miR-101-3p在雞毒支原體感染中的作用研究
發(fā)布時(shí)間：2018-12-13 03:24
【摘要】：雞毒支原體(Mycoplasma gallisepticum,簡稱MG)是雞慢性呼吸道疾病的病原體,主要通過定殖于宿主呼吸道建立慢性感染,引起呼吸道組織的損傷和一系列病理變化。該病廣泛分布于世界各地,給家禽養(yǎng)殖業(yè)造成了巨大的經(jīng)濟(jì)損失,迄今仍未找到有效的防控措施。深入研究MG的致病機(jī)制,有助于該病的預(yù)防及治療方法的尋找。大量研究表明,mi RNA在病原致病中發(fā)揮著重要的作用,本研究旨在探討高通量測序篩選出的gga-mi R-101-3p在MG感染中的作用,為闡明MG的致病機(jī)理提供科學(xué)依據(jù)。實(shí)驗(yàn)以雞毒支原體HS株(MG-HS)感染雞胚及DF-1細(xì)胞為對(duì)象,運(yùn)用Real-time Quantitative PCR(q PCR)方法檢測感染與非感染組織、細(xì)胞中g(shù)ga-mi R-101-3p及其靶基因的表達(dá)量;應(yīng)用雙熒光素酶報(bào)告基因系統(tǒng)對(duì)gga-mi R-101-3p的靶基因進(jìn)行驗(yàn)證;進(jìn)一步通過gga-mi R-101-3p的超表達(dá)和抑制實(shí)驗(yàn)來確證gga-mi R-101-3p與靶基因的調(diào)控關(guān)系。利用MTT法檢測DF-1細(xì)胞的增殖及流式細(xì)胞儀檢測細(xì)胞周期的變化,了解gga-mi R-101-3p對(duì)DF-1細(xì)胞生物學(xué)的影響。主要成果如下:1.檢測了gga-mi R-101-3p在MG-HS感染與非感染雞胚(12-20日齡)肺組織及DF-1細(xì)胞中的表達(dá)量。gga-mi R-101-3p在17 d、18 d、19 d MG-HS感染雞胚的肺組織中的表達(dá)量極顯著高于正常對(duì)照組(p0.01),而13 d、14 d時(shí)極顯著低于正常對(duì)照組(p0.01),20 d時(shí)顯著低于正常對(duì)照組(p0.05)。MG-HS感染DF-1細(xì)胞中g(shù)ga-mi R-101-3p在的表達(dá)量極顯著高于正常對(duì)照組(p0.01)。2.利用在線軟件mi RDB、Mir Target2和Target Scan等預(yù)測了gga-mi R-101-3p的靶基因,通過RNAhybrid等軟件對(duì)gga-mi R-101-3p與EZH2 3’UTR靶序列的堿基配對(duì)情況、二者結(jié)合形成RNA雙鏈的二級(jí)結(jié)構(gòu)和自由能,以及靶序列在不同物種間保守性等主要生物信息學(xué)指標(biāo)進(jìn)行了歸納分析,并結(jié)合功能分析初步確定EZH2為gga-mi R-101-3p的靶基因。3.通過雙熒光素酶報(bào)告系統(tǒng)分析,gga-mi R-101-3p能顯著抑制EZH2 3’UTR雙熒光素酶報(bào)告基因的熒光素酶活性,揭示了gga-mi R-101-3p是通過與EZH2 3’UTR結(jié)合而發(fā)揮作用,初步確定EZH2可能是gga-mi R-101-3p的靶基因。4.gga-mi R-101-3p超表達(dá)和抑制實(shí)驗(yàn)表明,在DF-1細(xì)胞中轉(zhuǎn)染gga-mi R-101-3p mimics能顯著抑制EZH2基因m RNA和蛋白的表達(dá);而轉(zhuǎn)染gga-mi R-101-3p inhibitor后,EZH2基因m RNA和蛋白的表達(dá)則顯著上調(diào)。進(jìn)一步證實(shí)了EZH2是gga-mi R-101-3p的靶基因,且gga-mi R-101-3p負(fù)向調(diào)控EZH2的表達(dá)。5.q PCR分析了EZH2在MG-HS感染與非感染雞胚(12-20日齡)肺組織、DF-1細(xì)胞中的表達(dá)量。結(jié)果顯示,18 d、19 d MG-HS感染雞胚的肺組織中EZH2的相對(duì)表達(dá)量極顯著低于與正常對(duì)照組(p0.01),20 d時(shí)極顯著高于正常對(duì)照組(p0.01);且在MG-HS感染DF-1細(xì)胞中,EZH2的表達(dá)量極顯著低于正常對(duì)照組(p0.01),說明EZH2與gga-mi R-101-3p的表達(dá)量呈明顯負(fù)相關(guān)。6.超表達(dá)gga-mi R-101-3p能顯著抑制DF-1細(xì)胞的增殖,且使細(xì)胞周期停滯在G1期,不能進(jìn)入S期進(jìn)行DNA的合成與復(fù)制。
[Abstract]:Mycoplasma gallinis (Mycoplasma gallisepticum,) is the pathogen of chronic respiratory diseases in chickens. Chronic infection is established by colonization of host respiratory tract, which results in the injury of respiratory tract tissue and a series of pathological changes. The disease is widely distributed all over the world, causing huge economic losses to poultry industry, so far no effective prevention and control measures have been found. Further study on the pathogenesis of MG is helpful to the prevention and treatment of MG. A large number of studies have shown that, mi RNA plays an important role in pathogenicity. The purpose of this study was to explore the role of gga-mi R-101-3p screened by high-throughput sequencing in MG infection, and to provide scientific basis for elucidating the pathogenesis of MG. The expression of gga-mi R-101-3p and its target genes in infected and non-infected tissues and cells were detected by Real-time Quantitative PCR (q PCR) method in chicken embryo and DF-1 cells infected with Mycoplasma Chicken HS strain (MG-HS). The target gene of gga-mi R-101-3p was verified by double luciferase reporter gene system, and the regulatory relationship between gga-mi R-101-3p and target gene was confirmed by gga-mi R-101-3p overexpression and inhibition experiments. MTT assay was used to detect the proliferation of DF-1 cells and flow cytometry was used to detect the changes of cell cycle. The effect of gga-mi R-101-3p on the cell biology