脐带间充质干细胞通过NR4A1调节原发性功能不全卵巢中卵泡膜间质细胞凋亡的作用研究

目的 探讨脐带间充质干细胞(UCMSCs)对原发性卵巢功能不全(POI)大鼠的治疗作用及卵巢中卵泡膜间质细胞表达的NR4A1在此治疗过程中的机制。方法 选用5周龄雌性wistar大鼠,随机分为对照组、POI组、POI+UCMSCs组,POI组腹腔注射顺铂(CP),对照组腹腔注射等量的0.9%生理盐水,注射7 d。POI+UCMSCs组待注射顺铂后,观察1周,尾静脉注射UCMSCs悬液。注射1 周后,酶联免疫吸附法检测 3 组大鼠血清中 E₂、FSH 水平,HE 染色观察卵巢的形态学改变,TUNEL检测大鼠卵巢中颗粒细胞的凋亡情况。体外培养大鼠卵泡膜间质细胞(TICs),并进行鉴定及检测NR4A1的表达,随机分组给予顺铂及UCMSCs处理,western blot检测NR4A1的表达变化,Annexin V-FITC细胞凋亡检测试剂盒检测细胞的凋亡情况。结果 与对照组相比,POI组大鼠卵巢中各级卵泡的数目明显减少(P<0.05),而“类黄体样”结构明显增多(P<0.05),UCMSCs处理后卵巢中卵泡的数目明显增加(P<0.05)。与对照组相比,POI组血清中E₂水平明显下降(P<0.01)、FSH水平升高(P<0.05)。与POI组相比,POI+UCMSCs组血清中E₂水平升高、FSH水平降低(P<0.05)。与对照组相比,POI组大鼠卵巢中凋亡的颗粒细胞数目明显增多。POI+UCMSCs组颗粒细胞凋亡的数目明显减少(P<0.01)。体外培养的TICs呈梭形,并且表达Cyp17a1和NR4A1,不表达FSHR。CP处理后,TICs表达的NR4A1水平降低,而CP+UCMSCs组NR4A1表达水平有所升高(P<0.05)。CP处理后能够增加TICs的凋亡情况,而CP+UCMSCs组凋亡比例明显下降(P<0.01)。结论 UCMSCs通过调节TICs表达的NR4A1水平,增加TICs对颗粒细胞凋亡的调节作用,改善POI卵巢的功能,这为临床上应用UCMSCs治疗POI提供一定的理论和实验依据。

Effect of umbilical cord mesenchymal stem cells on the regulation of apoptosis of theca cells by NR4A1 in primary ovarian insufficiency

Objective To investigate the therapeutic effects of umbilical cord mesenchymal stem cells (UCMSCs) on (primary ovarian insufficiency) POI rats and the mechanism of NR4A1 expression in the theca-interstitial cells (TICs) during the treatment. Methods Female Wistar rats of 5 weeks old were randomly divided into control group, POI group and POI+UCMSCs group. POI group was intraperitoneally injected with cis-platin (CP), and the control group was intraperitoneally injected with 0.9% saline, following with 7 days. POI+UCMSCs were treated with CP for 1 week, and then UCMSCs suspension was injected into the tail vein. One week later, the levels of E₂ and FSH in the serum of the three groups were detected by enzyme-linked immunosorbent assay (ELISA). The morphological changes of ovaries were observed by HE staining. The apoptosis of granulosacells was detected by TUNEL. In addition, TICs were cultured in vitro and NR4A1 was expressed in TICs. CP and UCMSCs were treated into TICs and western blot was used to detect the levels of NR4A1 expression. Annexin V-FITC apoptosis assay kit was used to detect the apoptosis of TICs. Results Compared with the control group, the ovary of POI group was atrophy and the number of follicles in the ovary was significantly reduced(P<0.05), following the “luteal-like” structure being increased significantly(P<0.05). The number of follicles in the ovary increased significantly after UCMSCs treatment(P<0.05). Compared with the control group,the serum E₂ level in the POI group was significantly decreased(P<0.01), while the FSH level was significantly increased(P<0.05). Compared withthe POI group, the serum E₂ level was increased and the FSH level was decreased by UCMSCs treatment(P<0.05). Compared with the control group, the apoptosis of the granulosa cells in the POI group was significantly increased, while the UCMSCs could decrease the apoptosis of the granulosa cells(P<0.01). In addition, TICs cultured in vitro were fusiform and Cyp17a1 and NR4A1 were expressed, but FSHR expressed negative(P<0.05). After CP treatment, the NR4A1level was significantly decreased, and the apoptosis of TICs was significantly increased. After treatment with UCMSCs, the expression of NR4A1 was increased, but the apoptosis of TICs was significantly decreased(P<0.01). Conclusion UCMSCs could restore the ovarian function of POI by regulating the NR4A1 level of TICs expression and increase the regulation of TICs on granulosa cell apoptosis. This could provide a certain theoretical and experimental basis about the treatment of UCMSCs in POI.