家兔卵巢组织冷冻保存、体外培养及移植的研究
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摘要
第一部分4种冷冻复苏方法对家兔卵巢组织形态学及卵母细胞PCNA表达的影响
目的:
观察4种冷冻复苏方法对家兔卵巢组织形态学及卵母细胞PCNA表达的影响,探讨适宜的卵巢组织冷冻复苏方案。
方法:
将12只健康日本大耳白家兔随机分为4组,分别采用PROH(PROH组)及DMSO(DMSO组)慢速程序化冷冻和DMSO+PROH(玻璃化组1)及DMSO+EG(玻璃化组2)玻璃化冷冻方法冻存家兔卵巢组织,液氮保存一月后复苏,分别行HE染色及免疫组化染色,观察冷冻复苏后卵巢组织结构和各级卵泡形态学的改变及始基和初级卵泡卵母细胞PCNA表达的变化。
结果:
1. 4种冷冻复苏方法使家兔卵巢组织始基卵泡和初级卵泡的形态正常率分别下降为80.08%和55.00%、70.53%和52.94%、67.55%和47.06%及69.65%和55.56%而其相应新鲜对照组始基卵泡和初级卵泡的形态正常率分别为90.14%和86.05%、91.45%和89.29%、91.76%和84.62%及92.15%和89.47%,差异均有显著性(P<0.05);PROH组始基卵泡形态正常率最高,达80.08%,与DMSO组(70.53%)、玻璃化组1 (67.55%)、玻璃化组2 (69.65%)比较,差异均有显著性(P<0.05)。各冷冻组合并后计算始基卵泡的形态正常率为72.70%,初级卵泡的形态正常率为52.77%,二者比较,差异有显著性(P<0.05)。4个冷冻组均可见卵巢组织结构受损的表现,镜下可见部分卵泡与周围间质细胞分离,间质细胞连接变得疏松,排裂紊乱,有裂隙形成。
2.免疫组化染色结果显示冷冻组中PROH组始基及初级卵泡卵母细胞PCNA的阳性率最高达76.98%,玻璃化组2次之,为72.44%,与新鲜对照组比较(78.04%)差异无显著性(P>0.05),而DMSO组(58.97%)及玻璃化组1(48.00%)PCNA阳性率较新鲜对照组明显降低(P<0.05)。
结论:
1. 4种冷冻复苏方法均对家兔卵巢组织结构造成一定程度的损伤,使各级卵泡的形态正常率明显下降,出现间质受损的表现。
2. PROH慢速程序化冷冻法明显优于DMSO慢速程序化法及玻璃化法,较适合卵巢组织中始基卵泡的保存。
3.冻存卵巢组织对初级卵泡的影响大于始基卵泡。
4. PROH慢速程序化冷冻及DMSO+EG玻璃化冷冻能较好地保持家兔卵巢组织中始基及初级卵泡卵母细胞的增殖活性。
第二部分慢速程序化冷冻及玻璃化冷冻方案对家兔卵巢组织超微结构、ER和MHC-Ⅱ类抗原的表达及卵泡细胞增殖活性的影响
目的:
观察PROH慢速程序化冷冻及DMSO+EG玻璃化冷冻对家兔卵巢组织超微结构、ER和MHC-Ⅱ类抗原表达及卵泡细胞增殖活性的影响,判断复苏后卵巢组织抗原性和免疫原性是否发生改变,为今后卵巢组织移植及体外培养的研究奠定基础。
方法:
1、将健康日本大耳白家兔9只随机分为3组,1组为新鲜对照组,另2组为冷冻组。冷冻组分别采用PROH慢速程序化冷冻及DMSO+EG玻璃化冻存方案冻存家兔卵巢组织。
2、复苏后透射电镜观察两种冷冻方法对家兔卵巢组织超微结构的影响。
3、SP免疫组化法观察两种方法对家兔卵巢组织ER及MHC-Ⅱ类抗原表达的影响。
4、机械性分离新鲜组及冷冻组复苏的组织块,分离下的卵泡按直径的大小再分为:小OGC组,直径100~150μm,有2~5层颗粒细胞包裹的小次级卵泡;大OGC组,直径200~300μm,有5层以上颗粒细胞包裹的大次级卵泡。每组分别取20个形态正常的OGC,行3H标记的胸腺嘧啶核苷掺入试验。
结果:
1、PROH组始基卵泡卵母细胞部分线粒体肿胀,呈空泡状;部分线粒体形态尚正常;细胞核形态正常,核膜完整,界线清晰;颗粒细胞核膜完整,胞质内线粒体形态正常,细胞间缝隙连接完好。DMSO+EG组,始基卵泡卵母细胞线粒体肿胀,细胞器崩解;细胞形态尚可,边界清晰,核膜完整。周围颗粒细胞内线粒体尚可,间质细胞线粒体肿胀明显,呈空泡状。
2、免疫组化结果显示,两冷冻组复苏后家兔卵巢组织ER的表达与新鲜对照组比较,无明显变化(P>0.05)。DMSO+EG玻璃化冷冻组MHC—Ⅱ类抗原的表达与新鲜对照组比较明显下降(P<0.05),PROH组无明显变化(P>0.05)。.
