Cortactin在幽門(mén)螺桿菌VacA致凋亡中的作用及其機(jī)制研究
發(fā)布時(shí)間：2018-08-13 09:15
【摘要】：研究背景和目的： 幽門(mén)螺桿菌（Helicobacter pylori, H.pylori）是人體內(nèi)最常見(jiàn)的病原菌之一，它感染全世界50%以上的人群，其感染與慢性胃炎、消化性潰瘍、胃黏膜相關(guān)的淋巴樣組織淋巴瘤和胃腺癌關(guān)系密切，世界衛(wèi)生組織已把H.pylori列為胃癌的首要致病因子。目前雖已取得部分較為直接的H.pylori與胃癌相關(guān)的證據(jù)，也有對(duì)其誘發(fā)胃癌的分子機(jī)制的研究，但仍有眾多問(wèn)題需要進(jìn)一步闡明。 有報(bào)道認(rèn)為H.pylori感染引起的凋亡異常在胃癌的發(fā)生發(fā)展中起主要作用[1]。體內(nèi)外實(shí)驗(yàn)均已證實(shí)H.pylori感染可導(dǎo)致胃粘膜上皮細(xì)胞凋亡，持續(xù)的大量細(xì)胞凋亡將使胃粘膜組織損傷，胃粘膜上皮細(xì)胞大量丟失，功能障礙，繼發(fā)大量的細(xì)胞增殖，，進(jìn)一步可發(fā)展為腸化生和不典型增生，抑凋亡基因或促凋亡基因在“高運(yùn)轉(zhuǎn)”狀況下可能發(fā)生改變，當(dāng)細(xì)胞凋亡與細(xì)胞增殖嚴(yán)重失衡，胃粘膜難免向腫瘤方向發(fā)展。 H.pylori的致病因子有多種，其中最主要的是空泡細(xì)胞毒素(vacuolating cytotoxin,VacA)和細(xì)胞毒素相關(guān)蛋白(cytotoxin associated protein A, CagA)。VacA是目前倍受關(guān)注的毒力因子之一，近來(lái)研究發(fā)現(xiàn)VacA的p37亞基與胃癌的發(fā)生密切相關(guān)[2]。 皮動(dòng)蛋白（cortical actin-binding protein，cortactin）是一種微絲骨架結(jié)合蛋白，cortactin與H.pylori定植、粘附、胞內(nèi)運(yùn)動(dòng)密切相關(guān)。最近研究顯示H.pylori使cortactin的SH3區(qū)失激活和去磷酸化，從而影響肌動(dòng)蛋白（actin）的重新分布，使細(xì)胞的緊密連接松弛，利于毒素蛋白入侵[3]；cortactin是信號(hào)傳導(dǎo)通路和細(xì)胞骨架重要的聯(lián)系分子，它可以提高細(xì)胞遷移的能力�，F(xiàn)在研究顯示它與細(xì)胞的運(yùn)動(dòng)、腫瘤侵襲轉(zhuǎn)移有著密切關(guān)系。 既往研究中使用的VacA蛋白一般有H.pylori分泌的VacA蛋白的粗純液，純化的H.pylori分泌的VacA蛋白和重組表達(dá)的VacA蛋白。H.pylori分泌的VacA蛋白的粗純液具有良好的抗原性，制備相對(duì)簡(jiǎn)單，但其蛋白成分復(fù)雜，不利于闡明其作用。純化的H.pylori分泌的VacA蛋白具有良好的抗原性,且成分單一，便于證實(shí)其生物學(xué)作用。 本研究的目的有二個(gè)，一是擬通過(guò)兩種方式獲得VacA蛋白，即直接純化H.pylori分泌的VacA蛋白，及通過(guò)重組表達(dá)獲得VacA蛋白，并比較這兩種蛋白致細(xì)胞凋亡和空泡效應(yīng)的差異，以此選擇生物學(xué)效應(yīng)好的一種蛋白用于細(xì)胞實(shí)驗(yàn)。二是首先明確cortactin在VacA致細(xì)胞凋亡過(guò)程中存在調(diào)控作用；其次是明確cortactin調(diào)控凋亡的機(jī)制，即是否通過(guò)影響凋亡相關(guān)蛋白而發(fā)揮作用。我們擬構(gòu)建cortactin過(guò)表達(dá)和cortactin siRNA干擾的AGS細(xì)胞穩(wěn)轉(zhuǎn)株，使用H.pylori分泌的VacA蛋白分別與人胃腺癌細(xì)胞（AGS細(xì)胞）、cortactin過(guò)表達(dá)的AGS細(xì)胞和cortactin siRNA干擾的AGS細(xì)胞穩(wěn)轉(zhuǎn)株共孵，通過(guò)流式細(xì)胞技術(shù)和免疫印跡法，檢測(cè)細(xì)胞凋亡及凋亡相關(guān)蛋白表達(dá)的差異，以揭示cortactin在VacA致胃上皮細(xì)胞凋亡過(guò)程中存在調(diào)控作用，并初步探討其機(jī)理。 方法： 1. H.pylori分泌VacA蛋白的純化。取凍存的H.pyloriATCC26695菌株，接種于skirrow氏培養(yǎng)基平皿，微需氧環(huán)境培養(yǎng)48h。