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目的探讨滋肾育胎丸对抗精子抗体(As Ab)阳性的不孕大鼠卵母细胞的干预作用。方法采用主动免疫建立As Ab阳性的免疫性不孕模型。将100只雌性SD大鼠随机分为空白对照组、模型组及滋肾育胎丸低、中、高剂量组(简称中药低、中、高剂量组),每组20只。造模22天开始灌胃给药,模型组给予5 m L/kg生理盐水灌胃,中药低、中、高剂量组分别给予0.075、0.100、0.150 g/m L滋肾育胎丸灌胃,每天1次,至36天。分别在造模21天及36天采用ELISA法测定As Ab和转化生长因子(transforming growth factor-β,TGF-β)水平。处死大鼠后取卵母细胞,采用实时定量聚合酶链反应(Real-time PCR)及Western blot分别检测TGF-βmRNA及其蛋白表达水平;采用流式细胞学(FCM)和末端终未标记(TUNEL)法测定卵母细胞凋亡率;采用细胞计数-8法(CCK-8)及5-乙炔基-2’脱氧尿嘧啶核苷(5-ethynyl-2’-deoxyuridine,Ed U)检测卵母细胞增殖水平;采用划痕实验及侵袭小室(Transwell)测定卵母细胞迁移与侵袭能力。结果与空白对照组比较,模型组卵母细胞凋亡增加,细胞增殖减少,迁移与侵袭能力减弱,TGF-βmRNA及其蛋白表达水平显著下调(P<0.05);与模型组比较,中药高剂量组卵母细胞凋亡减少,细胞增殖增加,迁移与侵袭能力增强,TGF-βmRNA及其蛋白表达水平显著上调(均P<0.05);且中药高剂量组作用明显优于中药低、中剂量组(P<0.05)。结论应用高剂量滋肾育胎丸干预免疫性不孕大鼠可明显上调其卵母细胞TGF-β水平,改善卵母细胞功能。
Objective To explore the intervention effect of Zishen Yutai Pill on the oocytes of infertile rats that are positive to As Ab. Methods As Ab positive immuno-infertility model was established by active immunization. 100 female SD rats were randomly divided into control group, model group and Zishen Yutai pill low, medium and high dose group (referred to as low, medium and high dose of traditional Chinese medicine group), 20 in each group. The rats in the model group were treated with 5 m L / kg saline by intragastric administration on the 22th day after model establishment. The rats in the low, medium and high dose groups were given 0.075, 0.100 and 0.15 g / 1 day, to 36 days. The levels of As Ab and transforming growth factor-β (TGF-β) were measured by ELISA at 21 days and 36 days respectively. The oocytes were killed after the rats were killed, and the mRNA and protein levels of TGF-β were detected by real-time PCR and Western blot respectively. Flow cytometry (FCM) and terminal unlabeled TUNEL method was used to detect the rate of oocyte apoptosis. The eggs were detected by CCK-8 and 5-ethynyl-2’-deoxyuridine (Ed U) The proliferation of blastocyst and blastoderm cells was measured by using scratch assay and Transwell assay. Results Compared with the blank control group, the apoptosis of oocyte in the model group was increased, the cell proliferation decreased, the migration and invasion ability weakened, and the expression of TGF-βmRNA and its protein were significantly decreased (P <0.05). Compared with the model group, The oocyte apoptosis decreased, cell proliferation increased, migration and invasion ability increased, TGF-βmRNA and its protein expression levels were significantly increased (all P <0.05); and Chinese medicine high-dose group was significantly better than the traditional Chinese medicine low and medium dose group (P <0.05). Conclusion Intervention of immunization of infertility rats with high-dose Zishen Yutai Pills can significantly increase the level of TGF-β in oocytes and improve oocyte function.