3、两冷冻组复苏后小OGC的cpm值与新鲜对照组比较,无明显差异(P>0.05),而大OGC的cpm值较新鲜对照组明显下降(P<0.05)。
结论:
1. PROH慢速程序化冷冻对家兔卵巢组织超微结构的影响小于DMSO+EG玻璃化冷冻。
2.两种冷冻复苏方法,对家兔卵巢组织ER的表达均无明显影响。
3. DMSO+EG玻璃化冷冻复苏明显降低了卵巢组织的免疫原性。
4.两种冷冻复苏方法对形态结构完整的小OGC增殖活力无明显影响,这些卵泡体外生长和成熟,可能更容易成功。多层颗粒细胞包裹的大OGC即便形态正常,其增殖活性也明显受到抑制。
第三部分家兔卵巢组织冷冻保存后体外培养的研究
目的:
探讨适宜的卵巢组织体外培养方法。
方法:
1.无菌条件下取新鲜家兔卵巢组织,复苏PROH慢速程序化冷冻保存的家兔卵巢组织,分别采用部分分离培养及组织块培养方法体外连续培养14d,作为新鲜部分分离培养组、新鲜组织块培养组、冷冻部分分离培养组、冷冻组织块培养组。
2.每隔1天倒置显微镜下观察培养情况,并收集组织培养液500μl待测E2,同时补入新鲜培养液500μl。采用酶免法检测各组E2分泌量的变化。
培养14d结束时机械性分离各组卵巢组织,将形态结构完整,颗粒细胞连续,折光性好的大OGC(直径200~300μm,有5层以上颗粒细胞包裹的大次级卵泡)和小OGC(直径100~150μm,有2~5层颗粒细胞包裹的小次级卵泡)每组分别各取20个,进行3H标记的胸腺嘧啶核苷掺入试验。
结果:
1.倒置显微镜下新鲜及冷冻部分分离培养组均可见单层颗粒细胞包裹的小卵泡,连续培养14d,未见明显生长,卵泡内出现黑色颗粒,有退化坏死的迹象。
2.新鲜及冷冻部分分离培养组,培养14d后机械性分离卵巢组织均可见窦卵泡形成,进一步分离得到GV期卵子,未得到MⅡ期卵子。
3.新鲜及冷冻组织块培养组,14d培养结束时未见到有明显窦腔的卵泡形成。
4.各培养组E2分泌量的变化:新鲜组织块组,第6天E2分泌量达高峰,第8天开始逐渐下降;冷冻组织块组E2分泌量持续下降;而新鲜及冷冻部分分离培养组E2分泌持续上升。
5. 3H标记胸腺嘧啶核苷掺入试验结果:新鲜及冷冻部分分离培养组和新鲜组织块组所得到的小OGC其cpm值均无明显差异(P>0.05),而冷冻组织块得到小OGC的cpm值较以上各组明显降低(P<0.05)。
6.冷冻组织块组培养结束时,未得到形态正常的大OGC。新鲜及冷冻部分分离培养所得到的形态正常的大OGC其cpm值无明显差异(P>0.05),而新鲜组织块组所得到的大OGC的cpm值较上两组明显下降(P<0.05)。
结论:
1.部分分离法体外培养新鲜及冷冻的家兔卵巢组织明显优于组织块培养法。体外培养14d,前者培养液中E2呈上升趋势,有窦卵泡形成;后者随培养时间的延长E2呈下降趋势,未见窦卵泡形成。
2.新鲜及冷冻组织部分分离培养所得到的形态正常的大小OGC均有良好的增殖活性,说明PROH慢速程序化冷冻较好地保存了存活卵泡的体外发育潜能。
3.该培养系统不适合家兔卵巢组织始基及初级卵泡的体外培养。
4.冷冻卵巢组织更不适合组织块培养,所得到的形态正常的小OGC增殖活性差,未得到形态正常的大OGC。
第四部分家兔卵巢组织移植的研究
目的:
探讨适宜的卵巢组织移植部位,观察移植成活物的生理功能。
方法:
1.选健康大耳白家兔20只随机分为对照组(5只)、去势组(5只)、移植组(10只)。
2.移植组选宫旁阔韧带为移植部位,将切下的双侧卵巢去除髓质,切成1×1×1mm3大小的组织块植入左侧阔韧带的浆膜下,移植术后隔日肌注HMG 10IU/天,至术后半月。去势组切除双侧卵巢。
3.术后第2天开始隔日行阴道脱落细胞学涂片,观察雌激素水平的变化,至术后一个半月。
4.术后一月开始,每只家兔每隔5日,连续3次取耳缘静脉血2ml,酶免法测定血清E2水平。
5.术后1个半月再次开腹,取对照组子宫内膜及卵巢;取去势组子宫内膜;取移植组的成活移植物及子宫内膜,做HE染色行组织形态学观察。
6.移植成活的卵巢组织及对照组卵巢组织行SP免疫组化染色,观察PCNA及VEGF表达的变化。
结果:
1.阴道脱落细胞学涂片结果:对照组阴道细胞涂片显示细胞大,为多边型,胞浆稀薄透明,核小固缩,为雌激素作用的表现;去势组术后4—6天开始,阴道细胞涂片显示细胞呈圆形,核大圆形或椭圆形,核浆比增加,为低雌激素水平的表现;移植组1只术后死亡,剩余家兔术后4—6天开始阴道细胞涂片显示低雌激素水平,7只家兔术后10—20天重新恢复雌激素作用的表现,另2只未恢复。
2. E2测定结果:移植组7只家兔术后1个月血清E2水平恢复正常,与正常对照组比较,差异无显著性(P>0.05);另2只家兔E2未恢复正常。去势组家兔E2水平明显降低,与对照组比较,差异有显著性(P<0.05)。
3. HE染色子宫内膜形态学观察:去势组:子宫内膜上皮细胞由单层柱状细胞组成,腺体数量少,形态萎缩,间质细胞排裂紧密。移植组:E2水平恢复的7只家兔,子宫内膜表层细胞呈锯齿状,由假复层柱状细胞构成,腺体数量多,排列紧密,形态饱满,间质细胞排列疏松,同对照组;另2只E2低水平的家兔子宫内膜同去势组。
4.移植组卵巢组织形态学观察:移植组6只阔韧带处找到成活的卵巢组织,外观见移植的卵巢组织被纤维组织及脂肪组织包裹,HE染色镜下可见形态正常的各级卵泡及大量新生血管。
5.免疫组化结果:移植组卵巢组织始基及初级卵泡卵母细胞PCNA表达阳性率为78.67%与对照组(77.55%)比较无明显差异(P>0.05),VEGF的表达部位及表达强度与对照组比较也无明显差异(P>0.05)。
结论:
1.子宫旁阔韧带处血供丰富,内环境接近卵巢生理状态,移植手术操作简便,较适合卵巢组织移植,结合移植术后应用促性腺激素,获得了较高的移植成活率。
2.宫旁阔韧带处移植成活的卵巢组织有很好的内分泌功能,能分泌生理剂量的E2,对阴道上皮及子宫内膜均能发挥良好的作用。
3.移植成活的卵巢组织卵母细胞有良好的增殖活性,并能很好地分泌VEGF,对维持血管的生成及正常结构有重要意义。
PartⅠ:Effects of four kinds of different frozen-thawed methods on the morphology of rabbit ovarian tissue and the PCNA expression of the oocytes in the primordial and the primary follicles
Objective:
To observe the effects of four kinds of different frozen-thawed methods on the morphology of rabbit ovarian tissue and the PCNA expression of the oocytes in the primordial and the primary follicles to investigate the optimal protocol of cryopreserving rabbit ovarian tissue.
