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Cortactin在幽门螺杆菌VacA致凋亡中的作用及其机制研究

发布时间:2018-08-13 09:15
【摘要】:研究背景和目的: 幽门螺杆菌(Helicobacter pylori, H.pylori)是人体内最常见的病原菌之一,它感染全世界50%以上的人群,其感染与慢性胃炎、消化性溃疡、胃黏膜相关的淋巴样组织淋巴瘤和胃腺癌关系密切,世界卫生组织已把H.pylori列为胃癌的首要致病因子。目前虽已取得部分较为直接的H.pylori与胃癌相关的证据,也有对其诱发胃癌的分子机制的研究,但仍有众多问题需要进一步阐明。 有报道认为H.pylori感染引起的凋亡异常在胃癌的发生发展中起主要作用[1]。体内外实验均已证实H.pylori感染可导致胃粘膜上皮细胞凋亡,持续的大量细胞凋亡将使胃粘膜组织损伤,胃粘膜上皮细胞大量丢失,功能障碍,继发大量的细胞增殖,,进一步可发展为肠化生和不典型增生,抑凋亡基因或促凋亡基因在“高运转”状况下可能发生改变,当细胞凋亡与细胞增殖严重失衡,胃粘膜难免向肿瘤方向发展。 H.pylori的致病因子有多种,其中最主要的是空泡细胞毒素(vacuolating cytotoxin,VacA)和细胞毒素相关蛋白(cytotoxin associated protein A, CagA)。VacA是目前倍受关注的毒力因子之一,近来研究发现VacA的p37亚基与胃癌的发生密切相关[2]。 皮动蛋白(cortical actin-binding protein,cortactin)是一种微丝骨架结合蛋白,cortactin与H.pylori定植、粘附、胞内运动密切相关。最近研究显示H.pylori使cortactin的SH3区失激活和去磷酸化,从而影响肌动蛋白(actin)的重新分布,使细胞的紧密连接松弛,利于毒素蛋白入侵[3];cortactin是信号传导通路和细胞骨架重要的联系分子,它可以提高细胞迁移的能力。现在研究显示它与细胞的运动、肿瘤侵袭转移有着密切关系。 既往研究中使用的VacA蛋白一般有H.pylori分泌的VacA蛋白的粗纯液,纯化的H.pylori分泌的VacA蛋白和重组表达的VacA蛋白。H.pylori分泌的VacA蛋白的粗纯液具有良好的抗原性,制备相对简单,但其蛋白成分复杂,不利于阐明其作用。纯化的H.pylori分泌的VacA蛋白具有良好的抗原性,且成分单一,便于证实其生物学作用。 本研究的目的有二个,一是拟通过两种方式获得VacA蛋白,即直接纯化H.pylori分泌的VacA蛋白,及通过重组表达获得VacA蛋白,并比较这两种蛋白致细胞凋亡和空泡效应的差异,以此选择生物学效应好的一种蛋白用于细胞实验。二是首先明确cortactin在VacA致细胞凋亡过程中存在调控作用;其次是明确cortactin调控凋亡的机制,即是否通过影响凋亡相关蛋白而发挥作用。我们拟构建cortactin过表达和cortactin siRNA干扰的AGS细胞稳转株,使用H.pylori分泌的VacA蛋白分别与人胃腺癌细胞(AGS细胞)、cortactin过表达的AGS细胞和cortactin siRNA干扰的AGS细胞稳转株共孵,通过流式细胞技术和免疫印迹法,检测细胞凋亡及凋亡相关蛋白表达的差异,以揭示cortactin在VacA致胃上皮细胞凋亡过程中存在调控作用,并初步探讨其机理。 方法: 1. H.pylori分泌VacA蛋白的纯化。取冻存的H.pyloriATCC26695菌株,接种于skirrow氏培养基平皿,微需氧环境培养48h。刮取适量H.pylori菌落混悬于skirrow氏液体培养基中,微需氧环境培养24h。收集约2L H.pylori菌液,收集上清液于4℃加入达50%饱和度的硫酸铵,充分沉淀后以离心,沉淀蛋白溶于5ml PBS缓冲液1中。蛋白样品依次采用HiTrap SP HP柱、HiTrap Phenyl HP柱、HiTrap Q HP柱进行纯化。纯化VacA蛋白采用免疫印迹法进行鉴定。 2.重组VacA蛋白的表达纯化。以H.pylori60190的VacA基因片段为模板,合成VacA基因片段,构建在pUC57上。随后载体质粒pQE30及VacA基因进行双酶切。质粒pQE30回收大片段与VacA基因进行连接,连接产物转化后,涂布于LB平板上,37℃恒温培养箱倒置培养过夜。挑取生长良好的菌落接种于LB培养液中培养至第二天上午,用小量质粒抽提试剂盒抽提质粒,并分别做酶切鉴定。鉴定正确的VacA质粒转化到M15感受态细胞,涂布A+平板,37℃培养过夜,挑单菌落到LB中,摇床里过夜活化培养。收集菌液,获得VacA重组蛋白,采用Ni2+-NTA树脂纯化法纯化VacA蛋白,SDS-PAGE电泳法鉴定该蛋白。 3. H.pylori分泌的VacA蛋白和重组VacA蛋白分别与AGS细胞共孵,流式细胞技术检测细胞凋亡,倒置显微镜观察细胞空泡变性,从而比较这两种方法获得的蛋白的生物学效应。 4. pLVX-siRNA2-Puro-hScramble慢病毒载体构建、慢病毒包装及稳转人胃癌AGS细胞株筛选。 5. H.pylori分泌的VacA蛋白分别与AGS细胞、cortactin过表达的AGS细胞和cortactin siRNA干扰的AGS细胞稳转株共孵,通过流式细胞技术和免疫印迹方法,在0h、6h、12h、24h检测细胞凋亡及凋亡蛋白表达。 结果: 1.纯化出H.pylori分泌VacA蛋白,SDS-PAGE分析其蛋白分子量与预期的一致,免疫印迹法鉴定该蛋白为VacA蛋白。 2.纯化出重组表达VacA蛋白,SDS-PAGE分析其蛋白分子量与预期的一致。 3. H.pylori分泌VacA蛋白能显著诱导AGS细胞凋亡和致AGS细胞空泡效应,而重组表达VacA蛋白无显著诱导AGS细胞凋亡和致AGS细胞空泡效应的作用。 4.成功构建出cortactin过表达的AGS细胞稳转株和cortactin siRNA干扰的AGS细胞稳转株。 5. cortactin过表达后,VacA蛋白致AGS细胞凋亡率增加,凋亡蛋白Bax表达增高,抗凋亡蛋白Bcl-2表达降低;沉默cortactin表达,VacA蛋白致AGS细胞凋亡率降低,凋亡蛋白Bax表达降低,抗凋亡蛋白Bcl-2表达增高。 结论: 1. H.pylori分泌的VacA蛋白虽然表达量少,但其生物活性强,纯化出的蛋白量足以用于后续研究。 2. cortactin促进VacA蛋白导致的胃上皮细胞凋亡增加,其机制可能与凋亡蛋白的调控有关。
