【作者】 唐丽丽；

【导师】 罗颂平；

【作者基本信息】 广州中医药大学 ， 中医妇科学， 2007， 博士

【摘要】 目的本课题通过建立SD和Wistar大鼠间同种异体卵巢移植模型，观察补肾复方“寿胎丸”对同种异体卵巢移植后急性排斥反应的作用。本实验旨在通过观察寿胎丸对大鼠同种异体卵巢移植模型组织形态学与超微结构的影响、内分泌激素的影响、T淋巴细胞亚群的影响及细胞因子IL-2、IFN-γ的影响，来探讨补肾中药复方“寿胎丸”抗卵巢移植急性排斥反应的机理。方法238只Wistar雌性大鼠随机分为7组，每组大鼠34只。①寿胎丸低剂量组；②寿胎丸中剂量组；③寿胎丸高剂量组；④CsA组：⑤CsA+寿胎丸中剂量组；⑥正常组：⑦模型组。⑥组为正常组，不予手术处理。①、②、③、④、⑤、⑦均建立同种异体卵巢移植模型，受体Wistar大鼠，供体SD大鼠。术后给药，寿胎丸低剂量组以寿胎丸水煎剂，按6.7g／kg／d剂量灌胃，连用28天；寿胎丸中剂量组以寿胎丸水煎剂，按13.4g／kg／d剂量灌胃，连用28天；寿胎丸高剂量组以寿胎丸水煎剂，按26.8g／kg／d剂量灌胃，连用28天；CsA组：以CsA按10mg／kg／d剂量灌胃，连用14天，然后减量，按6mg／kg／d剂量连用14天；CsA+寿胎丸中剂量组同时予CsA和寿胎丸水煎剂，CsA剂量同CsA组，寿胎丸水煎剂剂量同寿胎丸中剂量组；正常组及模型组以生理盐水，按5ml／kg／d剂量灌胃，连用28天。移植术前一日以及移植术后第5—14天，15—28天，29—35天中雌激素分泌最高峰当日取血检测血清E₂、P、FSH。各组于移植术后d7、d14、d21、d28、d35分批处死Wistar大鼠各5、8、8、8、5只。取血、双侧卵巢及脾脏：检测与移植排斥密切相关的血清Th1细胞因子：IL-2、IFN-γ；用流式细胞仪检测脾脏T淋巴细胞亚群CD3⁺、CD4⁺、CD8⁺的比值；光镜下计算各个发育阶段卵泡、黄体的数量，观察间质的变化，以及组织坏死或纤维化程度；电镜下观察移植卵巢超微结构变化。观察记录各组动物的体重、活动、皮毛、进食及粪便情况，比较分析药物的副反应。结果1．移植卵巢组织形态学及超微结构移植术后，各卵巢移植组大鼠动情周期恢复率由高到低依次是：CsA+寿胎丸中剂量组、CsA组、寿胎丸高剂量组、寿胎丸中剂量组、寿胎丸低剂量组、模型组。寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组移植卵巢体积和重量均较正常组卵巢有增加：且均高于模型组，差异有显著性（P＜0.01）。寿胎丸低、中剂量组移植卵巢重量低于寿胎丸高剂量组，差异有显著性（P＜0.05）。CsA+寿胎丸中剂量组大鼠移植卵巢的间质损害程度最轻。寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组的卵巢黄体、原始卵泡、初级生长卵泡、成熟卵泡数目均高于模型组，差异有显著性（P＜0.01）。寿胎丸中、高剂量组、CsA组、CsA+寿胎丸中剂量组的次级生长卵泡数目均高于模型组，差异有显著性（P＜0.01）。寿胎丸低、中剂量组初级生长卵泡数目均低于寿胎丸高剂量组，差异有显著性（P＜0.01）。寿胎丸低、中、高剂量组次级生长卵泡数目相比较，均有显著性差异，（P＜0.01）。CsA+寿胎丸中剂量组的原始卵泡和初级生长卵泡数目显著高于CsA组（P＜0.01）。移植术后21天，模型组卵巢超微结构呈现急性排斥反应的表现。寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组移植卵巢超微结构基本正常，新生毛细血管结构正常。移植术后35天，各组移植卵巢超微结构均呈现褪化现象，但CsA+寿胎丸中剂量组褪化程度较轻。2．血清内分泌激素E₂、P、FSH移植术前，各卵巢移植组血清E₂、P水平均低于正常组，差异有显著性（P＜0.01）。在移植术后5—14天血清E₂、P水平有大幅度升高，与移植前水平相比较，有显著性差异（P＜0.01）。移植术后15—28天，寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组血清E₂、P水平继续升高，均高于模型组，差异有显著性（P＜0.01）。CsA+寿胎丸中剂量组血清E₂、P水平最高，与正常组相比，无显著性差异。寿胎丸低剂量组血清E₂、P水平低于寿胎丸高剂量组，差异有显著性（P＜0.01）。移植术后29—35天，寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组血清E₂、P水平均高于模型组，差异有显著性（P＜0.01）。CsA+寿胎丸中剂量组血清E₂、P水平高于模型组、寿胎丸低、中、高剂量组及CsA组。寿胎丸低剂量组与寿胎丸高剂量组血清P水平相比，有显著性差异（P＜0.05）。移植术前各卵巢移植组血清FSH水平均高于正常组，差异有显著性（P＜0.01）。寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组移植术后FSH水平开始逐渐下降，移植术后5—14天、术后15—28天及术后29—35天血清FSH水平均低于移植前水平，差异有显著性。模型组大鼠移植术后各时间段的血清FSH水平比较，无显著性差异。3．脾脏T淋巴细胞亚群CD3⁺、CD4⁺、CD8⁺含量移植术后7天，各卵巢移植组大鼠脾脏CD3⁺、CD4⁺、CD8⁺含量比正常组明显升高，差异有显著性。移植术后14天，CsA+寿胎丸中剂量组CD3⁺含量较术后7天降低明显，差异有显著性（P＜0.05）。组间比较，寿胎丸中、高剂量组、CsA组、CsA+寿胎丸中剂量组CD3⁺含量均低于模型组，差异有显著性。寿胎丸高剂量组、CsA组、CsA+寿胎丸中剂量组CD4⁺含量较术后7天降低明显，差异有显著性。组间比较，寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组CD4⁺含量均低于模型组，差异有显著性。寿胎丸低、中、高剂量组大鼠脾脏CD8⁺水平高于正常组，差异有显著性（P＜0.05）。移植术后21天，寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组大鼠脾脏CD3⁺、CD4⁺含量继续下降，均低于模型组（P＜0.01）。寿胎丸低、中剂量组大鼠脾脏CD3⁺含量高于正常组，差异有显著性（P＜0.01）。寿胎丸低、中剂量组CD4⁺含量高于正常组、寿胎丸高剂量组、CsA组、CsA+寿胎丸中剂组，差异有显著性。移植术后28天，寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组大鼠脾脏CD3⁺、CD4⁺含量继续降低，均低于模型组（P＜0.01）。寿胎丸低剂量组CD3⁺、CD4⁺含量高于寿胎丸高剂量组、CsA组、CsA+寿胎丸中剂量组及正常组，差异有显著性。CsA组、CsA+寿胎丸中剂量组CD8⁺含量低于模型组，差异有显著性（P＜0.05）。