雌激素对去卵巢大鼠Wnt16、β-catenin、OPG、RANKL表达的影响

发布时间：2018-06-17 17:05

本文选题：雌激素 + Wnt16　；参考：《南京医科大学》2017年硕士论文

【摘要】：背景:雌激素是女性生长、发育过程中必不可少的类固醇激素,主要功能有促进女性生殖器官的发育和成熟,维持女性的第二性征;同时,对成骨细胞、破骨细胞、破骨前体细胞等也有一定调节作用。成骨细胞和/或破骨细胞功能紊乱是导致骨质疏松症发生的关键病理生理机制。骨质疏松症分为原发性和继发性两种,而原发性骨质疏松症常见于中老年人群中,尤以绝经后女性最为常见。目前研究认为,绝经后骨质疏松症与雌激素减少有关。2013年国际绝经指南指出,围绝经期和绝经早期给予绝经激素治疗(menopausal hormone therapy,MHT)可减缓骨量流失速度[1,2],但具体机制尚不明确。目前研究表明,Wnt/β-catenin途径、OPG/RANK/RANKL系统在绝经后骨质疏松症中起到一定程度的调节作用。Wnt16属于Wnt家族,由分泌性糖蛋白组成,具有高度保守型,参与调控成骨细胞、破骨细胞活性。Wnt16与配体结合后,激活Wnt16/β-catenin经典途径,引起成骨细胞相关基因表达活跃,促进骨形成,维持骨稳态平衡。RANKL是表达在成骨细胞和基质细胞上的膜结合蛋白,可被金属蛋白酶分解为可溶性RANKL(sRANKL),RANKL通过与破骨前体细胞、破骨细胞表面RANK特异受体结合,促进破骨前体细胞分化成熟,增强破骨细胞骨吸收活性。OPG属于肿瘤坏死因子(Tumor necrosis factor,TNF)受体超家族成员,是一种分泌型糖蛋白。OPG可以竞争性与RANKL相关诱导配体结合,拮抗RANK与RANKL之间的相互作用。因此,OPG可抑制破骨细胞分化、成熟,并诱导破骨细胞凋亡。然而,雌激素是否通过调节Wnt16/β-catenin途径、OPG/RANK/RANKL系统发挥骨保护作用尚不明确。本研究首先从骨组织微观形态上检验绝经后骨质疏松症造模是否成功,再从Wnt16/β-catenin途径、OPG/RANK/RANKL系统入手,从基因和蛋白表达层面比较,研究雌激素减缓绝经早期骨量流失的机制,最后通过雌激素作用4周与16周两个时间点的比较,揭示雌激素这种作用的时间依赖性。目的:本研究通过去卵巢Wistar大鼠建立绝经后骨质疏松症模型,外源性给予17β-雌二醇,观察雌激素对去卵巢大鼠Wnt16、β-catenin、OPG、RANKL表达水平的影响,探讨雌激素发挥骨保护作用的机制,为绝经早期女性雌激素补充治疗提供实验依据。方法:将40只12周龄雌性Wistar大鼠随机分为4组,即假手术组、去势组、实验组1、实验组2,每组10只。去势组、实验组1、实验组2在喂养1周后切除双侧卵巢组织,假手术组切除等体积腹部脂肪组织。术后2周,实验组1连续4周给予皮下注射17β-雌二醇100 μ g/kg,实验组2连续16周给予颈背部皮下注射17β-雌二醇100 μ g/kg,其余2组给与等量0.9%Nacl,每天1次,连续给药16周。16周后,经腹主动脉采血,ELISA法测定血清雌二醇水平;HE染色法观察骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)、骨小梁间距(Tb.SP);Western blot法检测大鼠胫骨平台骨皮质区RANKL、OPG、β-catenin表达水平;RT-PCR法测定大鼠胫骨骨皮质区Wnt16表达水平。结果:给药16周后,去势组与假手术组、实验组1、实验组2比较,Tb.N减少且排列疏、Tb.Th变薄且Tb.SP增宽(均P0.05),骨髓环境中脂肪细胞数量增多。假手术组、实验组2与去势组比较,OPG、β-atenin、Wnt16mRNA和血清E2水平显著升高,RANKL水平显著下降,差异均有统计学意义(均P0.05)。实验组1与去势组比较,β-catenin、血清E2水平显著升高,RANKL水平显著下降,差异均有统计学意义(均P0.05)。结论:17β-雌二醇可能通过上调Wnt16、β-catenin、OPG表达,下调RANKL表达,减缓绝经早期骨量丢失,发挥骨保护作用,预防绝经后骨质疏松症的发生。
[Abstract]:Background: estrogen is an essential steroid hormone in the growth and development of women. The main functions are to promote the development and maturation of female reproductive organs and to maintain the secondary sex of women; at the same time, it also has some regulatory effect on osteoblasts, osteoclasts and osteoclast cells. The dysfunction of osteoblasts and / or osteoclasts is caused by the dysfunction of osteoblasts and / or osteoclasts. The key pathophysiological mechanism of osteoporosis occurs. Osteoporosis is divided into two primary and secondary types. Primary osteoporosis is common in middle-aged and elderly people, especially postmenopausal women. The present study suggests that postmenopausal osteoporosis and estrogen reduction are related to the.2013 International Menopause guidelines, perimenopausal period. Menopausal hormone therapy (MHT) can slow down the velocity [1,2] of bone loss, but the specific mechanism is not clear. The present study shows that Wnt/ beta -catenin pathway, OPG/RANK/RANKL system plays a certain degree of regulation in postmenopausal osteoporosis and.Wnt16 belongs to the Wnt family, from the secretory glycoprotein. It is highly conservative and participates in the regulation of osteoblasts. After the binding of the osteoclast activity.Wnt16 to the ligand, it activates the classical pathway of Wnt16/ beta -catenin, causes the active expression of the related genes in osteoblasts, promotes the formation of bone, and maintains the homeostasis of bone, which is a membrane binding protein expressed on the osteoblast and matrix cells, and can be used as a metalloprotein. The enzyme is decomposed into soluble RANKL (sRANKL). RANKL combines with RANK specific receptor on the surface of osteoclast and osteoclast to promote the differentiation and maturation of osteoclast cells and enhance the bone resorption activity of osteoclasts..OPG is a member of the tumor necrosis factor (Tumor necrosis