补肾活血方调节雷公藤多甙致卵巢储备功能下降大鼠卵巢免疫功能的实验研究

发布时间：2018-03-05 13:37

本文选题：卵巢储备功能　切入点：免疫　出处：《南京中医药大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：通过观察雷公藤多甙(GTW)致卵巢储备功能下降(DOR)大鼠免疫细胞因子白细胞介素-2(IL-2)的水平和卵巢肿瘤坏死因子-α(TNF-α)的表达,进一步探讨补肾活血方改善卵巢储备功能的作用机制,为临床治疗DOR提供科学依据。方法：选取SD雌性健康大鼠48只,根据体重随机分为6组(n=8)：空白对照组(灌胃同体积的生理盐水)、GTW模型组(GTW50mg/kg/d)、六味地黄汤组(GTW50mg+六味地黄汤7.50g生药/kg/d)、补肾活血方低剂量组(GTW50mg+补肾活血方3.25g生药/kg/d)、补肾活血方中剂量组(GTW50mg+补肾活血方7.50g生药/kg/d)、补肾活血方高剂量组(GTW50mg+补肾活血方15.00g生药/kg/d)。连续15天灌胃给药,给药结束后1-4日内结合阴道细胞学涂片,将未出现明显动情周期的大鼠乙醚麻醉,予股动脉取血后颈椎脱臼处死,并立刻摘取大鼠双侧卵巢。观察各组大鼠动情周期、体重、卵巢质量、卵巢系数；采用放射免疫法检测大鼠血清中FSH、E2的变化；采用放射免疫法检测大鼠血清中免疫细胞因子IL-2的变化；HE染色检测大鼠卵巢组织形态学的改变；免疫组化法检测大鼠卵巢组织TNF-α水平的表达。结果：(1)模型组大鼠卵巢质量及卵巢系数较空白对照组降低；模型组大鼠血清中FSH明显高升,E2明显下降,与空白对照组比较均有显著差异(P值均为p0.001)；模型组大鼠血清中免疫细胞因子IL-2下降,与空白对照组比较均有显著差异(p0.01)；模型组大鼠卵巢组织中各期卵泡数均明显减少,卵泡间质内有不同程度的炎细胞浸润及纤维化；模型大鼠TNF-α呈低表达与空白对照组有显著差异(p0.05)。 (2)补肾活血方低、中、高剂量组与六味地黄汤组均可不同程度改善GTW大鼠的卵巢质量及卵巢系数；均能明显降低GTW大鼠血清FSH水平,与模型组比较均有显著差异(p0.001)；补肾活血方中、高剂量组与六味地黄汤组均可升高血清E2水平,与模型组比较均有差异(P值分别为p0.01、p0.05、p0.01);补肾活血方中、高剂量组与六味地黄汤组均可升高血清中免疫细胞因子IL-2水平,与模型组比较均有差异(均为p0.05)；补肾活血方中、高剂量组卵泡颗粒细胞和卵母细胞中的TNF-α表达较模型组改善,存在统计学差异(P0.05),与空白组比较无明显差异。结论：(1)补肾活血方可降调GTW大鼠血清中FSH水平,上调血清中E2水平,表明补肾活血方可改善卵巢储备功能,上调血清中免疫细胞因子IL-2,拮抗卵巢中卵泡的损伤,升高卵泡颗粒细胞和卵母细胞TNF-α的表达,可能是其防治DOR的又一作用机制之一。 (2)补肾活血方(补肾活血法)和六味地黄汤(补肾法)均能改善卵巢储备功能,抑制卵泡的损伤,但补肾活血方在改善卵泡颗粒细胞和卵母细胞TNF-α的表达方面优于六味地黄汤,与前期研究基础一致,反映出补肾活血方可通过多方面作用机制来改善DOR。
[Abstract]:Objective: to observe the level of interleukin-2 (IL-2) and the expression of tumor necrosis factor- 伪 (TNF- 伪) in rat ovarian reserve dysfunction induced by GTW (Tripterygium wilfordii). To explore the mechanism of Bushen Huoxue recipe in improving ovarian reserve function and provide scientific basis for clinical treatment of DOR. Methods: 48 SD female healthy rats were selected. According to body weight, they were randomly divided into six groups: blank control group (fed with normal saline of the same volume and GTW model group), GTW 50 mg / kg / kg / d group, Liuwei Dihuang decoction group with GTW 50 mg / kg / kg / kg / d, low dose group with low dose of Bushen Huoxue recipe (GTW 50 mg / kg / kg / kg / d), low dose group with low dose of Bushen Huoxue recipe (GTW 50 mg / kg / kg / kg / d), and the group with tonifying kidney. GTW 50mg Bushen Huoxue prescription 7.50g crude drug / kg 路kg 路d ~ (-1 / d) in medium dose group and 15.00 g / kg / kg / d GTW _ (50mg) Bushen / Huoxue prescription / high dose group were administrated by stomach for 15 days. Within 1-4 days after administration, combined with vaginal cytological smear, the rats without obvious estrous cycle were anesthetized with ether, and were killed by cervical dislocations after femoral artery blood extraction, and bilateral ovaries were removed immediately. The estrus cycle and body weight of the rats in each group were observed. The changes of serum FSHE _ 2 in rats were detected by radioimmunoassay, the changes of immunocyte factor IL-2 in serum by radioimmunoassay and the morphological changes of ovary by HE staining. The expression of TNF- 伪 in rat ovary was detected by immunohistochemistry. Results the ovarian weight and ovarian coefficient