目的 初探玻璃化冷冻后卵巢原位移植技术对其子代小鼠糖代谢水平的影响和机制.方法 建立卵巢玻璃化冷冻后原位移植出生子代小鼠模型,实验分为新鲜卵巢原位移植出生子代(新鲜组)和玻璃化冷冻卵巢原位移植出生子代(玻璃化冷冻组),另设自然交配出生子代为对照组,每组16只小鼠,检测子代小鼠出生到6月龄时各阶段体质量及6月龄成年子代鼠的空腹血糖、空腹胰岛素、糖耐量水平;取材6月龄子代小鼠肝脏组织,qRT-PCR检测生长因子受体结合蛋白10(growth factor receptor-bound protein 10,Grb10)mRNA的表达;Western blotting 检测Grb10、磷酸化蛋白激酶B(phosphate-protein kinase B,p-AKT)和磷酸化丝裂原活化蛋白激酶(phosphate-mitogen-activated protein kinase,p-MAPK)蛋白的表达.结果 玻璃化冷冻组2周龄和3周龄子代小鼠的体质量均低于对照组(P=0.007,P<0.001),差异均具有统计学意义;新鲜组4月、5月龄子代小鼠的体质量均低于对照组(P=0.023,P=0.013),差异均具有统计学意义.6月龄子代鼠新鲜组和玻璃化冷冻组小鼠空腹血糖均较对照组升高(P<0.001,P=0.002),差异均具有统计学意义;玻璃化冷冻组小鼠空腹胰岛素水平较对照组升高,差异具有统计学意义(P=0.017),但玻璃化冷冻组与新鲜组之间差异均无统计学意义(均P>0.05).葡萄糖注射后30 min时,新鲜组子代小鼠血糖水平显著高于玻璃化冷冻组(P<0.001),120 min时,新鲜组和玻璃化冷冻组子代小鼠血糖水平显著高于对照组(P=0.034,P=0.018),差异均具有统计学意义.新鲜组和玻璃化冷冻组子代肝脏内Grb10 mRNA和蛋白的表达显著高于对照组(均P<0.001),差异均具有统计学意义;玻璃化冷冻组和新鲜组子代肝脏内p-AKT(均P=0.024)和p-MAPK(P=0.019,P=0.002)蛋白的表达较对照组显著性降低,差异均具有统计学意义,玻璃化冷冻组与新鲜组之间差异均无统计学意义(均P>0.05).结论 卵巢玻璃化冷冻后原位移植技术影响子代小鼠生长和糖代谢,通过子代成年小鼠肝脏内印记基因Grb10过表达,抑制肝脏内胰岛素/胰岛素样生长因子-1信号通路,降低细胞利用葡萄糖水平,使成年子代小鼠出现糖代谢紊乱的现象.
Experimental study on the risk of glucose metabolism disorders in vitrified-frozen ovarian orthotopic transplantation technique mediated insulin/IGF-1 signaling pathway
Objective To explore the effect and mechanism of vitrified ovarian transplantation in situ on the glucose metabolism level of its offspring mice.Methods To establish mouse model of vitrified ovarian orthotopic transplantation,the mice were divided into three groups:fresh ovarian orthotopic transplantation offsprings group(fresh group),vitrified ovarian orthotopic transplantation offsprings group(vitrification group),natural mating birth offsprings group(control group),16 mice per group.The growth weight from birth to 6 months,fasting blood glucose level,glucose tolerance and serum insulin level in 6-month mice were detected in the three groups.The liver tissues of 6-month mice were obtained.The mRNA expression of growth factor receptor-bound protein 10(Grb10)was detected by qRT-PCR.The protein expressional levels of Grb10,phosphate-protein kinase B(p-AKT)and phosphate-mitogen-activated protein kinase(p-MAPK)were detected by Western blotting.Results The body weight of the mice was lower in the vitrification group than in control group at 2 and 3 weeks after birth(P=0.007,P<0.001),the differences were statistically significant.The body weight of the mice was lower in the fresh group than in control group at 4 and 5 months after birth(P=0.023,P=0.013),the differences were statistically significant.The fasting plasma glucose level of 6-month-old mice in the fresh group and the vitrification group was significantly higher than that in control group(P<0.001,P=0.002),the differences were statistically significant;the fasting insulin level of the mice in the vitrification group was higher than that in control group(P=0.017),the difference was statistically significant;however,no significant differences were found between the fresh and vitrification groups(all P>0.05).Thirty minutes after glucose injection,the fasting serum glucose level in fresh group was significantly higher than that in vitrification group(P<0.001),120 min after glucose injection,the fasting serum glucose level in fresh group and vitrification group were significantly higher than that in control group(P=0.034,P=0.018),the difference was statistically significant.Compared with control group,the protein and mRNA expressional levels of Grb10 increased significantly in vitrification group and fresh group(all P<0.001),the difference was statistically significant.The protein expressional levels of p-AKT(all P=0.024)and p-MAPK(P=0.019,P=0.002)decreased significantly in vitrification group and fresh group compared with control group,the differences were statistically significant,there were no significant differences between vitrification group and fresh group(all P>0.05).Conclusion Vitrified ovarian orthotopic transplantation can affect the growth of the offspring of the mice,overexpression of the mRNA and protein of the imprinted gene Grb10 in the adult mouse liver,glucose metabolism disorder in adult offspring mice,reduced the cellular glucose levels by inhibiting the insulin/insulin-like growth factors-1 signaling pathway in offspring.
VitrificationOvarian tissueGrowth factor receptor-binding protein 10Insulin/IGF-1 signaling pathway
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