论文部分内容阅读
目的:探讨miR-21在化疗性卵巢早衰大鼠模型中的治疗潜能及其可能机制。方法:体外构建miR-21慢病毒载体(LV-miR-21)。将大鼠随机分为空白对照组、模型组、空载组及miR-21组,通过腹腔注射环磷酰胺(CTX)建立化疗所致卵巢早衰大鼠模型。建模后往miR-21组大鼠双侧卵巢注射LV-miR-21,注射后第1、15、30、45、60天分批处死大鼠。阴道脱落细胞涂片检测大鼠动情周期;化学发光法与免疫放射法测定性激素水平;进行卵巢称重、计数各级卵泡数;TUNEL法测定卵巢组织颗粒细胞凋亡率;qRT-PCR、Western blot法检测miR-21靶基因PTEN、PDCD的mRNA及蛋白水平。结果:体外成功构建miR-21慢病毒载体。实验结束时,miR-21组有64%(16/25)大鼠恢复规律动情周期。注射后第15、30、45、60天,miR-21组的E_2水平、卵巢颗粒细胞凋亡率均高于模型组和空载组(P=0.000),FSH水平以及PTEN、PDCD4的mRNA、蛋白表达较相应时间点的模型组和空载组显著下降(P=0.000)。注射后第30、45、60天,miR-21组的卵巢重量显著高于模型组和空载组,但仍低于空白对照组;注射后第45、60天,miR-21组各级卵泡数均多于模型组和空载组(P=0.000);结论:miR-21在化疗诱导卵巢早衰大鼠模型中具有治疗潜能,其具体作用机制可能与下调靶基因PTEN、PDCD4有关。
Objective: To investigate the therapeutic potential of miR-21 in rat model of chemotherapy-induced premature ovarian failure and its possible mechanism. Methods: miR-21 lentiviral vector (LV-miR-21) was constructed in vitro. The rats were randomly divided into blank control group, model group, no-load group and miR-21 group. The rat model of premature ovarian failure induced by chemotherapy was established by intraperitoneal injection of cyclophosphamide (CTX). After modeling, LV-miR-21 was injected into both ovaries of miR-21 group and rats were sacrificed on the 1st, 15th, 30th, 45th and 60th days after injection. Vaginal exfoliated cell smears were used to detect the estrous cycle of rats; chemiluminescence and radioimmunoassay were used to determine the levels of sex hormones; ovaries were weighed and count the number of follicles at each stage; TUNEL method was used to determine the apoptosis rate of ovarian granulosa cells; qRT-PCR and Western blot Method to detect the mRNA and protein levels of miR-21 target gene PTEN and PDCD. Results: miR-21 lentiviral vector was successfully constructed in vitro. At the end of the experiment, 64% (16/25) of the miR-21 groups regained the regular estrous cycle. At the 15th, 30th, 45th, and 60th day after injection, the levels of E 2 and granulosa cell apoptosis in miR-21 group were significantly higher than those in model group and no-load group (P = 0.000), FSH, PTEN and PDCD4 mRNA, Compared with the model group and the no-load group, the protein expression was significantly decreased (P = 0.000). On the 30th, 45th and 60th day after injection, the ovary weight of miR-21 group was significantly higher than that of the model group and the no-load group, but still lower than that of the blank control group. On the 45th and 60th day after injection, (P = 0.000). Conclusion: miR-21 has therapeutic potential in chemotherapy-induced premature ovarian failure in rats. The specific mechanism may be related to the down-regulation of PTEN and PDCD4.