometrial cells from patients with endometriosis（EMS）.Methods From January to July 2012,a total of 50 samples from patients with EMS were included into study group.At the same time,another 88 samples form cases who were underwent operation because of cervical lesions,uterine septum,ovarian teratoma,ovarian cyst were included into control group.There were no significant differences on general information（eg.age）between two groups（P〉0.05）.The cell model was established.The cells were incubated without or with different concentration of metformin（1μmol/L,10μmol/L,100μmol/L and 1 000μmol/L）for 24 h,48h,72 h,and the influence of eutopic endometrial cell prooliferation was detected by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide（MTT）.The changes of cell cycle and cell apoptosis were observed by flow cytometry（FCM）.Expressions of Bcl-2,Bax in the cells were measured by enzyme linked immunosorbent assay（ELISA）method.The study protocol was approved by the Ethical Review Board of Investigation of Xingtai People′s Hospital.Informed consent was obtained from all participates.Results There was no significant difference between eutopic endometrial cell proliferation in control group incubated with or without metformin（1μmol/L,10μmol/L,100μmol/L and 1 000μmol/L）.The proliferation of eutopic endomentrial cells of EMS patients were inhibited by metformin in a dose-dependent manner.The proliferation of eutopic endometrium cell incubated without or with metformin（1μmol/L,10μmol/L,100μmol/L and 1 000μmol/L）for 48 h,72h were significantly inhibited（P〈0.05）.The inhibition rate was highest after 1 000μmol/L metformin（P〈0.01）.After intervention of metformin,eutopic endometrial cells in G1 phase were significantly higher than those of the control group（P〈0.05）.The expressions of Bcl-2 were all up-regulated after metformin treatment,while the expressions of Bax were all down-regulated.Conclusion Metformin can inhibit cell proliferation of eutopic endometrium of EMS,induce cell apoptosis and change its cell cycle′s proportion.