豚鼠卵巢组织片和分离的窦前卵泡玻璃化冷冻效果比较  

Comparative study between guinea pig ovarian fragments and isolated preantral follicles cryopreservation with vitrification

作　　者：张悦[1] 陈文铭[2] 谢迟迟 谢双双[2] 孙君慧 徐芝慧[2] 刘军[3] 周颖[2] 

机构地区：[1]广西医科大学附属第一医院生殖医学研究中心,广西南宁530021 [2]温州医科大学附属第一医院生殖医学中心,浙江温州325015 [3]澳大利亚莫纳什大学医学研究所

出　　处：《温州医学院学报》2014年第4期252-257,共6页Journal of Wenzhou Medical College

基　　金：国家自然科学基金资助项目(30370749);浙江省自然科学基金资助项目(LY13C120002);浙江省科技厅科研基金资助项目(2003C33001);温州市科技局对外合作项目(H20080061)

摘　　要：目的 :比较分析不同玻璃化冷冻配方对豚鼠卵巢组织片和单个窦前卵泡形态、存活率及体外发育能力的影响。方法:取5周龄雌性豚鼠卵巢,实验分新鲜对照组、组织片A液毒性组、组织片B液毒性组、卵泡A液毒性组和卵泡B液毒性组,及相应的冻融组,即组织片A液冻融组、组织片B液冻融组、卵泡A液冻融组、卵泡B液冻融组,共9组行毒性试验和冷冻保存。各组卵巢组织片行HE染色形态学观察,分离窦前卵泡经锥虫蓝染色活力测试并行体外培养。结果:A液冻融组和B液冻融组豚鼠卵巢组织片HE染色光镜观察可见大部分卵泡形态正常,A液组和B液组异常窦前卵泡数与新鲜对照组比较差异有统计学意义(P<0.05);在分离的窦前卵泡锥虫蓝染色活力测试中,冻融卵巢组织片A组的卵泡复苏率(为66.1%)显著低于(P<0.05)冻融卵泡B液组(为78.5%)和新鲜对照组(为87.5%);其余组别之间差异无统计学意义。体外培养卵泡发育情况组织片冷冻组比卵泡冷冻组差,以A液组尤甚,第6天存活卵泡数目不足(为20.0%),与新鲜对照组(为60.0%)及卵泡B液冻融组(为50.0%)相比较差异有统计学意义(P<0.05)。结论:玻璃化冷冻法能较好地冷冻保存豚鼠卵巢组织片和分离的窦前卵泡,而单个豚鼠窦前卵泡玻璃化冷冻优于卵巢组织片的玻璃化冷冻;B液较A液更适合豚鼠卵巢卵泡的玻璃化冷冻保存。窦前卵泡的体外培养证明玻璃化冷冻复苏卵泡具有一定的继续发育能力。Objective： To compare the follicle morphology, survival rate and in vitro growth potential of guinea pig ovarian fragments and isolated preantral follicles after vitrification. Methods： Five- weeks aged female guinea pigs were used. Two vitrification solutions （solution A and B） were applied to vitrify guinea pig ovarian fragments and isolated preantral follicles. The toxicity and cryopreservation efficiency of the two solutions were tested on both fragments and isolated preantral follicles. The ovaries were observed using hematoxylin and eosin staining; the viability of the follicles was evaluated by trypan blue. The isolated preantral follicle was cultured in vitro to evaluated the follicle development potential. Results： Based on morphological analyze, the majority follicles were normal in fragments cryopreservation, but the abnormal follicle were higher than fresh group, P〈0.05. The recover rates of follicle after being thawed using trypan blue were 66.1% in solution A fragment vitrification group, significantly lower than that in solution B follicle vitrification group （78.5%） and fresh group （87.5%）, P〈0.05. No significance was found among other groups. During in vitro culture, the vitrifiedfragments showed poorer develop potential than follicle vitrification groups, typically solution A fragment group, and the follicle survival rate by day 6 was lower than （20.0%）, significantly lower than fresh group （60.0%） and solution B follicle vitrification group （50.0%）, P〈0.05. Conclusion： The results show that the vitrification can preserve guinea pig ovarian fragments and isolated preantral follicles, the latter one achieved better results. Solution B was more efficient in preserving guinea pig follicles. The follicles show growth potential during in vitro culture after being thawed.

关 键 词：玻璃化 冷冻保护剂 豚鼠 窦前卵泡 

分 类 号：R318.52[医药卫生—生物医学工程]