单纯疱疹病毒Ⅰ型体外感染人口腔上皮细胞的初步探讨
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摘要
牙周病是人类常见病之一,是成人牙齿丧失的主要原因。牙周炎的发病机制一直是近年来牙周病学领域的研究热点。众所周知,牙菌斑为牙周病的始动因子,它与牙石,食物嵌塞,(牙合)创伤等局部因素和全身因素共同作用引起牙周病。但细菌致病学说并不能很好地解释牙周病的部位特异性以及间断爆发性等问题。同时基因检测等分子生物学技术问世和不断发展,许多学者研究发现在牙周炎患者病变位点的龈沟液和牙龈组织中常可检测到疱疹病毒,尤其是单纯疱疹病毒Ⅰ型。因此提出单纯疱疹病毒感染与各型牙周炎具有一定相关关系。单纯疱疹病毒在牙周炎的发生发展中又是通过怎样的途径发挥作用的。考虑到病毒感染宿主的第一步便是病毒吸附进入宿主细胞,而人口腔上皮细胞又是人类口腔中先天性防御系统的第一道屏障,因此在体外建立一个单纯疱疹病毒Ⅰ型感染人口腔上皮细胞的实验模型就具有其特殊的意思。本研究拟在体外大量扩增获得HSV-1,再用标化的病毒直接感染人口腔上皮细胞KB细胞系,建立HSV-1体外感染人口腔上皮细胞模型。并通过间接感染的方法“转移感染”Vero细胞,利用倒置显微镜、透射电镜技术和PCR技术对模型建立结果进行鉴定。
实验一HSV-1体外感染人口腔上皮细胞实验模型的建立
目的在体外建立HSV-1感染人口腔上皮细胞实验模型,并运用一定方法进行鉴定。
材料和方法在体外利用Vero细胞(非洲绿猴肾细胞)扩增HSV-1,并利用噬斑计数法测定病毒滴度(PFU/ml),用此病毒分别以MOI为0.5PFU/cell、1.0 PFU/cell、3.0 PFU/cell感染人口腔上皮细胞KB细胞,并以正常Vero细胞上清液代替HSV-1病毒液作为空白对照。分别于0h、24h、48h在倒置相差显微镜下观察细胞形态,于感染后48h收集细胞,制成超薄切片在透射电镜下观察超微结构。并用PCR技术检测感染后KB细胞基因组中是否包含HSV-1特异性的糖蛋白D基因片段。
结果在体外通过Vero细胞成功的扩增出大量HSV-1,并将其滴定标化为10~6PFU/ml。HSV-1体外感染KB细胞后于24h,48h在倒置显微镜下观察细胞形态,未观察到明显的细胞病变效应。透射电镜下,KB细胞核内及胞浆内均未见HSV-1病毒颗粒。而KB细胞基因组DNA经PCR扩增,凝胶电泳后可见一位于709bp位置的阳性条带,与预测结果一致。
结论HSV-1体外感染人口腔上皮KB细胞实验模型建立。KB细胞为HSV-1的非允许性细胞,HSV-1感染KB细胞后可能形成转化性感染或潜伏性感染。
实验二KB细胞的上清液对Vero细胞的“转移感染”及鉴定
目的通过“转移感染”的间接方法将前述实验中KB细胞的上清液感染对HSV-1敏感的Vero细胞,以此检验HSV-1是否成功感染人口腔上皮KB细胞。
材料和方法收集实验一模型中不同MOI组HSV-1体外感染KB细胞48h后的培养上清,并以之感染HSV-1的允许性细胞Vero细胞。分别于0h、24h、48h在倒置相差显微镜下观察细胞形态,于感染后48h收集细胞,制成超薄切片在透射电镜下观察超微结构和病毒形态。并在“转移感染”48h后计算细胞病变率。
结果倒置显微镜下,Vero细胞“转移感染”24h时可观察到细胞病变出现;48h时细胞病变率上升。扫描电镜下Vero细胞核内可见大量大小均一的典型HSV-1颗粒。通过对细胞病变率进行统计,得知各滴度组“转移感染”后Vero细胞病变率有显著性差异。
结论KB细胞中潜伏的病毒被活化并在Vero细胞中产生了感染性病毒颗粒,证明了HSV-1可在体外感染口腔上皮细胞,并表达其基因。
综上所述,本研究利用Vero细胞大量扩增HSV-1,并以此感染人口腔上皮细胞KB细胞,在体外建立HSV-1感染人口腔上皮细胞实验模型。并收集HSV-1感染人口腔上皮细胞后的上清液“转移感染”Vero细胞,通过倒置显微镜,透射电镜超薄切片技术及PCR技术进行检验。结果表明HSV-1能够直接感染口腔上皮KB细胞,形成转化性感染或潜伏性感染,并在KB细胞内表达其基因。
Periodontal disease,one of the most common human oral diseases in human race,is a kind of chronic infectious diseases that caused by the invasion of microorganism and resulting in the destruction of periodontal tissue.During the past 100 years,the majority of researchers considered that bacterial plaque was the most important etiological factor of periodontal disease.Since the mid 1990s,many studies have indicated that herpes simplex viruses(HSV)is often detected in periodontitis sites, especially herpes simplex virus type 1(HSV-1).These results indicated that HSV-1 may play a significant role in the development of periodontal disease.Some researchers presumed that the damage to the epithelium barrier of the host may be one of the most important pathogenesis of HSV-1-associated periodontal disease.However,the mechanism has rarely been researched for lack of the model that HSV infects human oral epithelial cells in vitro directly.In this study,we abundantly amplified HSV-1 strains in Vero cells which were used to infect human oral epithelial cells,and established the model that HSV-1 infected human oral epithelial cells in vitro.The present study provides a basis of experimental technique for the further study of the pathogenesis of herpesvirus-associated periodontal disease
