骨髓间充质干细胞移植对环磷酰胺化疗致卵巢损伤保护作用的体外研究

Experimental study on the protective effects of bone marrow mesenchymal stem cell transplantation on ovarian injury caused by chemotherapy with cyclophosphamide in vitro

目的研究骨髓间充质干细胞移植对环磷酰胺化疗所致卵巢损伤的保护作用。方法大鼠分为空白组(NG)、卵巢损伤组(DG)和卵巢损伤/骨髓干细胞移植组(D/T),观察大鼠的动情周期,测定性激素和抗穆勒氏管激素(AMH)表达水平,卵泡数量及卵巢组织学观察,评估3组大鼠的卵巢功能。结果实验过程中,NG组大鼠阴道涂片呈正常动情周期改变,DG组和D/T组大鼠在给予环磷酰胺后,动情周期紊乱。D/T组25%(4/16)大鼠移植细胞后1个月开始出现完整动情周期(6~8 d),其他大鼠随着时间进展,逐渐出现完整动情周期。DG组大鼠至移植3个月后,动情周期仍未恢复。造模前,3组大鼠雌二醇、卵泡刺激素、AMH表达水平和卵泡数量差异无统计学意义(P>0.05);移植后,DG组雌二醇表达水平显著降低(P<0.05),卵泡刺激素表达水平逐渐升高,并显著高于NG组(P<0.05);移植后15 d和1个月,D/T组的雌二醇表达水平显著低于NG组(P<0.05),卵泡刺激素表达水平显著高于NG组(P<0.05);移植后2个月和3个月,D/T组雌二醇表达水平逐渐升高,与NG组差异无统计学意义(P>0.05),卵泡刺激素的表达水平与NG组差异无统计学意义(P>0.05)。移植后各时间点,DG组的AMH表达水平显著低于NG组(P<0.05),DG组和D/T组大鼠卵泡数量显著低于NG组(P<0.05),而D/T组大鼠卵泡数量显著高于DG组(P<0.05)。移植后15 d、1个月和2个月,D/T组的AMH表达水平显著低于NG组(P<0.05),移植后3个月,D/T组的AMH表达水平与NG组差异无统计学意义(P>0.05)。卵巢形态学观察发现D/T组卵泡逐渐恢复至正常状态。结论骨髓间充质干细胞移植可以有效修复化疗药物环磷酰胺引起的卵巢损伤。

Objective To investigate the protective effects of bone marrow mesenchymal stem cell transplantation on ovarian injury caused by cyclophosphamide chemotherapy in vitro. Methods The rats were divided into blank group（ NG group）,ovariectomized group（ DG group） and ovarian injury/bone marrow stem cell transplantation group（ D/T group）. The ovarian function indexes including estrous cycle,the expression levels of sex hormones and anti-Mullerian hormone（ AMH）,the number of follicles and ovary histological changes were observed and compared among the three groups. Results During the experiment,the vaginal smear in NG group showed normal estrous cycle. The oestrous cycle in DG group and D/T group was disordered after treated by cyclophosphamide. The complete estrous cycle（ 6 ~ 8 d） began to appear on 1 month after cell transplantion in 25%（ 4/16） of D/T group,and complete estrous cycle appeared gradually in the other rats with time going on. The rats in DG group on 3 months after cell transplantion,the estrous cycle was still not recovered. Before modeling there were no significant differences in the levels of estradiol,follicle stimulating hormone（ FSH）,AMH and follicle count among the three groups（ P〉0. 05）,however,after transplantation,the levels of estradiol in DG group were significantly decreased（ P〈0. 05）,and the levels of FSH were increased gradually,moreover,which were significantly higher than those in NG group（ P〈0. 05）. The levels of estradiol in D/T group were significantly lower than those in NG group（ P〈0. 05） on 15 days and 1 month after transplantation,and the levels of FSH were significantly higher than those in NG group（ P〈0. 05）. The expression levels of estradiol in D/T group were increased gradually on 2 months and 3 months after transplantation,however,there were no significant differences in the expression levels of estradiol and FSH between D/T group and NG group（ P〉0. 05）. Moreover the levels of AMH in DG group in every time point after transplantation were significantly lower than those in NG group（ P〈0. 05）,and the numbers of follicle in DG group and D/T group were significantly lower than those in NG group（ P〈0. 05）. However the numbers of follicles in D/T group were significantly higher than those in DG group（ P〈0. 05）. On15 d,1 m and 2 m after transplantation,the expression levels of AMH in D/T group were significantly lower than those in NG group（ P〈0. 0 5）,but there were no significant differences in the expression levels of AMH on 3 months after transplantation between D/T group and NG group（ P〉0. 05）. The observation of ovarian morphology showed that the follicle in D/T group gradually returned to normal state. Conclusion The bone marrow mesenchymal stem cell transplantation can effectively repair the ovarian injury caused by cyclophosphamide chemotherapy.