本文選題：結(jié)核病 + 顆粒溶素　； 參考：《華中科技大學(xué)》2012年博士論文

【摘要】：[背景目的] 卡介苗作為預(yù)防結(jié)核病的唯一疫苗在全球廣泛使用,但對成人的保護效力存在很大波動,且不能清除潛伏感染者和罹患結(jié)核病患者體內(nèi)的細菌。臨床上耐多藥和廣泛耐藥結(jié)核病患者的大量出現(xiàn),抗結(jié)核新藥的研制步伐尚不能適應(yīng)疾病治療的需要。因此,開發(fā)治療型疫苗治療結(jié)核病的研究迫在眉睫。顆粒溶素是一種表達于人的細胞毒性T細胞(CTLs)及自然殺傷(NK)細胞的溶細胞性炎癥前因子,具有廣譜殺傷病原微生物包括結(jié)核分枝桿菌的作用。但是顆粒溶素本身并不能穿過細胞膜,只有在穿孔素的幫助下,它才能夠進入細胞內(nèi)從而發(fā)揮作用,這一特性阻礙了它抗胞內(nèi)細菌的潛在的臨床應(yīng)用。 復(fù)制缺陷(E1區(qū))的腺病毒載體是一種被廣泛應(yīng)用的運輸特定目的基因在真核細胞內(nèi)表達,已證實是非常具有潛力的預(yù)防傳染病,尤其是呼吸道感染性疾病的疫苗載體。因此,我們嘗試構(gòu)建表達人顆粒溶素(granulysin)的重組腺病毒(rAdhGA),并將其擴增、純化,測定病毒的滴度及其在動物體內(nèi)的分布；在體外細胞模型檢測了該重組腺病毒對被巨噬細胞吞噬的恥垢分枝桿菌的殺傷作用；評價rAdhGA對感染了結(jié)核分枝桿菌毒株H37Rv的BALB/c小鼠模型的治療效果。 [方法] 首先,將人顆粒溶素基因序列插入腺病毒穿梭質(zhì)粒后,與腺病毒骨架質(zhì)粒共同轉(zhuǎn)染HEK293細胞包裝出表達人顆粒溶素的重組腺病毒(rAdhGA)。然后,使用RT-PCR從基因水平證實該重組腺病毒在HEK293的表達人顆粒溶素mRNA; Western blot方法分別驗證該重組腺病毒在HEK293和巨噬細胞系U973中表達具有生物功能的人顆粒溶素蛋白；通過免疫熒光的方法檢測rAdhGA在巨噬細胞系U973中的表達；利用Clontech腺病毒純化試劑盒對驗證正確的重組腺病毒(rAdhGA)進行純化、擴增及利用腺病毒滴度測定試劑盒進行滴度測定。進一步地,通過免疫熒光的方法檢測rAdhGA經(jīng)呼吸道感染后在動物體內(nèi)的分布；離體實驗經(jīng)抗酸染色檢測該重組腺病毒對被巨噬細胞吞噬的恥垢分枝桿菌的殺傷作用。最后,通過組織荷菌量、組織病理學(xué)改變等指標評價rAdhGA對感染了結(jié)核分枝桿菌毒株H37Rv的BALB/c小鼠模型的治療效果。 [結(jié)果] 本研究成功構(gòu)建了表達顆粒溶素的重組腺病毒rAdhGA;制備該重組腺病毒的滴度為3.95×1010ifu/ml。rAdhGA滴鼻感染小鼠后主要分布在小鼠的肺部,并持續(xù)性高表達至少7天以上。rAdhGA可以直接殺傷被巨噬細胞U937吞噬的恥垢分枝桿菌；與對照組相比,高劑量的rAdhGA能明顯減少被H37Rv感染的BALB/c小鼠的肺部菌荷量。 [結(jié)論] 表達顆粒溶素的重組腺病毒rAdhGA具有很強的直接殺傷感染小鼠肺臟的結(jié)核分枝桿菌的作用,是一種有效的治療胞內(nèi)感染的結(jié)核分枝桿菌的疫苗。 [背景與目的] ECSM2(endothelial cell-specific molecule 2)是十多年前發(fā)現(xiàn)于人臍靜脈內(nèi)皮細胞(HUVEC)內(nèi)表達的多肽序列,隨后的多次研究證明其是血管內(nèi)皮細胞特異性表達基因。由于結(jié)構(gòu)的特異性,直到最近一段時間該基因的生物學(xué)功能才逐漸開始被了解,有限的研究數(shù)據(jù)表明該分子參與細胞的遷移與凋亡,但是隱藏在背后的信號傳導(dǎo)機制及該分子新的功能仍值得探索。 [方法] 在本實驗中,我們通過雜交瘤技術(shù)制備兔源性的ECSM2單克隆抗體,通過免疫印跡、免疫沉淀、去糖基化、免疫熒光染色和共聚焦顯微鏡等技術(shù)檢測ECSM2在細胞的定位及生物學(xué)特性；通過細胞聚集實驗和細胞跨孔實驗檢測ECSM2對成纖維細胞生長因子(bFGF)誘導(dǎo)的內(nèi)皮細胞遷移的影響；通過ERK上游MEK激活抑制劑的使用定義了ECSM2影響bFGF誘導(dǎo)的內(nèi)皮細胞遷移的信號傳導(dǎo)通路。 [結(jié)果] 在本實驗中,我們獲得了兔源性的ECSM2單克隆抗體,驗證了ECSM2是經(jīng)糖基化修飾的膜蛋白,僅在血管內(nèi)皮細胞(ECs)表達,特別集中于細胞連接處。細胞聚集實驗和細胞跨孔實驗均表明ECSM2促進細胞聚集,減弱成纖維細胞生長因子(bFGF)誘導(dǎo)的內(nèi)皮細胞遷移。