Methods:
12 healthy female rabbits were randomly divided into four groups. Rabbit ovarian tissues were cryopreserved by procedure slow-freezing protocols including PROH and DMSO method (PROH group, DMSO group) and vitrification protocols including DMSO+PROH and DMSO+EG methord(vitrification group 1, vitrification group 2). Thawed after cryopreserved by liquid nitrogen for one month, HE staining and immunohistochemistry staining were used to observe the changes of the structure of frozen-thawed ovarian tissues and the morphology of different stages of follicles and PCNA expression of the oocytes in the primordial and the primary follicles.
Results:
1. The rates of the healthy primordial follicles and primary follicles of rabbit ovarian tissue declined to 80.08% and 55.00%、70.53% and 52.94%、67.55%and47.06%、69.65% and 55.56% in four frozen-thawed groups respectively,while that of fresh control groups were 90.14%and 86.05%、91.45% and 89.29%、91.76%and 84.62%、92.15% and 89.47% respectively. The differences between the fresh control groups and the freezing groups were significant (P<0.05). In all freezing groups, the rate of the healthy primordial follicles in PROH group was the highest. Compared the rate of the healthy primordial follicles in PROH group with that of DMSO group(70.53%)、vitrification group1(67.55%) and vitrification group2 (69.65%), the difference was significant (P<0.05).
After merging the freezing groups, the rate of the healthy primordial follicles was 72.7%.The rate of the healthy primary follicles was 52.77%. The difference was significant (P<0.05).
In the four kinds of freezing groups, the ovarian tissue structures were damaged. Some follicles were separated with the stromal cells. The connections among stromal cells were losse, disordered, and the gaps had formed by microscope.
2. The results of immunohistochemistry staining: the PCNA positive rate of the oocytes in the primordial and the primary follicles in PROH group was the highest (76.98%), the second was vitrification group2 (72.44%). Compared with the fresh control group (78.04%), the difference was not significant(P>0.05), while the PCNA positive rate in DMSO group(58.97%) and vitrification group1(48.00%) declined significantly compared with that of the fresh control group(P<0.05).
Conclusions:
1. Four kinds of frozen-thawed protocols damaged the structure of rabbit ovarian tissue in a certain extent. The rates of the healthy follicles at all stages declined significantly and the damagement appeared in the srtomal cells.
2. PROH procedure slow- freezing method is the best among DMSO and two vitrification methods for cryoperserving primordial follicles of ovarian tissue.
3. Cryoperserving methods have more damagement to the primary follicles than to the primordial follicles.
4. PROH procedure slow-freezing method and DMSO+EG vitrification protocol are the optimal protocols for keeping the proliferative activity of the oocytes in the primordial follicles and the primary follicles.
PartⅡEffects of procedure slow-freezing protocol and vitrification protocol on ultra microstructure, ER and MHC-Ⅱantigen expression of rabbit ovarian tissues and proliferative activity of the follicles
Objective:
To investigate the effecst of PROH procedure slow-freezing protocol and DMSO+EG vitrification protocol on ultra microstructure, ER and MHC-Ⅱantigen expression of rabbit ovarian tissues and proliferative activity of the follicles.
Methods:
1. Nine healthy female rabbits were randomly divided into three groups, one group was the fresh control group and the other groups were the freezing groups. The freezing groups were cryopreserved by PROH procedure slow-freezing method and DMSO+EG vitrification method.
2. After thawing, the effects of two freezing methods on ultra microstructure of rabbit ovarian tissues were observed by transmission electron microscope.
3. The effects of two freezing methods on ER and MHC-Ⅱantigen expressions of rabbit ovarian tissues were observed by SP immunohistochemistry method.
4. Follicles were mechanically dissected from ovarian tissue. Twenty small OGCs(diameter100-150μm, wrapped up by 2~5 layers of granulosa cells)and twenty big OGCs(diameter200-300μm, wrapped up many layers of granulosa cells)with normal appearance from every groups were tested with 3H-TdR uptake method respectively.
Results:
1. In PROH group, partial chondriosomes of the oocyte from primordial follicle swelled which appeared vacuole, and some chondriosomes were normal. The nucleus appearance was normal, and nuclear membrane was integrity and clear. The nuclear membranes of granular cells were integrity; in which chondriosomes were normal and gap junctions among cells were intact. In DMSO+EG groups, the most chondriosomes in the oocyte from primordial follicle swelled and its organelles were disaggregated, but cell appearance was nomal.The nuclear membrane was integrity and clear. In surrounding granular cells chondriosomes were acceptable, and chondriosomes of stromal cells were obviously swollen and appeared vacuole.
2. The result of immunohistochemistry suggested that there was no difference on ER expression of rabbit tissues in two frozen-thawed groups and the fresh group (P>0.05). MHC-Ⅱantigen expression in DMSO+EG group was significantly decreased compared with the fresh group (P<0.05), but there was no significant difference in PROH group(P>0.05).
3. The proliferative activity of big OGCs in two frozen-thawed groups significantly decreased compared with the fresh group (P<0.05), but there were no significant difference in small OGCs (P>0.05).
Conclusions:
1. The effect of PROH procedure slow-freezing protocol on ultra microstructure of rabbit ovarian tissues was less than DMSO+EG vitrification protocol.
2. There was no significant effect on ER expression of rabbit ovarian tissues by two frozen-thawed methods.
3. The DMSO+EG vitrification method obviously degraded the immunogenicity of ovarian tissues.
4. Two frozen-thawed methods did not have significant effect on the proliferative activity of small OGCs with intact appearance and structure, which may easily develop and maturate in vitro. But big OGCs packed by multilayer granular cells appearing normal morphology, its proliferative activity were significantly suppressed.
PartⅢ:Research on in vitro culture of cryopreserved rabbit ovarian tissue
Objective:
To investigate the suitable method of in vitro culture of ovarian tissue.
Methods:
1. Took fresh rabbit ovarian tissues and thawed the frozen rabbit ovarian tissues preserved by PROH procedure slow-freezing method, then used partial-isolated culture method and tissue mass culture method to continually culture them in vitro for 14d as fresh partial-isolated culture group, fresh tissue mass culture group, frozen partial-isolated culture group and frozen tissue mass culture group.
2. The results of culture were observed by inverted microscope every two days. The tissue culture medium was collected for 500μl to detect the content of E2, and an identical amount of fresh culture medium was added. The secretion production of E2 was measured in each group by ELISA method.
