本文關(guān)鍵詞：從巨噬細(xì)胞Ipr1基因功能研究探討結(jié)核分枝桿菌感染固有免疫的調(diào)節(jié)機制　出處：《重慶醫(yī)科大學(xué)》2009年博士論文　論文類型：學(xué)位論文

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【摘要】： 2005年,哈佛大學(xué)的Pan等在小鼠巨噬細(xì)胞內(nèi)發(fā)現(xiàn)了一個介導(dǎo)巨噬細(xì)胞抗胞內(nèi)病原體的基因—胞內(nèi)病原體抗性基因1(intracellularpathogen resistance 1,Ipr1),該基因與結(jié)核病的易感性有著密切的聯(lián)系。表達(dá)Ipr1基因的小鼠,感染Mtb后巨噬細(xì)胞發(fā)生凋亡,細(xì)菌不易增殖。而IPR1基因缺陷的小鼠感染Mtb后巨噬細(xì)胞表現(xiàn)為壞死,從而利于細(xì)菌在宿主體內(nèi)的增殖與擴散。然而,Ipr1基因增強巨噬細(xì)胞抗結(jié)核分枝桿菌感染的機制還不清楚。 本課題通過RT-PCR法從C57BL/6J小鼠胸腺組織中獲取Ipr1基因,構(gòu)建真核表達(dá)質(zhì)粒pEGFP-Ipr1,觀察Ipr1基因在細(xì)胞水平調(diào)節(jié)巨噬細(xì)胞抗分枝桿菌的作用。構(gòu)建Ipr1和綠色熒光蛋白(Greenfluorescent protein,GFP)真核共表達(dá)穿梭質(zhì)粒pBGOI,然后將該穿梭質(zhì)粒電轉(zhuǎn)到卡介苗BCG中,構(gòu)建可靶向遞送pBGOI質(zhì)粒進(jìn)入巨噬細(xì)胞中進(jìn)行表達(dá)的重組卡介苗BCGi,并在細(xì)胞水平和小鼠體內(nèi)觀察該重組卡介苗的表達(dá)。通過重組卡介苗BCGi靶向遞送Ipr1基因于感染Mtb的小鼠體內(nèi),觀察重組卡介苗BCGi對小鼠Mtb感染的作用,運用基因芯片技術(shù)、蛋白芯片技術(shù)、Real-time PCR、ELISA檢測實驗組和對照組與免疫相關(guān)分子的表達(dá)差異,初步探討Ipr1基因在抗Mtb感染中的可能的作用機制。 第一部分Ipr1基因表達(dá)對巨噬細(xì)胞抗分枝桿菌感染的影響 目的:獲取Ipr1基因全長編碼序列,構(gòu)建Ipr1基因與EGFP基因融合表達(dá)載體,鑒定Ipr1基因在小鼠巨噬細(xì)胞株RAW264.7中的表達(dá)及細(xì)胞內(nèi)定位,觀察Ipr1基因的表達(dá)對小鼠巨噬細(xì)胞株RAW264.7細(xì)胞體外殺傷結(jié)核分枝桿菌H37Ra作用的影響。 方法:從C57BL/6J小鼠胸腺組織提取總RNA,以RT-PCR法調(diào)取Ipr1基因編碼序列,克隆至真核表達(dá)載體pEGFP-C1,獲得真核表達(dá)質(zhì)粒pEGFP-Ipr1,經(jīng)PCR、酶切鑒定正確后,脂質(zhì)體轉(zhuǎn)染pEGFP-Ipr1至小鼠巨噬細(xì)胞株RAW264.7,采用RT-PCR法,Western blotting法分別在轉(zhuǎn)錄水平及翻譯水平檢測Ipr1基因的表達(dá)及激光共聚焦顯微鏡觀察融合蛋白的表達(dá)及細(xì)胞內(nèi)定位。應(yīng)用G418壓力篩選穩(wěn)定表達(dá)Ipr1基因的小鼠巨噬細(xì)胞株RAW264.7細(xì)胞,獲得高表達(dá)Ipr1基因的巨噬細(xì)胞。體外感染分枝桿菌H37Ra,感染后24h及96h細(xì)胞裂解物細(xì)菌培養(yǎng)菌落計數(shù)(CFU)的方法觀察巨噬細(xì)胞抗結(jié)核分枝桿菌H37Ra感染活性。 結(jié)果:從C57BL/6J小鼠胸腺組織內(nèi)擴增出大小為1338bp片段。成功構(gòu)建了重組真核表達(dá)質(zhì)粒pEGFP-Ipr1,轉(zhuǎn)染小鼠巨噬細(xì)胞株RAW264.7,經(jīng)RT-PCR、Western blotting鑒定Ipr1基因在轉(zhuǎn)錄水平及翻譯水平獲得表達(dá),共聚焦顯微鏡觀察Ipr1融合綠色熒光蛋白表達(dá)產(chǎn)物定位于細(xì)胞核內(nèi)。經(jīng)G418壓力篩選后獲得穩(wěn)定表達(dá)Ipr1基因的RAW264.7細(xì)胞。細(xì)胞水平抗分枝桿菌H37Ra實驗結(jié)果顯示Ipr1基因表達(dá)的實驗組巨噬細(xì)胞裂解物細(xì)菌培養(yǎng)菌落計數(shù)(CFU)低于對照組細(xì)胞,差異具有統(tǒng)計學(xué)意義(p＜0.05)。 