Cx43（间隙连接蛋白43）和GATA-4（转录因子GATA结合蛋白4）在PCOS模型大鼠中表达及复方甘芍贴剂的干预作用

发布时间：2018-05-19 21:07

本文选题：多囊卵巢综合征 + 大鼠　；参考：《山西医科大学》2014年硕士论文

【摘要】：目的：多囊卵巢综合征(PCOS)是常见的女性排卵障碍性疾病，临床表现为月经稀发、不孕、多毛、肥胖等。但迄今病因未明，临床治疗效果欠佳。 Cx43是颗粒细胞膜表面的连接蛋白。GATA-4是一种转录调节因子。June等动物研究发现Cx43与颗粒细胞的正常发育有关。White等发现GATA-4与颗粒细胞的增殖分化密切相关。二者均与卵泡的正常发育有关。探讨Cx43和GATA-4在PCOS模型大鼠卵巢的表达及复方甘芍贴剂干预作用机制。本课题组前期研究结果证实，复方甘芍贴剂治疗PCOS模型大鼠，在血清性激素水平、光镜和电镜下的组织学变化均与目前临床PCOS推荐治疗药物达英-35的效果类似。本研究拟通过Cx43和GATA-4在研究组与对照组中的变化来探讨复方甘芍贴剂可能的治疗机制。方法：将48只6周龄的SD雌性大鼠,首先随机分为两组,正常对照组(12只)和实验对照组(36只),正常对照组同期灌服1%CMC(1ml/100g),1次/d,连续3周。实验对照组用来曲唑建立多囊卵巢综合征（PCOS）大鼠模型,采用化学发光法检测大鼠血清性激素水平,符合PCOS模型的大鼠进入下一步研究,将36只大鼠随机分为PCOS模型组（对照）、达英-35（炔雌醇环丙孕酮）对照组和外用复方甘芍贴剂研究组。达英-35（炔雌醇环丙孕酮）组为自9周龄起，按人与大鼠公斤体重折算系数给药，将药物溶于1%CMC(1ml/100g)给药，1次/d，连续3周；复方甘芍外用贴剂组64d龄时，脱毛后并给予贴剂，1贴/d，并且同期灌服1%CMC(1ml/100g),1次/d，连续3周；PCOS模型组和正常对照组大鼠同期灌服同体积1%CMC (1ml/100g)，1次/d，共3周。其中模型组和达英-35组因操作不当，，灌死两只，各剩下11只。剩余46只于84日龄取血清测睾酮(T),卵泡生成素(FSH),黄体生成素(LH),取双侧卵巢,左侧置于10%的甲醛液中固定24小时，用免疫组织化学观察Cx43和GATA-4在PCOS模型大鼠的表达情况;右侧卵巢放于-70度冰箱冻存,采用Western blot方法观察Cx43和GATA-4在实验大鼠卵巢颗粒细胞中的蛋白表达情况。采用SPSS17.0统计软件进行分析;数据均以均数±标准差表示,多样本间比较采用单因素方差分析，两两比较用LSD-t检验两组间均数间相关性分析采用Pearson相关。以P＜0.05为差异具有统计学意义。结果： 1.与正常对照组比较,正常对照组大鼠血清FSH水平为（2.41±0.23）IU/L，LH水平为（8.11±0.85）IU/L，T水平为（2.3±0.96）nmol/L,36只来曲唑诱导的多囊卵巢综合征模型大鼠血清FSH、LH、T水平分别为FSH（2.95±0.21）IU/L，LH（8.72±0.99）IU/L，T（3.18±1.46）nmol/L，提示造模成功。 2.免疫组织化学染色:（1）与正常组大鼠卵巢Cx43灰度值(149.59±9.25)比较, PCOS模型组大鼠卵巢Cx43灰度值(98.19±6.51)，差异具有显著性（P＜0.05）；与PCOS模型组大鼠卵巢Cx43灰度值(98.19±6.51)比较，复方甘芍贴剂组大鼠卵巢颗粒细胞膜中Cx43棕黄色颗粒阳性表达灰度值（144.28±4.72），差异具有显著性（P＜0.05）；与达英-35（炔雌醇环丙孕酮）对照组（143.79±9.81）相比，复方甘芍贴剂组大鼠卵巢颗粒细胞膜中Cx43棕黄色颗粒阳性表达灰度值（144.29±4.72），没有显著性差异（P0.05）。（2）与正常组大鼠卵巢灰度值GATA-4(129.37±9.99)比较, PCOS模型组大鼠卵巢GATA-4灰度值(162.91±9.38)，差异具有显著性（P＜0.05）；与PCOS模型组大鼠卵巢GATA-4灰度值(162.91±9.38)比较，复方甘芍贴剂组大鼠卵巢GATA-4棕黄色颗粒阳性表达灰度值（115.13±7.04），差异具有显著性（P＜0.05）；与达英-35（炔雌醇环丙孕酮）治疗组（112.37±7.90），复方甘芍贴剂组大鼠卵巢GATA-4棕黄色颗粒阳性表达灰度值（115.13±7.04），没有显著性差异（P0.05）。 3.采用Western blot方法检测结果:（1）与正常组大鼠卵巢（0.86±0.10）比较，PCOS模型组大鼠卵巢Cx43蛋白表达（0.66±0.15），差异具有显著性（P＜0.05）；与PCOS模型组大鼠卵巢Cx43(0.66±0.15)比较，复方甘芍贴剂组大鼠卵巢（0.86±0.10），差异具有显著性（P＜0.05）；与达英-35（炔雌醇环丙孕酮）治疗组（0.88±0.12），复方甘芍贴剂组大鼠卵巢GATA-4蛋白表达（0.86±0.10），没有显著性差异（P0.05）。（2）与正常组大鼠卵巢GATA-4（0.90±0.10）比较，PCOS模型组大鼠卵巢GATA-4蛋白表达（1.64±0.16），差异具有显著性（P＜0.05）；与PCOS模型组大鼠卵巢GATA-4比较，复方甘芍贴剂组大鼠卵巢GATA-4（1.00±0.09），差异具有显著性（P＜0.05）；与达英-35（炔雌醇环丙孕酮）治疗组（1.15±0.32）比较，复方甘芍贴剂组大鼠卵巢GATA-4蛋白表达（1.00±0.09），没有显著性差异（P0.05）。结论：1. PCOS模型大鼠卵巢颗粒细胞Cx43表达减少, GATA-4表达增加与多囊卵巢综合征的发病可能有关.2.复方甘芍贴剂治疗后, PCOS模型大鼠卵巢颗粒细胞Cx43表达增加,GATA-4表达下降,提示该贴剂可能通过调节Cx43与GATA-4水平,改善卵泡的发育,抑制其凋亡而发挥治疗作用。
[Abstract]:Objective: polycystic ovary syndrome (PCOS) is a common female ovulation disorder. The clinical manifestations of polycystic ovary syndrome are dilute menstruation, infertility, hairy, obesity and so on. But so far the etiology is not clear and the effect of clinical treatment is not good. Cx43 is a connexin.GATA-4 on the surface of granular cell membrane, a transcription regulator.June and other animal studies found Cx43 and granular cells The normal development