HCV NS5A对TLR3，TLR4表达的影响
摘要
目的丙型肝炎病毒(Hepatitis C virus, HCV)感染呈全球性流行发展,是欧美及日本等国家终未期肝脏疾病的主要原因之一。目前,全球HCV的感染率约为3%,大约1.7亿人感染HCV,每年新发丙型肝炎病例达到3.5万例。我国HCV感染率为1.5～3.5%,总体感染人群3000万以上。HCV感染后约80%成为慢性持续状态,慢性感染可导致肝脏慢性炎症坏死和纤维化,甚至可发展为肝硬化及肝细胞癌,对患者的健康和生命危害非常大,已成为严重的社会和公共卫生问题。丙型肝炎病毒非结构蛋白NS5A作为重要的非结构蛋白,近年来有关它的研究倍受关注,它具有抗凋亡、转录激活作用、干扰细胞内信号转导通路、调解病毒复制水平等多项生物学活性,提示其在干扰素疗效预测、病毒复制、细胞凋亡、肝细胞癌发生等多方面均具有重要作用。目前关于TLRs与机体相互作用的机制成为研究热点之一,其中Toll样受体3(TLR3)主要表达于树突状细胞,能够识别病毒的dsRNA,在抗病毒过程中发挥了重要作用。TLR4是最早发现的哺乳动物TLRs蛋白,机体多种细胞表面均能表达TLR4,它被认为与革兰氏阴性细菌及其内毒素的识别和激活有关。它们在肝脏疾病包括酒精性肝病、病毒性肝炎、肝纤维化等的发生、发展过程中发挥重要作用。
在本研究中,我们通过转染HCV NS5A蛋白表达质粒观察HCV NS5A蛋白对肝细胞内TLR3及TLR4 mRNA和蛋白表达的影响,为丙型肝炎的发病机制积累实验依据,并对丙型肝炎的治疗提供理论依据。
方法1.质粒转染：将QSG7701细胞种植于六孔板,待细胞融合60-70%时,按照说明书中的转染程序,分别将pcNS5A和pRc/CMV质粒转染入细胞,12小时后换含10%胎牛血清的DMEM培养基,转染48小时后收集细胞进行接下来的实验。
2.免疫细胞化学染色：用免疫细胞化学染色的方法检测TLR4蛋白质及NS5A蛋白质的表达。
3. RT-PCR:按RNA提取试剂盒说明书进行,提取上述转染的7701细胞总RNA,用紫外光分光光度计测定RNA的浓度。取2ugRNA,逆转录为cDNA进行PCR扩增,再用2%琼脂糖凝胶电泳观测TLR3 mRNA及TLR4 mRNA结果并照相。
4.间接免疫荧光：用间接免疫荧光的方法检测TLR3及TLR4蛋白质的表达。
5. Western blot:用Western blot的方法以β-actin为参照,检测TLR4蛋白质的表达。
结果1.转染pcNS5A质粒组的7701细胞内有HCV NS5A蛋白颗粒表达,呈棕黄色颗粒,主要分布在细胞浆中,以胞核周围最为明显,未转染组和转染pRc/CMV质粒组的7701细胞中则无HCV NS5A蛋白表达。
2.用免疫细胞化学染色检测TLR4蛋白的表达,结果显示转染pcNS5A质粒组内有TLR4蛋白质表达,呈棕黄色颗粒,分布在细胞膜中,.未转染组和转染pRc/CMV质粒组则无明显TLR4蛋白表达。说明HCV NS5A能够促进TLR4蛋白质的表达。
3.用RT-PCR检测TLR3及TLR4 mRNA的表达。结果表明,在GAPDH内参照表达一致情况下,pcNS5A质粒转染组TLR4 mRNA的表达量明显高于空载体组和未转染组,空载体组和未转染组mRNA的表达相似,结果说明HCV NS5A能够激活TLR4 mRNA的转录水平；然而,pcNS5A质粒转染组与空载体组和未转染组TLR3 mRNA的表达量无明显差别,说明HCV NS5A对TLR3mRNA的转录水平至少无上调作用。
4.用免疫荧光方法检测TLR3及TLR4蛋白质的表达。结果显示转染pcNS5A质粒组内有TLR4蛋白质表达,呈现绿色荧光颗粒,未转染组和转染pRc/CMV质粒组则无明显TLR4蛋白表达。说明HCV NS5A能够促进TLR4蛋白质的表达。转染pcNS5A质粒组内及未转染组和转染pRc/CMV质粒组的细胞内均无TLR3蛋白颗粒的表达,说明HCV NS5A对TLR3蛋白的表达无上调作用。
5.用Western blot检测TLR4蛋白质的表达,以β-actin为内参照。结果显示pcNS5A质粒转染组TLR4蛋白质的表达量明显高于空载体组和未转染组,空载体组和未转染组的表达相似。说明HCV NS5A能够促进TLR4蛋白质的表达。
结论1.HCV NS5A表达质粒能够转染至QSG7701细胞中,表达HCV NS5A蛋白。
2. HCV NS5A能激活TLR4 mRNA的转录水平。
3. HCV NS5A能促进TLR4蛋白质的表达。
4. HCV NS5A对TLR3 mRNA的转录水平及TLR3蛋白质的表达均无明显上调作用。
Background
Hepatitis C virus (HCV) infection is world-widely distributed, it is one of the main reasons of the end-stage liver diseases in Europe, America and Japan. According to the World Health Organization, the global rate of HCV infection is about 3%.The number of infections is more than 170 million, there are about 35,000 new cases every year. The hcv infection rate is in the range of 1.5～3.5% in China, and totally more than 30 million