BTV-HbC_3纯化及其感染肿瘤细胞机制初探
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摘要
本文发展了一种反向免疫共沉淀分离纯化病毒的新方法，用于从培养物中分离高纯度，高活性的蓝舌病毒。用透射电镜和凝胶过滤层析检测纯化产物病毒的结构完整性和纯度；组织培养技术检测产物的生物学活性并计算纯化得率。结果表明，纯化后的病毒悬液通过凝胶过滤层析仅出现一个紫外吸收峰，其余的未见明显的吸收峰，仅表现为靠近基线的波动；将纯化前后的病毒悬液用磷钨酸负染后直接透射电镜观察，可见到完整的病毒粒子及其干净的背景；在相同条件下，纯化前的BTV-HbC_3对Vero细胞的TCID_(50)是10~(-6.6)／ml，纯化后的病毒对Vero细胞的TCID_(50)是10~(-5.7)／ml；选取一定稀释度的复孔计数空斑，依据空斑计算公式求得不同稀释度下纯化前后病毒的滴度，通过比较纯化前后的病毒滴度计算该纯化方法的纯化得率，在10~(-3)，10~(-4)，10~(-5)这三个稀释度下，所计算纯化得率分别为69.6％，70.8％，50.4％。
经蓝舌病毒处理过的细胞(7402细胞，Hep-3B细胞)在普通光学显微镜下即可看到明显的形态改变。细胞在接种病毒24 hr后开始收缩变圆，间隔变大，细胞轮廓增强，胞内有粗大折光颗粒；36hr后细胞逐渐脱落，漂浮，直至破碎死亡。用MTT染色方法检测蓝舌病毒对这两株肝癌细胞株的抑制活性。在10°稀释度的蓝舌病毒作用下测得这两株细胞的抑制率分别为：51.45％和77.69％。
通过PI染色检测经蓝舌病毒处理后不同时间的细胞凋亡情况，其中感染病毒后48hr的7402，Hep-3B细胞的凋亡率分别为20.1％，33.0％。通过Hoechst33258染色后进行共聚焦显微镜观察，结果在这两株细胞中均观察到典型的凋亡细胞，主要表现为凋亡细胞出现核碎裂、边聚，碎裂的细胞核呈致密浓染的颗粒状或块状荧光，与此同时，完整核膜清晰可见。
A novel reverse co-immunoprecipitation method was used to get the purified BTV-HbC3 particles and gel filtration, electron microscopy(EM), TCID50 and plaque assay were used to analyze the purification results. Sephadex G-200 gel filtration analysis of the purified sample by absorbance values measured at 280nm , only one steep UV absorbance peak can be seen ; background of electron micrograph of the purified virus is clean and the complete structure of virus particles can be clearly seen; the 50% tissue culture infective dose on Vero cell line is 10-6.6/ml before purification and 10-5.7/ml after purification; purified and unpurified virus liters can be attained from plaque assay of BTV-HbC3 on Hep-3B cell line . Purification recovery rate can be gotten by comparing between virus titers of purified and unpurified. On 10-3, 10-4, 10-5 dilution degrees respectively , the recovery rates are 69.6 %, 70.8 %, 50.4 %. From the results described above we can draw the conclusion that the new method introduced can produ
ce large amounts of high purified viral particles with high infectivity so it can be applied to further research on developing a large scale production system.
BTV-HbC3 can selectively replicate in 7402, Hep-3B cell lines and induce cytopathic effects (CPE), and eventually cause complete lysis and death of cells. Between the two human liver cell lines, Hep-3B cells are more susceptible to BTV-HbC3 sinfection, the CPE of which occur quickly and can reach 90% at 36h p.i., while 7402 cells are the less sensitive. The inhibition activity of BTV-HbC3 on the two liver cell lines (7402, Hep-3B ) was tested by MTT. Under the same condition, the inhibition ratio of BTV is 77.69% on Hep-3B; 51.45% on 7402.