related to.White found that GATA-4 was closely related to the proliferation and differentiation of granulosa cells. All two were related to the normal development of follicle. The expression of Cx43 and GATA-4 in the ovary of the rat model of PCOS and the intervention mechanism of compound Gump peony patch were discussed. The results of the previous study in this group confirmed that the compound Gump peony patch was used to treat PCOS model rats, The level of serum sex hormone, the histological changes under light and electron microscopy are similar to that of the current clinical PCOS recommended medications, Da Ying -35. This study intends to explore the possible therapeutic mechanism of compound Gump peony patch by the changes of Cx43 and GATA-4 in the study group and the control group.
Methods: 48 SD female rats of 6 weeks old were randomly divided into two groups, the normal control group (12 rats) and the experimental control group (36 rats). The normal control group was given 1%CMC (1ml/100g), 1 /d and 3 weeks for 3 weeks. The experimental control group was used to establish the rat model of polycystic ovary syndrome (PCOS), and the serum sex hormone was detected by chemiluminescence. 36 rats were randomly divided into PCOS model group (control), Da Ying -35 (estriol proprop progesterone) control group and foreign compound Gump peony patch group. The group of Da British -35 (estriol proprop) group was from 9 weeks of age, and the drug was dissolving in 1%CMC by human and rat kg body weight conversion coefficient. (2) 36 rats were randomly divided into the model group (control), the control group of Da Ying (estriol proprop) and the compound Gump peony patch group. 1ml/100g) administration, 1 times /d, for 3 weeks. When the compound Gump peony external use patch group was 64D, it was depilated and given a patch, 1 /d, and 1%CMC (1ml/100g) and 1 /d for 3 weeks for the same period; PCOS model group and normal control group were given the same volume 1%CMC (1ml/100g), 1 /d, for 3 weeks. The model group and Da Ying -35 group were irrigated because of improper operation. The remaining 11 were left 11. The remaining 46 were serum testosterone (T), follicular hormone (FSH), luteinizing hormone (LH), bilateral ovary and 24 hour in the 10% formaldehyde solution. The expression of Cx43 and GATA-4 in PCOS model rats was observed by immunohistochemistry; the right ovary was stored in -70 degree refrigerator, and Western BL was used. Ot method was used to observe the protein expression of Cx43 and GATA-4 in the ovarian granulosa cells of experimental rats. The data were analyzed with SPSS17.0 statistical software. The data were expressed with mean standard deviation, and a single factor analysis of variance was used for the comparison. The correlation analysis between the two groups was compared with the LSD-t test, and the correlation analysis was correlated with Pearson. P < 0.05 was 0.05. The difference was statistically significant.