結(jié)論:成功獲取小鼠Ipr1基因全長編碼序列,構(gòu)建了Ipr1基因與EGFP基因融合表達(dá)質(zhì)粒pEGFP-Ipr1,Ipr1基因表達(dá)產(chǎn)物定位于細(xì)胞核內(nèi)。G418壓力篩選出高表達(dá)Ipr1基因的細(xì)胞。體外抗菌實驗表明Ipr1基因的表達(dá)增強了巨噬細(xì)胞殺傷胞內(nèi)吞噬的結(jié)核分枝桿菌H37Ra的能力。 第二部分靶向遞送pBGO1真核質(zhì)粒的重組BCGi的構(gòu)建及鑒定 目的:構(gòu)建Ipr1和綠色熒光蛋白(Green fluorescent protein,GFP)真核共表達(dá)穿梭質(zhì)粒pBGOI,并在人肺腺癌細(xì)胞株A549中進(jìn)行表達(dá)。構(gòu)建可靶向遞送pBGOI質(zhì)粒進(jìn)入巨噬細(xì)胞中進(jìn)行表達(dá)的重組卡介苗BCGi,并在細(xì)胞水平和小鼠體內(nèi)觀察該重組卡介苗的表達(dá)。 方法:將GFP基因、分枝桿菌復(fù)制子OriM和Ipr1基因同時克隆入雙啟動子真核共表達(dá)載體pBudCE4.1質(zhì)粒中,構(gòu)建pBOGI穿梭質(zhì)粒,脂質(zhì)體法轉(zhuǎn)染pBOGI質(zhì)粒于培養(yǎng)的A549細(xì)胞中,RT-PCR法、免疫組化、Western Blotting、熒光顯微鏡檢測Ipr1和GFP的表達(dá).將pBGOI電轉(zhuǎn)入卡介苗BCG中,構(gòu)建重組卡介苗BCGi,PCR進(jìn)行鑒定,擴增,將BCGi導(dǎo)入RAW264.7細(xì)胞后作RT-PCR、Western Blotting檢測目的基因的表達(dá)。重組BCGi滴鼻方式免疫BALB/c小鼠,以RT-PCR法,免疫組化法檢測肺脾組織中目的基因的表達(dá)。 結(jié)果:酶切和測序分析表明pBGOI真核共表達(dá)穿梭質(zhì)粒構(gòu)建成功pBGOI轉(zhuǎn)染A549細(xì)胞后,RT-PCR、Western Blotting法檢測到目的基因的表達(dá)。熒光顯微鏡觀察到轉(zhuǎn)染細(xì)胞中有綠色熒光蛋白表達(dá),免疫組化可以檢測到Ipr1蛋白的表達(dá)。菌落PCR鑒定重組卡介苗BCGi構(gòu)建成功,BCGi導(dǎo)入RAW264.7細(xì)胞,RT-PCR法檢測到目的基因的表達(dá)。重組BCGi滴鼻免疫的方式免疫BALB/c小鼠,RT-PCR法,免疫組化法檢測到肺脾組織中目的基因的表達(dá)。 結(jié)論:成功構(gòu)建真核共表達(dá)穿梭質(zhì)粒pBGOI,成功構(gòu)建重組卡介苗BCGi,為進(jìn)一步研究Ipr1的功能及作用機制奠定基礎(chǔ)。 第三部分Ipr1基因抗小鼠Mtb感染的作用及機制的初步探討 目的:重組卡介苗BCGi靶向遞送Ipr1基因于感染結(jié)核分枝桿菌的小鼠體內(nèi),觀察重組卡介苗BCGi對小鼠結(jié)核分枝桿菌感染的作用及可能的分子機制。 方法:用BCGi免疫感染了結(jié)核分枝桿菌Mtb的C3HeB/FeJ小鼠,3周后處死,檢測肺脾器官荷菌量、肺脾病理學(xué)改變、以及Ipr1在肺組織的表達(dá),并做小鼠肺組織基因芯片檢測、小鼠肺組織Real-timePCR檢測、小鼠血清細(xì)胞因子蛋白芯片檢測、小鼠血清IL-10、TNF-α和IFN-γ的檢測。 結(jié)果:重組BCGi組肺脾器官荷菌顯著降低。重組BCGi組小鼠肺組織病變范圍和程度輕于PBS組和BCG組。免疫組化檢測到小鼠肺組織中有Ipr1的表達(dá)�；蛐酒Y(jié)果中BCGi組比BCG組上調(diào)2倍的基因有:Igh-6、Lbp、Ltf、Ly96、Ncf4、Nfkb1、Nfkb2、Nfkbia、Nos2、Prg2、Sftpd、Stab1、Tlr4。Real-time PCR實驗結(jié)果Fas、Mcl1、Bcl2、Caspase3、iNOS、LRG47、NRAMP1基因上調(diào)。血清蛋白芯片結(jié)果,BCGi組比BCG組下調(diào)1.3倍的炎癥細(xì)胞因子有Eotaxin、Eotaxin-2、IL-1β、IL-3、IL-6、IL-10、IL-17、I-TAC、Leptin、TNFα。ELISA結(jié)果中重組BCGi組血清IFN-γ水平明顯高于BCG組。 結(jié)論:重組BCGi對小鼠結(jié)核病有一定的免疫治療作用。Ipr1基因通過增強固有免疫抗Mtb感染,特別是增強TLR4的活化途徑。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392.1

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