【作者】 王琳；

【导师】 徐键；

【作者基本信息】 浙江大学 ， 妇产科学， 2013， 硕士

【摘要】 背景：随着现代肿瘤治疗方案的发展,年轻女性肿瘤病人的生存率大大提高。但是,手术及其放化疗可引起卵巢内分泌功能及生育能力的丧失。如何保留年轻未育女性肿瘤患者的生殖功能越来越受到医学界的关注。其中,卵巢组织的异种移植是当前研究保留生殖能力的重要途径之一。但卵巢组织的移植往往会面临移植早期缺血缺氧对卵巢组织功能的损伤,导致卵泡消失,基质纤维化。因此,在移植早期使用促血管生成药物,促进新生血管生成,对于挽救卵巢组织功能有重要意义。血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)都是血管生成因子家族的一员,将其应用于人卵巢组织异种移植兔模型中的促血管生成还未见报道。因此,研究VEGF和bFGF在人卵巢组织异种移植早期血管新生中的作用,对于提高卵巢组织移植效果有重要意义。目的：研究VEGF和bFGF在人卵巢组织异种移植兔模型中移植早期促进血管新生,降低细胞凋亡,保留卵巢组织功能中的作用。方法：选择25只雌性新西兰大白兔作为人卵巢组织异种移植的受体,经去势处理后随机分配到5组中。新鲜人卵巢组织移植到兔背肌内,按以下不同处理分成五组：(A)卵巢组织移植前VEGF(50ng/ml)孵育+移植后连续7天皮下注射VEGF(50ng/ml)1ml;(B):卵巢组织移植前bFGF(100ng/ml)孵育+移植后连续7天皮下注射bFGF(100ng/ml)1ml;(C):卵巢组织移植前VEGF(50ng/ml)+bFGF(100ng/ml)孵育+移植后连续7天皮下注射VEGF(50ng/ml)+bFGF(100ng/ml)1ml; D组：卵巢组织移植前生理盐水孵育+移植后连续7天皮下注射生理盐水1m1；E组：对照组,不做处理。移植前3天起每天使用FTY720(2mg/kg)进行免疫抑制。移植后4周,A、B、C、D组开始隔日肌注尿促性腺素(HMG)10IU至移植后6周,所有移植物于移植后6周全部回收。采用放免技术检测兔外周血雌二醇(E2)水平；6周后卵巢组织回收率及肉眼形态；回收后移植物HE染色观察组织形态及纤维化坏死率；移植物内新生血管生成情况及细胞增殖情况分别用CD31和Ki-67进行免疫组化方法(En Vision二步法)检测；细胞凋亡用TUNEL方法检测。结果：1、使用VEGF和bFGF能更好地维持卵巢组织分泌雌二醇的能力；2、单独使用VEGF以及联合使用VEGF和bFGF都能较好地降低卵巢组织基质的纤维化和坏死率；3、单独使用VEGF或bFGF,以及联合使用两者可以提高移植卵巢组织中CD31的表达；4、移植后Ki-67广泛表达于各组卵巢组织中的基质细胞,联合使用VEGF和bFGF可见Ki-67零星染色于移植后的始基卵泡上的颗粒细胞；5、联合使用VEGF和bFGF能显著降低移植后卵巢组织中细胞的凋亡率。结论：1、在卵巢移植早期给予足量持续的促血管生成因子,能改善移植效果；2、VEGF与bFGF,特别是两药联合能够提高移植早期血供,降低细胞凋亡,减少基质纤维化,保存较多的卵巢功能。更多还原

【Abstract】 Background:Advances in cancer diagnosis and treatment have greatly enhanced life expectancy of young women who suffer from malignant diseases, but chemo-and radiotherapy often result in premature ovarian failure (POF), losing both endocrine and reproductive functions, because of the massive destruction of the ovarian reserve. How to preserve the reproductive function of young female cancer patients without kids gets more and more attention of the medical. The human ovarian tissue xenotransplantation is one of the most important ways to keep reproductive capability. Ovarian tissue transplantation often face the ischemic injury to ovarian tissue function at early time of transplant, leading to follicle disappear, stroma fibrosis. Therefore, using angiogenesis drugs in the early transplantation promotes new angiogenesis, it has important significance to save ovarian tissue function. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the member of angiogenesis factor family, the application in ovarian tissue xenograft rabbit model to promoting angiogenesis has not been reported. To investigate the benefits of treatments with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) with regard to the outcome of fresh human ovarian tissue xenotransplantation in rabbit has important significance.Objective:To investigate the benefits of early treatments with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) with regard to the outcome of fresh human ovarian tissue xenotransplantation in rabbit.Method:Fresh human ovarian tissue xenotransplanted into the back muscle of25castrated female New Zealand rabbits for6weeks with the immunosuppression of FTY720(2mg/kg/d). Rabbits were randomly divided into five experimental groups:(A) Pretransplantation grafts treatment with human recombinant VEGF:rabbits accepted hypodermic injection with lml human recombinant VEGF (50ng/ml) at transplant site7days after transplantation;(B) Pretransplantation grafts treatment with human recombinant bFGF:rabbits accepted hypodermic injection with lml human recombinant bFGF (100ng/ml) at transplant site7days after transplantation;(C) Pretransplantation grafts treatment by VEGF+bFGF:rabbits accepted hypodermic injection with lml VEGF (50ng/ml)+bFGF (100ng/ml) at transplant site7days after transplantation;(D)Pretransplantation grafts treatment by normal saline:rabbits accepted hypodermic injection with equivalent normal saline at transplant site7days after transplantation;(E) Pretransplantation grafts no treatment:rabbits received nothing as control. Four weeks after transplantation, human menopausal gonadotropin (HMG)10IU was administered every second day in group A, group B, group C and group D for2weeks. Graft survival was assessed by estradiol level, ovarian tissue recovery, histological analysis, TUNEL assay and immunohistochemical staining for Ki-67and CD31expression.Results:1、VEGF and bFGF could better extend the endocrine ability of ovarian tissue;2、Single use VEGF and combined use of VEGF and bFGF could reduce ovarian tissue stromal fibrosis and necrosis rate well;3、Single use VEGF or bFGF, and use a combination of both could improve ovarian tissue CD31expression;4、Ki-67expression dispersed in ovarian stroma after transplantation in five groups, and primordial follicles after transplantation showed a few Ki-67dyeing in granular cells in the group of the combination of VEGF and bFGF;5、The combination of VEGF and bFGF could significantly reduce ovarian tissue cell apoptosis rate.Conclusions:1、The use of angiogenesis factor at early time of ovarian transplantation could improve the effect of transplantation;2、VEGF and bFGF, especially two drug combination could improve blood supply and reduce early transplantation cell apoptosis, reduce stroma fibrosis and save more ovarian function.更多还原