从巨噬细胞Ipr1基因功能研究探讨结核分枝杆菌感染固有免疫的调节机制

发布时间：2018-01-01 06:05

  本文关键词：从巨噬细胞Ipr1基因功能研究探讨结核分枝杆菌感染固有免疫的调节机制　出处：《重庆医科大学》2009年博士论文　论文类型：学位论文

  更多相关文章： Ipr1基因 结核分枝杆菌 巨噬细胞

【摘要】： 2005年,哈佛大学的Pan等在小鼠巨噬细胞内发现了一个介导巨噬细胞抗胞内病原体的基因—胞内病原体抗性基因1(intracellularpathogen resistance 1,Ipr1),该基因与结核病的易感性有着密切的联系。表达Ipr1基因的小鼠,感染Mtb后巨噬细胞发生凋亡,细菌不易增殖。而IPR1基因缺陷的小鼠感染Mtb后巨噬细胞表现为坏死,从而利于细菌在宿主体内的增殖与扩散。然而,Ipr1基因增强巨噬细胞抗结核分枝杆菌感染的机制还不清楚。 本课题通过RT-PCR法从C57BL/6J小鼠胸腺组织中获取Ipr1基因,构建真核表达质粒pEGFP-Ipr1,观察Ipr1基因在细胞水平调节巨噬细胞抗分枝杆菌的作用。构建Ipr1和绿色荧光蛋白(Greenfluorescent protein,GFP)真核共表达穿梭质粒pBGOI,然后将该穿梭质粒电转到卡介苗BCG中,构建可靶向递送pBGOI质粒进入巨噬细胞中进行表达的重组卡介苗BCGi,并在细胞水平和小鼠体内观察该重组卡介苗的表达。通过重组卡介苗BCGi靶向递送Ipr1基因于感染Mtb的小鼠体内,观察重组卡介苗BCGi对小鼠Mtb感染的作用,运用基因芯片技术、蛋白芯片技术、Real-time PCR、ELISA检测实验组和对照组与免疫相关分子的表达差异,初步探讨Ipr1基因在抗Mtb感染中的可能的作用机制。 第一部分Ipr1基因表达对巨噬细胞抗分枝杆菌感染的影响 目的:获取Ipr1基因全长编码序列,构建Ipr1基因与EGFP基因融合表达载体,鉴定Ipr1基因在小鼠巨噬细胞株RAW264.7中的表达及细胞内定位,观察Ipr1基因的表达对小鼠巨噬细胞株RAW264.7细胞体外杀伤结核分枝杆菌H37Ra作用的影响。 方法:从C57BL/6J小鼠胸腺组织提取总RNA,以RT-PCR法调取Ipr1基因编码序列,克隆至真核表达载体pEGFP-C1,获得真核表达质粒pEGFP-Ipr1,经PCR、酶切鉴定正确后,脂质体转染pEGFP-Ipr1至小鼠巨噬细胞株RAW264.7,采用RT-PCR法,Western blotting法分别在转录水平及翻译水平检测Ipr1基因的表达及激光共聚焦显微镜观察融合蛋白的表达及细胞内定位。应用G418压力筛选稳定表达Ipr1基因的小鼠巨噬细胞株RAW264.7细胞,获得高表达Ipr1基因的巨噬细胞。体外感染分枝杆菌H37Ra,感染后24h及96h细胞裂解物细菌培养菌落计数(CFU)的方法观察巨噬细胞抗结核分枝杆菌H37Ra感染活性。 结果:从C57BL/6J小鼠胸腺组织内扩增出大小为1338bp片段。成功构建了重组真核表达质粒pEGFP-Ipr1,转染小鼠巨噬细胞株RAW264.7,经RT-PCR、Western blotting鉴定Ipr1基因在转录水平及翻译水平获得表达,共聚焦显微镜观察Ipr1融合绿色荧光蛋白表达产物定位于细胞核内。经G418压力筛选后获得稳定表达Ipr1基因的RAW264.7细胞。细胞水平抗分枝杆菌H37Ra实验结果显示Ipr1基因表达的实验组巨噬细胞裂解物细菌培养菌落计数(CFU)低于对照组细胞,差异具有统计学意义(p＜0.05)。 结论:成功获取小鼠Ipr1基因全长编码序列,构建了Ipr1基因与EGFP基因融合表达质粒pEGFP-Ipr1,Ipr1基因表达产物定位于细胞核内。G418压力筛选出高表达Ipr1基因的细胞。体外抗菌实验表明Ipr1基因的表达增强了巨噬细胞杀伤胞内吞噬的结核分枝杆菌H37Ra的能力。 第二部分靶向递送pBGO1真核质粒的重组BCGi的构建及鉴定 目的:构建Ipr1和绿色荧光蛋白(Green fluorescent protein,GFP)真核共表达穿梭质粒pBGOI,并在人肺腺癌细胞株A549中进行表达。构建可靶向递送pBGOI质粒进入巨噬细胞中进行表达的重组卡介苗BCGi,并在细胞水平和小鼠体内观察该重组卡介苗的表达。 