【作者】 王浩；

【导师】 李翠萍；

【作者基本信息】 湖南中医药大学 ， 中医妇科学， 2012， 博士

【摘要】 目的：通过观察益肾调周法对卵巢早衰造模大鼠动情周期的影响；对卵巢甾体类固醇激素生成能力(E2、P、T)的影响；对卵巢储备能力的测评(FSH、LH、INHB、AMH);对体液免疫(AOAB)和神经免疫(β-EP)的调节；对卵巢颗粒细胞凋亡相关蛋白表达的干预(Bcl-2、Fas、Fas-I);对细胞免疫相关细胞因子(VEGF、TNF-α、 INF-γ)的调节;血清中血脂的变化(TC、TG)以及对卵巢组织形态学观察等项目,从神经-内分泌-免疫网络的整体性和协调性方面探讨该法干预卵巢早衰的机制,探讨其量效关系,为中医药防治POF的方法提供科学的依据。方法：选SPF级健康雌性8周龄SD大鼠,通过阴道脱落细胞学涂片,选出有动情周期为受试动物,除空白组(A)外,其余大鼠均给予注射环磷酰胺致POF模型。环磷酰胺模型大鼠随机分为即模型对照组(B)、克龄蒙对照组(C)、滋肾填精汤高剂量组(D1)、滋肾填精汤低剂量组(D2)、温肾填精汤高剂量组(E1)、温肾填精汤低剂量组(E2)、益肾调周法高剂量组(F1)、益肾调周法低剂量组(F2)、益肾调周法高-低剂量组(G1)、益肾调周法低-高剂量组(G2),A,B组给予每日灌胃两次蒸馏水,中药各组实验当日冷开水配置所需动物灌胃浓度,低剂量,高剂量分别相当于临床等效药量的2、4倍,克龄蒙对照组采用相当于临床等效药量的4倍,每日灌胃一次。各组均连续用药20天。用药期间每日检测一次阴道脱落细胞,并记录。于实验末次给药后,实验大鼠断头采血,取血清。采用放射免疫计数仪测定血清中E2、T、P值;用酶联免疫测定试剂盒测定血清中FSH、LH、β-EP、 AOAB和VEGF、TNF-α、IFN-γ的表达;采用双抗体夹心酶联免疫吸附技术，检测AMH, INHB;氧化酶法测定血清TC、TG水平;取血后将大鼠,分离出下丘脑,酶联免疫测定试剂盒测定下丘脑β-EP水平;断头后迅速解剖同一侧卵巢,固定,脱水,石蜡包埋,连续切片,染色,最大剖面光镜下观察卵巢的各级卵泡数及黄体数的变化及病理改变;免疫组化法观察Bcl-2、Fas、Fas-L的蛋白表达。结果：1.益肾调周法对动情周期的影响：益肾调周法高-低剂量(G1)能够有效改善POF模型大鼠的紊乱的动情周期,恢复正常周期。2.益肾调周法对甾体类固醇激素的影响：益肾调周法高-低剂量组(G1)的血清E2,P值显著升高,而T值显著下降。3.益肾调周法对卵巢储备能力的影响：益肾调周法高-低剂量组(G1)血清FSH, LH的水平显著降低,而血清AMH, INHB的表达增强。4.益肾调周法对卵巢细胞免疫相关因子的影响：益肾调周法高-低剂量组((G1)血清TNF-α、FN-γ的表达显著降低,但是血清VEGF的表达增强不显著。5.益肾调周法对卵巢颗粒细胞凋亡相关蛋白表达的影响：益肾调周法高-低剂量组(G1)卵巢颗粒细胞Bcl-2蛋白的阳性表达增强显著,Fas蛋白的表达增强,而使Fas-L蛋白表达减弱。6.益肾调周法对体液免疫和神经免疫的影响：益肾调周法高-低剂量组(G1)血清AOAB浓度显著降低,血清和下丘脑的β-EP的含量显著提高。7.益肾调周法对血脂的影响：益肾调周法高-低剂量组(G1)血清中TC和TG的水平显著降低。8.益肾调周法对卵巢形态学的影响：益肾调周法高-低剂量组(G1)卵巢中初级卵泡,生长卵泡以及黄体的数量显著增加,炎性侵润减少。结论：1.腹腔注射环磷酰胺,符合POF的诊断标准,成功造模。2.益肾调周法高-低剂量(G1)有效改善紊乱的动情周期。3.益肾调周法高-低剂量组(G1)能够调节卵巢颗粒细胞的甾体类固醇激素的生成并且能够提高卵巢的储备能力。5.益肾调周法高-低剂量成功参与细胞免疫调节和体液免疫调节,加强对卵巢功能的神经调节,益肾调周法高-低剂量(G1)有效抑制卵巢细胞凋亡的发生。8.益肾调周法高-低剂量组(G1)有效降低血脂并促进卵泡发育。因此,益肾调周法能够通过调整NEI网络的多个环节以实现干预POF的综合效应。更多还原