The ovarian tissues were separated mechanically on D14th of culture. The cpm of small secondary follicles (small OGCs ,diameter 100-150μm, wrapped up by 2~5 layers of granulosa cells) and the cpm of big secondary follicles (big OGCs ,diameter 200-300μm , wrapped up many layers of granulosa cells) were measured by 3H-TdR uptake method.
Results:
1. Small follicles wrapped by monolayer granulosa cells in fresh and frozen partial-isolated culture group were found failed to increase in diameter and become dark and necrotic in appearance through inverted microscope after continual culture for 14 days.
2. There were antral follicles forming in the fresh and the frozen partial-isolated culture group after cultured for 14 days. The follicles of GV phase but not MⅡwere found after further separation in two partial-isolated culture groups .
3. There were no antral follicles forming in two tissue mass culture groups after cultured for 14 days.
4. There was highest secretion production of E2 on 6th day and then it began to decrease on 8th day in the fresh tissue mass group, while the secretion production of E2 decreased continually in the frozen tissue mass group, and kept increasing in two partial -isolated culture groups.
5. There were no significant differences of cpm in small OGCs among fresh and frozen partial -isolated culture groups and fresh tissue mass culture group.,but the cpm of small OGCs in frozen tissue mass culture group decreased significantly.
6. There were no big OGCs with normal morphology in the frozen mass culture group. There were no significant differences in the cpm of big OGCs between the fresh and frozen partial -isolated culture groups(P>0.05),while there was significant decrease of the cpm in big OGCs in the fresh tissue mass group compared with the above two groups(P<0.05).
Conclusions:
1. The partial -isolated culture method of in vitro culture of rabbit ovarian tissue was better than tissue mass cultural method. The secretion production of E2 increased and there were antral follicles forming in the former groups. The secretion production of E2 decreased and there were no antral follicles forming in the latter groups.
2. There were good proliferative competences of the big and small OGCs cultured from the fresh and frozen partial-isolated culture groups, which suggested PROH procedure slow-freezing method can maintain the development potentiality of survival follicles.
3. The culture system is not suitable for primordial and primary follicles in vitro development.
4. Tissue mass culture method is not suitable for the culture frozen ovarian tissue.
PartⅣ:Research on transplantation of the rabbit ovarian tissue
Objective:
To investigate the suitable transplantation position of ovarian tissue and observe the physiological function of transplanted survival.
Methods:
1. 20 healthy rabbits were chosen, and randomly divided into control group (5 rabbits), castration group (5 rabbits) and transplantation group(10 rabbits).
2. Broad ligament beside the uterus was chosen as the position of transplantation in the transplantation group and both ovaries were cut ,then medullary substance of two ovaries were removed, 1×1×1mm3 tissue were cut and implanted into the subserosa of the left broad ligament. After the operation,HMG 10IU /d were injected i.m every other day for half month. Both ovaries were cut in the castration group.
3. The change of E2 level was assessed by vaginal cytology from the second day to one and a half month after operation.
4. Venous blood of the ear was taken and the serum E2 level was assessed by ELISA in each rabbit three times every 5 day from one month after the operation.
5. One and a half month after operation, the endometrium and ovaries of the control group, the endometrium of the castration group and the endometrium and survival ovarian tissue of the transplantation group were taken and then HE staining was made to observe the morphology.
6. The expression of PCNA and VEGF were measured by immunohistochemistry in the ovarian tissue of the transplanted survival and the control group.
Results:
1. There were signs of low estrogen level in the vaginal cast-off cells in the castration group 4~6 days after operation. A rabbit died after the operation and 7 rabbits showed sighs of low estrogen level in the vaginal cast-off cells and recovered 10~20 days after operation in the transplantation group, but the other 2 rabbits didn’t recover.
2. The serum E2 level returned normal one month after operation in the 7 rabbits of the transplantation group, which had no significant difference compared with the control group(P>0.05). The serum E2 level didn’t return the normal level in the other 2 rabbits. The serum E2 level decreased significantly in the castration group compared with the control group(P<0.05).
3. The epithelial cells of endometrium were composed of monolayer columnar cells, and the glands were few and shrinked, the stromal cells arranged compactly in the castration group.In the transplantation group, the surface layer cells of the endometrium showed jagged and composed of pseudoambilayer columnar cells and there were large amounts of glands which arranged tightly and shaped well-stacked, the stromal cell arranged rarefactly in 7 rabbits similar to the control group. The morphology of endometrium in the other 2 rabbits with low E2 level was similar to the castration group.
4. The survival ovarian tissue of 6 rabbits was found in the broad ligament in the transplantation group. The transplanted ovarian tissues were wrapped by fibrous tissues and fatty tissues. And follicles of each phase with eumorphism and large amount of new vessels were found in the survival transplanted ovarian tissue .
5. The positive rate of PCNA expression in the oocytes of the primordial and the primary follicle in the transplantation group was 78.67%,which had no significant difference compared with the control group(77.55%)(P>0.05).There were no significant difference in the expression of VEGF between the two groups(P>0.05).
Conclusions:
1. There is abundant blood supply in broad ligament beside the uterus, and its internal environment is similar to the physiological condition of ovary. The operation is convenient and suitable for transplantation of ovarian tissue. The method of transplantation combining with using Gn after operation harvested a high survival rate.
2. There was smooth endocrinological function in the survival ovarian tissue in broad ligament beside the uterus and E2 with physiological dose could be secreted, which played an important role to the epithelium of vaginal and endometrium.
3. The oocytes in the survival ovarian tissues had smooth proliferative activity and had the ability to secrete VEGF, which played an important role in maintaining the production and structure of blood vessels.