方法:将GFP基因、分枝杆菌复制子OriM和Ipr1基因同时克隆入双启动子真核共表达载体pBudCE4.1质粒中,构建pBOGI穿梭质粒,脂质体法转染pBOGI质粒于培养的A549细胞中,RT-PCR法、免疫组化、Western Blotting、荧光显微镜检测Ipr1和GFP的表达.将pBGOI电转入卡介苗BCG中,构建重组卡介苗BCGi,PCR进行鉴定,扩增,将BCGi导入RAW264.7细胞后作RT-PCR、Western Blotting检测目的基因的表达。重组BCGi滴鼻方式免疫BALB/c小鼠,以RT-PCR法,免疫组化法检测肺脾组织中目的基因的表达。 结果:酶切和测序分析表明pBGOI真核共表达穿梭质粒构建成功pBGOI转染A549细胞后,RT-PCR、Western Blotting法检测到目的基因的表达。荧光显微镜观察到转染细胞中有绿色荧光蛋白表达,免疫组化可以检测到Ipr1蛋白的表达。菌落PCR鉴定重组卡介苗BCGi构建成功,BCGi导入RAW264.7细胞,RT-PCR法检测到目的基因的表达。重组BCGi滴鼻免疫的方式免疫BALB/c小鼠,RT-PCR法,免疫组化法检测到肺脾组织中目的基因的表达。 结论:成功构建真核共表达穿梭质粒pBGOI,成功构建重组卡介苗BCGi,为进一步研究Ipr1的功能及作用机制奠定基础。 第三部分Ipr1基因抗小鼠Mtb感染的作用及机制的初步探讨 目的:重组卡介苗BCGi靶向递送Ipr1基因于感染结核分枝杆菌的小鼠体内,观察重组卡介苗BCGi对小鼠结核分枝杆菌感染的作用及可能的分子机制。 方法:用BCGi免疫感染了结核分枝杆菌Mtb的C3HeB/FeJ小鼠,3周后处死,检测肺脾器官荷菌量、肺脾病理学改变、以及Ipr1在肺组织的表达,并做小鼠肺组织基因芯片检测、小鼠肺组织Real-timePCR检测、小鼠血清细胞因子蛋白芯片检测、小鼠血清IL-10、TNF-α和IFN-γ的检测。 结果:重组BCGi组肺脾器官荷菌显著降低。重组BCGi组小鼠肺组织病变范围和程度轻于PBS组和BCG组。免疫组化检测到小鼠肺组织中有Ipr1的表达。基因芯片结果中BCGi组比BCG组上调2倍的基因有:Igh-6、Lbp、Ltf、Ly96、Ncf4、Nfkb1、Nfkb2、Nfkbia、Nos2、Prg2、Sftpd、Stab1、Tlr4。Real-time PCR实验结果Fas、Mcl1、Bcl2、Caspase3、iNOS、LRG47、NRAMP1基因上调。血清蛋白芯片结果,BCGi组比BCG组下调1.3倍的炎症细胞因子有Eotaxin、Eotaxin-2、IL-1β、IL-3、IL-6、IL-10、IL-17、I-TAC、Leptin、TNFα。ELISA结果中重组BCGi组血清IFN-γ水平明显高于BCG组。 结论:重组BCGi对小鼠结核病有一定的免疫治疗作用。Ipr1基因通过增强固有免疫抗Mtb感染,特别是增强TLR4的活化途径。
[Abstract]:In 2005, the Harvard University Pan found a pathogen resistance gene mediated by macrophages against intracellular pathogens and intracellular gene 1 in mouse macrophages (intracellularpathogen resistance 1, Ipr1), the gene and susceptibility to tuberculosis. There is a close relationship between the expression of Ipr1 gene in mice, macrophage apoptosis after infection of Mtb. The bacteria is not easy proliferation. And IPR1 gene deficient mice after infection with Mtb macrophages showed necrosis, which help the bacteria multiply and spread between hosts. However, Ipr1 gene enhanced macrophage anti tuberculosis mechanism of Mycobacterium infection is not clear.