people in China have been infected. There are 80% of the infected persons progress to chronic hepatitis, Some of which developed to liver cirrhosis or hepatocellular carcinoma. HCVNS5A as an important non-structural protein has become the focus of research because of its multi-functions in HCV replication, pathogenesis. Currently, the mechanism of interaction between TLRs and the host immnunity becomes more and more interested. TLR3 was mainly expressed in dendritic cells, it can identify the virus dsRNA and play an important role in the process of anti-virus. TLR4 is the first protein found in mammalian TLRs. The surface of much cells such as monocytes, plasma cells, neutrophils, endothelial cells can express TLR4, it is considered to be relevant to gram-negative bacteria and endotoxin recognition and activation, and also found to play an important role in alcoholic liver disease, viral hepatitis, liver fibrosis.
In this study, plasmid pcNS5A was transfected to QSG7701 cells. We detect the impact of HCV NS5A on the expression of TLR3 mRNA, TLR4 mRNA and protein in the liver cells. It can afford the experimental evidence for the pathogenesis of the hepatitis C.
Methods
1. Plasmid transfection:We planted QSG7701 cells in six-well plates before transfection, until 60%-70% cells merging, put pcNS5A or pRc/ CMV plasmids into cells according to the procedure of Lipofectin2000, changed for 10% of fetal bovine serum medium after 12 hours, harvested after 48 hours.
2.Immuno- cytochemistry:We detected the expression of TLR4 and NS5A protein by immunocytochemistry method.
3. RT-PCR:transfected cells were extracted total RNA in accordance with the procedure of RNA extraction kit. We measured the concentration and purity of RNA by UV spectrophotometer, took 2μgRNA to make cDNA on the basis of reverse transcription, the products were augmented by PCR and separated by 2% agarose gel electrophoresis.
4. Indirect immunofluorescence method:We used indirect immunofluorescence method to detect TLR4 protein expression.
5. Western blot:We detected TLR4 protein expression by Western blot method, usingβ-actin as a reference.
Results
1. The cells transfected with plasmid pcNS5A expressed HCV NS5A protein which stained as brown particle. It is distributed in the cytoplasm, obviously around the nucleus. Non-transfection cells and that of transfected pRc/CMV plasmid did not express HCV NS5A protein.