移植术后35天，寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组大鼠脾脏CD3⁺含量、CD4⁺含量仍较低，均低于模型组，差异有显著性（P＜0.01）。寿胎丸低剂量组的大鼠脾脏CD3⁺、CD4⁺含量高于正常组，差异有显著性。各组CD8⁺水平相比，无显著性差异。4．血清细胞因子IL-2、IFN-γ各卵巢移植组大鼠移植术后7天的血清IL-2水平较高，均高于正常组水平，差异有显著性。移植术后14天时各卵巢移植组血清IL-2水平有所下降，寿胎丸中、高剂量组、CsA+寿胎丸中剂量、CsA组血清IL-2水平均低于模型组，差异有显著性。移植术后21天，寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组血清IL-2水平继续下降，均低于模型组水平，差异有显著性。移植术后28天，CsA+寿胎丸中剂量组大鼠血清IL-2水平低于模型组，差异有显著性（P＜0.05）。移植术后35天，各组大鼠血清IL-2水平组间比较，无显著性差异。移植术后7天，各卵巢移植组大鼠血清IFN-γ水平均较正常组升高，差异有显著性。移植术后14天，模型组血清IFN-γ水平较正常组升高，差异有显著性（P＜0.01）。移植术后21天，CsA组、CsA+寿胎丸中剂量血清IFN-γ水平低于术后7天水平，差异有显著性（P＜0.05）。组间比较，模型组血清IFN-γ水平较正常组升高，差异有显著性（P＜0.05）。移植术后28天，寿胎丸低、中、高剂量组、CsA组、CsA+寿胎丸中剂量组血清IFN-γ水平低于术后7天水平，差异有显著性。组间比较，寿胎丸中、高剂量组、CsA+寿胎丸中剂量组、CsA组血清IFN-γ水平低于模型组，差异有显著性。移植术后35天，CsA+寿胎丸中剂量组血清IFN-γ水平低于模型组，差异有显著性（P＜0.05）。结论1．寿胎丸有助于移植术后大鼠动情周期的恢复，与CsA联合应用，协同作用明显。寿胎丸促进移植卵巢的成活，使其重量和体积增加，寿胎丸高剂量组在促进移植卵巢重量增加方面优于寿胎丸低、中剂量组。寿胎丸可以减轻移植卵巢急性排斥反应引起的间质损害程度，与CsA联合应用，有协同作用。寿胎丸有促进移植卵巢黄体及各阶段卵泡发育的作用，尤其是对原始卵泡及初级生长卵泡的发育有更明显的作用，并且存在一定的量效关系。寿胎丸与CsA联合应用，可以减轻移植卵巢超微结构的异常改变。2．寿胎丸可促使移植卵巢恢复内分泌功能，减缓其内分泌功能的丢失，与CsA联合应用，有协同作用；寿胎丸低、中、高剂量间有一定的量效关系。寿胎丸可促使移植卵巢与垂体建立负反馈调节反应。3．寿胎丸抑制了大鼠同种异体卵巢移植模型T淋巴细胞的增殖，尤其是抑制了辅助性T淋巴细胞的增殖，而且寿胎丸低剂量和中剂量的作用弱于寿胎丸高剂量，呈现一定的量效关系。4．寿胎丸与CsA可抑制卵巢移植术后大鼠Th1型细胞因子的分泌，降调血清IL-2、IFN-γ水平，两者有协同作用。寿胎丸可能是通过以上各方面机理来起到抗大鼠同种异体卵巢移植模型急性排斥反应的作用。更多还原

【Abstract】 Objective:To observe the effect and mechanism of ShouTaiWan in treating acute rejection ofovarian allotransplantation in SD rats and Wistar rats. To observe the effect of ShouTaiWanon ovarian histology and morphology, serum endocrine hormone, T lymphocyte subsets,and cytokines interleukin-2（IL-2） and interferon-γ（IFN-γ） of these rats.Methods:238 adult female Wistar rats were randomly divided into seven groups:①lowdose of ShouTaiWan group,②middle dose of ShouTaiWan group,③high dose ofShouTaiWan group,④CsA group,⑤CsA+ShouTaiWan group,⑥normal group,⑦modelgroup. No surgery was performed on the rats in the normal group. Ovarianallotransplantation surgery was performed on the rats of other groups.After surgery, rats in group①were treated with ShouTaiWan decoction at 6.7g/kg/din 28 days. Rats in group②were treated with ShouTaiWan decoction at 13.4g/kg/d in28 days. Rats in group③were treated with ShouTaiWan decoction at 26.8g/kg/d in 28days. Rats in group④received a 14-day course of cyclosporine A （CsA） at 10mg/kg/d, andthen another 14-day course of CsA at 6mg/kg/d. Rats in group⑤were treated withequivalent CsA as group④,and with equivalent ShouTaiWan decoction as group②.Normal group and model group were treated with 0.9% NaCl at 5ml/kg/d in 28 days.Serum estradiol （E₂）, progestogen （P） and follicle stimulating hormone （FSH） ofrecipient rats were measured before Ovarian allotransplantation surgery and in the 5th-14th,15th-28th, 29th-35th days postoperative. The Wistar rats were killed and evaluated at d7,d14, d21, d28 and d35 after transplantation. Number of rat spleen T lymphocyte and itssubsets were determined by flow cytometry. ELISA measured serum IL-2 and