目的:
观察4种冷冻复苏方法对家兔卵巢组织形态学及卵母细胞PCNA表达的影响,探讨适宜的卵巢组织冷冻复苏方案。
方法:
将12只健康日本大耳白家兔随机分为4组,分别采用PROH(PROH组)及DMSO(DMSO组)慢速程序化冷冻和DMSO+PROH(玻璃化组1)及DMSO+EG(玻璃化组2)玻璃化冷冻方法冻存家兔卵巢组织,液氮保存一月后复苏,分别行HE染色及免疫组化染色,观察冷冻复苏后卵巢组织结构和各级卵泡形态学的改变及始基和初级卵泡卵母细胞PCNA表达的变化。
结果:
1. 4种冷冻复苏方法使家兔卵巢组织始基卵泡和初级卵泡的形态正常率分别下降为80.08%和55.00%、70.53%和52.94%、67.55%和47.06%及69.65%和55.56%而其相应新鲜对照组始基卵泡和初级卵泡的形态正常率分别为90.14%和86.05%、91.45%和89.29%、91.76%和84.62%及92.15%和89.47%,差异均有显著性(P<0.05);PROH组始基卵泡形态正常率最高,达80.08%,与DMSO组(70.53%)、玻璃化组1 (67.55%)、玻璃化组2 (69.65%)比较,差异均有显著性(P<0.05)。各冷冻组合并后计算始基卵泡的形态正常率为72.70%,初级卵泡的形态正常率为52.77%,二者比较,差异有显著性(P<0.05)。4个冷冻组均可见卵巢组织结构受损的表现,镜下可见部分卵泡与周围间质细胞分离,间质细胞连接变得疏松,排裂紊乱,有裂隙形成。
2.免疫组化染色结果显示冷冻组中PROH组始基及初级卵泡卵母细胞PCNA的阳性率最高达76.98%,玻璃化组2次之,为72.44%,与新鲜对照组比较(78.04%)差异无显著性(P>0.05),而DMSO组(58.97%)及玻璃化组1(48.00%)PCNA阳性率较新鲜对照组明显降低(P<0.05)。
结论:
1. 4种冷冻复苏方法均对家兔卵巢组织结构造成一定程度的损伤,使各级卵泡的形态正常率明显下降,出现间质受损的表现。
2. PROH慢速程序化冷冻法明显优于DMSO慢速程序化法及玻璃化法,较适合卵巢组织中始基卵泡的保存。
3.冻存卵巢组织对初级卵泡的影响大于始基卵泡。
4. PROH慢速程序化冷冻及DMSO+EG玻璃化冷冻能较好地保持家兔卵巢组织中始基及初级卵泡卵母细胞的增殖活性。
第二部分慢速程序化冷冻及玻璃化冷冻方案对家兔卵巢组织超微结构、ER和MHC-Ⅱ类抗原的表达及卵泡细胞增殖活性的影响
目的:
观察PROH慢速程序化冷冻及DMSO+EG玻璃化冷冻对家兔卵巢组织超微结构、ER和MHC-Ⅱ类抗原表达及卵泡细胞增殖活性的影响,判断复苏后卵巢组织抗原性和免疫原性是否发生改变,为今后卵巢组织移植及体外培养的研究奠定基础。
方法:
1、将健康日本大耳白家兔9只随机分为3组,1组为新鲜对照组,另2组为冷冻组。冷冻组分别采用PROH慢速程序化冷冻及DMSO+EG玻璃化冻存方案冻存家兔卵巢组织。
2、复苏后透射电镜观察两种冷冻方法对家兔卵巢组织超微结构的影响。
3、SP免疫组化法观察两种方法对家兔卵巢组织ER及MHC-Ⅱ类抗原表达的影响。
4、机械性分离新鲜组及冷冻组复苏的组织块,分离下的卵泡按直径的大小再分为:小OGC组,直径100~150μm,有2~5层颗粒细胞包裹的小次级卵泡;大OGC组,直径200~300μm,有5层以上颗粒细胞包裹的大次级卵泡。每组分别取20个形态正常的OGC,行3H标记的胸腺嘧啶核苷掺入试验。
结果:
1、PROH组始基卵泡卵母细胞部分线粒体肿胀,呈空泡状;部分线粒体形态尚正常;细胞核形态正常,核膜完整,界线清晰;颗粒细胞核膜完整,胞质内线粒体形态正常,细胞间缝隙连接完好。DMSO+EG组,始基卵泡卵母细胞线粒体肿胀,细胞器崩解;细胞形态尚可,边界清晰,核膜完整。周围颗粒细胞内线粒体尚可,间质细胞线粒体肿胀明显,呈空泡状。
2、免疫组化结果显示,两冷冻组复苏后家兔卵巢组织ER的表达与新鲜对照组比较,无明显变化(P>0.05)。DMSO+EG玻璃化冷冻组MHC—Ⅱ类抗原的表达与新鲜对照组比较明显下降(P<0.05),PROH组无明显变化(P>0.05)。.
3、两冷冻组复苏后小OGC的cpm值与新鲜对照组比较,无明显差异(P>0.05),而大OGC的cpm值较新鲜对照组明显下降(P<0.05)。
结论:
1. PROH慢速程序化冷冻对家兔卵巢组织超微结构的影响小于DMSO+EG玻璃化冷冻。
2.两种冷冻复苏方法,对家兔卵巢组织ER的表达均无明显影响。
3. DMSO+EG玻璃化冷冻复苏明显降低了卵巢组织的免疫原性。
4.两种冷冻复苏方法对形态结构完整的小OGC增殖活力无明显影响,这些卵泡体外生长和成熟,可能更容易成功。多层颗粒细胞包裹的大OGC即便形态正常,其增殖活性也明显受到抑制。
第三部分家兔卵巢组织冷冻保存后体外培养的研究
目的:
探讨适宜的卵巢组织体外培养方法。
方法:
1.无菌条件下取新鲜家兔卵巢组织,复苏PROH慢速程序化冷冻保存的家兔卵巢组织,分别采用部分分离培养及组织块培养方法体外连续培养14d,作为新鲜部分分离培养组、新鲜组织块培养组、冷冻部分分离培养组、冷冻组织块培养组。
2.每隔1天倒置显微镜下观察培养情况,并收集组织培养液500μl待测E2,同时补入新鲜培养液500μl。采用酶免法检测各组E2分泌量的变化。
培养14d结束时机械性分离各组卵巢组织,将形态结构完整,颗粒细胞连续,折光性好的大OGC(直径200~300μm,有5层以上颗粒细胞包裹的大次级卵泡)和小OGC(直径100~150μm,有2~5层颗粒细胞包裹的小次级卵泡)每组分别各取20个,进行3H标记的胸腺嘧啶核苷掺入试验。
结果:
1.倒置显微镜下新鲜及冷冻部分分离培养组均可见单层颗粒细胞包裹的小卵泡,连续培养14d,未见明显生长,卵泡内出现黑色颗粒,有退化坏死的迹象。
2.新鲜及冷冻部分分离培养组,培养14d后机械性分离卵巢组织均可见窦卵泡形成,进一步分离得到GV期卵子,未得到MⅡ期卵子。
3.新鲜及冷冻组织块培养组,14d培养结束时未见到有明显窦腔的卵泡形成。
4.各培养组E2分泌量的变化:新鲜组织块组,第6天E2分泌量达高峰,第8天开始逐渐下降;冷冻组织块组E2分泌量持续下降;而新鲜及冷冻部分分离培养组E2分泌持续上升。
5. 3H标记胸腺嘧啶核苷掺入试验结果:新鲜及冷冻部分分离培养组和新鲜组织块组所得到的小OGC其cpm值均无明显差异(P>0.05),而冷冻组织块得到小OGC的cpm值较以上各组明显降低(P<0.05)。
6.冷冻组织块组培养结束时,未得到形态正常的大OGC。新鲜及冷冻部分分离培养所得到的形态正常的大OGC其cpm值无明显差异(P>0.05),而新鲜组织块组所得到的大OGC的cpm值较上两组明显下降(P<0.05)。
结论:
1.部分分离法体外培养新鲜及冷冻的家兔卵巢组织明显优于组织块培养法。体外培养14d,前者培养液中E2呈上升趋势,有窦卵泡形成;后者随培养时间的延长E2呈下降趋势,未见窦卵泡形成。
2.新鲜及冷冻组织部分分离培养所得到的形态正常的大小OGC均有良好的增殖活性,说明PROH慢速程序化冷冻较好地保存了存活卵泡的体外发育潜能。
3.该培养系统不适合家兔卵巢组织始基及初级卵泡的体外培养。
4.冷冻卵巢组织更不适合组织块培养,所得到的形态正常的小OGC增殖活性差,未得到形态正常的大OGC。