【Abstract】 Objective:Observation the effect of Yishen tune-periods method to oestrous cycle in POF model rats and ovarian steroid hormone generating capacity in POF model rats (E2、P、T); Observation the effect of Yishen tune-periods method to ovarian reserve capacity (FSH、LH、 INHB、AMH); Observation the moderate effect of Yishen tune-periods method to humoral immunity (AOAB) and neuroimmunology (P-EP); Observation the effect of Yishen tune-periods method to the apoptosis related proteins of granule cells (Bcl-2、Fas、Fas-1); Observation the moderate effect of Yishen tune-periods method to cellular immune related factors (VEGF、TNF-α INF-γ); Observation the change of Yishen tune-periods method to serum lipid (TC、TG) and Observation the effect of Yishen tune-periods method to ovarian morphology,in order to discuss interaction mechanism between POF and Yishen tune-periods method considering integrity and coordination of nerve-endocrine-immune network,and dose-effect relationship,it can provide the scientific basis in prevention and treatment POF with Chinese medicine.Method:choose SPF female healthy8weeks SD rats, the vaginal exfoliated cytology of each rat was inspected to elect rats with normal estrous cycle for animal subjects, except the blank group (A), the rest of animal subjects are given by the injection of cyclophosphamide to establish POF model. POF model rats were randomly divided into namely model group (B),Climen group (C), Zishentianjing soup high-dose group (D1), Zishentianjing soup low-dose group (D2),Wenshentianjing soup high-dose group (E1),Wenshentianjing soup low-dose group(E2),Yishen tune-periods method high-dose group (F1),Yishen tune-periods method low-dose group (F2), Yishen tune-periods method high-low dose group (G1) and Yishen tune-periods method low-high dose group (G2). A, B group were poured distilled water into stomach twice daily, each Chinese traditional medicine group was poured into stomach with the cold water for required concentration, low dose, high doses were equivalent to the clinical equivalent quantity2,4times respectively, Climen group (C) is equivalent to the clinical equivalent4times of quantity, each group was poured once daily, Consecutive medication last20days. The vaginal exfoliated cytology of each rat was inspected and record.After the last medication, all rats were taken blood by decollation.after doing it,measure level of E2、P、T in serum using calculating instrument of radio-immunity method; detect level of FSH、LH、β-EP、AOAB and expression of VEGF、TNF-α IFN-yin serum using ELISA; detect level of AMH, INHB in serum using double antibody sandwich of ELISA; measure level of TC、TG in serum using CHO. Decollation to separate hypothalamus and measure content of β-EP with hypothalamus using ELISA;spaying and fixation with paraffins,slicing,red dying to observe quantity of follicle at all levels and pathological change of follicle;measure protein expression of Bcl-2、Fas、Fas-L in serum using immunohistochemical methodResult:1. The effect of Yishen tune-periods method to oestrous cycle:Disorder oestrous cycle was improved in Yishen tune-periods method high-lowdose group (G1) dramatically.2. The effect of Yishen tunc-periods method to ovarian steroid hormone generating capacity:The level of E2, P in serum of Yishen tune-periods method high-lowdose group (G1) was elevated dramatically and the level of T in same group was reduced dramatically.3. The effect of Yishen tune-periods method to ovarian reserve capacity:The level of FSH、LH in serum of Yishen tune-periods method high-low dose group (G1) drops dramatically,the expression of INHB、 AMH in serum was strengthened apparently.4. The moderate effect of Yishen tune-periods method to cellular immune related factors:The expression of TNF-αand INF-ydrops apparently in serum of Yishen tune-periods method high-low dose group (G1), and the expression of VEGF are strengthened in same group indistinctively.5.The effect of Yishen tune-periods method to the apoptosis related proteins of granule cells:Not only the expression of Bcl-2in serum of Yishen tune-periods method high-low dose group (G1) was strengthened distinctively, but also Fas did, in the contrary, the expression of Fas-1drops apparently.6.The moderate effect of Yishen tune-periods method to humoral immunityand neuroimmunology:The concentration of AOAB in serum of Yishen tune-periods method high-low dose group (G1) drop apparently,and the concentration of (3-EP in serum and hypothalamus are elevated apparently in same group.7. The change of Yishen tune-periods method to serum lipid:The level of both TC and TG drop apparently in serum of Yishen tune-periods method high-low dose group (G1).8.The effect of Yishen tune-periods method to ovarian morphology:The quantity of primary follicle,growing follicle and corpus luteum are incr-eased apparently in ovary of Yishen tune-periods method high-low dose group (G1),and inflammatory assault embellish are redused apparently in same group.Conclusion:1. It is succeeding by the injection of cyclophosphamide to establish POF model.2. Yishen tune-periods method with high-low dose can improve the disorder oestrous cycle.3. Yishen tune-periods method with high-low dose can adjust ovarian steroid hormone generating capacity.4. Yishen tune-periods method with high-low dose can improve ovarian reserve capacity.5. Yishen tune-periods method with high-low dose can participate in immune adjustment and follicle growth.6. Yishen tune-periods method with high-low dose can control the apoptosis of ovarian granulose cell.7. Yishen tune-periods method with high-low dose can weaken immune response and enhance neuroregulation act on ovary.8. Yishen tune-periods method with high-low dose can reduce the contect of serum lipid.9. Yishen tune-periods method with high-low dose can encourage follicular development.Therefore, Yishen tune-periods method can adjust the NEI network through multiple links in order to realize the comprehensive effect on the intervention of POF.更多还原