Experiment one:Establishment of the model that HSV-1 infected human oral epithelial cells in vitro.
Objective:To abundantly amplify HSV-1 strains and establish the model that HSV-1 infected human oral epithelial cells in vitro.
Materials and Methods:HSV-1 strains were abundantly amplified in Vero cells and viral titer was detected by plaque forming experiment.KB cells were infected with HSV-1 at an MOI of 0.5,1.0 or 3.0 PFU/cell.The morphology of the cells was inspected for microscopically detectable alterations through inverted microscope when HSV-1 infected KB cells for 0 h,24 h,48 h respectively.Infected KB cells were collected and then observed through TEM.Nucleic acid of the virus was detected by PCR.
Result:HSV-1 strains were abundantly amplified in Vero cells and the titer was standardized to 10~6 PFU/ml.The infected KB cells in three groups didn't display obvious cytopathic effect(CPE)under inverted microscope at either 24 h or 48 h under inverted microscope.There were no mature virus particles in endochylema or nucleus under TEM.The glycoprotein D gene order of HSV-1 could be detected in infected KB cells by PCR.
Conclusion:The model that HSV-1 infected human oral epithelial cells in vitro was established.KB cells are the non-permissive cell of HSV-1.HSV-1 infects KB cells leading to transforming infection or virus incubated in the cells causing latent infection.
Experiment Two:Transfer infection of HSV-1 to Vero cells
Objective:To checkout indirectly that whether the model of HSV-1 infected human oral epithelial cells in vitro is successful or not.
Materials and Methods:After HSV-1 infected KB cells for 48 h,the culture supernatant of infected groups and blank control group were collected.The collected culture supematant were incubated on Vero monolayer.The morphology of the cells was observed through inverted microscope when HSV-1 transfer infected Vero cells for 0 h,24 h,48 h respectively.Infected Vero cells were collected and then observed through TEM.We scored CPE ratio of infected Vero cells in the three groups when transfer infected for 48 h.
Result:Compared with control group,Vero cells which infected by the culture supernatant of infected KB cells showed typical CPE at 24 h i.p. under inverted microscope.The amount of CPE was increased at 48 h i.p. Numerous immature virus particles were prominent within the nucleus under TEM.Significant difference of CPE ratio of infected Vero cells in three different groups.