of DF-1 cells was investigated. The main results are as follows: 1. The expression of gga-mi R-101-3p in lung tissues and DF-1 cells of MG-HS infected and non-infected chicken embryos (12-20 days old) was detected. Gga-mi R-101-3p was expressed at 17 days and 18 days after infection. The expression of MG-HS in the lung tissue of chicken embryo infected with MG-HS on day 19 was significantly higher than that of the normal control group (p0.01), and was significantly lower than that of the control group at the 14th day of 13 days (p0.01). The expression of gga-mi R-101-3p in DF-1 cells infected with MG-HS was significantly higher than that in normal controls (p0.01). The target genes of gga-mi R-101-3p were predicted by on-line software mi RDB,Mir Target2 and Target Scan, and the base pairs of gga-mi R-101-3p and EZH2 3'UTR target sequences were analyzed by RNAhybrid and other software. The secondary structure and free energy of RNA double strands and the conservation of target sequences among different species were summarized and analyzed. Combined with functional analysis, EZH2 was identified as the target gene of gga-mi R-101-3p. Through the analysis of double luciferase report system, gga-mi R-101-3p can significantly inhibit the luciferase activity of EZH2 3'UTR double luciferase reporter gene, which indicates that gga-mi R-101-3p acts by combining with EZH2 3'UTR. EZH2 may be the target gene of gga-mi R-101-3p. The overexpression and inhibition of 4.gga-mi R-101-3p in DF-1 cells showed that transfection of gga-mi R-101-3p mimics could significantly inhibit the expression of EZH2 gene m RNA and protein. After transfection of gga-mi R-101-3p inhibitor, the expression of m RNA and protein of EZH2 gene was significantly up-regulated. It was further confirmed that EZH2 was the target gene of gga-mi R-101-3p, and gga-mi R-101-3p negatively regulated the expression of EZH2. 5.q PCR was used to analyze the lung tissues of MG-HS infected and non-infected chicken embryos (12-20 days old). The amount of expression in DF-1 cells. The results showed that the relative expression of EZH2 in the lung tissue of chicken embryo infected with MG-HS on day 18 and 19 was significantly lower than that of the control group (p0.01), and at 20 days it was significantly higher than that of the normal control group (p0.01). The expression of EZH2 in DF-1 cells infected with MG-HS was significantly lower than that in normal controls (p0.01), indicating that the expression of EZH2 and gga-mi R-101-3p was negatively correlated. 6. Overexpression of gga-mi R-101-3p could significantly inhibit the proliferation of DF-1 cells and arrest the cell cycle in G1 phase, and could not enter S phase for DNA synthesis and replication.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.31
本文編號(hào)：2375782
[Abstract]:Mycoplasma gallinis (Mycoplasma gallisepticum,) is the pathogen of chronic respiratory diseases in chickens. Chronic infection is established by colonization of host respiratory tract, which results in the injury of respiratory tract tissue and a series of pathological changes. The disease is widely distributed all over the world, causing huge economic losses to poultry industry, so far no effective prevention and control measures have been found. Further study on the pathogenesis of MG is helpful to the prevention and treatment of MG. A large number of studies have shown that, mi RNA plays an important role in pathogenicity. The purpose of this study was to explore the role of gga-mi R-101-3p screened by high-throughput sequencing in MG infection, and