第四部分家兔卵巢组织移植的研究
目的:
探讨适宜的卵巢组织移植部位,观察移植成活物的生理功能。
方法:
1.选健康大耳白家兔20只随机分为对照组(5只)、去势组(5只)、移植组(10只)。
2.移植组选宫旁阔韧带为移植部位,将切下的双侧卵巢去除髓质,切成1×1×1mm3大小的组织块植入左侧阔韧带的浆膜下,移植术后隔日肌注HMG 10IU/天,至术后半月。去势组切除双侧卵巢。
3.术后第2天开始隔日行阴道脱落细胞学涂片,观察雌激素水平的变化,至术后一个半月。
4.术后一月开始,每只家兔每隔5日,连续3次取耳缘静脉血2ml,酶免法测定血清E2水平。
5.术后1个半月再次开腹,取对照组子宫内膜及卵巢;取去势组子宫内膜;取移植组的成活移植物及子宫内膜,做HE染色行组织形态学观察。
6.移植成活的卵巢组织及对照组卵巢组织行SP免疫组化染色,观察PCNA及VEGF表达的变化。
结果:
1.阴道脱落细胞学涂片结果:对照组阴道细胞涂片显示细胞大,为多边型,胞浆稀薄透明,核小固缩,为雌激素作用的表现;去势组术后4—6天开始,阴道细胞涂片显示细胞呈圆形,核大圆形或椭圆形,核浆比增加,为低雌激素水平的表现;移植组1只术后死亡,剩余家兔术后4—6天开始阴道细胞涂片显示低雌激素水平,7只家兔术后10—20天重新恢复雌激素作用的表现,另2只未恢复。
2. E2测定结果:移植组7只家兔术后1个月血清E2水平恢复正常,与正常对照组比较,差异无显著性(P>0.05);另2只家兔E2未恢复正常。去势组家兔E2水平明显降低,与对照组比较,差异有显著性(P<0.05)。
3. HE染色子宫内膜形态学观察:去势组:子宫内膜上皮细胞由单层柱状细胞组成,腺体数量少,形态萎缩,间质细胞排裂紧密。移植组:E2水平恢复的7只家兔,子宫内膜表层细胞呈锯齿状,由假复层柱状细胞构成,腺体数量多,排列紧密,形态饱满,间质细胞排列疏松,同对照组;另2只E2低水平的家兔子宫内膜同去势组。
4.移植组卵巢组织形态学观察:移植组6只阔韧带处找到成活的卵巢组织,外观见移植的卵巢组织被纤维组织及脂肪组织包裹,HE染色镜下可见形态正常的各级卵泡及大量新生血管。
5.免疫组化结果:移植组卵巢组织始基及初级卵泡卵母细胞PCNA表达阳性率为78.67%与对照组(77.55%)比较无明显差异(P>0.05),VEGF的表达部位及表达强度与对照组比较也无明显差异(P>0.05)。
结论:
1.子宫旁阔韧带处血供丰富,内环境接近卵巢生理状态,移植手术操作简便,较适合卵巢组织移植,结合移植术后应用促性腺激素,获得了较高的移植成活率。
2.宫旁阔韧带处移植成活的卵巢组织有很好的内分泌功能,能分泌生理剂量的E2,对阴道上皮及子宫内膜均能发挥良好的作用。
3.移植成活的卵巢组织卵母细胞有良好的增殖活性,并能很好地分泌VEGF,对维持血管的生成及正常结构有重要意义。
PartⅠ:Effects of four kinds of different frozen-thawed methods on the morphology of rabbit ovarian tissue and the PCNA expression of the oocytes in the primordial and the primary follicles
Objective:
To observe the effects of four kinds of different frozen-thawed methods on the morphology of rabbit ovarian tissue and the PCNA expression of the oocytes in the primordial and the primary follicles to investigate the optimal protocol of cryopreserving rabbit ovarian tissue.
Methods:
12 healthy female rabbits were randomly divided into four groups. Rabbit ovarian tissues were cryopreserved by procedure slow-freezing protocols including PROH and DMSO method (PROH group, DMSO group) and vitrification protocols including DMSO+PROH and DMSO+EG methord(vitrification group 1, vitrification group 2). Thawed after cryopreserved by liquid nitrogen for one month, HE staining and immunohistochemistry staining were used to observe the changes of the structure of frozen-thawed ovarian tissues and the morphology of different stages of follicles and PCNA expression of the oocytes in the primordial and the primary follicles.
Results:
1. The rates of the healthy primordial follicles and primary follicles of rabbit ovarian tissue declined to 80.08% and 55.00%、70.53% and 52.94%、67.55%and47.06%、69.65% and 55.56% in four frozen-thawed groups respectively,while that of fresh control groups were 90.14%and 86.05%、91.45% and 89.29%、91.76%and 84.62%、92.15% and 89.47% respectively. The differences between the fresh control groups and the freezing groups were significant (P<0.05). In all freezing groups, the rate of the healthy primordial follicles in PROH group was the highest. Compared the rate of the healthy primordial follicles in PROH group with that of DMSO group(70.53%)、vitrification group1(67.55%) and vitrification group2 (69.65%), the difference was significant (P<0.05).