BTV-HbC3 can induce apoptosis in 7402, Hep-3B cell lines. The result of flow cytometry assay shows the apoptosis ratio of 48hr p.i. is 20.1% on 7402; 33.0% on Hep-3B. The result of confocal microscopy also indicates typical apoptosis induced by BTV-HbC3 in two human liver cell lines.
经蓝舌病毒处理过的细胞(7402细胞，Hep-3B细胞)在普通光学显微镜下即可看到明显的形态改变。细胞在接种病毒24 hr后开始收缩变圆，间隔变大，细胞轮廓增强，胞内有粗大折光颗粒；36hr后细胞逐渐脱落，漂浮，直至破碎死亡。用MTT染色方法检测蓝舌病毒对这两株肝癌细胞株的抑制活性。在10°稀释度的蓝舌病毒作用下测得这两株细胞的抑制率分别为：51.45％和77.69％。
通过PI染色检测经蓝舌病毒处理后不同时间的细胞凋亡情况，其中感染病毒后48hr的7402，Hep-3B细胞的凋亡率分别为20.1％，33.0％。通过Hoechst33258染色后进行共聚焦显微镜观察，结果在这两株细胞中均观察到典型的凋亡细胞，主要表现为凋亡细胞出现核碎裂、边聚，碎裂的细胞核呈致密浓染的颗粒状或块状荧光，与此同时，完整核膜清晰可见。
A novel reverse co-immunoprecipitation method was used to get the purified BTV-HbC3 particles and gel filtration, electron microscopy(EM), TCID50 and plaque assay were used to analyze the purification results. Sephadex G-200 gel filtration analysis of the purified sample by absorbance values measured at 280nm , only one steep UV absorbance peak can be seen ; background of electron micrograph of the purified virus is clean and the complete structure of virus particles can be clearly seen; the 50% tissue culture infective dose on Vero cell line is 10-6.6/ml before purification and 10-5.7/ml after purification; purified and unpurified virus liters can be attained from plaque assay of BTV-HbC3 on Hep-3B cell line . Purification recovery rate can be gotten by comparing between virus titers of purified and unpurified. On 10-3, 10-4, 10-5 dilution degrees respectively , the recovery rates are 69.6 %, 70.8 %, 50.4 %. From the results described above we can draw the conclusion that the new method introduced can produ
ce large amounts of high purified viral particles with high infectivity so it can be applied to further research on developing a large scale production system.
BTV-HbC3 can selectively replicate in 7402, Hep-3B cell lines and induce cytopathic effects (CPE), and eventually cause complete lysis and death of cells. Between the two human liver cell lines, Hep-3B cells are more susceptible to BTV-HbC3 sinfection, the CPE of which occur quickly and can reach 90% at 36h p.i., while 7402 cells are the less sensitive. The inhibition activity of BTV-HbC3 on the two liver cell lines (7402, Hep-3B ) was tested by MTT. Under the same condition, the inhibition ratio of BTV is 77.69% on Hep-3B; 51.45% on 7402.
BTV-HbC3 can induce apoptosis in 7402, Hep-3B cell lines. The result of flow cytometry assay shows the apoptosis ratio of 48hr p.i. is 20.1% on 7402; 33.0% on Hep-3B. The result of confocal microscopy also indicates typical apoptosis induced by BTV-HbC3 in two human liver cell lines.
