本文關(guān)鍵詞：利用RNAi技術(shù)抑制ERβ表達(dá)對(duì)人成骨細(xì)胞株hFOB 1.19中TGF-β1和BMP-2表達(dá)的影響　出處：《中南大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： ERβ 人成骨細(xì)胞 RNA干擾 TGF-β1 BMP-2

【摘要】：目的雌激素受體(Estrogen receptor, ER)基因作為影響骨代謝較重要的候選基因之一,包括ERα與ERβ兩種,且有研究表明,ER基因與骨骼生長(zhǎng)有關(guān),ER的基因多態(tài)性決定骨骼生長(zhǎng)和性成熟,基因突變導(dǎo)致骨質(zhì)丟失和骨骼生長(zhǎng)異常等。關(guān)于ERα基因與骨代謝的關(guān)系已有較多研究,而ERβ基因的研究則較少。并且多種骨生長(zhǎng)因子在骨形成和骨改建過(guò)程通過(guò)調(diào)控細(xì)胞的活動(dòng)起著十分重要的作用,其中以TGF-β1和BMP-2最為重要。目前RNA干(?)(RNA interference, RNAi)已成為研究基因功能和細(xì)胞信號(hào)通路的有力工具,它可通過(guò)內(nèi)源性表達(dá)或外源性介導(dǎo)siRNA以序列特異性的方式有效抑制內(nèi)源性基因的表達(dá)。因此,本研究的主要內(nèi)容包括：①采用逆轉(zhuǎn)錄病毒載體作為RNAi的工具,針對(duì)人ERβ基因特異的沉默位點(diǎn),構(gòu)建3種shRNA逆轉(zhuǎn)錄病毒重組質(zhì)粒,并包裝成高效的逆轉(zhuǎn)錄病毒；②探討運(yùn)用ERβ-shRNA逆轉(zhuǎn)錄病毒載體介導(dǎo)的RNAi技術(shù)抑制人成骨細(xì)胞株hFOB 1.19中ERβ表達(dá)的效率；③通過(guò)抗性篩選得到穩(wěn)定感染ERβ-shRNA逆轉(zhuǎn)錄病毒載體的hFOB 1.19細(xì)胞,探討ERβ穩(wěn)定抑制后對(duì)hFOB 1.19細(xì)胞增殖的影響,并在雌激素干預(yù)下,研究ERβ穩(wěn)定抑制后對(duì)TGF-β1和BMP-2表達(dá)的影響,為探討ERβ基因如何通過(guò)人成骨細(xì)胞調(diào)節(jié)骨代謝提供理論基礎(chǔ)。 方法①利用軟件設(shè)計(jì)3種特異性ERβ-shRNA,體外合成后將其定向克隆入pRNAT-H1.4/Retro逆轉(zhuǎn)錄病毒質(zhì)粒中,并包裝成逆轉(zhuǎn)錄病毒；②對(duì)體外培養(yǎng)的人成骨細(xì)胞株hFOB 1.19進(jìn)行形態(tài)學(xué)觀察及HE染色；利用ERβ-shRNA逆轉(zhuǎn)錄病毒載體瞬時(shí)感染hFOB 1.19細(xì)胞后,通過(guò)流式細(xì)胞儀檢測(cè)感染效率,并使用半定量RT-PCR和Western blot技術(shù)檢測(cè)對(duì)ERβmRNA和蛋白表達(dá)的抑制效率；③取ERβ抑制效率最高的感染ERβ-shRNA逆轉(zhuǎn)錄病毒載體的hFOB 1.19細(xì)胞,通過(guò)抗性篩選,將得到穩(wěn)定感染的hFOB 1.19細(xì)胞進(jìn)行擴(kuò)大培養(yǎng),通過(guò)半定量RT-PCR和Western blot技術(shù)檢測(cè)ERβ穩(wěn)定抑制的效率；并利用MTT法檢測(cè)ERβ穩(wěn)定抑制后對(duì)細(xì)胞增殖的影響；隨后在雌激素干預(yù)下,使用半定量RT-PCR和Western blot技術(shù)檢測(cè)ERβ穩(wěn)定抑制后對(duì)TGF-β1和BMP-2表達(dá)的影響。 結(jié)果①經(jīng)鑒定,我們成功構(gòu)建了3種ERβ-shRNA逆轉(zhuǎn)錄病毒重組質(zhì)粒,并包裝成高效的逆轉(zhuǎn)錄病毒,分別為ERβ-shRNA-1、ERβ-shRNA-2、ERβ-shRNA-3逆轉(zhuǎn)錄病毒載體；②體外培養(yǎng)的人成骨細(xì)胞株hFOB 1.19具有典型的成骨樣細(xì)胞形態(tài)；流式細(xì)胞儀檢測(cè)結(jié)果顯示,ERβ-shRNA逆轉(zhuǎn)錄病毒載體瞬時(shí)感染hFOB 1.19細(xì)胞的效率均高達(dá)70%以上,表明逆轉(zhuǎn)錄病毒具有良好的細(xì)胞感染能力；ERβ-shRNA-1、ERβ-shRNA-2、ERβ-shRNA-3逆轉(zhuǎn)錄病毒載體對(duì)hFOB1.19細(xì)胞中ERβmRNA的抑制率分別為(54.56±0.95)%、(69.60±51.12)%、(76.49±1.15)%,蛋白的抑制率分別為(59.21±4.44)%、(78.35±2.00)%、(85.60±2.66)%(均P0.05)；③我們成功篩選出穩(wěn)定感染ERβ-shRNA-3逆轉(zhuǎn)錄病毒載體的hFOB 1.19細(xì)胞,ERβmRNA和蛋白的抑制率分別為(83.23±2.45)%和(93.11±O.57)%(均P0.05),MTT法檢測(cè)顯示ERβ穩(wěn)定抑制后對(duì)細(xì)胞的增殖沒(méi)有明顯影響(P0.05),因此表明我們成功建立了ERβ穩(wěn)定抑制的hFOB 1.19細(xì)胞模型；在雌激素干預(yù)下,ERβ穩(wěn)定抑制后對(duì)TGF-β1 mRNA和蛋白的上調(diào)率分別為(26.65±3.81)%和(23.79±3.76)%, BMP-2 mRNA和蛋白的上調(diào)率分別為(16.62±1.71)%和(18.08±3.20)%(均P0.05)。 結(jié)論①成功構(gòu)建了3種ERβ-shRNA逆轉(zhuǎn)錄病毒載體；②3種ERβ-shRNA逆轉(zhuǎn)錄病毒載體均能顯著抑制人成骨細(xì)胞株hFOB 1.19中ERβ的表達(dá),其中ERβ-shRNA-3逆轉(zhuǎn)錄病毒載體最有效；③成功建立了ERβ穩(wěn)定抑制的hFOB 1.19細(xì)胞模型；④在雌激素干預(yù)下,ERβ穩(wěn)定抑制后可上調(diào)hFOB 1.19細(xì)胞中TGF-β1和BMP-2的表達(dá)水平,提示ERβ通過(guò)調(diào)節(jié)TGF-β1和BMP-2的表達(dá)在骨代謝中發(fā)揮作用。
