重组表达GP120分子对RA外周血CD4～+T细胞抑制的研究
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摘要
类风湿关节炎(rheumatoid arthritis,RA)是一种原因不明的,以慢性进行性多关节损害为特征的自身免疫性疾病。在疾病的发展中T细胞起着重要的作用。细胞因子分析有证据表明:即CD4+T细胞在疾病的开始可能是关键性的,已确定的滑膜炎可能依赖T细胞的辅助。通过单克隆抗体或封闭与B细胞的相互作用而清除CD4+T细胞能够阻断自身免疫病。
Gp120是人免疫缺陷病毒(humanimmunodeficiencs,HIV)的衣壳蛋白,由基因env编码,其分子量为120KD,负责结合CD4受体和共受体,决定细胞嗜性。在病毒侵入人体T细胞的过程中发挥重要的作用,同时它还存在游离态的形式,通过一种类似于超抗原作用的途径,在体内非特异性地激活一些主要的免疫细胞,从而大大增强了HIV对人体的危害作用。
目的旨在研究重组表达的gp120分子对RA患者外周血CD4+T细胞活性的影响,为RA的生物治疗寻找新的途径。
方法在原有的gp120基因原核表达载体的基础上,构建重组gp120真核表达质粒,再以阳离子聚合物来包裹真核质粒pcDNA3-gp120,转染NIH3T3小鼠成纤维细胞系,经G418筛选阳性克隆后,免疫组化鉴定gp120表达,将表达gp120的NIH3T3细胞系与RA病人外周血淋巴细胞共同培养,同时以转染空质粒载体无gp120表达的NIH3T3细胞系与RA病人外周血淋巴细胞共同培养作对照分组,最后采用ELISA法测定培养上清中的sIL-2R(可溶性白介素2受体)的表达情况,来分析gp120分子对RA外周血CD4+T细胞的抑制作用。
Rheumatoid arthritis(RA) is an autoimmune disease of unknown etiology characterized by chronic inflammation and progressive joint destrution. T cell play an important role in development of disease.Cell factor evidence shows I.e. possibly CD4+T are critical in the beginning of disease ,already definite arthromeningitis depends on aid of T cell possibly it could intercept autoallergic disease through the interaction of monoclonal antibody or block and B cell to clean up CD4+T.
gp120 is capsid protein of HIV virus encoded by gene ,envmolecular weight is120KD.Playing an important part in viral invasion human body T cell,at the same time existing free state style,Which activate many fundamental immunocells nonspecificly in vivo,through an way similar to super antigen thus strengthen demic harmful effect of HIV.
Objective Study the effect of peripheral-blood lymphocyte inhibition (PBL) induced by the recombination expression of GP120 in patients with rheumtoid arthritis ,to search a new way of biotherapy of RA.
Method we construct recombination gp120 eukaryotic expression plasmid on the ground of built-in gp120 gene prokaryotic expression vector,then parcel eucaryon plasmid pcDNA3-gp120 with cationic polymerization, transfect NIH3T3 mouse collagenoblast cell line,bolting masculine Clon with G418,detect the expression of gp120 by immunohistochemistry,coculture NIH3T3 cell line expressing gp120 and peripheral-blood activated lymphocyte in patients with rheumtoid
Gp120是人免疫缺陷病毒(humanimmunodeficiencs,HIV)的衣壳蛋白,由基因env编码,其分子量为120KD,负责结合CD4受体和共受体,决定细胞嗜性。在病毒侵入人体T细胞的过程中发挥重要的作用,同时它还存在游离态的形式,通过一种类似于超抗原作用的途径,在体内非特异性地激活一些主要的免疫细胞,从而大大增强了HIV对人体的危害作用。
目的旨在研究重组表达的gp120分子对RA患者外周血CD4+T细胞活性的影响,为RA的生物治疗寻找新的途径。
方法在原有的gp120基因原核表达载体的基础上,构建重组gp120真核表达质粒,再以阳离子聚合物来包裹真核质粒pcDNA3-gp120,转染NIH3T3小鼠成纤维细胞系,经G418筛选阳性克隆后,免疫组化鉴定gp120表达,将表达gp120的NIH3T3细胞系与RA病人外周血淋巴细胞共同培养,同时以转染空质粒载体无gp120表达的NIH3T3细胞系与RA病人外周血淋巴细胞共同培养作对照分组,最后采用ELISA法测定培养上清中的sIL-2R(可溶性白介素2受体)的表达情况,来分析gp120分子对RA外周血CD4+T细胞的抑制作用。
Rheumatoid arthritis(RA) is an autoimmune disease of unknown etiology characterized by chronic inflammation and progressive joint destrution. T cell play an important role in development of disease.Cell factor evidence shows I.e. possibly CD4+T are critical in the beginning of disease ,already definite arthromeningitis depends on aid of T cell possibly it could intercept autoallergic disease through the interaction of monoclonal antibody or block and B cell to clean up CD4+T.
gp120 is capsid protein of HIV virus encoded by gene ,envmolecular weight is120KD.Playing an important part in viral invasion human body T cell,at the same time existing free state style,Which activate many fundamental immunocells nonspecificly in vivo,through an way similar to super antigen thus strengthen demic harmful effect of HIV.
