本文選題：雌激素 + Wnt16�。� 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:雌激素是女性生長(zhǎng)、發(fā)育過(guò)程中必不可少的類固醇激素,主要功能有促進(jìn)女性生殖器官的發(fā)育和成熟,維持女性的第二性征;同時(shí),對(duì)成骨細(xì)胞、破骨細(xì)胞、破骨前體細(xì)胞等也有一定調(diào)節(jié)作用。成骨細(xì)胞和/或破骨細(xì)胞功能紊亂是導(dǎo)致骨質(zhì)疏松癥發(fā)生的關(guān)鍵病理生理機(jī)制。骨質(zhì)疏松癥分為原發(fā)性和繼發(fā)性兩種,而原發(fā)性骨質(zhì)疏松癥常見(jiàn)于中老年人群中,尤以絕經(jīng)后女性最為常見(jiàn)。目前研究認(rèn)為,絕經(jīng)后骨質(zhì)疏松癥與雌激素減少有關(guān)。2013年國(guó)際絕經(jīng)指南指出,圍絕經(jīng)期和絕經(jīng)早期給予絕經(jīng)激素治療(menopausal hormone therapy,MHT)可減緩骨量流失速度[1,2],但具體機(jī)制尚不明確。目前研究表明,Wnt/β-catenin途徑、OPG/RANK/RANKL系統(tǒng)在絕經(jīng)后骨質(zhì)疏松癥中起到一定程度的調(diào)節(jié)作用。Wnt16屬于Wnt家族,由分泌性糖蛋白組成,具有高度保守型,參與調(diào)控成骨細(xì)胞、破骨細(xì)胞活性。Wnt16與配體結(jié)合后,激活Wnt16/β-catenin經(jīng)典途徑,引起成骨細(xì)胞相關(guān)基因表達(dá)活躍,促進(jìn)骨形成,維持骨穩(wěn)態(tài)平衡。RANKL是表達(dá)在成骨細(xì)胞和基質(zhì)細(xì)胞上的膜結(jié)合蛋白,可被金屬蛋白酶分解為可溶性RANKL(sRANKL),RANKL通過(guò)與破骨前體細(xì)胞、破骨細(xì)胞表面RANK特異受體結(jié)合,促進(jìn)破骨前體細(xì)胞分化成熟,增強(qiáng)破骨細(xì)胞骨吸收活性。OPG屬于腫瘤壞死因子(Tumor necrosis factor,TNF)受體超家族成員,是一種分泌型糖蛋白。OPG可以競(jìng)爭(zhēng)性與RANKL相關(guān)誘導(dǎo)配體結(jié)合,拮抗RANK與RANKL之間的相互作用。因此,OPG可抑制破骨細(xì)胞分化、成熟,并誘導(dǎo)破骨細(xì)胞凋亡。然而,雌激素是否通過(guò)調(diào)節(jié)Wnt16/β-catenin途徑、OPG/RANK/RANKL系統(tǒng)發(fā)揮骨保護(hù)作用尚不明確。本研究首先從骨組織微觀形態(tài)上檢驗(yàn)絕經(jīng)后骨質(zhì)疏松癥造模是否成功,再?gòu)腤nt16/β-catenin途徑、OPG/RANK/RANKL系統(tǒng)入手,從基因和蛋白表達(dá)層面比較,研究雌激素減緩絕經(jīng)早期骨量流失的機(jī)制,最后通過(guò)雌激素作用4周與16周兩個(gè)時(shí)間點(diǎn)的比較,揭示雌激素這種作用的時(shí)間依賴性。目的:本研究通過(guò)去卵巢Wistar大鼠建立絕經(jīng)后骨質(zhì)疏松癥模型,外源性給予17β-雌二醇,觀察雌激素對(duì)去卵巢大鼠Wnt16、β-catenin、OPG、RANKL表達(dá)水平的影響,探討雌激素發(fā)揮骨保護(hù)作用的機(jī)制,為絕經(jīng)早期女性雌激素補(bǔ)充治療提供實(shí)驗(yàn)依據(jù)。方法:將40只12周齡雌性Wistar大鼠隨機(jī)分為4組,即假手術(shù)組、去勢(shì)組、實(shí)驗(yàn)組1、實(shí)驗(yàn)組2,每組10只。去勢(shì)組、實(shí)驗(yàn)組1、實(shí)驗(yàn)組2在喂養(yǎng)1周后切除雙側(cè)卵巢組織,假手術(shù)組切除等體積腹部脂肪組織。術(shù)后2周,實(shí)驗(yàn)組1連續(xù)4周給予皮下注射17β-雌二醇100 μ g/kg,實(shí)驗(yàn)組2連續(xù)16周給予頸背部皮下注射17β-雌二醇100 μ g/kg,其余2組給與等量0.9%Nacl,每天1次,連續(xù)給藥16周。16周后,經(jīng)腹主動(dòng)脈采血,ELISA法測(cè)定血清雌二醇水平;HE染色法觀察骨小梁厚度(Tb.Th)、骨小梁數(shù)目(Tb.N)、骨小梁間距(Tb.SP);Western blot法檢測(cè)大鼠脛骨平臺(tái)骨皮質(zhì)區(qū)RANKL、OPG、β-catenin表達(dá)水平;RT-PCR法測(cè)定大鼠脛骨骨皮質(zhì)區(qū)Wnt16表達(dá)水平。結(jié)果:給藥16周后,去勢(shì)組與假手術(shù)組、實(shí)驗(yàn)組1、實(shí)驗(yàn)組2比較,Tb.N減少且排列疏、Tb.Th變薄且Tb.SP增寬(均P0.05),骨髓環(huán)境中脂肪細(xì)胞數(shù)量增多。假手術(shù)組、實(shí)驗(yàn)組2與去勢(shì)組比較,OPG、β-atenin、Wnt16mRNA和血清E2水平顯著升高,RANKL水平顯著下降,差異均有統(tǒng)計(jì)學(xué)意義(均P0.05)。實(shí)驗(yàn)組1與去勢(shì)組比較,β-catenin、血清E2水平顯著升高,RANKL水平顯著下降,差異均有統(tǒng)計(jì)學(xué)意義(均P0.05)。結(jié)論:17β-雌二醇可能通過(guò)上調(diào)Wnt16、β-catenin、OPG表達(dá),下調(diào)RANKL表達(dá),減緩絕經(jīng)早期骨量丟失,發(fā)揮骨保護(hù)作用,預(yù)防絕經(jīng)后骨質(zhì)疏松癥的發(fā)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R580

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