IFN-γlevelin recipient rats. The histological and morphological alteration of ovarian grafs was studiedwith microscope. Ovary ultrastructure was dectected by electric microscope. Generalizedcase of rats in every group was observed to analyze the side effect of medicines. Results:1. Histology, morphology and ultrastructure of ovarian graftsRate of rats restoring estrous cycle from high to low was : CsA+ShouTaiWan group,CsA group, high dose of ShouTaiWan group, middle dose of ShouTaiWan group, low doseof ShouTaiWan group, model group.Weight and volume of ovarian grafts in low dose of ShouTaiWan group, middle doseof ShouTaiWan group, high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWan group were significantly higher than those of normal group and modelgroup（P＜0.01）.Numbers of corpus luteum, primordial follicles, primary follicles and graafian folliclesin low dose of ShouTaiWan group, middle dose of ShouTaiWan group, high dose ofShouTaiWan group, CsA group, and CsA+ShouTaiWan group were significantly higherthan those of model group（P＜0.01）. Numbers of primordial follicles and primary folliclesin CsA+ShouTaiWan group was significantly higher than those of CsA group （P＜0.01）.21 days after operation, ultrastructure of ovarian grafts in model group was in accordwith that of acute rejection, ultrastructure of ovarian grafts in low dose of ShouTaiWangroup, middle dose of ShouTaiWan group, high dose of ShouTaiWan group, CsA group,and CsA+ShouTaiWan group was normal, and the structure of newborn capillary vesselwas normal too. 35 days after operation, ovary ultrastructure of CsA+ShouTaiWan groupwas degenerated more slightly than other groups.2. Serum endocrine hormone E₂, P, and FSHThe mean levels of serum E₂ and P in normal group were significantly higher thanthose of other six groups before transplantation （P＜0.01）. 5-14 days after transplantation,the mean levels of serum E₂ and P in every group were significantly higher than thosebefore transplantation （P＜0.01）.15-28 days after transplantation, The mean levels of serum E₂ and P in low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly higher than those of modelgroup（P＜0.01）. The levels of serum E₂ and P in low dose of ShouTaiWan group weresignificantly lower than those of high dose of ShouTaiWan group （P＜0.01）.29-35 days after transplantation, The mean levels of serum E₂ and P in low dose ofShouTaiWan group, middle dosc of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly higher than those of modelgroup（P＜0.01）. The mean levels of serum E₂ and P in CsA+ShouTaiWan group weresignificantly higher than those in low dose of ShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group, CsA group and model group. Thelevel of serum P in low dose of ShouTaiWan group were significantly lower than those ofhigh dose of ShouTaiWan group （P＜0.05）.The mean level of serum FSH in normal group was significantly lower than that ofother six groups before transplantation（P＜0.01）. 