上呼吸道上皮细胞的原代培养及其与11型人乳头状瘤病毒结合的研究
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摘要
目的：建立原代培养人呼吸道鳞状上皮细胞与假复层纤毛柱状上皮细胞的方法,比较11型人乳头状瘤病毒与两种上皮细胞的结合能力是否存在差异,并对细胞表面硫酸乙酰肝素分子在病毒与细胞表面的结合过程发挥的作用进行初步探讨。
方法：取成人正常上呼吸道粘膜,利用无血清培养基对鳞状上皮细胞进行原代培养,无血清培养基以及气-液界面培养联合应用进行假复层纤毛柱状上皮细胞的原代培养及诱导分化；所得细胞与11型人乳头状瘤病毒样颗粒进行结合反应,免疫印迹法检测病毒样颗粒与细胞是否发生结合,ELISA检测结合于细胞表面的病毒样颗粒的吸光值,并比较病毒样颗粒与两种上皮细胞的结合能力是否存在统计学差异；用硫酸乙酰肝素的竞争性抑制物肝素及其特异性酶-乙酰肝素酶干预病毒样颗粒与细胞的结合,检测干预前后病毒-细胞结合率的变化,推测硫酸乙酰肝素在病毒-细胞结合过程中所起的作用。
结果：
1.利用无血清培养基培养的人呼吸道鳞状上皮细胞,呈扁平、多角形,核居中,可见1-2个核仁,胞浆透明,无颗粒,细胞连接成片后呈典型的鹅卵石路面状,经抗人角蛋白多克隆抗体染色呈阳性,证实为上皮细胞；气-液界面培养的假复层纤毛柱状上皮,以及气-液界面培养所得到的假复层纤毛柱状上皮细胞纤毛分化良好,接近生理状态。
2.免疫印迹检测显示：11型人乳头状瘤病毒样颗粒与呼吸道鳞状上皮细胞及假复层纤毛柱状上皮细胞结合后,用抗11型人乳头状瘤病毒样颗粒的单克隆抗体可检测到阳性条带,说明病毒样颗粒与两种上皮细胞均可发生结合。ELISA检测结合于细胞表面的病毒样颗粒的吸光值,鳞状上皮细胞组A450为(0.81±0.12,n=6),假复层纤毛柱状上皮细胞A450为(0.76±0.10,n=6),两组数据经组间t-检验,差别无统计学意义。
3.肝素以及乙酰肝素酶对细胞表面的预处理都可抑制11型人乳头状瘤病毒样颗粒与呼吸道上皮细胞的结合,且该抑制作用在一定范围内与干预物的浓度呈正相关；当干预物超过一定浓度后,病毒颗粒与细胞的结合会稳定在一个较低水平,但不会完全被阻断。
结论：
1.无血清培养基以及气-液界面培养法是原代培养呼吸道鳞状上皮细胞与假复层纤毛柱状上皮细胞的有效方法,培养细胞纯度高、分化好、细胞形态与生理状态接近。
2.11型人乳头状瘤病毒可在体外与呼吸道上皮细胞结合,且与两种上皮细胞的结合能力无差异,因此,11型人乳头状瘤病毒在呼吸道内不同区域感染率的差异不是在病毒-细胞结合阶段产生。
3.细胞表面的硫酸乙酰肝素分子参与11型人乳头状瘤病毒与呼吸道上皮细胞的结合过程,并发挥十分重要的作用,硫酸乙酰肝素可能是11型人乳头状瘤病毒的细胞表面受体。
Objective. To establish an efficient method for direct isolation and culture of primary human respiratory epithelial cells from normal tissues, and investigate the attachment of human papillomavirus-11 (HPV-11) to respiratory epithelial cells, and discuss the effect of cellular heparan sulfate on virus-cell interaction.
Methods.1. Human respiratory epithelial cells were isolated by 0.25%trypsin-EDTA, and cultured in a serum-free medium, and induced ciliated in an air-liquid interface system.2. The binding of HPV-11 virus-like particles (VLPs) to respiratory epithelial cells was detected by immunoblot, and quantified by the absorbance of bound particles measured by ELISA. The absorbance of squamous and ciliated cells was quantitively analysed using SPSS software.3. We further investigated the effect of heparin and heparitinase on the binding of HPV-11 virus-like particles (VLPs) to respiratory epithelial cells.
Results.1. Primary laryngeal epithelial cells adhered to the cell flask after 6-7 days and formed a characteristic epithelioid shape showing a pavement-like arrangement. The epithelial phenotype of cells was also confirmed by expression of cytokeratins 5 and 8 on cell cytospins by immunofluorescence. Respiratory epithelial cells were observed with cilia appearance two weeks after air-liquid interface culture.2. HPV-11VLPs were capable of binding to surface of respiratory epithelial cells. There was no significant difference in the binding of HPV-11 to suamous and ciliated cells.3. The binding of HPV-11 and respiratory cells was reduced by heparin and heparitinase, and was in direct proportion with the concentration.