Objective Study the effect of peripheral-blood lymphocyte inhibition (PBL) induced by the recombination expression of GP120 in patients with rheumtoid arthritis ,to search a new way of biotherapy of RA.
Method we construct recombination gp120 eukaryotic expression plasmid on the ground of built-in gp120 gene prokaryotic expression vector,then parcel eucaryon plasmid pcDNA3-gp120 with cationic polymerization, transfect NIH3T3 mouse collagenoblast cell line,bolting masculine Clon with G418,detect the expression of gp120 by immunohistochemistry,coculture NIH3T3 cell line expressing gp120 and peripheral-blood activated lymphocyte in patients with rheumtoid
引文
1. Blaschke S,Middel P,et al. Expression if activation induced, T cell-derived,and chemokine-related cytokine/lymphotactim and its functional role in rheumatoid arthritis. Arthritis Rheum, 2003,48:1858-1872
2. Fox D A.The role T cells in The immunopathogenesis of rheumatoid arthritis:new perspectives. Arthritis Rheum,1997,40:598-609
3. David Davis, Heike Trischmann. Antibodies raised to short synthetic peptides with sequences derived from HIV-1 SF2 gp120 can both neutralize and enhance HIV-1 SF13: A later variant isolated from the same host. Journal of Medical Virology, 2001,64:207-216.
4. Sanjay C. Keswani, Michelle Polley. Schwann cell chemokine receptors mediate HIV-1 gp120 toxicity to sensory neurons. Annals of Neurology, 2003,54:287-296 .
5. 蒋 明 ,DAVID YU, 林 孝 义 , 朱 立 平 主 编 . 中 华 风 湿 病 学 . 华 夏 出 版社,2004,189-196
6. Roncarolo MG, Baccheta C, Bordignon C, Narula S, Levings MK. Type 1 T regulatory cells. Immunol Rev 2001,182:68–79
7. 施桂英主编. 关节炎概要.北京:中国医药科技出版社,2005,71-80
8. Geng Meiyu, Li Fuchuan. The potential molecular targets of marine sulfated polymannuroguluronate interfering with HIV-1 entry: Interaction between SPMG and HIV-1 rgp120 and CD4 molecule. Antiviral Research, 2003,59:127–135
9. 江方正, 金宁一, 金洪涛等. HIV-1 表面蛋白 gp120 DNA 疫苗质粒的构建与表达. 中国生物制品学杂志, 2002,15:257-25
10. 杨贵波, 邵一鸣. 人类免疫缺陷病毒与黏膜免疫. World Chin J Digestol, 28;13(14):1750-175
11. Garson H. Tishkoff and Lois T. Hunt. Unexpected Molecular Mimicry among Peptides MHC Class II, Blood-Clotting Factor X, and HIV-1 Envelope Glycoprotein GP120. Thrombosis Research, 2000,98:343-346
12. 杜冰, 钱旻. gp120 对体内免疫细胞的作用. 国外医学.免疫学分册,2003,26:89-9
1. Datta SK. Major peptide autoepitopes for nucleosome-centered T and B cell interaction in human and murine lupus. Ann NY Acad Sci 2003;987:79–90.
2. CopeAP. Exploring the reciprocal relationship between immunity and inflammation in chronic inflammatory arthritis. Rheumatology 2003; 42:716–31.
3. Gonzalez G, Montero E, Leon K, Cohen IR, Lage A. Autoimmunization to epidermal growth factor, a component of the immunological homunculus. Autoimmun Rev 2002;1:89–95.
4. Chatenoud L, Salomon B, Bluestone JA. Suppressor T cells—they’re back and critical for regulation of autoimmunity! Immunol Rev 2001; 182:149–63.
5. Kumar V, Sercarz E. An integrative model of regulation centered on recognition of TCR peptide/MHC complexes. Immunol Rev 2001; 182:113–21.
6. Durkin HG, Waksman BH. Thymus and tolerance. Is regulation the major function of the thymus? Immunol Rev 2001;182:33–57.
7. Shevach EM, McHugh RS, Piccirillo CA, Thornton AM. Control of T-cell activation by CD4+ CD25+ suppressor T cells. Immunol Rev 2001;182:58–67.
8. Annacker O, Pimenta-Araujo R, Burlen-Defranoux O, Bandeira A. On the ontogeny and physiology of regulatory T cells. Immunol Rev 2001;182:5–17.