5-14,15-18,29-35 days after transplantation,the mean levels of serum FSH in in low dose of ShouTaiWan group, middle dose ofShouTaiWan group, high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWangroup were significantly lower than those before transplantation. The change of serum FSHin model group after operation was not significant.3. Rat spleen T lymphocyte and its subsets7 days after transplantation, The mean levels of spleen CD3⁺, CD4⁺ and CD8⁺ innormal group was significantly lower than those of other six groups.14 days after transplantation, The mean level of spleen CD3⁺ in CsA+ShouTaiWangroup was significantly lower than those in the 7th day after transplantation. The meanlevels of spleen CD3⁺ in middle dose of ShouTaiWan group, high dose of ShouTaiWangroup, CsA group, and CsA+ShouTaiWan group were significantly lower than that ofmodel group. The levels of spleen CD4⁺ in high dose of ShouTaiWan group, CsA group,and CsA+ShouTaiWan group were significantly lower than those in the 7th day aftertransplantation. The levels of spleen CD4⁺ in low dose of ShouTaiWan group, middle doseof ShouTaiWan group, high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWan group were significantly lower than that of model group. The mean levels ofspleen CD8⁺ in low dose of ShouTaiWan group, middle dose of ShouTaiWan group, andhigh dose of ShouTaiWan group were significantly higher than that of normal group（P＜0.05）.21 days after transplantation, the mean levels of spleen CD3⁺ and CD4⁺ in low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly lower than those of modelgroup （P＜0.01）. The levels of spleen CD3⁺ in low dose of ShouTaiWan group and middledose of ShouTaiWan group were significantly higher than that of normal group（P＜0.01）.The levels of CD4⁺ in low dose of ShouTaiWan group and middle dose of ShouTaiWangroup were significantly higher than those of normal group, high dose of ShouTaiWangroup, CsA group, and CsA+ ShouTaiWan group.28 days after transplantation, the levels of spleen CD3⁺ and CD4⁺ in low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly lower than those of model group （P＜0.01）. The levels of spleen CD3⁺ and CD4⁺ in low dose of ShouTaiWan groupwere significantly higher than those in high dose of ShouTaiWan group, CsA group, CsA+ShouTaiWan group and normal group.35 days after transplantation, the levels of spleen CD3⁺ and CD4⁺ in low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly lower than those of modelgroup （P＜0.01）. The levels of spleen CD3⁺ and CD4⁺ in low dose of ShouTaiWan groupwere significantly higher than those of normal group. The difference of spleen CD8⁺between seven groups was not significant.4. Serum cytokines IL-2 and IFN-γ7 days after transplantation, the level of serum IL-2 in normal group was significantlylower than those of other six groups. 14 days after transplantation, the mean level of serumIL-2 in middle dose of ShouTaiWan group, high dose of ShouTaiWan group, CsA group,and CsA+ShouTaiWan group were significantly lower than that of model group. 