利用RNAi技术抑制ERβ表达对人成骨细胞株hFOB 1.19中TGF-β1和BMP-2表达的影响

发布时间：2018-01-05 12:02

本文关键词：利用RNAi技术抑制ERβ表达对人成骨细胞株hFOB 1.19中TGF-β1和BMP-2表达的影响　出处：《中南大学》2011年博士论文　论文类型：学位论文

更多相关文章： ERβ 人成骨细胞 RNA干扰 TGF-β1 BMP-2

【摘要】：目的雌激素受体(Estrogen receptor, ER)基因作为影响骨代谢较重要的候选基因之一,包括ERα与ERβ两种,且有研究表明,ER基因与骨骼生长有关,ER的基因多态性决定骨骼生长和性成熟,基因突变导致骨质丢失和骨骼生长异常等。关于ERα基因与骨代谢的关系已有较多研究,而ERβ基因的研究则较少。并且多种骨生长因子在骨形成和骨改建过程通过调控细胞的活动起着十分重要的作用,其中以TGF-β1和BMP-2最为重要。目前RNA干(?)(RNA interference, RNAi)已成为研究基因功能和细胞信号通路的有力工具,它可通过内源性表达或外源性介导siRNA以序列特异性的方式有效抑制内源性基因的表达。因此,本研究的主要内容包括：①采用逆转录病毒载体作为RNAi的工具,针对人ERβ基因特异的沉默位点,构建3种shRNA逆转录病毒重组质粒,并包装成高效的逆转录病毒；②探讨运用ERβ-shRNA逆转录病毒载体介导的RNAi技术抑制人成骨细胞株hFOB 1.19中ERβ表达的效率；③通过抗性筛选得到稳定感染ERβ-shRNA逆转录病毒载体的hFOB 1.19细胞,探讨ERβ稳定抑制后对hFOB 1.19细胞增殖的影响,并在雌激素干预下,研究ERβ稳定抑制后对TGF-β1和BMP-2表达的影响,为探讨ERβ基因如何通过人成骨细胞调节骨代谢提供理论基础。方法①利用软件设计3种特异性ERβ-shRNA,体外合成后将其定向克隆入pRNAT-H1.4/Retro逆转录病毒质粒中,并包装成逆转录病毒；②对体外培养的人成骨细胞株hFOB 1.19进行形态学观察及HE染色；利用ERβ-shRNA逆转录病毒载体瞬时感染hFOB 1.19细胞后,通过流式细胞仪检测感染效率,并使用半定量RT-PCR和Western blot技术检测对ERβmRNA和蛋白表达的抑制效率；③取ERβ抑制效率最高的感染ERβ-shRNA逆转录病毒载体的hFOB 1.19细胞,通过抗性筛选,将得到稳定感染的hFOB 1.19细胞进行扩大培养,通过半定量RT-PCR和Western blot技术检测ERβ稳定抑制的效率；并利用MTT法检测ERβ稳定抑制后对细胞增殖的影响；随后在雌激素干预下,使用半定量RT-PCR和Western blot技术检测ERβ稳定抑制后对TGF-β1和BMP-2表达的影响。结果①经鉴定,我们成功构建了3种ERβ-shRNA逆转录病毒重组质粒,并包装成高效的逆转录病毒,分别为ERβ-shRNA-1、ERβ-shRNA-2、ERβ-shRNA-3逆转录病毒载体；②体外培养的人成骨细胞株hFOB 1.19具有典型的成骨样细胞形态；流式细胞仪检测结果显示,ERβ-shRNA逆转录病毒载体瞬时感染hFOB 1.19细胞的效率均高达70%以上,表明逆转录病毒具有良好的细胞感染能力；ERβ-shRNA-1、ERβ-shRNA-2、ERβ-shRNA-3逆转录病毒载体对hFOB1.19细胞中ERβmRNA的抑制率分别为(54.56±0.95)%、(69.60±51.12)%、(76.49±1.15)%,蛋白的抑制率分别为(59.21±4.44)%、(78.35±2.00)%、(85.60±2.66)%(均P0.05)；③我们成功筛选出稳定感染ERβ-shRNA-3逆转录病毒载体的hFOB 1.19细胞,ERβmRNA和蛋白的抑制率分别为(83.23±2.45)%和(93.11±O.57)%(均P0.05),MTT法检测显示ERβ稳定抑制后对细胞的增殖没有明显影响(P0.05),因此表明我们成功建立了ERβ稳定抑制的hFOB 1.19细胞模型；在雌激素干预下,ERβ稳定抑制后对TGF-β1 mRNA和蛋白的上调率分别为(26.65±3.81)%和(23.79±3.76)%, BMP-2 mRNA和蛋白的上调率分别为(16.62±1.71)%和(18.08±3.20)%(均P0.05)。结论①成功构建了3种ERβ-shRNA逆转录病毒载体；②3种ERβ-shRNA逆转录病毒载体均能显著抑制人成骨细胞株hFOB 1.19中ERβ的表达,其中ERβ-shRNA-3逆转录病毒载体最有效；③成功建立了ERβ稳定抑制的hFOB 1.19细胞模型；④在雌激素干预下,ERβ稳定抑制后可上调hFOB 1.19细胞中TGF-β1和BMP-2的表达水平,提示ERβ通过调节TGF-β1和BMP-2的表达在骨代谢中发挥作用。
[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.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346

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