2. By the immunohistochemistry method, it is found the cells transfected with plasmid pcNS5 A expressed TLR4 protein. It is mainly distributed on the cell membrane. Non-transfection cells and that of transfected pRc /CMV plasmid did not express TLR4 protein.
3. TLR4 mRNA expression in pcNS5A transfected cells is higher than in the untransfected or pRc/CMV plasmid transfected cells. The level of TLR4 mRNA expression between the pRc/CMV plasmid transfected cells and the untransfected cells is similar. This shows that HCV NS5A can stimulate the transcription of TLR4 mRNA. TLR3mRNA expression between pcNS5A transfected cells, blank group and the group of transfected pRc/CMV plasmid is similar. This shows that HCV NS5A doesn't stimulate the transcription of TLR3 mRNA.
4. By the indirect immunofluorescence method, It is found the cells transfected with plasmid pcNS5A expressed higher level of TLR4 protein than non-transfection group and the group of transfected pRc/CMV plasmid. There is no difference inTLR3 protein expression between the cells of transfected with plasmid pcNS5A, in non-transfection cells and that of transfected pRc/CMV plasmid. This shows that HCV NS5A can't stimulate the expression of TLR3 protein.
5. By western blot method, we found that the TLR4 protein expression in pcNS5A transfected cells was much higher than in the untransfected cells or in the cells transfected with pRc/CMV plasmid. TLR4 protein expression level between the cells transfected with pRc/CMV plasmid and the untransfected cells is similar. This shows HCV NS5A up-regulated the expression of TLR4 protein.
Conclusion:
1. HCV NS5A expression plasmid was transfected to QSG7701 cells successfully, and HCVNS5 A protein expression was detected.
2. HCV NS5A stimulates the transcription of TLR4 mRNA.
3. HCV NS5A promote the expression of TLR4 protein.
4. HCV NS5A doesn't stimulate the transcription of TLR3 mRNA and the expression of TLR3 protein.
在本研究中,我们通过转染HCV NS5A蛋白表达质粒观察HCV NS5A蛋白对肝细胞内TLR3及TLR4 mRNA和蛋白表达的影响,为丙型肝炎的发病机制积累实验依据,并对丙型肝炎的治疗提供理论依据。
方法1.质粒转染：将QSG7701细胞种植于六孔板,待细胞融合60-70%时,按照说明书中的转染程序,分别将pcNS5A和pRc/CMV质粒转染入细胞,12小时后换含10%胎牛血清的DMEM培养基,转染48小时后收集细胞进行接下来的实验。
2.免疫细胞化学染色：用免疫细胞化学染色的方法检测TLR4蛋白质及NS5A蛋白质的表达。
3. RT-PCR:按RNA提取试剂盒说明书进行,提取上述转染的7701细胞总RNA,用紫外光分光光度计测定RNA的浓度。取2ugRNA,逆转录为cDNA进行PCR扩增,再用2%琼脂糖凝胶电泳观测TLR3 mRNA及TLR4 mRNA结果并照相。
4.间接免疫荧光：用间接免疫荧光的方法检测TLR3及TLR4蛋白质的表达。
5. Western blot:用Western blot的方法以β-actin为参照,检测TLR4蛋白质的表达。
结果1.转染pcNS5A质粒组的7701细胞内有HCV NS5A蛋白颗粒表达,呈棕黄色颗粒,主要分布在细胞浆中,以胞核周围最为明显,未转染组和转染pRc/CMV质粒组的7701细胞中则无HCV NS5A蛋白表达。
2.用免疫细胞化学染色检测TLR4蛋白的表达,结果显示转染pcNS5A质粒组内有TLR4蛋白质表达,呈棕黄色颗粒,分布在细胞膜中,.未转染组和转染pRc/CMV质粒组则无明显TLR4蛋白表达。说明HCV NS5A能够促进TLR4蛋白质的表达。
3.用RT-PCR检测TLR3及TLR4 mRNA的表达。结果表明,在GAPDH内参照表达一致情况下,pcNS5A质粒转染组TLR4 mRNA的表达量明显高于空载体组和未转染组,空载体组和未转染组mRNA的表达相似,结果说明HCV NS5A能够激活TLR4 mRNA的转录水平；然而,pcNS5A质粒转染组与空载体组和未转染组TLR3 mRNA的表达量无明显差别,说明HCV NS5A对TLR3mRNA的转录水平至少无上调作用。