After merging the freezing groups, the rate of the healthy primordial follicles was 72.7%.The rate of the healthy primary follicles was 52.77%. The difference was significant (P<0.05).
In the four kinds of freezing groups, the ovarian tissue structures were damaged. Some follicles were separated with the stromal cells. The connections among stromal cells were losse, disordered, and the gaps had formed by microscope.
2. The results of immunohistochemistry staining: the PCNA positive rate of the oocytes in the primordial and the primary follicles in PROH group was the highest (76.98%), the second was vitrification group2 (72.44%). Compared with the fresh control group (78.04%), the difference was not significant(P>0.05), while the PCNA positive rate in DMSO group(58.97%) and vitrification group1(48.00%) declined significantly compared with that of the fresh control group(P<0.05).
Conclusions:
1. Four kinds of frozen-thawed protocols damaged the structure of rabbit ovarian tissue in a certain extent. The rates of the healthy follicles at all stages declined significantly and the damagement appeared in the srtomal cells.
2. PROH procedure slow- freezing method is the best among DMSO and two vitrification methods for cryoperserving primordial follicles of ovarian tissue.
3. Cryoperserving methods have more damagement to the primary follicles than to the primordial follicles.
4. PROH procedure slow-freezing method and DMSO+EG vitrification protocol are the optimal protocols for keeping the proliferative activity of the oocytes in the primordial follicles and the primary follicles.
PartⅡEffects of procedure slow-freezing protocol and vitrification protocol on ultra microstructure, ER and MHC-Ⅱantigen expression of rabbit ovarian tissues and proliferative activity of the follicles
Objective:
To investigate the effecst of PROH procedure slow-freezing protocol and DMSO+EG vitrification protocol on ultra microstructure, ER and MHC-Ⅱantigen expression of rabbit ovarian tissues and proliferative activity of the follicles.
Methods:
1. Nine healthy female rabbits were randomly divided into three groups, one group was the fresh control group and the other groups were the freezing groups. The freezing groups were cryopreserved by PROH procedure slow-freezing method and DMSO+EG vitrification method.
2. After thawing, the effects of two freezing methods on ultra microstructure of rabbit ovarian tissues were observed by transmission electron microscope.
3. The effects of two freezing methods on ER and MHC-Ⅱantigen expressions of rabbit ovarian tissues were observed by SP immunohistochemistry method.
4. Follicles were mechanically dissected from ovarian tissue. Twenty small OGCs(diameter100-150μm, wrapped up by 2~5 layers of granulosa cells)and twenty big OGCs(diameter200-300μm, wrapped up many layers of granulosa cells)with normal appearance from every groups were tested with 3H-TdR uptake method respectively.
Results:
1. In PROH group, partial chondriosomes of the oocyte from primordial follicle swelled which appeared vacuole, and some chondriosomes were normal. The nucleus appearance was normal, and nuclear membrane was integrity and clear. The nuclear membranes of granular cells were integrity; in which chondriosomes were normal and gap junctions among cells were intact. In DMSO+EG groups, the most chondriosomes in the oocyte from primordial follicle swelled and its organelles were disaggregated, but cell appearance was nomal.The nuclear membrane was integrity and clear. In surrounding granular cells chondriosomes were acceptable, and chondriosomes of stromal cells were obviously swollen and appeared vacuole.
2. The result of immunohistochemistry suggested that there was no difference on ER expression of rabbit tissues in two frozen-thawed groups and the fresh group (P>0.05). MHC-Ⅱantigen expression in DMSO+EG group was significantly decreased compared with the fresh group (P<0.05), but there was no significant difference in PROH group(P>0.05).
3. The proliferative activity of big OGCs in two frozen-thawed groups significantly decreased compared with the fresh group (P<0.05), but there were no significant difference in small OGCs (P>0.05).
Conclusions:
1. The effect of PROH procedure slow-freezing protocol on ultra microstructure of rabbit ovarian tissues was less than DMSO+EG vitrification protocol.
2. There was no significant effect on ER expression of rabbit ovarian tissues by two frozen-thawed methods.
3. The DMSO+EG vitrification method obviously degraded the immunogenicity of ovarian tissues.
4. Two frozen-thawed methods did not have significant effect on the proliferative activity of small OGCs with intact appearance and structure, which may easily develop and maturate in vitro. But big OGCs packed by multilayer granular cells appearing normal morphology, its proliferative activity were significantly suppressed.
PartⅢ:Research on in vitro culture of cryopreserved rabbit ovarian tissue
Objective:
To investigate the suitable method of in vitro culture of ovarian tissue.
Methods:
1. Took fresh rabbit ovarian tissues and thawed the frozen rabbit ovarian tissues preserved by PROH procedure slow-freezing method, then used partial-isolated culture method and tissue mass culture method to continually culture them in vitro for 14d as fresh partial-isolated culture group, fresh tissue mass culture group, frozen partial-isolated culture group and frozen tissue mass culture group.
2. The results of culture were observed by inverted microscope every two days. The tissue culture medium was collected for 500μl to detect the content of E2, and an identical amount of fresh culture medium was added. The secretion production of E2 was measured in each group by ELISA method.
The ovarian tissues were separated mechanically on D14th of culture. The cpm of small secondary follicles (small OGCs ,diameter 100-150μm, wrapped up by 2~5 layers of granulosa cells) and the cpm of big secondary follicles (big OGCs ,diameter 200-300μm , wrapped up many layers of granulosa cells) were measured by 3H-TdR uptake method.
Results:
1. Small follicles wrapped by monolayer granulosa cells in fresh and frozen partial-isolated culture group were found failed to increase in diameter and become dark and necrotic in appearance through inverted microscope after continual culture for 14 days.
2. There were antral follicles forming in the fresh and the frozen partial-isolated culture group after cultured for 14 days. The follicles of GV phase but not MⅡwere found after further separation in two partial-isolated culture groups .
3. There were no antral follicles forming in two tissue mass culture groups after cultured for 14 days.
4. There was highest secretion production of E2 on 6th day and then it began to decrease on 8th day in the fresh tissue mass group, while the secretion production of E2 decreased continually in the frozen tissue mass group, and kept increasing in two partial -isolated culture groups.
5. There were no significant differences of cpm in small OGCs among fresh and frozen partial -isolated culture groups and fresh tissue mass culture group.,but the cpm of small OGCs in frozen tissue mass culture group decreased significantly.