Conclusion:The latent viruses in KB cells were activated and produced infectious viral particles.
We conclude from this study that:The model that HSV-1 infected human oral epithelial cells in vitro could be established.HSV-1 infects KB cells leading to transforming infection or latent infection.HSV-1 could transfer its DNA genes or fragments of its genome into chromosome of KB cells and steadily express them in progeny virus after cell division.
实验一HSV-1体外感染人口腔上皮细胞实验模型的建立
目的在体外建立HSV-1感染人口腔上皮细胞实验模型,并运用一定方法进行鉴定。
材料和方法在体外利用Vero细胞(非洲绿猴肾细胞)扩增HSV-1,并利用噬斑计数法测定病毒滴度(PFU/ml),用此病毒分别以MOI为0.5PFU/cell、1.0 PFU/cell、3.0 PFU/cell感染人口腔上皮细胞KB细胞,并以正常Vero细胞上清液代替HSV-1病毒液作为空白对照。分别于0h、24h、48h在倒置相差显微镜下观察细胞形态,于感染后48h收集细胞,制成超薄切片在透射电镜下观察超微结构。并用PCR技术检测感染后KB细胞基因组中是否包含HSV-1特异性的糖蛋白D基因片段。
结果在体外通过Vero细胞成功的扩增出大量HSV-1,并将其滴定标化为10~6PFU/ml。HSV-1体外感染KB细胞后于24h,48h在倒置显微镜下观察细胞形态,未观察到明显的细胞病变效应。透射电镜下,KB细胞核内及胞浆内均未见HSV-1病毒颗粒。而KB细胞基因组DNA经PCR扩增,凝胶电泳后可见一位于709bp位置的阳性条带,与预测结果一致。
结论HSV-1体外感染人口腔上皮KB细胞实验模型建立。KB细胞为HSV-1的非允许性细胞,HSV-1感染KB细胞后可能形成转化性感染或潜伏性感染。
实验二KB细胞的上清液对Vero细胞的“转移感染”及鉴定
目的通过“转移感染”的间接方法将前述实验中KB细胞的上清液感染对HSV-1敏感的Vero细胞,以此检验HSV-1是否成功感染人口腔上皮KB细胞。
材料和方法收集实验一模型中不同MOI组HSV-1体外感染KB细胞48h后的培养上清,并以之感染HSV-1的允许性细胞Vero细胞。分别于0h、24h、48h在倒置相差显微镜下观察细胞形态,于感染后48h收集细胞,制成超薄切片在透射电镜下观察超微结构和病毒形态。并在“转移感染”48h后计算细胞病变率。
结果倒置显微镜下,Vero细胞“转移感染”24h时可观察到细胞病变出现;48h时细胞病变率上升。扫描电镜下Vero细胞核内可见大量大小均一的典型HSV-1颗粒。通过对细胞病变率进行统计,得知各滴度组“转移感染”后Vero细胞病变率有显著性差异。
结论KB细胞中潜伏的病毒被活化并在Vero细胞中产生了感染性病毒颗粒,证明了HSV-1可在体外感染口腔上皮细胞,并表达其基因。
综上所述,本研究利用Vero细胞大量扩增HSV-1,并以此感染人口腔上皮细胞KB细胞,在体外建立HSV-1感染人口腔上皮细胞实验模型。并收集HSV-1感染人口腔上皮细胞后的上清液“转移感染”Vero细胞,通过倒置显微镜,透射电镜超薄切片技术及PCR技术进行检验。结果表明HSV-1能够直接感染口腔上皮KB细胞,形成转化性感染或潜伏性感染,并在KB细胞内表达其基因。
Periodontal disease,one of the most common human oral diseases in human race,is a kind of chronic infectious diseases that caused by the invasion of microorganism and resulting in the destruction of periodontal tissue.During the past 100 years,the majority of researchers considered that bacterial plaque was the most important etiological factor of periodontal disease.Since the mid 1990s,many studies have indicated that herpes simplex viruses(HSV)is often detected in periodontitis sites, especially herpes simplex virus type 1(HSV-1).These results indicated that HSV-1 may play a significant role in the development of periodontal disease.Some researchers presumed that the damage to the epithelium barrier of the host may be one of the most important pathogenesis of HSV-1-associated periodontal disease.However,the mechanism has rarely been researched for lack of the model that HSV infects human oral epithelial cells in vitro directly.In this study,we abundantly amplified HSV-1 strains in Vero cells which were used to infect human oral epithelial cells,and established the model that HSV-1 infected human oral epithelial cells in vitro.The present study provides a basis of experimental technique for the further study of the pathogenesis of herpesvirus-associated periodontal disease
Experiment one:Establishment of the model that HSV-1 infected human oral epithelial cells in vitro.