4.用免疫荧光方法检测TLR3及TLR4蛋白质的表达。结果显示转染pcNS5A质粒组内有TLR4蛋白质表达,呈现绿色荧光颗粒,未转染组和转染pRc/CMV质粒组则无明显TLR4蛋白表达。说明HCV NS5A能够促进TLR4蛋白质的表达。转染pcNS5A质粒组内及未转染组和转染pRc/CMV质粒组的细胞内均无TLR3蛋白颗粒的表达,说明HCV NS5A对TLR3蛋白的表达无上调作用。
5.用Western blot检测TLR4蛋白质的表达,以β-actin为内参照。结果显示pcNS5A质粒转染组TLR4蛋白质的表达量明显高于空载体组和未转染组,空载体组和未转染组的表达相似。说明HCV NS5A能够促进TLR4蛋白质的表达。
结论1.HCV NS5A表达质粒能够转染至QSG7701细胞中,表达HCV NS5A蛋白。
2. HCV NS5A能激活TLR4 mRNA的转录水平。
3. HCV NS5A能促进TLR4蛋白质的表达。
4. HCV NS5A对TLR3 mRNA的转录水平及TLR3蛋白质的表达均无明显上调作用。
Background
Hepatitis C virus (HCV) infection is world-widely distributed, it is one of the main reasons of the end-stage liver diseases in Europe, America and Japan. According to the World Health Organization, the global rate of HCV infection is about 3%.The number of infections is more than 170 million, there are about 35,000 new cases every year. The hcv infection rate is in the range of 1.5～3.5% in China, and totally more than 30 million people in China have been infected. There are 80% of the infected persons progress to chronic hepatitis, Some of which developed to liver cirrhosis or hepatocellular carcinoma. HCVNS5A as an important non-structural protein has become the focus of research because of its multi-functions in HCV replication, pathogenesis. Currently, the mechanism of interaction between TLRs and the host immnunity becomes more and more interested. TLR3 was mainly expressed in dendritic cells, it can identify the virus dsRNA and play an important role in the process of anti-virus. TLR4 is the first protein found in mammalian TLRs. The surface of much cells such as monocytes, plasma cells, neutrophils, endothelial cells can express TLR4, it is considered to be relevant to gram-negative bacteria and endotoxin recognition and activation, and also found to play an important role in alcoholic liver disease, viral hepatitis, liver fibrosis.
In this study, plasmid pcNS5A was transfected to QSG7701 cells. We detect the impact of HCV NS5A on the expression of TLR3 mRNA, TLR4 mRNA and protein in the liver cells. It can afford the experimental evidence for the pathogenesis of the hepatitis C.
Methods
1. Plasmid transfection:We planted QSG7701 cells in six-well plates before transfection, until 60%-70% cells merging, put pcNS5A or pRc/ CMV plasmids into cells according to the procedure of Lipofectin2000, changed for 10% of fetal bovine serum medium after 12 hours, harvested after 48 hours.
2.Immuno- cytochemistry:We detected the expression of TLR4 and NS5A protein by immunocytochemistry method.
3. RT-PCR:transfected cells were extracted total RNA in accordance with the procedure of RNA extraction kit. We measured the concentration and purity of RNA by UV spectrophotometer, took 2μgRNA to make cDNA on the basis of reverse transcription, the products were augmented by PCR and separated by 2% agarose gel electrophoresis.