[Abstract]:Objective to estrogen receptor (Estrogen receptor ER) gene as a candidate gene affecting bone metabolism is one of the more important, including ER alpha and ER beta two, and studies have shown that the ER gene and the growth of bones, the gene polymorphism of ER determines the skeletal growth and maturation, gene mutation leads to bone loss and abnormal bone growth etc. there have been many about. The research on the relationship between gene and bone metabolism of ER alpha, ER beta gene research is less. And a variety of bone growth factors and bone formation in bone remodeling by regulating cell activity plays a very important role, especially in TGF- beta 1 and BMP-2 are the most important. At present, RNA (?) (RNA interference RNAi) has become a powerful tool to study gene function and cell signaling pathways, it can be in a sequence specific manner inhibit endogenous gene expression by endogenous or exogenous expression mediated by siRNA. Therefore, this research The main contents include: using retroviral vectors as RNAi tool for silencing locus specific to human ER gene, construct 3 shRNA retroviral recombinant plasmid, and packaged into efficient retrovirus; to assess the efficiency of the use of ER beta -shRNA retroviral vector mediated RNAi inhibition of human osteoblastic cell line the expression of hFOB ER beta 1.19; through the resistance screening stable infection ER beta -shRNA retroviral vector hFOB 1.19 cells, to explore the effects of ER beta stability after inhibition on proliferation of hFOB 1.19 cells, and the estrogen intervention, ER beta stable inhibition effect on the expression of TGF- beta 1 and BMP-2, in order to explore the ER beta gene in human osteoblasts through regulating bone metabolism and provide a theoretical basis.
Methods using the software design of 3 kinds of specific ER beta -shRNA synthesis in vitro after cloned into pRNAT-H1.4/Retro retroviral plasmid, and packaged into retrovirus; II on cultured human osteoblast cell line hFOB 1.19 were observed by ER and HE staining; beta -shRNA retroviral vector transiently infected hFOB 1.19 cells then, by detecting the infection efficiency by flow cytometry, and the inhibition efficiency using semi quantitative RT-PCR and Western blot used to detect the expression of ER beta mRNA and ER beta protein; takes the highest inhibition efficiency with ER beta -shRNA retroviral vector hFOB 1.19 cells by resistance screening, hFOB infection will be stable expanding culture 1.19 cells, inhibition efficiency by detection of ER beta stable semi quantitative RT-PCR and Western blot technology; and using MTT method to detect ER beta stable inhibition effect on cell proliferation After estrogen intervention, the effects of ER beta stable inhibition on the expression of TGF- beta 1 and BMP-2 were detected by semi quantitative RT-PCR and Western blot.