引文
1. Jeggo MH, Gumm ID and Taylor WE Clinical and serological response of sheep to serial challenge with different bluetongue vires types. Res Vet Sci, 1983, 34:205-211
2. Mertens PPC , Burroughs JN and Anderson J. Purification and Properties of Virus Particles, Infectious Subviral Particles, and Cores of Bluetongue Virus Serotypes 1 and 4. Virology, 1987, 157:375-386
3．董长垣，等人．湖北省蓝舌病毒血清流行病学调查，医药研究和卫生管理，中国人口出版社，1998，p135
4．董长垣，陈东娥，陈晓，等人．蓝舌病毒HbC株的分离和生物学特征的研究，临床医学研究，中国人口出版社，1998，12：180-182
5．陈晓，严银钫，董长垣，等人．蓝舌病毒HbC株的增殖和血清学特点，湖北医科大学学报，1999，20(3)：179-180
6．董长垣，陈晓，严银钫，等人．蓝舌病毒HbC株体外增殖和基因组特征，湖北医科大学学报，1999，20(1)：7-9，120
7．张芃伟，董长垣，涂攀，等人．检测蓝舌病毒技术的建立，中国病毒学，2001，16(4)：393
8. Coffey MC, Strong LE, Forsyth PA. Reovirus Therapy of Tumors with Activated Ras Pathway. Science, 1998,282(13):1332-1334
9. Martin SA., and Zweerink HJ: Isolation and characterization of two types of bluetongue virus particles. Virology, 1972, 50:495-506
10. Verwoerd DW, Els HJ, De Villiers EM and Huismans H. Structure of the bluetongue virus capsid. J Virol, 1972, 10:783-794
11. Huismans H and Els HJ. Characterization of the tubules associated with the replication of three different orbiviruses. Virology, 1979, 92:397-406
12．[美]F．奥斯伯，R．布伦特，R．E．金斯顿，等人．精编分子生物学实验指南．pp．435-437；394-400；373-376．科学出版社．1999．
13. Karber G.. Beitrag zur kollektiven behandelung pharmakologischer reihenversuche. Arch Exp Pathol Pharmakol. 1931, 162:480-483
14．傅继华．病毒学实用实验技术，pp 62-64；64-66，山东科学技术出版社，2001
15．王健伟，姜慧英，温乐英，等人．蓝舌病毒L3基因的克隆与表达，中华实验和临床病毒学杂志，1999，13：213-216
16．马洪超，苏艳，孙淑芳，等人．蓝舌病毒Z1株VP7基因的克隆及序列分析，病毒学报，2000，16：34-37
17．王健伟，姜慧英，李晓成，等人．在昆虫细胞中高效表达蓝舌病毒VP7蛋白作为组特异性诊断抗原，病毒学报，1999，15：238-243
18. Mosmaan T. Colorimetric Assay Cellular Growth and Survival: Application to Proliferation and Cytotoxity Assays. J Immunol Methods, 1983, 65:55
19．薛彬，Edeman A，Thorbecke G J．介绍一种用颜色显示法测细胞的生长及增殖．北京医科大学学报，1987，19(2)：125
20. Chappell JD. Identification of Carbohydrate-binding Domains in the Attachment Proteins of Type 1 and Type 3 Reoviruses. J Virol, 2000, 74:8472-8479
21. Bos JL. Ras Oncogene in Human Cancer: A Review. Cancer Research. 1989, 9: 4682-4689
22. Lowe PN, Skinner RH. Regulation of Ras Signal Transduction in Normal and Tansformed Cells. Cell Signal, 1994, 6(2): 109-123
23. Robinson MJ, Cobb MH. Mitogen-activated Protein Kinase Pathway. Curr. Opin.Cell. Biol, 1997, 9(2): 180-186
24. Walther W, Stein U. Viral vectors for gene transfer: a review of their use in the treatment of human disease. Drags, 2000, 60:249
25. Ahlert T, Schirrmacher V. Isolation of a human melanoma adapted Newcastle disease virus mutant with highly selective replication patterns. Cancer Research, 1900, 50:5962
