摘要
目的探讨卵巢新型玻璃化冷冻法——细针穿刺浸入式玻璃化冷冻法(needle immersed vitrification,NIV)保存与移植重建化疗性卵巢损伤大鼠模型的卵巢功能效果。方法 8～9周龄SPF级封闭群雌性Wistar大鼠52只,体重250～300 g;选择至少有2次正常动情周期的50只大鼠纳入实验研究。随机取10只作为供体,采用NIV法冷冻保存卵巢组织并复苏。剩余40只大鼠根据处理方法不同随机分为3组:环磷酰胺组(C组,n=14)及环磷酰胺/移植组(C/T组,n=12)大鼠腹腔注射环磷酰胺,共21 d,建立化疗性卵巢功能损伤模型,C/T组将复苏的卵巢组织移植于大鼠左侧卵巢囊内;对照组(NS组,n=14)大鼠每日腹腔注射生理盐水,连续21 d。比较各组大鼠动情周期恢复情况、卵巢组织重量以及卵巢组织形态学改变、各级卵泡数目。结果 C/T组1只大鼠于移植术后2 d死亡,其余大鼠均存活至实验完成。各组大鼠在注射结束后4周体重趋于一致,差异无统计学意义(P>0.05)。NS组大鼠实验期间均具有规律、正常的动情周期,C组和C/T组大鼠在注射药物期间均出现动情周期紊乱。注射结束后,C组动情周期中位恢复时间为9 d(95%CI:7.9～10.1 d),C/T组为6 d(95%CI:4.9～7.1 d),两组比较差异有统计学意义(χ2=6.571,P=0.010)。C组在注射结束后4周卵巢组织重量较前减轻;C/T组大鼠移植卵巢组织均存活生长良好,其重量呈逐渐增加趋势。组织学观察示,C组与NS组、C/T组相比始基卵泡及初级卵泡数目均显著减少(P<0.05),C/T组与NS组比较差异无统计学意义(P>0.05);各组次级卵泡和窦状卵泡数目比较,差异均无统计学意义(P>0.05)。结论将NIV法冷冻保存的正常卵巢组织移植于化疗性卵巢损伤大鼠体内后,能显著缩短动情周期恢复时间,移植卵巢组织生长良好,各级卵泡计数接近正常水平,初步提示具有化疗后重建卵巢内分泌和生育能力的作用。
Objective To investigate the effects of ovarian tissue cryopreservation by needle immersed vitrification （NIV） method and subsequently orthotopic transplantation on ovarian function reconstruction in chemotherapy-induced ovary damage rat model. Methods A total of 52 matured virginal female Wistar rats at age of 8-9 weeks housed in specific-pathogen- free facilities, weighing 250-300 g. Vaginal smears were obtained daily, 50 rats having at least 2 consecutive normal estrous cycles were included in the experiment. Ten rats were selected as donors randomly, and NIV method was used for cryopreserving ovarian tissues. The remaining 40 rats were divided into 3 groups according to different treatments： cyclophosphamide group （C group, n=14）, cyclophosphamide/transplantation group （C/T group, n=12）, and control group （NS group, n=14）. In C group and C/T group, the rats received peritoneal injection of cyclophosphamide every day for 21 days to establish the chemotherapy-induced ovary damage models; and then the frozen-thawed ovarian tissues orthotopically transplanted into the left ovarian bursae in C/T group. The rats received peritoneal injections of 0.9% saline solution every day for 21 days in NS group. Estrous cycle recovery time, ovary weight, morphology change of ovarian tissues, and follicle count were compared among 3 groups. Results One rat died at 2 days after transplantation in C/T group; the other rats survived to the completion of the experiment. At 4 weeks after the end of injection, no significant difference in body weight was found among 3 groups （P 〉 0.05）. The rats of NS group had regularestrous cycle, but cyclic changes in vaginal smears were observed in C group and C/T group during cyclophosphamide treatment. The median estrous cycle recovery was 9 days （95%CI： 7.9-10.1 days） in C group, and was 6 days （95%CI： 4.9-7.1 days） in C/T group, showing significant difference （χ2=6.571, P=0.010）. The ovarian weight showed an obvious downtrend in C group at 4 weeks after cyclophosphamide treatment, and an upward trend was observed in CIT group. The ovarian grafts survived and grew well in C/T group. Primordium follicles and primary follicles in C/T group and NS group were significantly more than those in C group （P 〈 0.05）, but no significant difference was found between NS group and C/T group （P 〉 0.05）. There was no significant difference in secondary follicles and antral follicles among the 3 groups （P 〉 0.05）. Condusion The method of ovarian tissue cryopreservation by NIV and subsequently orthotopic transplantation can significantly shorten the estrous cycle recovery time in chemotherapy-induced ovary damage rat model. Ovarian grafts grow well, follicle count is similar to normal level. So it has the potential ability of ovarian endocrine and fertility reconstructinn after chemotherapy.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2013年第9期1116-1121,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(81001032)~~
关键词
化疗性卵巢损伤
卵巢组织冷冻保存
玻璃化冷冻法
原位移植
大鼠
Chemotherapy-induced ovary damage
Ovarian tissue cryopreservation
Vitrification
Orthotopictransplantation
Rat
