经血间充质干细胞通过IGF-1信号通路改善小鼠卵巢早衰

Menstrual blood mesenchymal stem cells improve premature ovarian failure in mice via IGF-1 signaling

目的探讨经血间充质干细胞(MenSCs)移植改善化疗所致卵巢早衰(POF)小鼠卵巢功能的效果和分子机制.方法48只C57BJ雌鼠被随机分成3组:对照组、POF组和干细胞组.环磷酰胺和白消安腹腔注射构建POF小鼠;干细胞组小鼠在化疗药物注射后3 d开始鼠尾静脉注射MenSCs.采用qRT-PCR定量分析卵巢内生长因子受体结合蛋白10(GRB10)的mRNA表达;采用Western blotting定量分析卵巢内GRB10、叉头状转录因子O亚家族蛋白3(FOXO3a)、p-FOXO3a、胰岛素样生长因子受体(IGF-1R)、p-IGF-1R、蛋白激酶B(AKT)、p-AKT的蛋白表达.结果对照组、POF组、干细胞组的血清雌二醇浓度分别为(222.29±35.13)、(90.87±28.76)、(193.24±48.74)pg/mL,3组之间差异有统计学意义(F=19.332,P<0.001);POF组低于对照组(P<0.001)和干细胞组(P<0.001).对照组、POF组、干细胞组小鼠超排的成熟卵子数分别为(40.00±10.07)、(9.00±7.42)、(24.60±10.24)个,3组之间差异有统计学意义(F=13.792,P=0.001);对照组(P<0.001)和干细胞组(P=0.021)高于POF组,且对照组高于干细胞组(P=0.023).Western blotting检测对照组、POF组、干细胞组卵巢内GRB10蛋白条带灰度值分别为1.22±0.05、1.82±0.13、1.10±0.09,3组之间差异有统计学意义(F=94.340,P<0.001);POF组高于对照组(P<0.001)和干细胞组(P<0.001).3组中p-FOXO3a的蛋白条带灰度值分别为0.80±0.08、0.33±0.06、0.76±0.05;3组之间差异有统计学意义(F=98.673,P<0.001);POF组低于对照组(P<0.001)和干细胞组(P<0.001).3组小鼠中p-IGF-1R的蛋白条带灰度值分别为0.31±0.10、0.32±0.08、0.42±0.01,3组之间差异有统计学意义(F=3.882,P=0.044);干细胞组p-IGF-1R蛋白含量高于对照组(P=0.023)和POF组(P=0.039).对照组、POF组、干细胞组小鼠的p-AKT蛋白条带灰度值分别为0.57±0.02、0.51±0.05、0.64±0.03,3组之间差异有统计学意义(F=22.227,P<0.001);干细胞组蛋白含量高于对照组(P=0.005)和POF组(P<0.001),对照组高于POF组(P=0.004).结论MenSCs移植后,可能是通过干细胞旁分泌的胰岛素样生长因子1(IGF-1)与IGF-1R结合,促进p-IGF-1R的表达以及下调卵巢靶细胞GRB10的表达,激活IGF-1信号通路,促进磷脂酰肌醇3-激酶(PI3K)-AKT-FOXO3a途径下游因子p-FOXO3a的表达,从而激活原始卵泡发育,改善POF.

Objective To investigate the effect and molecular mechanism of menstrual blood mesenchymal stem cells(MenSCs)transplantation on the ovarian function in mice with premature ovarian failure(POF)induced by chemotherapy.Methods A total of 48 C57BJ female mice were randomly divided into control,POF and MenSCs groups.The POF models were induced by intraperitoneal injection of cyclophosphamide combined with busulfan,and the MenSCs models were established by injecting MenSCs via tail vein 3 days after chemotherapy.The mRNA expression of growth factor receptor-binding protein 10(GRB10)in ovaries was detected with qRT-PCR.The protein expressions of GRB10,forkhead box O3(FOXO3a),p-FOXO3a,insulin-like growth factor receptor(IGF-1R),p-IGF-1R,protein kinase B(AKT)and p-AKT were detected with Western blotting.Results The concentration of serum estradiol in the control group,POF group,and MenSCs group was(222.29±35.13),(90.87±28.76),and(193.24±48.74)pg/mL,respectively(F=19.332,P<0.001),and it was lower in POF group than in control group(P<0.001)and MenSCs group(P<0.001).The number of superovulated mature eggs was 40.00±10.07,9.00±7.42,and 24.60±10.24,respectively(F=13.792,P=0.001),and it was higher in control group(P<0.001)and MenSCs group(P=0.021)than in POF group,and higher in control group than in MenSCs group(P=0.039).The gray value of GRB10 protein bands was 1.22±0.05,1.82±0.13,and 1.10±0.09,respectively(F=94.340,P<0.001),and it was higher in POF group than in control group(P<0.001)and MenSCs group(P<0.001).The gray value of p-FOXO3a protein bands was 0.80±0.08,0.33±0.06,and 0.76±0.05,respectively(F=98.673,P<0.001),and it was lower in POF group than in control group(P<0.001)and MenSCs group(P<0.001).The gray value of p-IGF-1R protein bands was 0.31±0.10,0.32±0.08,and 0.42±0.01,respectively(F=3.882,P=0.044),and it was higher in MenSCs group than in control group(P=0.023)and POF group(P=0.039).The gray value of p-AKT protein bands was 0.57±0.02,0.51±0.05,and 0.64±0.03,respectively(F=22.227,P<0.001),and it was higher in MenSCs group than in control group(P=0.005)and POF group(P<0.001),and higher in control group than in POF group(P=0.004).Conclusion Via the binding of IGF-1 and IGF-1R,MenSCs transplantation may upregulate p-IGF-1R expression while downregulate GRB10 expression,activate IGF-1 signaling pathway,upregulate p-FOXO3a expression,and thereby activates the development of primordial follicles and improves POF.