26. Rommelaere J, tattershall P. Oncosuppression by parvovirus. In Handbook of Parvoviruses. CRC Press, 1990, 41
2. Mertens PPC , Burroughs JN and Anderson J. Purification and Properties of Virus Particles, Infectious Subviral Particles, and Cores of Bluetongue Virus Serotypes 1 and 4. Virology, 1987, 157:375-386
3．董长垣，等人．湖北省蓝舌病毒血清流行病学调查，医药研究和卫生管理，中国人口出版社，1998，p135
4．董长垣，陈东娥，陈晓，等人．蓝舌病毒HbC株的分离和生物学特征的研究，临床医学研究，中国人口出版社，1998，12：180-182
5．陈晓，严银钫，董长垣，等人．蓝舌病毒HbC株的增殖和血清学特点，湖北医科大学学报，1999，20(3)：179-180
6．董长垣，陈晓，严银钫，等人．蓝舌病毒HbC株体外增殖和基因组特征，湖北医科大学学报，1999，20(1)：7-9，120
7．张芃伟，董长垣，涂攀，等人．检测蓝舌病毒技术的建立，中国病毒学，2001，16(4)：393
8. Coffey MC, Strong LE, Forsyth PA. Reovirus Therapy of Tumors with Activated Ras Pathway. Science, 1998,282(13):1332-1334
9. Martin SA., and Zweerink HJ: Isolation and characterization of two types of bluetongue virus particles. Virology, 1972, 50:495-506
10. Verwoerd DW, Els HJ, De Villiers EM and Huismans H. Structure of the bluetongue virus capsid. J Virol, 1972, 10:783-794
11. Huismans H and Els HJ. Characterization of the tubules associated with the replication of three different orbiviruses. Virology, 1979, 92:397-406
12．[美]F．奥斯伯，R．布伦特，R．E．金斯顿，等人．精编分子生物学实验指南．pp．435-437；394-400；373-376．科学出版社．1999．
13. Karber G.. Beitrag zur kollektiven behandelung pharmakologischer reihenversuche. Arch Exp Pathol Pharmakol. 1931, 162:480-483
14．傅继华．病毒学实用实验技术，pp 62-64；64-66，山东科学技术出版社，2001
15．王健伟，姜慧英，温乐英，等人．蓝舌病毒L3基因的克隆与表达，中华实验和临床病毒学杂志，1999，13：213-216
16．马洪超，苏艳，孙淑芳，等人．蓝舌病毒Z1株VP7基因的克隆及序列分析，病毒学报，2000，16：34-37
17．王健伟，姜慧英，李晓成，等人．在昆虫细胞中高效表达蓝舌病毒VP7蛋白作为组特异性诊断抗原，病毒学报，1999，15：238-243
18. Mosmaan T. Colorimetric Assay Cellular Growth and Survival: Application to Proliferation and Cytotoxity Assays. J Immunol Methods, 1983, 65:55
19．薛彬，Edeman A，Thorbecke G J．介绍一种用颜色显示法测细胞的生长及增殖．北京医科大学学报，1987，19(2)：125
20. Chappell JD. Identification of Carbohydrate-binding Domains in the Attachment Proteins of Type 1 and Type 3 Reoviruses. J Virol, 2000, 74:8472-8479
21. Bos JL. Ras Oncogene in Human Cancer: A Review. Cancer Research. 1989, 9: 4682-4689
22. Lowe PN, Skinner RH. Regulation of Ras Signal Transduction in Normal and Tansformed Cells. Cell Signal, 1994, 6(2): 109-123
23. Robinson MJ, Cobb MH. Mitogen-activated Protein Kinase Pathway. Curr. Opin.Cell. Biol, 1997, 9(2): 180-186
24. Walther W, Stein U. Viral vectors for gene transfer: a review of their use in the treatment of human disease. Drags, 2000, 60:249
25. Ahlert T, Schirrmacher V. Isolation of a human melanoma adapted Newcastle disease virus mutant with highly selective replication patterns. Cancer Research, 1900, 50:5962
26. Rommelaere J, tattershall P. Oncosuppression by parvovirus. In Handbook of Parvoviruses. CRC Press, 1990, 41