The Ipr1 gene was obtained from C57BL/6J mice thymus by RT-PCR method, to construct eukaryotic expression plasmid pEGFP-Ipr1, Ipr1 gene was observed in cell level regulation of macrophage antimycobacterial effect. The construction of Ipr1 and green fluorescent protein (Greenfluorescent protein, GFP) eukaryotic co expression shuttle plasmid pBGOI, then the shuttle plasmid electric to BCG BCG, construction of targeted delivery of pBGOI plasmid for expression of recombinant BCG BCGi into macrophages, and expression in cells and mice to observe the recombinant BCG. The recombinant BCG BCGi targeting mice Ipr1 gene delivery to Mtb infection, observation of recombinant BCG BCGi on Mtb infected mice, using gene chip protein chip technology, Real-time technology, PCR, ELISA expression between experimental groups and control groups were detected with immune related molecules, preliminary study Ip The possible mechanism of the R1 gene in the anti Mtb infection.
The effect of Ipr1 gene expression on anti Mycobacterium infection of macrophages in part 1
Objective: to obtain full length encoding sequence of Ipr1 gene, Ipr1 gene and EGFP fusion gene expression vector, expression and identification of Ipr1 gene in mouse macrophage cell line RAW264.7 in the location, to observe the effect of expression of Ipr1 gene of Mycobacterium tuberculosis H37Ra killing effect on RAW264.7 cells of mouse macrophage cell line.
Methods: total RNA was extracted from the thymus tissue of C57BL/6J mice with RT-PCR gene transfer of Ipr1 encoding sequence was cloned into eukaryotic expression vector pEGFP-C1 to obtain eukaryotic expression plasmid pEGFP-Ipr1 by PCR, after the identification of enzyme digestion, liposome transfection pEGFP-Ipr1 to mouse macrophage cell line RAW264.7, using the RT-PCR method, Western blotting method respectively in positioning detection of Ipr1 gene transcription and translation level of expression and laser confocal microscopy. The fusion protein expression and intracellular. Application of G418 pressure screening stable expression of murine macrophage RAW264.7 cell Ipr1 gene, to obtain the high expression of Ipr1 gene in vitro. Macrophages infected with Mycobacterium tuberculosis H37Ra infection, 24h and 96h cell lysates of bacterial colonies counting (CFU) method to observe the macrophage against Mycobacterium tuberculosis H37Ra infection activity.
Results: the amplification from C57BL/6J mice thymus in a fragment of 1338bp was successfully constructed. The recombinant eukaryotic expression plasmid pEGFP-Ipr1, transfected mouse macrophage cell line RAW264.7 by RT-PCR, Western, blotting identification of the Ipr1 gene was expressed at the transcription level and translation level, confocal microscopy Ipr1 fusion green fluorescent protein was located in the nucleus. Under the pressure of G418 after screening for stable expression of Ipr1 gene in RAW264.7 cells. The results of cell level anti Mycobacterium H37Ra showed that Ipr1 gene expression in the experimental group of macrophage lysate bacteria culture colony count (CFU) were lower than the control group, the difference was statistically significant (P < 0.05).
Conclusion: the successful acquisition of full-length cDNA encoding mouse Ipr1 gene sequence, construct Ipr1 fusion gene and EGFP gene expression plasmid pEGFP-Ipr1,.G418 pressure nucleus screened the high expression of Ipr1 gene product positioning cell expression of Ipr1 gene. The in vitro antibacterial experiment showed that Ipr1 gene expression and enhance the ability of macrophage phagocytosis of intracellular killing of Mycobacterium tuberculosis H37Ra.
