小鼠卵巢玻璃化冻融过程中FSH干预对血管生成相关因子表达的影响

Effect of FSH Interpose on the VEGF and αvβ3 Expression during Cryopreservation by Vitrification

目的在小鼠卵巢玻璃化冻融过程中给予重组人卵泡雌激素(r FSH)干预,通过观察其对卵泡刺激素受体(follicle-stimulating hormone receptor,FSH-R)及与血管生成相关的Integrinαvβ3、Angpt-2、Kindlin-2蛋白表达的影响,提供FSH干预对卵巢血管生成相关因子影响的依据。方法将4周龄C57BL/6J小鼠卵巢随机分为新鲜对照组(FCG)、玻璃化冻存对照组(VCG)、0.3IU·m L-1FSH干预玻璃化冻存组(FSH-VG)。对3组卵巢内各级卵泡进行计数;通过免疫组化方法观察并分析各组FSH-R、Angpt-2、Kindlin-2在卵巢不同细胞中的定位;观察粘附分子Integrinαvβ3在各组卵巢细胞中表达,并进行亚基表达量的分析。结果FSH-VG组的原始卵泡、初级卵泡、正常卵泡及总卵泡数均高于VCG组,但低于FCG组(P均<0.05),闭锁卵泡数VCG组高于FCG及FSH-VG组(P均<0.05);FSH-R与Angpt-2阳性表达呈现FSH-VG与VCG组均低于FCG组(P<0.05),但FSH-VG组高于VCG组(P<0.05);Kindlin-2阳性表达在FSH-VG组与FCG组之间差异无统计学意义(P>0.05),但均高于VCG组(P<0.01或<0.05);Integrinβ3蛋白表达量FCG与FSH-VG组间差异无统计学意义(P>0.05),但均高于VCG组(P<0.05),Integrinαv的表达3组间差异无统计学意义(P>0.05)。结论玻璃化冻融过程中添加FSH可以上调FSH-R及粗血管生成的相关因子Integrinβ3、Angpt-2和Kindlin-2蛋白的表达,提高正常卵泡数。

Objective To investigate the influence of r FSH interpose on the FSH、Integrin αvβ3,Angpt- 2and Kindlin- 2 proteins expression during cryopreservation by vtrification. Methods Four- week- old C57 BL /6J mice in anestrus. Ovaries were randomized into fresh control group( FCG),vitrification control group( VCG) and FSH intervented vitrification group( FSH- VG). For FCG and vitrification freezing groups( FSH- VG and VCG),the different stage of follicles were counted. The expression of FSH- R,Angpt- 2and Kindlin- 2 in different cells of ovary were observed and analyzed by immunohistochemical technique. Adhesion molecule( Integrin αvβ3) expression was observed in each group ovary cells. Results Follicle count statistics showed that primordial follicles,primary follicles,normal follicles and the total number of follicles in FSH- VG were significantly higher than those in VCG group,but they were significantly lower than those in FCG group( P < 0. 05). The number of follicles for atresia in VCG group was significantly higher than that in FCG and FSH- VG group( P < 0. 05). The expression of FSH- R and Angpt- 2 statistics showed that FSH-VG and VCG were significantly lower than FCG group( P < 0. 05) while FSH- VG were higher than the VCG group( P < 0. 05). The expression of Kindlin- 2 showed no significant difference between the FSH- VG and FCG group. The expression of Kindlin- 2 in FCG and FSH- VG was significantly higher than that in VCG group( P < 0. 01 or < 0. 05). The expression of Integrin β3 statistics showed FSH- VG was significantly higher than that in VCG( P < 0. 05). The expression of αv was not significantly different for either VCG or FSH-VG compared with FCG( P > 0. 05). Conclusion Vitrification freezing and thawing process of adding FSH may increase the expression of FSH- R,Integrin β3,Angpt- 2,Kindlin- 2 protein. It laid the foundation for FSH interventions to improve cryopreservation transplant angiogenesis speed.