刮取適量H.pylori菌落混懸于skirrow氏液體培養(yǎng)基中，微需氧環(huán)境培養(yǎng)24h。收集約2L H.pylori菌液，收集上清液于4℃加入達(dá)50%飽和度的硫酸銨,充分沉淀后以離心,沉淀蛋白溶于5ml PBS緩沖液1中。蛋白樣品依次采用HiTrap SP HP柱、HiTrap Phenyl HP柱、HiTrap Q HP柱進(jìn)行純化。純化VacA蛋白采用免疫印跡法進(jìn)行鑒定。 2.重組VacA蛋白的表達(dá)純化。以H.pylori60190的VacA基因片段為模板，合成VacA基因片段，構(gòu)建在pUC57上。隨后載體質(zhì)粒pQE30及VacA基因進(jìn)行雙酶切。質(zhì)粒pQE30回收大片段與VacA基因進(jìn)行連接，連接產(chǎn)物轉(zhuǎn)化后，涂布于LB平板上，37℃恒溫培養(yǎng)箱倒置培養(yǎng)過(guò)夜。挑取生長(zhǎng)良好的菌落接種于LB培養(yǎng)液中培養(yǎng)至第二天上午，用小量質(zhì)粒抽提試劑盒抽提質(zhì)粒，并分別做酶切鑒定。鑒定正確的VacA質(zhì)粒轉(zhuǎn)化到M15感受態(tài)細(xì)胞，涂布A+平板，37℃培養(yǎng)過(guò)夜，挑單菌落到LB中，搖床里過(guò)夜活化培養(yǎng)。收集菌液，獲得VacA重組蛋白，采用Ni2+-NTA樹(shù)脂純化法純化VacA蛋白，SDS-PAGE電泳法鑒定該蛋白。 3. H.pylori分泌的VacA蛋白和重組VacA蛋白分別與AGS細(xì)胞共孵，流式細(xì)胞技術(shù)檢測(cè)細(xì)胞凋亡，倒置顯微鏡觀察細(xì)胞空泡變性，從而比較這兩種方法獲得的蛋白的生物學(xué)效應(yīng)。 4. pLVX-siRNA2-Puro-hScramble慢病毒載體構(gòu)建、慢病毒包裝及穩(wěn)轉(zhuǎn)人胃癌AGS細(xì)胞株篩選。 5. H.pylori分泌的VacA蛋白分別與AGS細(xì)胞、cortactin過(guò)表達(dá)的AGS細(xì)胞和cortactin siRNA干擾的AGS細(xì)胞穩(wěn)轉(zhuǎn)株共孵，通過(guò)流式細(xì)胞技術(shù)和免疫印跡方法，在0h、6h、12h、24h檢測(cè)細(xì)胞凋亡及凋亡蛋白表達(dá)。 結(jié)果: 1.純化出H.pylori分泌VacA蛋白，SDS-PAGE分析其蛋白分子量與預(yù)期的一致，免疫印跡法鑒定該蛋白為VacA蛋白。 2.純化出重組表達(dá)VacA蛋白，SDS-PAGE分析其蛋白分子量與預(yù)期的一致。 3. H.pylori分泌VacA蛋白能顯著誘導(dǎo)AGS細(xì)胞凋亡和致AGS細(xì)胞空泡效應(yīng)，而重組表達(dá)VacA蛋白無(wú)顯著誘導(dǎo)AGS細(xì)胞凋亡和致AGS細(xì)胞空泡效應(yīng)的作用。 4.成功構(gòu)建出cortactin過(guò)表達(dá)的AGS細(xì)胞穩(wěn)轉(zhuǎn)株和cortactin siRNA干擾的AGS細(xì)胞穩(wěn)轉(zhuǎn)株。 5. cortactin過(guò)表達(dá)后，VacA蛋白致AGS細(xì)胞凋亡率增加，凋亡蛋白Bax表達(dá)增高，抗凋亡蛋白Bcl-2表達(dá)降低；沉默cortactin表達(dá)，VacA蛋白致AGS細(xì)胞凋亡率降低，凋亡蛋白Bax表達(dá)降低，抗凋亡蛋白Bcl-2表達(dá)增高。 結(jié)論： 1. H.pylori分泌的VacA蛋白雖然表達(dá)量少，但其生物活性強(qiáng)，純化出的蛋白量足以用于后續(xù)研究。 2. cortactin促進(jìn)VacA蛋白導(dǎo)致的胃上皮細(xì)胞凋亡增加，其機(jī)制可能與凋亡蛋白的調(diào)控有關(guān)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R573
本文編號(hào)：2180542
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R573
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 常輝;左錢(qián)飛;敬海明;鄒全明;蘭春慧;陳東風(fēng);;幽門(mén)螺桿菌分泌與重組表達(dá)的VacA蛋白的分離純化及其致細(xì)胞空泡效應(yīng)與凋亡的對(duì)比研究[J];軍事醫(yī)學(xué);2014年09期
本文編號(hào)：2180542
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