[Abstract]:Background and purpose:
Helicobacter pylori (H.pylori) is one of the most common pathogens in the human body. It infects more than 50% of the world population. Its infection is closely related to chronic gastritis, peptic ulcer, lymphoid tissue lymphoma associated with gastric mucosa and gastric adenocarcinoma. The World Health Organization has listed H.pylori as the primary pathogenic factor of gastric cancer. Although some direct evidence of H.pylori's association with gastric cancer has been obtained and the molecular mechanism of H.pylori's induction of gastric cancer has been studied, there are still many problems to be further clarified.
It has been reported that the abnormal apoptosis caused by H.pylori infection plays a major role in the occurrence and development of gastric cancer [1].In vivo and in vitro experiments have proved that H.pylori infection can induce apoptosis of gastric mucosal epithelial cells. Persistent apoptosis of a large number of cells will cause gastric mucosal tissue damage, a large number of gastric mucosal epithelial cells lost, dysfunction, and secondary large number of cells. Proliferation can further develop into intestinal metaplasia and atypical hyperplasia. Apoptosis-suppressing or apoptosis-promoting genes may change under the condition of "high-functioning". When apoptosis and cell proliferation are seriously imbalanced, gastric mucosa will inevitably develop toward tumor.
Vacuolating cytotoxin (VacA) and cytotoxin associated protein A (CagA) are the most important pathogenic factors of H. pylori. VacA is one of the virulence factors that have attracted much attention at present. Recent studies have found that the P37 subunit of VacA is closely related to the occurrence of gastric cancer [2].
Recent studies have shown that H. pylori inactivates and dephosphorylates the SH3 region of cortactin, thereby affecting the redistribution of actin and relaxing the tight junction of cells. It is beneficial to the invasion of toxin protein [3]; cortactin is an important link molecule between signal transduction pathway and cytoskeleton, which can improve the ability of cell migration.
