【作者】 冯翠娥；

【导师】 丛林；

【作者基本信息】 安徽医科大学 ， 妇产科学， 2010， 硕士

【摘要】 第一部分促性腺激素释放激素激动剂对玻璃化冻存兔卵巢组织的影响目的观察注射GnRH-a组和未注射GnRH-a组兔的冻融卵巢组织内各级卵泡的形态、数量、凋亡率有无差异,初步探讨GnRH-a预处理对兔卵巢组织冷冻损伤的影响。方法(1)选择动情周期正常的新西兰雌性大耳白兔18只,随机分成两组:GnRH-a组兔注射GnRH-a,对照组兔不注射GnRH-a,每组各9只。(2)GnRH-a组每15天给予0.25mg/kg醋酸亮丙瑞林(GnRH-a)皮下注射,共两次,30天后手术摘取双侧卵巢,一侧卵巢经处理后立即行玻璃化冷冻(groupA组),另一侧卵巢不冷冻投入4%多聚甲醛中固定(groupB组);对照组母兔每15天给予生理盐水0.3ml皮下注射,共两次,30天后手术摘取双侧卵巢,一侧卵巢经处理后立即行玻璃化冷冻(groupC组),另一侧卵巢不冷冻直接投入4%多聚甲醛中固定(groupD组)。(3)groupA组和groupC组玻璃化冷冻方法相同,均采用改良于Kagawa N [1]的方法。(4)HE染色,组织学分析各组卵巢组织中各级卵泡形态和数量的变化;TUNEL法检测各组卵巢组织中卵泡的凋亡情况。结果(1)组织学观察:groupA、groupB、groupC和groupD组形态正常的卵泡比例分别为71.37%、84.85%、66.57%、82.93%,冻存组正常形态卵泡比例均较新鲜组低(71.37%vs84.85%;66.57%vs82.93%;P<0.05),groupA组正常形态卵泡比例较groupC组高,差异有显著性(71.37%vs66.57%,P<0.05),groupB组正常形态卵泡比例较groupD组高,差异无显著性(84.85%vs82.93%,P>0.05),注射GnRH-a组冻融卵巢组织原始卵泡正常形态卵泡比例较未注射GnRH-a组冻融卵巢组织高(P<0.05)。(2)凋亡情况:GroupA组,GroupB组,GroupC组和GroupD组中Tunel阳性细胞比例分别为13.91%,9.21%,12.58%和8.16%,经冷冻保存后卵泡凋亡率有所增加(13.91%vs9.21%;12.58%vs8.16%,P<0.05),但在各冷冻组之间差异无显著性(13.91%vs12.58%,P>0.05)。结论(1)冻融过程对各级卵泡可造成一定程度的损伤。(2)GnRH-a预处理改变了卵泡池各级卵泡的构成比,增加了原始卵泡比例,有利于卵巢组织增加对冻融过程的耐受。(3)GnRH-a预处理对卵巢组织细胞凋亡无影响,冻融过程诱导卵泡凋亡的发生,可能是冻融损伤的机制之一。第二部分促性腺激素释放激素激动剂预处理对兔卵巢组织自体移植的影响目的初步探讨GnRH-a预处理对冻融兔卵巢组织自体移植后卵巢功能恢复、卵泡生长和血供建立可能产生的影响。方法(1)选取动情周期正常的新西兰雌性大耳白兔22只,随机分成三组:A组(n=8):给予0.25mg/kg醋酸亮丙瑞林皮下注射;B组(n=8):皮下注射生理盐水;C组(n=6):假手术组。(2)A、B组双侧卵巢手术取出后进行玻璃化冷冻(同实验一),4周后一部分自体移植于兔两侧腹股沟区肌肉层内,另一部分用4%多聚甲醛固定用于组织学观察。C组取兔下腹部纵切口,暴露双侧卵巢后即关腹。(3)手术后通过阴道上皮细胞观察各组动物动情周期的恢复时间及恢复率,分别于去势前、去势后4周、移植后第30天对各组动物进行耳缘静脉采血,测定血清FSH和E2水平。(4)移植后30天取出移植物,进行组织学观察。结果(1)手术后观察:A组(GnRH组)有2只,B组(对照组)有1只兔在去势手术后因感染、失血、护理不当等原因死亡,其余兔身体状况良好。(2)阴道上皮细胞观察:A组兔有4只恢复动情周期,动情周期恢复天数平均为20.00±2.16天,B组兔有5只恢复动情周期,动情周期恢复天数平均为15.80±1.30天,A组兔动情周期恢复较B组迟,差异有显著性(P<0.01);(3)血清学观察:A组去势前E2和FSH水平均较B组低,差异有显著性(P<0.05);A、B两组移植后E2浓度均较去势后高,而FSH浓度均较去势后低,差异有显著性(P<0.05);A、B两组卵巢组织移植后E2浓度显著低于C组假手术组(P<0.05),而FSH浓度显著高于C组(P<0.05),A、B两组移植后E2和FSH浓度比较,差异无显著性(P>0.05)。(4)组织学观察:卵巢组织冷冻复苏后,GnRH组与对照组卵巢组织比较,正常形态卵泡比例GnRH组较对照组高,差异有显著性(P<0.05);移植物内卵泡比较,GnRH组与对照组正常形态卵泡比例比较无显著性(P>0.05),正常形态卵泡比例均较移植前显著降低(P<0.01),仅见少数原始卵泡和初级卵泡。结论(1)腹股沟区大腿内侧肌肉层作为移植部位,简单易行,便于监测;(2)各冻存组的卵巢自体移植后可以存活并具有内分泌功能;(3)GnRH-a组卵巢功能恢复所需要的时间更长,可能是由于GnRH-a抑制了卵泡启动;(4)GnRH-a预处理不能减少移植组织缺血-再灌注损伤。更多还原