9. Sakaguchi S, Sakaguchi N, Shimizu J, Yamazaki S, Sakihama T, Itoh M, et al. Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 2001;182:18–32.
10. Weiner HL. Induction and mechanism of action of transforming growth factor-béta-secreting Th3 regulatory cells. Immunol Rev 2001;182:207–14.
11. Mason D. Some quantitative aspects of T-cell repertoire selection: the requirement for regulatory T cells. Immunol Rev 2001;182:80–8.
12. Goldrath AW, Bevan MJ. Selecting and maintaining a diverse T cell repertoire. Nature 1999;402:255–62.
13. Goldschneider I, Cone RE.A central role for peripheral dendritic cells in the induction of acquired thymic tolerance. Trends Immunol 2003; 24:77–81.
14. Coutinho A, Hori S, Carvalho T, Caramalho I, Demengeot J. Regulatory T cells: the physiology of autoreactivity in dominant tolerance and ‘quality control’ of immune responses. Immunol Rev 2001;182:89–98.
15. Roncarolo MG, Baccheta C, Bordignon C, Narula S, Levings MK. Type 1 T regulatory cells. Immunol Rev 2001;182:68–79.
16. Trani J, Moore DJ, Jarrett BP,Markmann JW, Lee MK, Singer A, et al.CD25+ immunoregulatory CD4 T cells mediate acquired central transplantation tolerance. J Immunol 2003;170:279–86.
17. Posselt AM, Barker CF, Friedman AL, Naji A. Prevention of autoimmune diabetes in the BB rat by intrathymic islet transplantation at birth. Science 1992;256:1321–4.
18. Shimizu J, Moriizumi E. CD4+CD25– T cells in aged mice are hyporesponsive and exhibit suppressive activity. J Immunol 2003; 170:1675–82.
19. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;299:1057–61.
20. Powrie F, LeachMW,Mauze S, Caddle LB, Coffman RL. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C B17 scid mice. Int Immunol 1993;5: 1461–71.
21. Weiner HL. The mucosal milieu creates tolerogenic dendritic cells and Tr1 and Th3 regulatory cells. Nat Immunol 2001;8:671–2.
22. Wing K, Ekmark A, Karlsson H, Rudin A, Suri-Payer E. Characterization of human CD25+CD4+ T cells in thymus, cord and adult blood. Immunology 2002; 106: 190–9.
23. Romagnoli P, Hudrisier D, van Meerwijk JP. Preferential recognition of self antigens despite normal thymic deletion of CD4(+)CD25(+) regulatory T cells. J Immunol 2002;168:1644–8.
24. Taams LS, Vukmanovic-Stejic M, Smith J, Dunne PJ, Fletcher JM, Plunkett FJ, et al. Antigen specific T cell suppression by human CD4+CD25+ regulatory T cells. Eur J Immunol 2002;32:1621–30.
25. Tung KS, Agersborg SS, Alard P, Garza KM, Lou YH. Regulatory T-cell endogenous antigen and neonatal environment in the prevention and induction of autoimmune disease. Immunol Rev 2001;182:135–48.
2. Fox D A.The role T cells in The immunopathogenesis of rheumatoid arthritis:new perspectives. Arthritis Rheum,1997,40:598-609
3. David Davis, Heike Trischmann. Antibodies raised to short synthetic peptides with sequences derived from HIV-1 SF2 gp120 can both neutralize and enhance HIV-1 SF13: A later variant isolated from the same host. Journal of Medical Virology, 2001,64:207-216.
4. Sanjay C. Keswani, Michelle Polley. Schwann cell chemokine receptors mediate HIV-1 gp120 toxicity to sensory neurons. Annals of Neurology, 2003,54:287-296 .
5. 蒋 明 ,DAVID YU, 林 孝 义 , 朱 立 平 主 编 . 中 华 风 湿 病 学 . 华 夏 出 版社,2004,189-196
6. Roncarolo MG, Baccheta C, Bordignon C, Narula S, Levings MK. Type 1 T regulatory cells. Immunol Rev 2001,182:68–79
7. 施桂英主编. 关节炎概要.北京:中国医药科技出版社,2005,71-80
8. Geng Meiyu, Li Fuchuan. The potential molecular targets of marine sulfated polymannuroguluronate interfering with HIV-1 entry: Interaction between SPMG and HIV-1 rgp120 and CD4 molecule. Antiviral Research, 2003,59:127–135
9. 江方正, 金宁一, 金洪涛等. HIV-1 表面蛋白 gp120 DNA 疫苗质粒的构建与表达. 中国生物制品学杂志, 2002,15:257-25
10. 杨贵波, 邵一鸣. 人类免疫缺陷病毒与黏膜免疫. World Chin J Digestol, 28;13(14):1750-175
11. Garson H. Tishkoff and Lois T. Hunt. Unexpected Molecular Mimicry among Peptides MHC Class II, Blood-Clotting Factor X, and HIV-1 Envelope Glycoprotein GP120. Thrombosis Research, 2000,98:343-346
12. 杜冰, 钱旻. gp120 对体内免疫细胞的作用. 国外医学.免疫学分册,2003,26:89-9
1. Datta SK. Major peptide autoepitopes for nucleosome-centered T and B cell interaction in human and murine lupus. Ann NY Acad Sci 2003;987:79–90.