factor, TNF), and a secretory glycoprotein.OPG can compete. Sex and RANKL induced ligand binding and antagonistic interaction between RANK and RANKL. Therefore, OPG can inhibit osteoclast differentiation, maturation, and induce osteoclast apoptosis. However, it is not clear whether estrogen plays the role of bone protection in the OPG/RANK/RANKL system by regulating the Wnt16/ beta -catenin pathway. This study first studied the microstructure of the bone tissue. To examine whether the model of postmenopausal osteoporosis is successful, and then from the Wnt16/ beta -catenin pathway and the OPG/RANK/RANKL system, the mechanism of estrogen reduction in the early menopause bone loss is studied from the level of gene and protein expression. Finally, the effect of estrogen is revealed by comparing the 4 weeks of estrogen action to the two time points of 16 weeks. Objective: to establish a postmenopausal osteoporosis model in ovariectomized Wistar rats and to give exogenous 17 beta estradiol to observe the effect of estrogen on the expression of Wnt16, beta -catenin, OPG and RANKL in ovariectomized rats, and to explore the mechanism of estrogen to play the protective role of bone in order to provide an estrogen supplement for women in the early menopause. Methods: 40 12 week old female Wistar rats were randomly divided into 4 groups, namely, sham operation group, castration group, experimental group 1, experimental group 2, each group 10. The ovariectomized group, the experimental group 1, the experimental group 2 removed bilateral ovarian tissue after feeding 1 weeks, and the sham operation group excised the abdominal adipose tissue. 2 weeks after the operation, the experimental group was given 1 hypodermic injection 4 weeks 1 consecutive 4 weeks giving 1 subcutaneous injection 1. 7 beta estradiol 100 g/kg, the experimental group was given 17 beta estradiol 100 mu on the back of the neck for 16 weeks, the rest 2 groups were given equal amount of 0.9%Nacl, 1 times a day, and after 16 weeks for 16 weeks.16 weeks, the serum estradiol level was measured by the abdominal aorta, ELISA method was used to determine the level of serum estradiol, and the number of bone trabecula (Tb.N) and trabecular bone of bone were observed by HE staining method. Distance (Tb.SP); Western blot method was used to detect the expression level of RANKL, OPG, and beta -catenin in the cortical area of tibial plateau of rats; RT-PCR method was used to determine the level of Wnt16 expression in the tibial cortical area of rats. Results: after 16 weeks, the ovariectomized group was compared with the sham operation group, the experimental group 1, and the experimental group 2, the Tb.N decreased and the arrangement was sparse, Tb.Th thinned and Tb.SP broadened (P0.05), bone marrow ring. In the sham operation group, the level of OPG, beta -atenin, Wnt16mRNA and serum E2 increased significantly, and the level of RANKL decreased significantly (P0.05) in the sham group compared with the castration group. The experimental group was compared with the castrated group (P0.05). The level of serum E2 in the experimental group was significantly higher than that in the castrated group. The level of serum E2 was significantly increased, and the level of RANKL decreased significantly. The difference was statistically significant. The difference was statistically significant. Study significance (P0.05). Conclusion: 17 beta estradiol may increase the expression of Wnt16, beta -catenin, OPG, down regulate the expression of RANKL, slow down the loss of bone mass in the early menopause, play the role of bone protection, and prevent postmenopausal osteoporosis.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580

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