骨髓间充质干细胞对卵巢颗粒细胞凋亡的作用研究

发布时间：2017-12-31 11:24

本文关键词：骨髓间充质干细胞对卵巢颗粒细胞凋亡的作用研究　出处：《福建医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：探讨大鼠BMSCs对大鼠卵巢颗粒细胞凋亡的作用。方法：1.从SD大鼠骨髓中提取MSCs,经体外扩增、纯化并进行鉴定。2.体外实验,采用3周龄左右的雌性SD大鼠,皮下注射60单位的孕马血清,48小时后无菌条件下取出双侧卵巢,用皮试针针头在体视镜下刺破各卵泡,冲出颗粒细胞。颗粒细胞在体外扩增、纯化并进行鉴定。用MTT法得出顺铂对颗粒细胞的半抑制浓度及最佳干预时间。采用半抑制浓度的30%即5mg/L作为实验的干预浓度,干预时间为72h。体外实验分为：G0组颗粒细胞；G5组颗粒细胞+5mg/L顺铂；GM5组颗粒细胞+5mg/L顺铂+BMSCs。GM5组采用Millicell共培养小室把颗粒细胞和BMSCs分上下两层进行共培养,BMSCs分泌的细胞因子可以自由通过。实验各组干预72h后,通过流式细胞术检测各组颗粒细胞的凋亡率,RT-PCR技术检测各组颗粒细胞凋亡相关基因的表达情况。3.体内实验分为四组：空白对照组、MSCs组、雌激素组、青年组。前三组采用9-10月龄的SD雌性大鼠各15只,青年组采用15只3-4月龄的SD雌性大鼠。通过阴道脱落细胞监测前三组围绝经模型鼠进入围绝经后,空白组通过尾静脉输注2mL的生理盐水,一周后重复注射等量生理盐水；MSCs组经尾静脉输注2ml含1～2×106MSCs,一周后注射相同剂量MSCs细胞悬液；雌激素组灌用生理盐水溶解的尼尔雌醇,按0.158mg/(kg. d)灌胃给药,连续给药8w；青年组采用3月龄左右的SD大鼠经尾静脉注射2ml的生理盐水,一周后重复注射等量生理盐水。实验各组干预后第一、三个月,分别处死各组SD大鼠5只,取左侧卵巢经固定、切片做TUNEL凋亡检测,在显微镜下统计颗粒细胞的凋亡率。结果：体外实验结果显示：G0、G5、GM5各组凋亡率分别为0.59%、13.04%、4.84%。凋亡相关基因表达情况分别为：Survivn GOGM5G5; P21 G5GOGM5; Cmy-c GOGM5G5; Bax G5GOGM5; Bcl-2 GOGM5G5。(G5组与GM5组各基因的表达相对量进行比较,其中P21, Cmy-c, Bax统计值P＜0.05有统计学意义；而G5组与GM5组Bcl-2, Survivn统计值P＞0.05无统计学意义)。体内实验结果显示在干预一个月后实验各组卵巢颗粒细胞的凋亡率分别为：空白组9.90±3.23%、MSCs组6.10±2.63%(P=0.075)、雌激素组6.60±3.13%(P=0.139)、青年组1.53±1.44%(P=0.001)；干预三个月后实验各组卵巢颗粒细胞的凋亡率分别为：空白组14.60±3.51%、MSC组8.29±3.35%(P=0.02)、雌激素组10.53±2.29%(P=0.062)、青年组3.56±1.44%(P＜0.01)(P为实验组与空白组卵巢颗粒细胞凋亡率统计值,P＜0.05差异有统计学意义)。结论：大鼠BMSCs在体内可以保护围绝经大鼠的卵巢颗粒细胞,减少其凋亡；在体外BMSCs可以保护顺铂诱导大鼠卵巢颗粒细胞的凋亡。综合体内、外实验结果：大鼠BMSCs具有抑制大鼠卵巢颗粒细胞凋亡的作用。
[Abstract]:Objective: to investigate the effect of rat BMSCs on apoptosis of rat ovarian granulosa cells. Methods 1. MSCs were extracted from SD rat bone marrow, amplified in vitro, purified and identified. 2. In vitro experiment, female SD rats of about 3 weeks old were used. After 48 hours of hypodermic injection of 60 units of pregnant horse serum, both ovaries were removed under aseptic condition. The follicles were punctured with the needle of skin test needle under stereoscopic microscope, and granulosa cells were expanded in vitro. Purification and identification. The semi-inhibitory concentration of cisplatin on granulosa cells and the optimal intervention time were obtained by MTT method. 30% mg / L of semi-inhibitory concentration was used as the experimental intervention concentration. The intervention time was 72 hours. G5 granulosa cells were treated with 5 mg / L cisplatin. Granulosa cells were co-cultured in 5 mg / L cisplatin BMSCs.GM5 group (GM5 group) with Millicell co-culture chamber. The granulosa cells and BMSCs were co-cultured in two layers. The cytokines secreted by BMSCs were free to pass through. After 72 hours of intervention, the apoptotic rate of granulosa cells in each group was detected by flow cytometry. RT-PCR technique was used to detect the