Objective:To abundantly amplify HSV-1 strains and establish the model that HSV-1 infected human oral epithelial cells in vitro.
Materials and Methods:HSV-1 strains were abundantly amplified in Vero cells and viral titer was detected by plaque forming experiment.KB cells were infected with HSV-1 at an MOI of 0.5,1.0 or 3.0 PFU/cell.The morphology of the cells was inspected for microscopically detectable alterations through inverted microscope when HSV-1 infected KB cells for 0 h,24 h,48 h respectively.Infected KB cells were collected and then observed through TEM.Nucleic acid of the virus was detected by PCR.
Result:HSV-1 strains were abundantly amplified in Vero cells and the titer was standardized to 10~6 PFU/ml.The infected KB cells in three groups didn't display obvious cytopathic effect(CPE)under inverted microscope at either 24 h or 48 h under inverted microscope.There were no mature virus particles in endochylema or nucleus under TEM.The glycoprotein D gene order of HSV-1 could be detected in infected KB cells by PCR.
Conclusion:The model that HSV-1 infected human oral epithelial cells in vitro was established.KB cells are the non-permissive cell of HSV-1.HSV-1 infects KB cells leading to transforming infection or virus incubated in the cells causing latent infection.
Experiment Two:Transfer infection of HSV-1 to Vero cells
Objective:To checkout indirectly that whether the model of HSV-1 infected human oral epithelial cells in vitro is successful or not.
Materials and Methods:After HSV-1 infected KB cells for 48 h,the culture supernatant of infected groups and blank control group were collected.The collected culture supematant were incubated on Vero monolayer.The morphology of the cells was observed through inverted microscope when HSV-1 transfer infected Vero cells for 0 h,24 h,48 h respectively.Infected Vero cells were collected and then observed through TEM.We scored CPE ratio of infected Vero cells in the three groups when transfer infected for 48 h.
Result:Compared with control group,Vero cells which infected by the culture supernatant of infected KB cells showed typical CPE at 24 h i.p. under inverted microscope.The amount of CPE was increased at 48 h i.p. Numerous immature virus particles were prominent within the nucleus under TEM.Significant difference of CPE ratio of infected Vero cells in three different groups.
Conclusion:The latent viruses in KB cells were activated and produced infectious viral particles.
We conclude from this study that:The model that HSV-1 infected human oral epithelial cells in vitro could be established.HSV-1 infects KB cells leading to transforming infection or latent infection.HSV-1 could transfer its DNA genes or fragments of its genome into chromosome of KB cells and steadily express them in progeny virus after cell division.
引文
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[1]Choi BK,Park SH,Yoo YJ,et al.Detection of major putative periodontopathogens in Korean advced adult periodontitis patients using a nucleic acid-based approach[J].J Periodontol.2000;71:1387-1394.
[2]金奇主编.医学分子病毒学.北京:科学出版社.2001;711-735.
[3]Zakay-Rones Z,Hochman N,Rones Y.Immunological response to herpes simplex virus in human gingival fluid.J Periodontol.1982;53(1):42-45.
[4]Parra B,Slots J.Detection of human viruses in periodontal pockets using polymerase chain reaction[J].Oral Microbiol Immunol.1996;11(5):289-293.
[5]Contreras A,Zadeh HH,Nowzari H,et al.Herpesvirus infection of inflammatory cells in human periodontitis.Oral Microbiol Immunol. 1999; 14(4): 206-212.