通過在內(nèi)皮細胞過表達或沉默ECSM2的實驗證實,ECSM2通過成纖維細胞生長因子受體(FGFR)-胞外信號調(diào)節(jié)激酶(ERK)-粘著斑激酶(FAK)這一信號通路調(diào)節(jié)bFGF介導(dǎo)的細胞遷移,FAK的酪氨酸磷酸化(活化)和ERK依賴的FAK的絲氨酸磷酸化之間的平衡在這一信號通路中至關(guān)重要的,最后我們提出了一個模型闡述ECSM2如何影響bFGF及其受體誘導(dǎo)的內(nèi)皮細胞(EC)遷移。 [結(jié)論] ECSM2是一個新的位于內(nèi)皮細胞連接處的蛋白,能夠通過FGFR-ERK-FAK途徑抑制bFGF介導(dǎo)的細胞遷移。這些發(fā)現(xiàn)預(yù)示著ECSM2很有可能是受體酪氨酸激酶(RTK)-、整合素(integrin)-及內(nèi)皮細胞結(jié)合部位介導(dǎo)信號的重要參與者,并可能對與血管內(nèi)皮功能障礙和受損EC結(jié)合信號相關(guān)的疾病有重要的影響。
[Abstract]:[background purpose]
As the only vaccine to prevent tuberculosis, Bacillus Calmette Guerin is widely used in the world, but there is a great fluctuation in the protection effectiveness of adults, and it can not remove the latent infection and the bacteria in the patients with tuberculosis. Therefore, the development of a therapeutic vaccine for the treatment of tuberculosis is imminent. Particle lysin is a cytotoxic pre - inflammatory factor expressed in human cytotoxic T cells (CTLs) and natural killer (NK) cells. It has a broad spectrum of killing pathogenic microbes, including Mycobacterium tuberculosis. But the particle lysin itself does not. Through the cell membrane, it is only able to enter the cell with the help of perforin, which hinders its potential clinical application of intracellular bacteria.
The adenovirus vector of the replicative defect (E1 region) is a widely used transport specific target gene expressed in eukaryotic cells, which has been proved to be a very potential vector for preventing infectious diseases, especially respiratory infections. Therefore, we attempt to construct recombinant adenoviruses (rAdhGA) expressing human particle lysin (granulysin), and It amplified, purified, measured the titer of the virus and its distribution in the animal. In vitro cell model was used to detect the killing effect of the recombinant adenovirus on macrophage phagocytic Mycobacterium phagocytic Mycobacterium, and to evaluate the therapeutic effect of rAdhGA on the BALB/c mouse model infected with Mycobacterium tuberculosis strain H37Rv.