Construction and identification of the second part of recombinant BCGi targeting the delivery of pBGO1 eukaryotic plasmids
Objective: to construct Ipr1 and green fluorescent protein (Green fluorescent, protein, GFP) eukaryotic expression plasmid pBGOI, and expressed in human lung adenocarcinoma cell line A549. Construction of targeted delivery of pBGOI plasmid for expression of recombinant BCG into macrophages and BCGi expression at the cell level and observe the recombinant mice BCG.
Methods: the GFP gene of Mycobacterium replicon OriM and Ipr1 gene were cloned into the dual promoter eukaryotic co expression vector plasmid pBudCE4.1. The constructed shuttle plasmid pBOGI transfected pBOGI plasmid in cultured A549 cells, RT-PCR assay, immunohistochemistry, Western, Blotting, Ipr1 and GFP expression were detected by fluorescence microscopy. PBGOI electroporated into BCG BCG, recombinant BCG BCGi, PCR were identified, amplified, BCGi into RAW264.7 cells after RT-PCR, to detect the expression of target gene Western Blotting. Recombinant BCGi intranasally immunization of BALB/c mice with RT-PCR method and the expression of immunohistochemical staining of lung and spleen tissue gene.
Results: enzyme digestion and sequencing analysis showed that the shuttle plasmid pBGOI was successfully constructed and transfected into A549 cells, RT-PCR pBGOI eukaryotic expression, the expression of Western Blotting detected the target gene. Fluorescence microscopy observed the expression of green fluorescence protein in transfected cells, immunohistochemistry detected the expression of Ipr1 protein. Colony PCR identification of recombinant BCG vaccine BCGi BCGi was successfully constructed and transfected into RAW264.7 cell by RT-PCR method to detect the expression of target gene. The recombinant BCGi intranasal immunization methods BALB/c mice were immunized by RT-PCR, immunohistochemistry method to detect the expression of target gene in the tissues of lung and spleen.
Conclusion: the recombinant eukaryotic shuttle plasmid pBGOI was successfully constructed and recombinant BCG BCGi was successfully constructed, which laid the foundation for further study of the function and mechanism of Ipr1.
Preliminary study on the effect and mechanism of the third part of Ipr1 gene against Mtb infection in mice
Objective: recombinant BCG BCGi targeted Ipr1 gene delivery in mice infected with Mycobacterium tuberculosis, to observe the effect and possible molecular mechanism of recombinant BCG BCGi on Mycobacterium tuberculosis infection in mice.
Methods: by immunization with BCGi infected with Mycobacterium tuberculosis Mtb C3HeB/FeJ mice were killed after 3 weeks, the lung and spleen organ bacterial load detection, pathological changes of lung and spleen pathology, and the expression of Ipr1 in lung tissue and the lung tissue of mice, gene chip detection, detection of lung tissue in Real-timePCR mice, mice serum cytokine detection protein chip serum IL-10, TNF-, IFN- alpha and gamma detection.
Results: the recombinant BCGi group lung spleen CFU significantly decreased in BCGi group. The lung tissue of mice in the scope and extent of the recombinant light in the PBS group and BCG group. The expression of Ipr1 was detected with immunohistochemistry in tissue microarray results. There are 2 times as many genes in BCGi group than in the BCG group were: Igh-6, Lbp Ltf, Ly96, Ncf4, Nfkb1, Nfkb2, Nfkbia, Nos2, Prg2, Sftpd, Stab1, Tlr4.Real-time, PCR Mcl1, the experimental results of Fas, Bcl2, Caspase3, iNOS, LRG47, NRAMP1 genes up-regulated. Serum protein microarray results, inflammatory cytokines BCGi were 1.3 times lower than the BCG group of Eotaxin, Eotaxin-2, IL-1 IL-3, IL-6, beta, IL-10, IL-17, I-TAC, Leptin, IFN- in serum gamma level of BCGi group was significantly higher than BCG group of recombinant TNF alpha.ELISA results.
Conclusion: recombinant BCGi has a certain immunotherapy effect on mice tuberculosis, and the.Ipr1 gene is enhanced by enhanced inherent immunity against Mtb infection, especially to enhance the activation pathway of TLR4.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392.1

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