VacA protein used in previous studies usually contains crude purified H.pylori-secreted VacA protein, purified H.pylori-secreted VacA protein and recombinant H.pylori-secreted VacA protein. The crude purified H.pylori-secreted VacA protein has good antigenicity and relatively simple preparation, but its protein composition is complex, which is not conducive to clarifying its role. The secreted VacA protein has good antigenicity and single component, which is easy to confirm its biological function.
There are two purposes of this study. One is to obtain VacA protein by two ways, that is, to purify the VacA protein secreted by H. pylori directly, and to obtain VacA protein by recombinant expression, and to compare the difference of cell apoptosis and vacuole effect between the two proteins, so as to select a protein with good biological effect for cell experiment. Cortactin plays a regulatory role in the process of vacA-induced apoptosis; secondly, it clarifies the mechanism of cortactin-induced apoptosis, that is, whether it plays a role by affecting apoptosis-related proteins. Cells (AGS cells), AGS cells overexpressed by cortactin and stable AGS cells transfected by cortactin siRNA interference were co-incubated. The differences of apoptosis and apoptosis-related protein expression were detected by flow cytometry and immunoblotting to reveal the regulatory role of cortactin in the process of gastric epithelial cell apoptosis induced by vacA and to explore its mechanism.
Method:
1. Purification of VacA protein secreted by H. pylori. Strain H. pylori ATCC26695 was frozen and inoculated in skirrow's medium for 48h in micro-aerobic environment. A suitable amount of H. pylori colony was scraped and suspended in skirrow's liquid medium for 24h in micro-aerobic environment. 2L H. pylori liquid was collected and the supernatant was added with 50% saturated sulfur at 4 C. The protein samples were purified by HiTrap SP HP column, HiTrap Phenyl HP column and HiTrap Q HP column. The purified VacA protein was identified by Western blot.
2. Expression and purification of recombinant vacA protein. VacA gene fragment was synthesized from H. pylori 60190 and constructed on pUC57. The vector plasmids pQE30 and VacA were digested by double enzyme digestion. The recombinant plasmids pQE30 were ligated to vacA gene. The conjugated product was transformed and coated on LB plate and cultured inverted in 37 C incubator. Overnight. Colonies with good growth were inoculated in LB culture medium until the next morning. Plasmids were extracted with a small amount of Plasmid Extraction Kit and identified by enzyme digestion. The correct vacA plasmids were transformed into M15 competent cells, coated with A + plate, cultured overnight at 37 C, single colony was selected into LB and cultured overnight in shaking bed. VacA recombinant protein was purified by Ni2 +-NTA resin and identified by SDS-PAGE electrophoresis.
3. VacA protein and recombinant vacA protein secreted by H. pylori were co-incubated with AGS cells respectively. Apoptosis was detected by flow cytometry and cell vacuole degeneration was observed by inverted microscope. The biological effects of the two proteins were compared.
4. Construction of pLVX-siRNA 2-Puro-hScramble lentiviral vector, packaging of lentiviruses and screening of stable human gastric cancer AGS cell lines.
5. VacA protein secreted by H. pylori was co-incubated with AGS cells, AGS cells overexpressed by cortactin and AGS cells stably transfected by cortactin siRNA interference respectively. Apoptosis and apoptosis protein expression were detected by flow cytometry and immunoblotting at 0, 6, 12 and 24 h.
Result:
1. VacA protein secreted by H. pylori was purified. The molecular weight of H. pylori was analyzed by SDS-PAGE. The protein was identified as VacA protein by Western blot.
2. the recombinant VacA protein was purified and analyzed by SDS-PAGE. The molecular weight of the protein was consistent with the expected value.
3. H. pylori secretion of VacA protein can significantly induce apoptosis and vacuolation of AGS cells, but the recombinant expression of VacA protein can not significantly induce apoptosis and vacuolation of AGS cells.
4. Steady AGS cell lines overexpressed by cortactin and stable AGS cell lines interfered by cortactin siRNA were successfully constructed.
5. After overexpression of cortactin, the apoptosis rate of AGS cells was increased, the expression of Bax protein was increased, and the expression of Bcl-2 protein was decreased.
Conclusion:
1. The VacA protein secreted by H. pylori is low in expression, but its biological activity is strong. The purified protein can be used for further study.
2. cortactin promotes the apoptosis of gastric epithelial cells induced by vacA protein, which may be related to the regulation of apoptotic protein.
【学位授予单位】:第三军医大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R573

【参考文献】

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1 常辉;左钱飞;敬海明;邹全明;兰春慧;陈东风;;幽门螺杆菌分泌与重组表达的VacA蛋白的分离纯化及其致细胞空泡效应与凋亡的对比研究[J];军事医学;2014年09期



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