6. There were no big OGCs with normal morphology in the frozen mass culture group. There were no significant differences in the cpm of big OGCs between the fresh and frozen partial -isolated culture groups(P>0.05),while there was significant decrease of the cpm in big OGCs in the fresh tissue mass group compared with the above two groups(P<0.05).
Conclusions:
1. The partial -isolated culture method of in vitro culture of rabbit ovarian tissue was better than tissue mass cultural method. The secretion production of E2 increased and there were antral follicles forming in the former groups. The secretion production of E2 decreased and there were no antral follicles forming in the latter groups.
2. There were good proliferative competences of the big and small OGCs cultured from the fresh and frozen partial-isolated culture groups, which suggested PROH procedure slow-freezing method can maintain the development potentiality of survival follicles.
3. The culture system is not suitable for primordial and primary follicles in vitro development.
4. Tissue mass culture method is not suitable for the culture frozen ovarian tissue.
PartⅣ:Research on transplantation of the rabbit ovarian tissue
Objective:
To investigate the suitable transplantation position of ovarian tissue and observe the physiological function of transplanted survival.
Methods:
1. 20 healthy rabbits were chosen, and randomly divided into control group (5 rabbits), castration group (5 rabbits) and transplantation group(10 rabbits).
2. Broad ligament beside the uterus was chosen as the position of transplantation in the transplantation group and both ovaries were cut ,then medullary substance of two ovaries were removed, 1×1×1mm3 tissue were cut and implanted into the subserosa of the left broad ligament. After the operation,HMG 10IU /d were injected i.m every other day for half month. Both ovaries were cut in the castration group.
3. The change of E2 level was assessed by vaginal cytology from the second day to one and a half month after operation.
4. Venous blood of the ear was taken and the serum E2 level was assessed by ELISA in each rabbit three times every 5 day from one month after the operation.
5. One and a half month after operation, the endometrium and ovaries of the control group, the endometrium of the castration group and the endometrium and survival ovarian tissue of the transplantation group were taken and then HE staining was made to observe the morphology.
6. The expression of PCNA and VEGF were measured by immunohistochemistry in the ovarian tissue of the transplanted survival and the control group.
Results:
1. There were signs of low estrogen level in the vaginal cast-off cells in the castration group 4~6 days after operation. A rabbit died after the operation and 7 rabbits showed sighs of low estrogen level in the vaginal cast-off cells and recovered 10~20 days after operation in the transplantation group, but the other 2 rabbits didn’t recover.
2. The serum E2 level returned normal one month after operation in the 7 rabbits of the transplantation group, which had no significant difference compared with the control group(P>0.05). The serum E2 level didn’t return the normal level in the other 2 rabbits. The serum E2 level decreased significantly in the castration group compared with the control group(P<0.05).
3. The epithelial cells of endometrium were composed of monolayer columnar cells, and the glands were few and shrinked, the stromal cells arranged compactly in the castration group.In the transplantation group, the surface layer cells of the endometrium showed jagged and composed of pseudoambilayer columnar cells and there were large amounts of glands which arranged tightly and shaped well-stacked, the stromal cell arranged rarefactly in 7 rabbits similar to the control group. The morphology of endometrium in the other 2 rabbits with low E2 level was similar to the castration group.
4. The survival ovarian tissue of 6 rabbits was found in the broad ligament in the transplantation group. The transplanted ovarian tissues were wrapped by fibrous tissues and fatty tissues. And follicles of each phase with eumorphism and large amount of new vessels were found in the survival transplanted ovarian tissue .
5. The positive rate of PCNA expression in the oocytes of the primordial and the primary follicle in the transplantation group was 78.67%,which had no significant difference compared with the control group(77.55%)(P>0.05).There were no significant difference in the expression of VEGF between the two groups(P>0.05).
Conclusions:
1. There is abundant blood supply in broad ligament beside the uterus, and its internal environment is similar to the physiological condition of ovary. The operation is convenient and suitable for transplantation of ovarian tissue. The method of transplantation combining with using Gn after operation harvested a high survival rate.
2. There was smooth endocrinological function in the survival ovarian tissue in broad ligament beside the uterus and E2 with physiological dose could be secreted, which played an important role to the epithelium of vaginal and endometrium.
3. The oocytes in the survival ovarian tissues had smooth proliferative activity and had the ability to secrete VEGF, which played an important role in maintaining the production and structure of blood vessels.
引文
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[1]. Eppig JJ, Schroeder AC. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol Reprod.1989, 41: 268-276.
[2]. Eppig JJ, O’Brien MJ .Development in vitro of mouse oocytes from primordial follicles. Reprod Bio.1996,54:197-207.
[3]. O’Brien MJ, Pentola JK, Eppig JJ , et al. A revised protocol for on vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod. 2003, 68 :1682-1686.
[4]. Abir R, Fisch B, Nitke S, et al.Morphological study of fully and partially isolated early human follicles.Fertil Steril. 2001,75(1):141-146
[5]. Lucyna KK.Recent achievements in vitro culture and preservation of ovarian follicles in mammals. Biol Reprod.2006,6(1):3-16
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[9]. Luisier JP, Matton P, Dufour JJ, et al. Growth rates of follicles in the ovary of the cow. J Reprod Fertil. 1987,81 :301-307
[10].王利红,陈子江,高选.人卵巢组织冷冻保存的实验研究.现代妇产科进展.2005,14(1):16-19
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[12].司徒镇强军正.主编细胞培养.西安:世界图书出版公司,1996.141
[13]. Newton H, Illingworth P. In vitro growth of murine preantral follicles after isolation from cryopreserved ovarian tissue.Hum Reprod.2001,16(3):423-429
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[4]. Schnorr J, Oehninger S, Toner J, et al. Functional studies of subcutaneous ovarian transplants in non-human primates:steroido genesis, endometrial development, ovulation, menstrual patterns and gamete morphology. Hum Reprod,2002,17:612-619
[5]. Donnez PJ, Dolmans MM, Demylle D, et al. Livebirth after arthotopic transplantation of cryopreserved ovarian tissue. Lancet, 2004, 364(9443) 1405-1410
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[8]. Nisolle M, Godin PA, Casanas-Roux F, et al.Histological and ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in nude mice. Fertil Steril .2000,74:122–129.
[9]. Wolner-Hanssen P, H?gglund L, Ploman F, et al. Autotransplantation of cryopreserved ovarian tissue to the right forearm 41/2 years after autologous stem cell transplantation.Acta Obstet Gynecol Scand .2005,84:695–699
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