Results: after identification, we successfully constructed 3 kinds of ER beta -shRNA retroviral recombinant plasmid, and packaged into efficient retrovirus, respectively ER beta -shRNA-1, ER beta -shRNA-2, ER beta -shRNA-3 retroviral vector; in vitro cultured human osteoblast cell line hFOB 1.19 cells with typical morphology; flow cytometry showed that the efficiency of ER beta -shRNA retroviral vector transiently infected hFOB 1.19 cells were above 70%, showed that the retrovirus has good cell infection ability; ER beta -shRNA-1, ER beta -shRNA-2, inhibition of ER beta mRNA hFOB1.19 cell rate of ER beta -shRNA-3 retroviral vector respectively. (54.56 + 0.95)% and (69.60 + 51.12)% and (76.49 + 1.15)%, protein inhibition rates were (59.21 + 4.44)% and (78.35 + 2)% and (85.60 + 2.66)% (P0.05); we successfully screened stable beta -shRNA-3 retrovirus infection ER Virus vector hFOB 1.19 cells, inhibition of ER beta mRNA and protein respectively (83.23 + 2.45)% and (93.11 + O.57)% (P0.05), MTT assay showed no obvious effect on cell proliferation after inhibition of ER beta stability (P0.05), so that we have successfully established a stable beta ER inhibition of hFOB 1.19 cell model; in estrogen intervention, ER beta stability after inhibition of TGF- beta 1 and mRNA on the expression of the protein were (26.65 + 3.81)% and (23.79 + 3.76)%, BMP-2 mRNA and protein were up-regulated (16.62 + 1.71)% and (18.08 + 3.20)% (P0.05).
Conclusion the successfully constructed 3 ER beta -shRNA retroviral vector; the expression of 3 kinds of ER beta -shRNA retroviral vector can significantly inhibit human osteoblast cell line hFOB 1.19 beta ER, the most effective ER beta -shRNA-3 retroviral vector; the successfully established ER beta stable inhibition of hFOB 1.19 cell model; in the estrogen intervention, ER beta stable inhibition after expression in hFOB 1.19 cells TGF- beta 1 and BMP-2, expression of ER beta by regulating TGF- beta 1 and BMP-2 play a role in bone metabolism.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 孟和,陳學(xué)輝,王起山,崔芳巖,潘玉春;短雙鏈RNA對(duì)雞胚盤細(xì)胞外源綠熒光蛋白基因表達(dá)的影響[J];動(dòng)物學(xué)報(bào);2004年02期

2 ;QSAR of Estrogen of Bisphenol A with 3D Vector of Atomic Property Correlation[J];結(jié)構(gòu)化學(xué);2007年08期

3 袁婺洲,Bodmer R,朱傳炳,王躍群,李永清,吳秀山;利用RNAi技術(shù)研究果蠅心臟發(fā)育基因的功能[J];遺傳學(xué)報(bào);2002年01期

4 湯富酬,薛友紡;RNA干涉與基因沉默[J];遺傳;2001年02期

5 張利生,陳大元;RNA干涉及其應(yīng)用前景[J];遺傳;2003年03期

6 吳潔;邱勇;張樂(lè);孫燕芳;陳新;;青少年特發(fā)性脊柱側(cè)凸患者雌激素受體基因多態(tài)性與骨密度的關(guān)系[J];中國(guó)骨質(zhì)疏松雜志;2006年03期

，

本文編號(hào)：1383017