2. CopeAP. Exploring the reciprocal relationship between immunity and inflammation in chronic inflammatory arthritis. Rheumatology 2003; 42:716–31.
3. Gonzalez G, Montero E, Leon K, Cohen IR, Lage A. Autoimmunization to epidermal growth factor, a component of the immunological homunculus. Autoimmun Rev 2002;1:89–95.
4. Chatenoud L, Salomon B, Bluestone JA. Suppressor T cells—they’re back and critical for regulation of autoimmunity! Immunol Rev 2001; 182:149–63.
5. Kumar V, Sercarz E. An integrative model of regulation centered on recognition of TCR peptide/MHC complexes. Immunol Rev 2001; 182:113–21.
6. Durkin HG, Waksman BH. Thymus and tolerance. Is regulation the major function of the thymus? Immunol Rev 2001;182:33–57.
7. Shevach EM, McHugh RS, Piccirillo CA, Thornton AM. Control of T-cell activation by CD4+ CD25+ suppressor T cells. Immunol Rev 2001;182:58–67.
8. Annacker O, Pimenta-Araujo R, Burlen-Defranoux O, Bandeira A. On the ontogeny and physiology of regulatory T cells. Immunol Rev 2001;182:5–17.
9. Sakaguchi S, Sakaguchi N, Shimizu J, Yamazaki S, Sakihama T, Itoh M, et al. Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 2001;182:18–32.
10. Weiner HL. Induction and mechanism of action of transforming growth factor-béta-secreting Th3 regulatory cells. Immunol Rev 2001;182:207–14.
11. Mason D. Some quantitative aspects of T-cell repertoire selection: the requirement for regulatory T cells. Immunol Rev 2001;182:80–8.
12. Goldrath AW, Bevan MJ. Selecting and maintaining a diverse T cell repertoire. Nature 1999;402:255–62.
13. Goldschneider I, Cone RE.A central role for peripheral dendritic cells in the induction of acquired thymic tolerance. Trends Immunol 2003; 24:77–81.
14. Coutinho A, Hori S, Carvalho T, Caramalho I, Demengeot J. Regulatory T cells: the physiology of autoreactivity in dominant tolerance and ‘quality control’ of immune responses. Immunol Rev 2001;182:89–98.
15. Roncarolo MG, Baccheta C, Bordignon C, Narula S, Levings MK. Type 1 T regulatory cells. Immunol Rev 2001;182:68–79.
16. Trani J, Moore DJ, Jarrett BP,Markmann JW, Lee MK, Singer A, et al.CD25+ immunoregulatory CD4 T cells mediate acquired central transplantation tolerance. J Immunol 2003;170:279–86.
17. Posselt AM, Barker CF, Friedman AL, Naji A. Prevention of autoimmune diabetes in the BB rat by intrathymic islet transplantation at birth. Science 1992;256:1321–4.
18. Shimizu J, Moriizumi E. CD4+CD25– T cells in aged mice are hyporesponsive and exhibit suppressive activity. J Immunol 2003; 170:1675–82.
19. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;299:1057–61.
20. Powrie F, LeachMW,Mauze S, Caddle LB, Coffman RL. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C B17 scid mice. Int Immunol 1993;5: 1461–71.
21. Weiner HL. The mucosal milieu creates tolerogenic dendritic cells and Tr1 and Th3 regulatory cells. Nat Immunol 2001;8:671–2.
22. Wing K, Ekmark A, Karlsson H, Rudin A, Suri-Payer E. Characterization of human CD25+CD4+ T cells in thymus, cord and adult blood. Immunology 2002; 106: 190–9.
23. Romagnoli P, Hudrisier D, van Meerwijk JP. Preferential recognition of self antigens despite normal thymic deletion of CD4(+)CD25(+) regulatory T cells. J Immunol 2002;168:1644–8.
24. Taams LS, Vukmanovic-Stejic M, Smith J, Dunne PJ, Fletcher JM, Plunkett FJ, et al. Antigen specific T cell suppression by human CD4+CD25+ regulatory T cells. Eur J Immunol 2002;32:1621–30.
25. Tung KS, Agersborg SS, Alard P, Garza KM, Lou YH. Regulatory T-cell endogenous antigen and neonatal environment in the prevention and induction of autoimmune disease. Immunol Rev 2001;182:135–48.