一、表达颗粒溶素的重组腺病毒基因治疗结核病小鼠模型二、内皮细胞特异性分子2（ECSM2）的细胞定位及功能学研究

发布时间：2018-05-24 17:01

本文选题：结核病 + 颗粒溶素　；参考：《华中科技大学》2012年博士论文

【摘要】：[背景目的] 卡介苗作为预防结核病的唯一疫苗在全球广泛使用,但对成人的保护效力存在很大波动,且不能清除潜伏感染者和罹患结核病患者体内的细菌。临床上耐多药和广泛耐药结核病患者的大量出现,抗结核新药的研制步伐尚不能适应疾病治疗的需要。因此,开发治疗型疫苗治疗结核病的研究迫在眉睫。颗粒溶素是一种表达于人的细胞毒性T细胞(CTLs)及自然杀伤(NK)细胞的溶细胞性炎症前因子,具有广谱杀伤病原微生物包括结核分枝杆菌的作用。但是颗粒溶素本身并不能穿过细胞膜,只有在穿孔素的帮助下,它才能够进入细胞内从而发挥作用,这一特性阻碍了它抗胞内细菌的潜在的临床应用。复制缺陷(E1区)的腺病毒载体是一种被广泛应用的运输特定目的基因在真核细胞内表达,已证实是非常具有潜力的预防传染病,尤其是呼吸道感染性疾病的疫苗载体。因此,我们尝试构建表达人颗粒溶素(granulysin)的重组腺病毒(rAdhGA),并将其扩增、纯化,测定病毒的滴度及其在动物体内的分布；在体外细胞模型检测了该重组腺病毒对被巨噬细胞吞噬的耻垢分枝杆菌的杀伤作用；评价rAdhGA对感染了结核分枝杆菌毒株H37Rv的BALB/c小鼠模型的治疗效果。 [方法] 首先,将人颗粒溶素基因序列插入腺病毒穿梭质粒后,与腺病毒骨架质粒共同转染HEK293细胞包装出表达人颗粒溶素的重组腺病毒(rAdhGA)。然后,使用RT-PCR从基因水平证实该重组腺病毒在HEK293的表达人颗粒溶素mRNA; Western blot方法分别验证该重组腺病毒在HEK293和巨噬细胞系U973中表达具有生物功能的人颗粒溶素蛋白；通过免疫荧光的方法检测rAdhGA在巨噬细胞系U973中的表达；利用Clontech腺病毒纯化试剂盒对验证正确的重组腺病毒(rAdhGA)进行纯化、扩增及利用腺病毒滴度测定试剂盒进行滴度测定。进一步地,通过免疫荧光的方法检测rAdhGA经呼吸道感染后在动物体内的分布；离体实验经抗酸染色检测该重组腺病毒对被巨噬细胞吞噬的耻垢分枝杆菌的杀伤作用。最后,通过组织荷菌量、组织病理学改变等指标评价rAdhGA对感染了结核分枝杆菌毒株H37Rv的BALB/c小鼠模型的治疗效果。 [结果] 本研究成功构建了表达颗粒溶素的重组腺病毒rAdhGA;制备该重组腺病毒的滴度为3.95×1010ifu/ml。rAdhGA滴鼻感染小鼠后主要分布在小鼠的肺部,并持续性高表达至少7天以上。rAdhGA可以直接杀伤被巨噬细胞U937吞噬的耻垢分枝杆菌；与对照组相比,高剂量的rAdhGA能明显减少被H37Rv感染的BALB/c小鼠的肺部菌荷量。 [结论] 表达颗粒溶素的重组腺病毒rAdhGA具有很强的直接杀伤感染小鼠肺脏的结核分枝杆菌的作用,是一种有效的治疗胞内感染的结核分枝杆菌的疫苗。 [背景与目的] ECSM2(endothelial cell-specific molecule 2)是十多年前发现于人脐静脉内皮细胞(HUVEC)内表达的多肽序列,随后的多次研究证明其是血管内皮细胞特异性表达基因。由于结构的特异性,直到最近一段时间该基因的生物学功能才逐渐开始被了解,有限的研究数据表明该分子参与细胞的迁移与凋亡,但是隐藏在背后的信号传导机制及该分子新的功能仍值得探索。 [方法] 在本实验中,我们通过杂交瘤技术制备兔源性的ECSM2单克隆抗体,通过免疫印迹、免疫沉淀、去糖基化、免疫荧光染色和共聚焦显微镜等技术检测ECSM2在细胞的定位及生物学特性；通过细胞聚集实验和细胞跨孔实验检测ECSM2对成纤维细胞生长因子(bFGF)诱导的内皮细胞迁移的影响；通过ERK上游MEK激活抑制剂的使用定义了ECSM2影响bFGF诱导的内皮细胞迁移的信号传导通路。 [结果] 在本实验中,我们获得了兔源性的ECSM2单克隆抗体,验证了ECSM2是经糖基化修饰的膜蛋白,仅在血管内皮细胞(ECs)表达,特别集中于细胞连接处。细胞聚集实验和细胞跨孔实验均表明ECSM2促进细胞聚集,减弱成纤维细胞生长因子(bFGF)诱导的内皮细胞迁移。通过在内皮细胞过表达或沉默ECSM2的实验证实,ECSM2通过成纤维细胞生长因子受体(FGFR)-胞外信号调节激酶(ERK)-粘着斑激酶(FAK)这一信号通路调节bFGF介导的细胞迁移,FAK的酪氨酸磷酸化(活化)和ERK依赖的FAK的丝氨酸磷酸化之间的平衡在这一信号通路中至关重要的,最后我们提出了一个模型阐述ECSM2如何影响bFGF及其受体诱导的内皮细胞(EC)迁移。 [结论] ECSM2是一个新的位于内皮细胞连接处的蛋白,能够通过FGFR-ERK-FAK途径抑制bFGF介导的细胞迁移。这些发现预示着ECSM2很有可能是受体酪氨酸激酶(RTK)-、整合素(integrin)-及内皮细胞结合部位介导信号的重要参与者,并可能对与血管内皮功能障碍和受损EC结合信号相关的疾病有重要的影响。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R52;R-332

【参考文献】

一、表达颗粒溶素的重组腺病毒基因治疗结核病小鼠模型 二、内皮细胞特异性分子2（ECSM2）的细胞定位及功能学研究

一、表达颗粒溶素的重组腺病毒基因治疗结核病小鼠模型二、内皮细胞特异性分子2（ECSM2）的细胞定位及功能学研究