4. Indirect immunofluorescence method:We used indirect immunofluorescence method to detect TLR4 protein expression.
5. Western blot:We detected TLR4 protein expression by Western blot method, usingβ-actin as a reference.
Results
1. The cells transfected with plasmid pcNS5A expressed HCV NS5A protein which stained as brown particle. It is distributed in the cytoplasm, obviously around the nucleus. Non-transfection cells and that of transfected pRc/CMV plasmid did not express HCV NS5A protein.
2. By the immunohistochemistry method, it is found the cells transfected with plasmid pcNS5 A expressed TLR4 protein. It is mainly distributed on the cell membrane. Non-transfection cells and that of transfected pRc /CMV plasmid did not express TLR4 protein.
3. TLR4 mRNA expression in pcNS5A transfected cells is higher than in the untransfected or pRc/CMV plasmid transfected cells. The level of TLR4 mRNA expression between the pRc/CMV plasmid transfected cells and the untransfected cells is similar. This shows that HCV NS5A can stimulate the transcription of TLR4 mRNA. TLR3mRNA expression between pcNS5A transfected cells, blank group and the group of transfected pRc/CMV plasmid is similar. This shows that HCV NS5A doesn't stimulate the transcription of TLR3 mRNA.
4. By the indirect immunofluorescence method, It is found the cells transfected with plasmid pcNS5A expressed higher level of TLR4 protein than non-transfection group and the group of transfected pRc/CMV plasmid. There is no difference inTLR3 protein expression between the cells of transfected with plasmid pcNS5A, in non-transfection cells and that of transfected pRc/CMV plasmid. This shows that HCV NS5A can't stimulate the expression of TLR3 protein.
5. By western blot method, we found that the TLR4 protein expression in pcNS5A transfected cells was much higher than in the untransfected cells or in the cells transfected with pRc/CMV plasmid. TLR4 protein expression level between the cells transfected with pRc/CMV plasmid and the untransfected cells is similar. This shows HCV NS5A up-regulated the expression of TLR4 protein.
Conclusion:
1. HCV NS5A expression plasmid was transfected to QSG7701 cells successfully, and HCVNS5 A protein expression was detected.
2. HCV NS5A stimulates the transcription of TLR4 mRNA.
3. HCV NS5A promote the expression of TLR4 protein.
4. HCV NS5A doesn't stimulate the transcription of TLR3 mRNA and the expression of TLR3 protein.
引文
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[2]Lemaitre B, Nicolas E, Michaut L, et al. The dorsoventral regulatory gene cassette spatzle Toll cactus control the potent antifungal response in Drosophila adults. Cell,1996,86: 973-983
[3]Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature,1997,388:394-397
[4]Chuang T, Ulevitch RJ. Identification of hTLR10:a novel human Toll-like receptor preferentially expressed in immune cells. Biochim Biophys Acta,2001,1518:157-161
[5]Chuang TH, Ulevitch RI. Cloning and characterization of a subfamily of human to toll-like receptors:hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw,2000,11:372-378
[6]Rock FL, Hardiman G, Timam TC, et al. A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci USA,1998,95:588-593
[7]Takeuchi O, Kawai T, Sanjo H, et al. TLR6:A novel member of an expanding toll-like receptor family. Gene,1999,231:59-65
[8]Takeda K, Akira S. Toll-like receptors in innate immunity. Int Immunol,2005,17:1-14
[9]Parker LC, Prince LR, Sabroe I. Translational mini-review series on Toll-like receptors: Netwoeks regulated by Toll-like receptors mediate innate and adaptive immunity [J]. Clin Exp Immunol,2007,147:199-207
[10]Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol,2004,9:987-995
[11]Akria S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity. Nat Immunol,2001,2:675-680
[12]Datta SK, Redecke V, Prilliman KP, et al. A subset of Toll-like receptor ligands induces cross-presentation by bone marrow-derived dendritic cells. J Immunol,2003,170:4102-4110
[13]Zhang X, Shan P, Qureshi S, et al. TLR4 deficiency confers susceptibility to lethal oxidant lung injury[J]. J Immunol,2005,175:4834-4838
[14]Medzhitov R, Janeway C Jr. Innate immune recognition:mechanisms and pathways. Immunol Rev,2000,173:89-97
[15]Akia S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett,2003,85:85-95
[16]Alexopoulou L, Holt AC, Medzhitov R, et al. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature,2001,413:732-738
[17]Hoshino K, Takeuchi O, Kawai T, et al. Cutting edge:Toll-like receptor 4 (TLR4) deficient mice are hyporesponsive to lipopolysaccharide:evidence for TLR4 as the Lps gene product. J Immunol,1999,162:3749-3752
[18]Netea MG, Van Deuren M, Kullberg BJ, et al. Does the shape of lipid A determine the interaction of LPS with Toll-like receptors? Trends Immunol,2002,23:135-139
[19]Ohashi K, Burkart V, Flohe S, et al. Cutting edge:heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol,2000,164:558-561
[20]Gao B, Tsan MF. Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages. J Biol Chem,2003,278:174-179
[21]Li T, Hu J, Thomas JA, et al. Differential induction of apop tosis by LPS and taxol in monocytic cells [J]. Mol Immunol,2005,42:1049-1055.