expression of apoptosis-related genes in granulosa cells. In vivo experiments were divided into four groups: blank control group and estrogen group. The first three groups were treated with 15 female SD rats aged 9-10 months. In the young group, 15 female SD rats aged 3-4 months were used. The first three groups of perimenopausal model rats were monitored by vaginal exfoliation cells after entering the perimenopausal period, and the blank group was infused with 2 mL of normal saline via caudal vein. After one week, the same amount of saline was injected repeatedly. In the MSCs group, 2 ml of MSCs cells were injected into the tail vein and 2 脳 106 MSCs were injected with the same dose of MSCs cell suspension one week later. In estrogen group, nilestriol dissolved in normal saline was administered intragastrically at 0.158 mg / kg 路d for 8 weeks. In the young group, 2 ml saline was injected through caudal vein in 3 months old SD rats, and the same amount of saline was injected repeatedly one week later. Five SD rats in each group were killed. The left ovary was fixed and the apoptosis rate of granulosa cells was measured by TUNEL. Results: the results of in vitro experiment showed that the apoptosis rate of G5 group was 0.59% and 13.04% respectively. 4.84. The expression of apoptosis-related genes was: survivvn GOGM5G5; P21 G5GOGM5; Cmy-c GOGM5G5; Bax G5GOGM5; The relative amount of gene expression in Bcl-2 GOGM5G5.(G5 group was compared with that in GM5 group, including P21, Cmy-c. The statistical value of Bax was statistically significant (P < 0. 05). And Bcl-2 in G5 group and GM5 group. There was no significant difference in Survivn statistical value (P > 0. 05). The results of in vivo experiment showed that the apoptosis rate of ovarian granulosa cells in each group was 9.90 卤3.23% in blank group after one month of intervention. MSCs group (6.10 卤2.63) and estrogen group (6.60 卤3.13). The young group (1.53 卤1.44) was 0.0001g. Three months after intervention, the apoptosis rate of ovarian granulosa cells was 14.60 卤3.51 in blank group and 8.29 卤3.35 in MSC group. In estrogen group (10.53 卤2.29) and young group (3.56 卤1.44), the apoptotic rate of ovarian granulosa cells in experimental group and blank group was 10.53 卤2.29 and 3.56 卤1.44, respectively (P < 0.01). P < 0.05 the difference was statistically significant. Conclusion: rat BMSCs can protect ovarian granulosa cells and reduce apoptosis of peri-menopausal rats in vivo. In vitro, BMSCs could protect rat ovarian granulosa cells from apoptosis induced by cisplatin. In vivo and in vitro, BMSCs could inhibit the apoptosis of rat ovarian granulosa cells.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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