Angiogenin参与冻融人卵巢组织微循环重建效果分析

The microcirculation reconstruction effect of angiogenin on cryopreserved human ovarian tissue

目的用新型玻璃化冷冻方法冻融人卵巢组织,在体外培养该组织时添加angiogenin,观察其对微血管生成的影响,并将培养后的组织进行异种移植,观察两周后移植物的血循环情况。方法收集人卵巢组织并用NIV法进行冻融,通过免疫组化染色比较冻融前后组织中angiogenin的表达情况,将冻融的卵巢组织放在angiogenin浓度分别为0,100,200及400ng/ml的培养液中培养7天,取出组织进行CD34免疫组化染色,计数并比较不同angiogenin浓度组卵巢组织微血管密度差异,确定较适宜的培养浓度后,在适宜浓度下培养冻融人卵巢组织0,2,5,7天,观察不同培养时间组卵巢组织的微循环重建效果,将体外培养后的卵巢组织移植到SCID小鼠皮下,一周后取出移植物,观察微血管密度变化及细胞凋亡情况。结果冻融后卵巢组织间质中的angiogenin表达量下降,卵巢组织在angiogenin浓度为200ng/ml的培养液中生长的卵巢组织微血管密度最高。添加angiogenin后培养5天的卵巢组织微血管密度较培养2天及培养7天组高。卵巢组织短期培养后移植至SCID小鼠皮下一周,与对照组比较,添加angiogenin 200ng/ml培养后的卵巢移植物微血管密度增高,间质细胞凋亡减少。结论 angiogenin可促进冻融人卵巢组织微循环重建,可为女性的生育能力保存提供有力的支持。

Objective To investigate the microcirculation reconstruction effect of angiogenin on cryopreserved human ovarian tissue in vitro and xenotransplantation.Methods The human ovarian tissues were collected and freeze thawed with NIV.The expression of angiogenin was detected with immunohistochemistry before and after freeze thawing.The freeze thawing ovarian tissues were cultured in angiogenin（0,100,200 and 400ng/ml）for 7days.The tissues were stained with CD34 immunohistochemistry to observe the effect of microcirculation of reconstruction.The human ovarian tissue cultured in vitro was implanted in the skin of SCID mice.1week after implantation,the vessel density and apoptosis of ovarian tissue were observed.Results Ovarian tissues were culture for 7days in four groups according to the concentration of Angiogenin,0,100,200 and 400ng/ml.The micro vascular density increase as Angiogenin concentration increase,but same at 200 and 400ng/ml.Angiogenesis in 5day group was more effective than that of 2days and 7days.After a short term culture,the ovarian tissue were xenotransplanted to SCID mice.One week later,the micro vascular density of 200ng/ml group was higher than control group,and the apoptosis rate of this group was significantly lower than that of control group.Conclusion The 200ng/ml of Angiogenin may be the best concentrate for in vitro culture ovarian tissue and xenontransplanted ovarian tissue.The density of micro vascular in 5day group is the highest.Apoptosis in angiogenin intervention group is significantly lower than control group.Our research provides the research clue to optimize and improve the female fertility preservation system.