in the model group were lower than those in the blank control group, and the serum FSH in the model group was significantly higher than that in the control group. Compared with the blank control group, there were significant differences (P < 0.05) between the model group and the blank control group, the decrease of serum immunocyte factor IL-2 in the model group and the significant difference between the model group and the blank control group, the number of follicles in the ovarian tissue of the model group were significantly decreased, and the number of follicles in the ovarian tissue of the model group was significantly lower than that of the blank control group. There were different degrees of inflammatory cell infiltration and fibrosis in the interstitial follicle, and the low expression of TNF- 伪 in the model rats was significantly different from that in the blank control group (p 0.05). (2) Bushen Huoxue recipe was low, medium and high dose groups and Liuwei Dihuang decoction group could improve ovary quality and ovarian coefficient of GTW rats in different degree, and could obviously reduce the level of serum FSH in GTW rats. There were significant differences between the model group and the model group (P 0.001), in the high dosage group and the Liuwei Dihuang decoction group, the serum E2 level was increased, and the difference P values were respectively p0.01 (P < 0.05) and 0.05% (P < 0.01), respectively, in the Bushen Huoxue recipe, and in the Bushen Huoxue recipe, the serum E _ 2 level was significantly higher in the high dose group than in the model group. The levels of IL-2 in serum of high dose group and Liuwei Dihuang decoction group were significantly higher than those of model group (all p0.05), and the expression of TNF- 伪 in follicular granulosa cells and oocytes in high dose group was better than that in model group (all p0.05), and the expression of TNF- 伪 in follicular granulosa cells and oocytes in high dose group was better than that in model group. There was statistical difference (P 0.05), and there was no significant difference between the control group and the control group. Conclusion (1) Bushen Huoxue decoction can decrease the level of FSH in serum and increase the level of E2 in serum of GTW rats, indicating that Bushen Huoxue decoction can improve the function of ovarian reserve, upregulate the immune cytokine IL-2 in serum, and antagonize the damage of follicle in ovary. The increased expression of TNF- 伪 in follicular granulosa cells and oocytes may be one of its mechanisms in the prevention and treatment of DOR. (2) both Bushen Huoxue recipe (Bushen Huoxue method) and Liuwei Dihuang decoction (Kidney tonifying method) can improve ovarian reserve function and inhibit follicle injury, but Bushen Huoxue recipe is superior to Liuwei Dihuang decoction in improving the expression of TNF- 伪 in follicular granulosa cells and oocytes. Consistent with the previous study, Bushen Huoxue decoction can improve DORs through various mechanisms.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285.5

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