1. compared with the normal control group, the serum FSH level of the normal control group was (2.41 + 0.23) IU/L, the level of LH was (8.11 + 0.85) IU/L, the level of T was (2.3 + 0.96) nmol/L. The serum FSH, LH, T level of the rat model of polycystic ovary syndrome induced by letrozole was FSH (2.95 + 0.21) IU/L, LH (8.72 + 0.99), indicating the formation of the model. Work.
2. immunohistochemical staining: (1) compared with the Cx43 gray value (149.59 + 9.25) in the normal group, the Cx43 gray value of the ovary in the PCOS model group was significantly (98.19 + 6.51), and the difference was significant (P < 0.05). Compared with the Cx43 gray value (98.19 + 6.51) in the rat ovary of the PCOS model group (98.19 + 6.51), the Cx43 brown and yellow in the granulosa cell membrane of the compound Gump patch group was Cx43 brown and yellow. The gray value of the positive expression of color particles was (144.28 + 4.72), and the difference was significant (P < 0.05). Compared with the control group (143.79 + 9.81) of -35 (estriol proproparone) in the control group (143.79 + 9.81), the positive expression of Cx43 brown yellow granules in the granulosa cell membrane of the compound Gump patch group was (144.29 + 4.72), and there was no significant difference (P0.05). (2) and the normal group The gray value of rat ovaries was GATA-4 (129.37 + 9.99). The GATA-4 gray value of the ovary in the PCOS model group was (162.91 + 9.38), and the difference was significant (P < 0.05). Compared with the GATA-4 gray value (162.91 + 9.38) of the rat ovary of the PCOS model group (162.91 + 9.38), the positive expression of the GATA-4 brown yellow granules in the ovary of the compound Gump patch group was (115.13 + 7.04), and the difference was different. It was significant (P < 0.05); with the treatment group (112.37 + 7.90) of Da Ying -35 (estriol propropyl progesterone), the positive expression of GATA-4 brown yellow granules in the ovary of the compound Gump peony patch group was (115.13 + 7.04), and there was no significant difference (P0.05).
3. Western blot method was used to detect the results of (1) compared with the normal group of rat ovary (0.86 + 0.10), the expression of Cx43 protein in the ovary of the PCOS model group was (0.66 + 0.15), the difference was significant (P < 0.05). Compared with the Cx43 (0.66 + 0.15) of the rat ovary (0.66 + 0.15) in the model group, the difference was significant (P < 0) (P < 0) (P < 0). 5): with the treatment group (0.88 + 0.12) of -35 (0.88 + 0.12), there was no significant difference in the expression of GATA-4 protein (P0.05). (2) compared with the normal group of rat ovary (0.90 + 0.10), the expression of GATA-4 protein in the ovary of the PCOS model group was (1.64 + 0.16), and the difference was significant (P < 0.05). Compared with the rat ovarian GATA-4 in the PCOS model group, the ovary was GATA-4 (1 + 0.09) in the compound Gump patch group (P < 0.05). Compared with the treatment group (1.15 + 0.32) of Da Ying -35 (1.15 + 0.32), the expression of GATA-4 egg white in the ovary of the compound Gump patch group was (1 + 0.09), and there was no significant difference (P0.05).
Conclusion: 1. PCOS model rat ovarian granulosa cells Cx43 expression decreased, GATA-4 expression increased and the pathogenesis of polycystic ovary syndrome may be related to the.2. compound glycyrrhizin patch treatment, PCOS model rat ovarian granulosa cells Cx43 expression increased, GATA-4 expression decreased, suggesting that the patch can improve the follicle by regulating the level of Cx43 and GATA-4. Development, inhibition of its apoptosis and play a therapeutic role.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.75

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