Conclusions.1. The application of serum-free medium and air-liquid interface system in primary culture of human respiratory epithelial cells was effectively efficient.2. The disparity of HPV infection in upper airway was not caused by virus-cell attachment.3. The binding of HPV and respiratory epithelial cells required cellular heparan sulfate that possibly was the receptor of HPV.
方法：取成人正常上呼吸道粘膜,利用无血清培养基对鳞状上皮细胞进行原代培养,无血清培养基以及气-液界面培养联合应用进行假复层纤毛柱状上皮细胞的原代培养及诱导分化；所得细胞与11型人乳头状瘤病毒样颗粒进行结合反应,免疫印迹法检测病毒样颗粒与细胞是否发生结合,ELISA检测结合于细胞表面的病毒样颗粒的吸光值,并比较病毒样颗粒与两种上皮细胞的结合能力是否存在统计学差异；用硫酸乙酰肝素的竞争性抑制物肝素及其特异性酶-乙酰肝素酶干预病毒样颗粒与细胞的结合,检测干预前后病毒-细胞结合率的变化,推测硫酸乙酰肝素在病毒-细胞结合过程中所起的作用。
结果：
1.利用无血清培养基培养的人呼吸道鳞状上皮细胞,呈扁平、多角形,核居中,可见1-2个核仁,胞浆透明,无颗粒,细胞连接成片后呈典型的鹅卵石路面状,经抗人角蛋白多克隆抗体染色呈阳性,证实为上皮细胞；气-液界面培养的假复层纤毛柱状上皮,以及气-液界面培养所得到的假复层纤毛柱状上皮细胞纤毛分化良好,接近生理状态。
2.免疫印迹检测显示：11型人乳头状瘤病毒样颗粒与呼吸道鳞状上皮细胞及假复层纤毛柱状上皮细胞结合后,用抗11型人乳头状瘤病毒样颗粒的单克隆抗体可检测到阳性条带,说明病毒样颗粒与两种上皮细胞均可发生结合。ELISA检测结合于细胞表面的病毒样颗粒的吸光值,鳞状上皮细胞组A450为(0.81±0.12,n=6),假复层纤毛柱状上皮细胞A450为(0.76±0.10,n=6),两组数据经组间t-检验,差别无统计学意义。
3.肝素以及乙酰肝素酶对细胞表面的预处理都可抑制11型人乳头状瘤病毒样颗粒与呼吸道上皮细胞的结合,且该抑制作用在一定范围内与干预物的浓度呈正相关；当干预物超过一定浓度后,病毒颗粒与细胞的结合会稳定在一个较低水平,但不会完全被阻断。
结论：
1.无血清培养基以及气-液界面培养法是原代培养呼吸道鳞状上皮细胞与假复层纤毛柱状上皮细胞的有效方法,培养细胞纯度高、分化好、细胞形态与生理状态接近。
2.11型人乳头状瘤病毒可在体外与呼吸道上皮细胞结合,且与两种上皮细胞的结合能力无差异,因此,11型人乳头状瘤病毒在呼吸道内不同区域感染率的差异不是在病毒-细胞结合阶段产生。
3.细胞表面的硫酸乙酰肝素分子参与11型人乳头状瘤病毒与呼吸道上皮细胞的结合过程,并发挥十分重要的作用,硫酸乙酰肝素可能是11型人乳头状瘤病毒的细胞表面受体。
Objective. To establish an efficient method for direct isolation and culture of primary human respiratory epithelial cells from normal tissues, and investigate the attachment of human papillomavirus-11 (HPV-11) to respiratory epithelial cells, and discuss the effect of cellular heparan sulfate on virus-cell interaction.
Methods.1. Human respiratory epithelial cells were isolated by 0.25%trypsin-EDTA, and cultured in a serum-free medium, and induced ciliated in an air-liquid interface system.2. The binding of HPV-11 virus-like particles (VLPs) to respiratory epithelial cells was detected by immunoblot, and quantified by the absorbance of bound particles measured by ELISA. The absorbance of squamous and ciliated cells was quantitively analysed using SPSS software.3. We further investigated the effect of heparin and heparitinase on the binding of HPV-11 virus-like particles (VLPs) to respiratory epithelial cells.
Results.1. Primary laryngeal epithelial cells adhered to the cell flask after 6-7 days and formed a characteristic epithelioid shape showing a pavement-like arrangement. The epithelial phenotype of cells was also confirmed by expression of cytokeratins 5 and 8 on cell cytospins by immunofluorescence. Respiratory epithelial cells were observed with cilia appearance two weeks after air-liquid interface culture.2. HPV-11VLPs were capable of binding to surface of respiratory epithelial cells. There was no significant difference in the binding of HPV-11 to suamous and ciliated cells.3. The binding of HPV-11 and respiratory cells was reduced by heparin and heparitinase, and was in direct proportion with the concentration.