[method]
First, after inserting the human granulomotin gene sequence into the adenovirus shuttle plasmid, the recombinant adenovirus (rAdhGA) was transfected with the adenovirus skeleton plasmids together with the HEK293 cells. Then, RT-PCR was used to confirm the expression of the recombinant adenovirus in HEK293 by RT-PCR, and the Western blot method was tested respectively. The recombinant adenovirus was expressed in HEK293 and macrophage line U973 to express the human granulomotin protein with biological function; the expression of rAdhGA in the macrophage system U973 was detected by immunofluorescence, and the Clontech adenovirus purification kit was used to purify the correct recombinant adenovirus (rAdhGA) and to amplify and utilize the adenovirus. The titer test kit was used to measure the titer. Further, the immunofluorescence method was used to detect the distribution of rAdhGA in the animals after respiratory infection. In vitro test, the killing effect of the recombinant adenovirus on macrophage phagocytic Mycobacterium phagocytic Mycobacterium was detected by antiacid staining. Finally, the tissue load and histopathology were changed. The effect of rAdhGA on BALB/c mice infected with Mycobacterium tuberculosis H37Rv was evaluated by variable index.
[results]
The recombinant adenovirus expressing granulosa lysin rAdhGA was successfully constructed in this study, and the titer of the recombinant adenovirus was mainly distributed in the lungs of mice with 3.95 1010ifu/ml.rAdhGA dripping nose infection, and the persistent high expression of.RAdhGA for at least 7 days could kill Mycobacterium phagocytic Mycobacterium phagocytic Mycobacterium phagocytosis directly. Compared with the control group, high dose of rAdhGA significantly reduced the lung bacterial load of H37Rv infected BALB/c mice.
[Conclusion]
Recombinant adenovirus expressing granulosa lysin rAdhGA has a strong direct killing effect on the infection of Mycobacterium tuberculosis in the lungs of mice, and is an effective vaccine for the treatment of Mycobacterium tuberculosis in the intracellular infection.
[background and purpose]
ECSM2 (endothelial cell-specific molecule 2) is a polypeptide sequence expressed in human umbilical vein endothelial cells (HUVEC) more than 10 years ago. Subsequent studies have shown that it is a specific expression gene of vascular endothelial cells. The biological function of the gene is gradually understood until the last segment of the structure. Limited data indicate that the molecule is involved in cell migration and apoptosis, but the signal transduction mechanism behind it and the new function of the molecule are still worth exploring.
[method]
In this experiment, rabbit derived ECSM2 monoclonal antibodies were prepared by hybridoma technique. The localization and biological characteristics of ECSM2 in cells were detected by immunoblotting, immunoprecipitation, de glycosylation, immunofluorescence staining and confocal microscopy, and the detection of ECSM2 to fibroblasts through cell aggregation experiment and cell cross hole test. The effect of growth factor (bFGF) induced migration of endothelial cells, and the use of MEK activation inhibitors on the upstream of ERK defines the signal transduction pathway that ECSM2 affects the migration of bFGF induced endothelial cells.
[results]
In this experiment, we obtained a rabbit derived ECSM2 monoclonal antibody, which proved that ECSM2 was a glycosylated membrane protein, expressed only in vascular endothelial cells (ECs), especially at the cell junction. Cell aggregation experiments and cell cross hole experiments showed that ECSM2 promoted fine cell aggregation and weakened fibroblast growth factor (bFGF) induced. Migration of endothelial cells. Through the overexpression or silencing of ECSM2 in endothelial cells, ECSM2 regulates bFGF mediated cell migration through the signaling pathway of fibroblast growth factor receptor (FGFR) - extracellular signal regulated kinase (ERK) - adhesion kinase (FAK), FAK tyrosine phosphorylation (activation) and ERK dependent FAK serine phosphoric acid The balance between chemistry is crucial in this signaling pathway. Finally, we present a model to illustrate how ECSM2 affects the migration of bFGF and its receptor induced endothelial cells (EC).
[Conclusion]
ECSM2 is a new protein located at the junction of endothelial cells and can inhibit bFGF mediated cell migration through the FGFR-ERK-FAK pathway. These findings suggest that ECSM2 is very likely to be an important participant in the receptor tyrosine kinase (RTK) - integrin (integrin) - and endothelial cell binding sites, and may be associated with vascular endothelial function. Obstacles and impaired EC have important effects on signal related diseases.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R52;R-332

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