二甲双胍治疗对多囊卵巢综合征模型大鼠卵巢中Fas/FasL表达的影响
Effects of metformin therapy on the expressions of Fas and Fas ligand in rat ovaries with polycystic ovary syndrome

摘要：目的 探讨二甲双胍(dimethylbiguanide, DB)治疗前后多囊卵巢综合征(polycystic ovary syndrome, PCOS)模型大鼠卵巢组织中Fas(factor associated suicide)和FasL(factor associated suicide ligand)蛋白的表达变化，为研究DB临床治疗PCOS的分子机制提供实验资料。方法 选取30只85天龄SD雌性大鼠，随机分为PCOS组(10只)、DB治疗组(10只)、对照组(10只)。前两组通过皮下注射脱氢表雄酮(dehydroepiandrosterone, DHEA)建立PCOS大鼠模型，PCOS组和对照组喂服安慰剂，同期DB治疗组喂服二甲双胍。14d后取出各组大鼠卵巢称重，观察卵巢大体解剖和组织学结构变化(HE染色)，并取外周血测定血清胰岛素(insulin, INS)、黄体生成素(luteinizing hormone, LH)和睾酮(testosterone, T)水平。采用免疫组织化学SP法和Western blot法检测3组卵巢组织中Fas蛋白和FasL蛋白的表达变化。结果 ①PCOS组大鼠卵巢的重量、体积、平均卵巢面积、最大卵泡直径和卵泡膜厚度均比对照组增加；DB治疗组大鼠以上各指标均比PCOS组降低，所有差异均有统计学意义(P均<0.05)；②PCOS组和DB治疗组大鼠血清INS、LH和T值均比对照组增高，且DB治疗组大鼠血清T值比PCOS组降低,所有差异均有统计学意义(P均<0.05)；③免疫组化显示，Fas蛋白和FasL蛋白主要表达于囊状卵泡颗粒细胞胞质内，且在PCOS组囊状扩张卵泡中近卵泡腔的1～2层颗粒细胞中表达较强，而在初级卵泡卵母细胞胞质和透明带表达较弱；④Western blot显示，PCOS组大鼠卵巢组织中Fas和FasL蛋白的表达较对照组增高，但DB治疗组Fas蛋白的表达较对照组和PCOS组均降低，而FasL蛋白的表达却较对照组增加，所有差异均具有统计学意义(P均<0.05)。结论 Fas/FasL的表达改变是DHEA诱导的PCOS组大鼠卵泡异常凋亡的主要通路之一，可以通过DB治疗逆转卵巢多囊性改变。
ABSTRACT: Objective To evaluate the effects of metformin therapy on the expressions of factor associated suicide (Fas) and Fas ligand in rat ovaries with polycystic ovary syndrome (PCOS) induced by DHEA so as to explore the molecular mechanism of metformin treatment of PCOS. Methods Totally 30 SD female rats aged 85 days were randomly divided into PCOS group (n=10), metformin (dimethylbiguanide, DB) therapy group (n=10), and control group (n=10). DHEA hypodermic injection was adopted to establish PCOS rat model in the first two groups, followed by administration of the placebo or metformin respectively, and similarly dissolvent injection and placebo in control group rats were introduced. After 14 days’ treatment, ovaries taken from the rats in each group were weighed, and their structural changes were observed with HE staining. The serum levels of insulin, luteinizing hormone and testosterone were tested by ELISA, and the changes of Fas and Fas ligand expressions were detected by immunohistochemistry and Western blot. Results ① We found significant increases in weight, volume, average ovarian surface area, maximum follicular diameter and follicular theca thickness of the rats in PCOS group compared with those in control group (all P<0.05). Remarkable declines in all the items above occurred in DB therapy group compared with PCOS group (all P<0.05). ② The serum INS, LH and T in PCOS and DB therapy groups were markedly increased compared with those in the control group, and a sharp decrease in serum T was detected in the rats of DB therapy group in comparison with PCOS group (all P<0.05). ③ Fas and FasL were