21 daysafter transplantation, the levels of serum IL-2 in low dose of ShouTaiWan group, middledose of ShouTaiWan group, high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWan group were significantly lower than that of model group. 28 days aftertransplantation, the level of serum IL-2 in CsA+ShouTaiWan group were significantlylower than that of model group （P＜0.05）. 35 days after transplantation, the difference ofserum IL-2 between seven groups was not significant.7 days after transplantation, the level of serum IFN-γin normal group wassignificantly lower than those of other six groups. 14 days after transplantation, the level ofserum IFN-γin model group was significantly higher than that of normal group（P＜0.01）.21 days after transplantation, The levels of serum IFN-γin CsA group and CsA+ShouTaiWan group were significantly lower than those in the 7th day after transplantation（P＜0.05）. 28 days after transplantation, The levels of serum IFN-γin low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly lower than those in the 7thday after transplantation. The levels of serum IFN-γin middle dose of ShouTaiWan group,high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWan group weresignificantly lower than that of model group. 35 days after transplantation, the levels ofserum IFN-γin CsA+ShouTaiWan group were significantly lower than that of modelgroup（P＜0.05）.Conclusion:1. ShouTaiWan helps the recovery of rat estrous cycle, and has obvious synergy combined with CsA. ShouTaiWan raise the survival rate, weight and size of allograftingovaries. High-dose group is superior to middle-dose group and low-dose group.ShouTaiWan can reduce the ovarian interstitial damage because of acute rejection, and hasobvious synergy combined with CsA. ShouTaiWan promote the development of ovarianluteal and various stages of follicle, especially promoting the growth of primordial folliclesand primary follicle. And there is a dose-response relationship. ShouTaiWan, combinedwith CsA, can reduce abnormal changes in the ultrastructure of ovarian grafts.2. ShouTaiWan can restore the endocrine function of ovarian grafts, reduce the loss oftheir endocrine function, and has obvious synergy combined with CsA. There was a certainamount of high-dose-effect relationship. ShouTaiWan can promote the establishment ofnegative feedback response between ovarian grafts and pituitary gland.3. ShouTaiWan inhibits the proliferation of T lymphocytes in allotransplantationmodel, particularly inhibiting the proliferation of auxiliary T lymphocytes, high-dose groupis superior to middle-dose group and low-dose group, showing a dose-responserelationship.4. ShouTaiWan can inhibit secretion of Th1-type cytokines in rats after ovariantransplantation, decrease the levels of serum IL-2 and IFN-γ, and has obvious synergycombined with CsA.ShouTaiWan maybe play the role of preventing acute rejection of allotransplantationin rats through all these mechanisms.更多还原