Conclusions.1. The application of serum-free medium and air-liquid interface system in primary culture of human respiratory epithelial cells was effectively efficient.2. The disparity of HPV infection in upper airway was not caused by virus-cell attachment.3. The binding of HPV and respiratory epithelial cells required cellular heparan sulfate that possibly was the receptor of HPV.
引文
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24. Rommel O, Dillner J, Fligge C, Bergsdorf C, Wang X, Selinka HC, Sapp M:Heparan sulfate proteoglycans interact exclusively with conformationally intact HPV L1 assemblies:basis for a virus-like particle ELISA. J Med Virol 2005,75:114-121.
25. Chen XS, Garcea RL, Goldberg I, Casini G, Harrison SC:Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16. Mol Cell 2000,5:557-567.
26. Modis Y, Trus BL, Harrison SC:Atomic model of the papillomavirus capsid. EMBO J 2002,21:4754-4762.
27. Day PM, Thompson CD, Buck CB, Pang YY, Lowy DR, Schiller JT: Neutralization of human papillomavirus with monoclonal antibodies reveals different mechanisms of inhibition.J Virol 2007,81:8784-8792.
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29. Sibbet G, Romero-Graillet C, Meneguzzi G, Campo MS:alpha6 integrin is not the obligatory cell receptor for bovine papillomavirus type 4. J Gen Virol 2000,81:327-334.
30. Johnson KM, Kines RC, Roberts JN, Lowy DR, Schiller JT, Day PM: Role of heparan sulfate in attachment to and infection of the murine female genital tract by human papillomavirus.J Virol 2009,83:2067-2074.
31. Mudhakir D, Harashima H:Learning from the viral journey:how to enter cells and how to overcome intracellular barriers to reach the nucleus. AAPS J 2009,11:65-77.
32. Sieczkarski SB, Whittaker GR:Dissecting virus entry via endocytosis. J Gen Virol 2002,83:1535-1545.
33. Day PM, Lowy DR, Schiller JT:Papillomaviruses infect cells via a clathrin-dependent pathway. Virology 2003,307:1-11.
34. Bousarghin L, Touze A, Sizaret PY, Coursaget P:Human papillomavirus types 16,31, and 58 use different endocytosis pathways to enter cells.J Virol 2003,77:3846-3850.
35. Hindmarsh PL, Laimins LA:Mechanisms regulating expression of the HPV 31 L1 and L2 capsid proteins and pseudovirion entry. Virol J 2007, 4:19.
36. Smith JL, Campos SK, Ozbun MA:Human papillomavirus type 31 uses a caveolin 1- and dynamin 2-mediated entry pathway for infection of human keratinocytes. J Virol 2007,81:9922-9931.36. Laniosz V, Holthusen KA, Meneses PI:Bovine papillomavirus type 1:from clathrin to caveolin.J Virol 2008,82:6288-6298.
37. Smith JL, Campos SK, Wandinger-Ness A, Ozbun MA:Caveolin-1-dependent infectious entry of human papillomavirus type 31 in human keratinocytes proceeds to the endosomal pathway for pH-dependent uncoating.J Virol 2008,82:9505-9512.
38. Laniosz V, Holthusen KA, Meneses PI:Bovine papillomavirus type 1: from clathrin to caveolin. J Virol 2008,82:6288-6298.
39. Abban CY, Bradbury NA, Meneses PI:HPV16 and BPV1 infection can be blocked by the dynamin inhibitor dynasore. Am J Ther 2008,15:304-311.
40. Spoden G, Freitag K, Husmann M, Boller K, Sapp M, Lambert C, Florin L:Clathrin-and caveolin-independent entry of human papillomavirus type 16--involvement of tetraspanin-enriched microdomains (TEMs). PLoS One 2008,3:e3313.
41. Hemler ME:Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu Rev Cell Dev Biol 2003,19:397-422.
42. Levy S, Shoham T:Protein-protein interactions in the tetraspanin web. Physiology (Bethesda) 2005,20:218-224.
43. Day PM, Lowy DR, Schiller JT:Heparan sulfate-independent cell binding and infection with furin-precleaved papillomavirus capsids.J Virol 2008,82:12565-12568.
44. Richards RM, Lowy DR, Schiller JT, Day PM:Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection. Proc Natl Acad Sci USA 2006,103:1522-1527.
45. Kamper N, Day PM, Nowak T, Selinka HC, Florin L, Bolscher J, Hilbig L, Schiller JT, Sapp M:A membrane-destabilizing peptide in capsid protein L2 is required for egress of papillomavirus genomes from endosomes.J Virol 2006,80:759-768.
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