摘要目的：探讨二甲双胍对孕激素耐药的子宫内膜癌细胞的调节作用及可能的作用机制。方法（1）采用醋酸甲羟孕酮（MPA）持续作用、浓度递增（1～15μmol/L）方法体外诱导子宫内膜癌细胞系Ishikawa细胞，建立孕激素耐药的子宫内膜癌细胞系Ishikawa/MPA细胞。（2）采用活细胞计数（CCK-8）法检测不同浓度MPA（1、5、10、20、40、60μmol/L）、二甲双胍（1、10、20、40、60、70、80 mmol/L）作用24 h后Ishikawa和Ishikawa/MPA细胞的增殖情况。（3）实验分为4组，即MPA组（加入20μmol/L的MPA）、二甲双胍组（加入20 mmol/L的二甲双胍）、MPA+二甲双胍组（加入20μmol/L的MPA和20 mmol/L的二甲双胍）、对照组（未加药物），采用蛋白印迹（western blot）法检测4组Ishikawa和Ishikawa/MPA细胞中PR-B蛋白的表达。结果（1）采用MPA持续作用、浓度递增方法体外诱导Ishikawa细胞，历经近1年时间成功建立了孕激素耐药的子宫内膜癌细胞系Ishikawa/MPA细胞。Ishikawa和Ishikawa/MPA细胞的倍增时间分别是（47±3）和（43±4）h，两者比较，差异无统计学意义（t=0.349，P=0.572）。（2）CCK-8法检测显示，不同浓度的MPA作用后，低浓度（1、5μmol/L）的MPA对Ishikawa/MPA细胞增殖具有促进作用，其抑制率分别为-3%、-13%，高浓度（10、20、40、60μmol/L）的MPA对Ishikawa/MPA细胞增殖的抑制作用不明显，其抑制率分别为4%、3%、9%、40%；而不同浓度MPA对Ishikawa细胞增殖均具有显著的抑制作用，其抑制率分别为16%、30%、41%、55%、65%、66%。在同一浓度下，两种细胞的抑制率分别比较，差异均有统计学意义（P<0.01）。不同浓度（1、10、20、40、60、70、80 mmol/L）的二甲双胍作用后，Ishikawa细胞和Ishikawa/MPA细胞的增殖均明显受到抑制，且随着二甲双胍浓度的增加其抑制作用均逐渐增强（χ2=72.9，P=0.000；χ2=76.3，P=0.000）。其中， Ishikawa细胞的抑制率分别为-6%、19%、37%、54%、70%、72%、83%，Ishikawa/MPA细胞的抑制率分别为-10%、20%、56%、89%、97%、98%、99%。（3）western blot法检测显示，Ishikawa细胞中，二甲双胍组、MPA+二甲双胍组细胞中PR-B蛋白的表达水平分别为（53.5±4.0）%、（37.7±5.2）%，分别与对照组[（23.4±3.0）%]比较，差异均有统计学意义（P<0.01）；Ishikawa/MPA细胞中，二甲双胍组、MPA+二甲双胍组细胞中PR-B蛋白的表达水平分别为（38.6±1.7）%、（36.3±2.5）%，分别与对照组[（6.4±1.6）%]比较，差异也均有统计学意义（P<0.01）。结论二甲双胍对孕激素耐药的子宫内膜癌细胞的耐药性有调节作用，其可能的作用机制为通过提高PR-B蛋白的表达水平以改善耐药细胞对孕激素的耐药性。

abstractsObjective To investigate the effect and mechanism of metformin on endometrial carcinoma subline of progestin-resistance and find whether metformin could regulate progestin-resistance in endometrial carcinoma. Methods Ishikawa endometrial carcinoma cells were cultured for a long period in the presence of the synthetic medroxyprogesterone 17-acetate (MPA) to generate a subline refractory to the&nbsp;growth-suppressive effects of MPA,named Ishikawa/MPA cells. The effect of MPA (1, 5, 10, 20, 40 and 60 μmol/L) or metformin (1, 10, 20, 40, 60, 70 and 80 mmol/L) on proliferation of the Ishikawa/MPA and parental Ishikawa cells was detected by cell counting kit-8 (CCK-8) method. Western blot was used to detect the effect of metformin and(or)MPA on the expression of PR-B in the Ishikawa/MPA and Ishikawa cells. Results The Ishikawa/MPA showed that growth stimulation rather than inhibition in the Ishikawa cells after low MPA concentration treatment. The doubling time of Ishikawa/MPA cell lines were (43±4) hours and that of Ishikawa cell line were (47 ± 3) hours. No changes in doubling time were observed (t=0.349,P=0.572). Low concentration (1 and 5μmol/L) of MPA could promote the growth of Ishikawa/MPA cells (by 3% and 13%). High concentration (10, 20, 40 and 60 μmol/L) of MPA could inhibited the growth of Ishikawa/MPA cells (by 4%, 3%, 9%and 40%) and the growth of Ishikawa cells (by 41%, 55%, 65%and 66%). At the same concentration, the difference of inhibition rates between the two cell lines were statistically significant (P<0.01). Metformin (1, 10, 20, 40, 60, 70 and 80 mmol/L) could inhibite the growth of Ishikawa/MPA (by-10%, 20%, 56%, 89%, 97%, 98%and 99%) greater than those in parental Ishikawa cells (by-6%, 19%, 37%, 54%, 70%, 72%and 83%), and the inhibitory effect was dose-dependent manner. Western blot assay showed that for Ishikawa cells, the protein expression levels of PR-B in metformin group and MPA+metformin group were respectively (53.5±4.0)%and (37.7±5.2)%, which were higher than that in the control group [(23.4 ± 3.0)%], and there were significant the differences (P<0.01). For Ishikawa/MPA cells, the protein expression levels of PR-B in metformin group and MPA+metformin group were respectively (38.6 ± 1.7)%,(36.3 ± 2.5)%,which were higher than those in the control group [(6.4 ± 1.6)%], and there were also significant differences (P<0.01). Conclusion Metformin may regulate the progestin-resistance in endometrial carcinoma by increasing the expression of PR-B.