to provide scientific basis for elucidating the pathogenesis of MG. The expression of gga-mi R-101-3p and its target genes in infected and non-infected tissues and cells were detected by Real-time Quantitative PCR (q PCR) method in chicken embryo and DF-1 cells infected with Mycoplasma Chicken HS strain (MG-HS). The target gene of gga-mi R-101-3p was verified by double luciferase reporter gene system, and the regulatory relationship between gga-mi R-101-3p and target gene was confirmed by gga-mi R-101-3p overexpression and inhibition experiments. MTT assay was used to detect the proliferation of DF-1 cells and flow cytometry was used to detect the changes of cell cycle. The effect of gga-mi R-101-3p on the cell biology of DF-1 cells was investigated. The main results are as follows: 1. The expression of gga-mi R-101-3p in lung tissues and DF-1 cells of MG-HS infected and non-infected chicken embryos (12-20 days old) was detected. Gga-mi R-101-3p was expressed at 17 days and 18 days after infection. The expression of MG-HS in the lung tissue of chicken embryo infected with MG-HS on day 19 was significantly higher than that of the normal control group (p0.01), and was significantly lower than that of the control group at the 14th day of 13 days (p0.01). The expression of gga-mi R-101-3p in DF-1 cells infected with MG-HS was significantly higher than that in normal controls (p0.01). The target genes of gga-mi R-101-3p were predicted by on-line software mi RDB,Mir Target2 and Target Scan, and the base pairs of gga-mi R-101-3p and EZH2 3'UTR target sequences were analyzed by RNAhybrid and other software. The secondary structure and free energy of RNA double strands and the conservation of target sequences among different species were summarized and analyzed. Combined with functional analysis, EZH2 was identified as the target gene of gga-mi R-101-3p. Through the analysis of double luciferase report system, gga-mi R-101-3p can significantly inhibit the luciferase activity of EZH2 3'UTR double luciferase reporter gene, which indicates that gga-mi R-101-3p acts by combining with EZH2 3'UTR. EZH2 may be the target gene of gga-mi R-101-3p. The overexpression and inhibition of 4.gga-mi R-101-3p in DF-1 cells showed that transfection of gga-mi R-101-3p mimics could significantly inhibit the expression of EZH2 gene m RNA and protein. After transfection of gga-mi R-101-3p inhibitor, the expression of m RNA and protein of EZH2 gene was significantly up-regulated. It was further confirmed that EZH2 was the target gene of gga-mi R-101-3p, and gga-mi R-101-3p negatively regulated the expression of EZH2. 5.q PCR was used to analyze the lung tissues of MG-HS infected and non-infected chicken embryos (12-20 days old). The amount of expression in DF-1 cells. The results showed that the relative expression of EZH2 in the lung tissue of chicken embryo infected with MG-HS on day 18 and 19 was significantly lower than that of the control group (p0.01), and at 20 days it was significantly higher than that of the normal control group (p0.01). The expression of EZH2 in DF-1 cells infected with MG-HS was significantly lower than that in normal controls (p0.01), indicating that the expression of EZH2 and gga-mi R-101-3p was negatively correlated. 6. Overexpression of gga-mi R-101-3p could significantly inhibit the proliferation of DF-1 cells and arrest the cell cycle in G1 phase, and could not enter S phase for DNA synthesis and replication.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.31
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