[6] Kamma JJ, Slots J. Herpesviral-bacterial interactions in aggressive periodontitis [J]. J Clin Periodontol. 2003; 30(5): 420-426.
[7] Contreras A, Slots J . Typing of herpes simplex virus from human periodontium [J] . Oral Microbiol Immunol , Oral Microbiol Immunol. 2001; 16(1): 63-64.
[8] N: shiyama SA, Nakano V, Velasquez-Melendez G, Avila-Campos MJ. Occurrence of herpes simplex virus 1 and three periodontal bacteria in patients with chronic periodontitis and necrotic pulp. Can J Microbiol. 2008; 54(4): 326-330.
[9] Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000. 2006; 42: 80-87.
[10] Holt SC, Ebersole JL. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the "red complex", a prototype polybacterial pathogenic consortium in periodontitis. Periodontol 2000. 2005; 38: 72-122.
[11] Slots J, Sugar C, Kamma JJ.Cytomegalovirus periodontal presence is associated with subgingival Dialister pneumosintes and alveolar bone loss. Oral Microbiol Immunol. 2002; 17(6): 369-374.
[12] Contreras A, Umeda M, Chen C, Bakker I, Morrison JL, Slots J. Relationship between herpesviruses and adult periodontitis and periodontopathic bacteria.J Periodontol.1999;70(5):478-484.
[13]Slots J.Herpesviral-bacterial synergy in the pathogenesis of human periodontitis.Curr Opin Infect Dis.2007;20(3):278-283.
[14]Taguchi Y,Imai H.Expression of b-defensin-2 in human gingival epithelial cells in response to challenge with Porphyromonas gingivalis in vitro[J].J Periodont Res.2006;41:334-339.
[15]Vankeerberghen A,Nuytten H,Dierickx K,et al.Differential induction of human beta-defensin expression by periodontal commensals and pathogens in periodontal pocket epithelial cells[J].J Periodontol.2005;76(8):1293-1303.
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[19]Hazrati E,Galen B,Lu W,Wang W,Ouyang Y,Keller MJ,Lehrer RI.Herold BC.Human alpha-and beta-defensins block multiple steps in herpes simplex virus infection.J Immunol.2006;177(12):8658-8666.
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6 Parra B,Slots J.Detection of human viruses in periodontal pockets using polymerase chain reaction.Oral Microbiol Immunol.1996;11(5):289-293.
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17 Cappuyns I,Gugerli P,Mombelli A.Viruses in periodontal disease -areview.Oral Dis.2005;11(4):219-229.
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34 Tanner AC, Izard J. Tannerella forsythia, a periodontal pathogen entering the genomic era. Periodontol 2000. 2006; 42: 88-113.
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36 Contreras A, Umeda M, Chen C, et al . Relationship between herpesviruses and adult periodontitis and periodontopathic bacteria. J Periodontol. 1999; 70(5): 478-484.
37 Nishiyama SA, Nakano V, Velasquez-Melendez G , et al . Occurrence of herpes simplex virus 1 and three periodontal bacteria in patients with chronic periodontitis and necrotic pulp. Can J Microbiol. 2008; 54(4): 326-330.
38 Slots J. Herpesviral-bacterial synergy in the pathogenesis of human periodontitis. Curr Opin Infect Dis. 2007; 20(3): 278-283.
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41 Taguchi Y, Imai H. Expression of b-defensin-2 in human gingival epithelial cells in response to challenge with Porphyromonas gingivalis in vitro.J Periodont Res. 2006; 41(4): 334-339.
42 Vankeerberghen A, Nuytten H, Dierickx K, et al. Differential induction of human beta-defensin expression by periodontal commensals and pathogens in periodontal pocket epithelial cells. J Periodontol. 2005; 76(8): 1293-1303.
43 Beverly A, Dale L, Page Fredericks. Antimicrobial Peptides in the Oral Environment: Expression and Function in Health and Disease.Curr Issues Mol Biol. 2005; 7(2): 119-133.
44 Lehrer RI, Daher K, Ganz T , et al. Direct inactivation of viruses by MCP-1 and MCP-2, natural peptide antibiotics from rabbit leukocytes. J Virol. 1985; 54(2): 467-472.