[22]Li S, Strelow A, Fontana EJ, et al. IRAK-4:a novel member of the IRAK family with the properties of an IRAK-kinase. Proc Natl Acad Sci USA,2002,99:5567-5572
[23]Suzuki N, Suzuki S, Duncan GS, et al. Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. Nature,2002,416:750-756
[24]Shama S, tenoever BR, Grandvaux N, et al. Triggering the interferon antiviral response through an IKK-related pathway. Science,2003,300:1148-1151
[25]Sun R, Gao B. Negative regulation of liver regeneration by innate immunity(natural killer cells/interferon-gamma). Gastroenterology,2004,127:1525-1539
[26]Matsumoto M, Funami K, Oshiumi H, et al. Toll-like receptor3:a link between toll-like receptor, interferon and viruses. Microbiol Immunol,2004,48:147-154
[27]Matsumoto M, Kikkawa S, Kohase M, et al. Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochern Biophys Res Commun,2002,293:1364-1369
[28]Li K, Foy E, Ferreon JC,et al. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci USA,2005,102:2992-2997
[29]Masaru Moriyama, Naoya Kato, Motoyuki Otsuka, et al. Interferon-beta is activated by hepatitis C virus NS5B and inhibited by NS4A, NS4B, and NS5A. Hep Intl,2007,1:302-310
[30]Nan Wang, Yuqiong Liang, Santhana Devaraj, et al. Toll-like receptor 3 mediates establishment of an antiviral state against hepatitis C virus in hepatoma cells. Journal of Virology,2009,83:9824-9834
[31]Chang S, Dolganiue A, Szabo G. Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins. J Leukoe Biol,2007,82: 479-487
[32]Riordan SM, Skinner NA, Kurtovic J, et al. Toll-like receptor expression in chronic hepatitis C:correlation with pro-inflammatory cytokine levels and liver injury. Inflamm Res,2006, 55:279-285
[33]Wisniewska-Ligier M, Wozniakowska-Gesicka T, Glowaka E, et al. Involvement of innate immunity in the pathogenesis of chronic hepatitis C in children. Scand J Immunol,2006,64: 425-432
[34]Thomas A, Laxton C, Rodman J, et al. Investigating Toll-like receptor agonists for potential to treat hepatitis C virus infection. Antimicrob Agents Chemother,2007,51:2969-2978
[35]Machida K, Cheng KT, Sung VM, et al. Hepatitis C virus induces Toll-like receptor 4 expression, leading to enhanced production of beta interferon and interleukin-6. J Virol, 2006,80:866-874
[36]Duesberg U, Von Dem Bussche A, Kirrching C, et al. Cell activation by synthetic lipopeptides of the hepatitis C virus (HCV)-core protein is mediated by Toll- like receptors (TLRs)2 and 4[J]. Immunol Lett,2002,84:89-95