【Abstract】 PartⅠExperimental Study on the Effect of the GnRH-a on Vitrification of Rabbit Ovarian TissueObjective:Observe differences of the injection of GnRH-a group and non-injection of GnRH-a group of the frozen-thawed ovarian tissue in rabbits at follicle morphology, quantity and apoptosis rate, to explore the possible effect of GnRHa pretreatment on rabbit ovarian tissue cryopreservation.Methods: (1) 18 female New Zealand white rabbits with normal estrous cycle were randomly divided into two groups:GnRH-a group(n=9) and non-injection of GnRH-a group (n=9).(2) GnRH-a group:injection of 2.5mg GnRH-a was treated every 15 days twice before ovarectomy. Then after ovarectomy ,one side of the ovary was vitrificated immediately after treatment(group A), the Other side of the ovary is not be frozen but drop into 4% paraformaldehyde fixed as control(group B); non-injection of GnRH-a group: injection of 0.3ml saline every 15 days,30 days later the ovarectomy was treated , one side of the ovary was vitrificated immediately after treatment(group C), the Other side of the ovary was not be frozen but drop into 4% paraformaldehyde fixed as control(groupD).(3) groupA and groupC have the same vitrification method using modified Noriko.K’s . (4) Histologically examined the morphological of follicles and qauntity of various stage follicles;Detected the apoptosis positive follicles in ovarian tissue by TUNEL.Result: (1) Histological examination: the proportion of morphologically normal follicles in groupA, groupB, groupC and groupD were 71.37%, 84.85%, 66.57% and 82.93%, the proportion of morphologically normal follicles cryopreserved group were lower than those of fresh group(71.37%vs84.85%;66.57%vs82.93%;P<0.05), group A was significantly higher than group C on the proportion of morphologically normal follicles(71.37%vs66.57%,P<0.05);group B was higher than group D on the the proportion of morphologically normal follicles with no significant statistical difference(84.85%vs82.93%,P>0.05),The proportion of primordial follicle in GnRH-a group was higher than the non-injection GnRH-a group(P<0.05). (2) The percentages of TUNEL-positive follicles of group A, groupB, groupC and groupD were 13.91% ,9.21% ,12.58% and 8.16%. The percentages of TUNEL-positive follicles of frozen group was significantly higher than fresh group (13.91%vs9.21% ;12.58%vs8.16%,P<0 05). The percentages of TUNEL-positive follicles was not significant between groupA and groupC (P> 0.05).Conclusion: (1) Freezing and thawing process could cause a certain degree of damage on follicles;(2) The GnRHa pretreatment changed the proportion of the follicle pool with increasing percentage of primordial follicle, It is beneficial for ovarian tissue to diminish the frozen-thawed injury.(3) The freezing and thawing process induced apoptosis occurred. The GnRH-a pretreatment did not influence the apoptosis. PartⅡExperimental Study on the Effect of the GnRH-a on Autotransplantation of Rabbit Ovarian TissueObjective: To explore the possible effect of GnRH-a pretreatment on cryopreserved rabbit ovarian tissue after autotransplantation.Methods: (1) 22 female New Zealand white rabbits with normal estrous cycle were randomly divided into 3 groups: group A(n=8): injection of 2.5mg GnRH-a was treated every 15 days twice before ovarectomy;group B(n=8): injection of saline was treated as control;group C(n=6): Sham-operated group.(2) group A and group B were operated to remove both ovaries and then vitrification (same as partⅠ), After 4 weeks, a part of ovarian pieces after freezing and thawing were autografted at bilateral superficial muscles of groin area.Another part were fixed with 4% paraformaldehyde for histological examination. group C :Sham-operated group.(3) observed the restoration rate and the interval of regular oestrous cyclic by vaginal smear and detected the serum level of E2 and FSH .Result: (1) There were two rabbits in group A and one rabbit in group B died for infections, blood loss or nursing improper,the remaining rabbits in good health.(2) Observation of vaginal smear: there are four rabbits in group A resume estrous cyclic and there are five rabbits in group B resume estrous cyclic, Interval of regular estrous cyclic in group A(20.00±2.16 days)was significantly longer than group B(15.80±1.30 days)(P<0.01) (3) The serum level of E2 before ovarectomy was higher than those after transplantation (P <0.05) and the FSH level was lower than those after transplantation (P <0.05), The level of E2 after transplantation in groupA and groupB was significantly lower than groupC and the level of FSH after transplantation in groupA and groupB was significantly higher than groupC (P <0.05). The level of E2 and FSH between groupA and groupB after transplantation was not significant. (P> 0.05). (4) Histological examination: After freezing and thawing,the proportion of normal morphology follicle in GnRH-a group was higher than those in control,the difference between two groups was significant(P<0.05); the proportion of normal morphology follicle in grafts was not significant between GnRH-a group and control. (P>0.05), the proportion of normal morphology follicle in grafts was significantly lower than before transplantation (P <0.01).only a small number of primordial follicles and primary follicles were exist in grafts.Conclusion: (1) frozen-thawed ovarian tissue after autotransplantation could survival with the hormone secretion ability. (2) superficial muscle of groin area was a good implantation site for its convenience,plentiful blood supplying and easy monitoring.(3) the ovarian function in GnRH-a group need more time to recover, May be due to GnRH-a inhibit the follicle growth; (4) The GnRH-a pretreatment did not decrease the Ischemia - reperfusion injury.更多还原