45 Daher KA, Selsted ME, Lehrer RI. Direct inactivation of viruses by human granulocyte defensins. J Virol. 1986; 60(3): 1068-1074.
46 Hazrati E, Galen B, Lu W , et al. Human alpha- and beta-defensins block multiple steps in herpes simplex virus infection. J Immunol. 2006; 177(12): 8658-8666.
[1]Choi BK,Park SH,Yoo YJ,et al.Detection of major putative periodontopathogens in Korean advced adult periodontitis patients using a nucleic acid-based approach[J].J Periodontol.2000;71:1387-1394.
[2]金奇主编.医学分子病毒学.北京:科学出版社.2001;711-735.
[3]Zakay-Rones Z,Hochman N,Rones Y.Immunological response to herpes simplex virus in human gingival fluid.J Periodontol.1982;53(1):42-45.
[4]Parra B,Slots J.Detection of human viruses in periodontal pockets using polymerase chain reaction[J].Oral Microbiol Immunol.1996;11(5):289-293.
[5]Contreras A,Zadeh HH,Nowzari H,et al.Herpesvirus infection of inflammatory cells in human periodontitis.Oral Microbiol Immunol. 1999; 14(4): 206-212.
[6] Kamma JJ, Slots J. Herpesviral-bacterial interactions in aggressive periodontitis [J]. J Clin Periodontol. 2003; 30(5): 420-426.
[7] Contreras A, Slots J . Typing of herpes simplex virus from human periodontium [J] . Oral Microbiol Immunol , Oral Microbiol Immunol. 2001; 16(1): 63-64.
[8] N: shiyama SA, Nakano V, Velasquez-Melendez G, Avila-Campos MJ. Occurrence of herpes simplex virus 1 and three periodontal bacteria in patients with chronic periodontitis and necrotic pulp. Can J Microbiol. 2008; 54(4): 326-330.
[9] Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000. 2006; 42: 80-87.
[10] Holt SC, Ebersole JL. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the "red complex", a prototype polybacterial pathogenic consortium in periodontitis. Periodontol 2000. 2005; 38: 72-122.
[11] Slots J, Sugar C, Kamma JJ.Cytomegalovirus periodontal presence is associated with subgingival Dialister pneumosintes and alveolar bone loss. Oral Microbiol Immunol. 2002; 17(6): 369-374.
[12] Contreras A, Umeda M, Chen C, Bakker I, Morrison JL, Slots J. Relationship between herpesviruses and adult periodontitis and periodontopathic bacteria.J Periodontol.1999;70(5):478-484.
[13]Slots J.Herpesviral-bacterial synergy in the pathogenesis of human periodontitis.Curr Opin Infect Dis.2007;20(3):278-283.
[14]Taguchi Y,Imai H.Expression of b-defensin-2 in human gingival epithelial cells in response to challenge with Porphyromonas gingivalis in vitro[J].J Periodont Res.2006;41:334-339.
[15]Vankeerberghen A,Nuytten H,Dierickx K,et al.Differential induction of human beta-defensin expression by periodontal commensals and pathogens in periodontal pocket epithelial cells[J].J Periodontol.2005;76(8):1293-1303.
[16]Beverly A,Dale L,Page Fredericks.Antimicrobial Peptides in the Oral Environment:Expression and Function in Health and Disease[J].Curr Issues Mol Biol.2005;7(2):119-133.
[17]Lehrer RI,Daher K,Ganz T,Selsted ME.Direct inactivation of viruses by MCP-1 and MCP-2,natural peptide antibiotics from rabbit leukocytes.J Virol.1985;54(2):467-472.
[18]Daher KA,Selsted ME,Lehrer RI.Direct inactivation of viruses by human granulocyte defensins.J Virol.1986;60(3):1068-1074.
[19]Hazrati E,Galen B,Lu W,Wang W,Ouyang Y,Keller MJ,Lehrer RI.Herold BC.Human alpha-and beta-defensins block multiple steps in herpes simplex virus infection.J Immunol.2006;177(12):8658-8666.