过敏毒素C3a在肾小球足细胞中的分泌性表达试验
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摘要
目的建立过敏毒素C3a分泌性表达慢病毒体系和分泌性高表达过敏毒素C3a的足细胞株,为研究过敏毒素C3a对足细胞的作用创造条件。方法人工合成过敏素素C3a分泌性表达基因;以重组慢病毒为媒介,将过敏毒素C3a分泌性表达基因导入到人足细胞系(human podocytes,HPC);利用药物筛选和连续梯度稀释克隆法获得稳定整合有过敏毒素C3a转基因结构的HPC细胞株;利用实时定量PCR从mRNA水平、酶联免疫吸附测定方法从蛋白质水平对过敏毒素C3a的表达和分泌情况进行鉴定。结果成功建立了过敏毒素C3a分泌性表达慢病毒体系;获得了分泌性高表达过敏毒素C3a的人肾小球足细胞株。结论所建立的过敏毒素C3a分泌性表达慢病毒体系和分泌性高表达过敏毒素C3a的足细胞株,为进一步分析过敏毒素C3a对足细胞的作用创造了条件。
Objective To pave the way for investigation of the roles of anaphylatoxin C3 a in glomerular podocytes,in the present study,an anaphylatoxin C3 a secrectory expression lentiviral system and a podocytes strain highly expressing C3 a in a secretory manner was constructed. Methods Anaphylotoxin C3 a secretory expression unit was synthesized and cloned into a lentivirus expression vector. After the sequence was confirmed by direct sequencing,recombinant lentivirus was packaged in 293 T cells. Infection of HPC,a human glomerular podocytes cell line,with the recombinant lentivirus was carried out. Blasticidin resistant cell clones were screened out. Significantly increased expression and secretion of anaphylatoxin C3 a was proved by real-time PCR and enzyme linked immunosorbent assay. Results An anaphylotoxin C3 a secrectory expression lentiviral system was correctly established. A podocyte strain highly expressing anaphylotoxin C3 a in a secretory manner was obtained. Conclusion An anaphylatoxin C3 a secrectory expression lentiviral system and a podocytes strain highly expressing C3 a in a secretory manner were successfully constructed. The podocytes strain was very useful for study of the function of C3 a/C3 aR axis in podocytes. The anaphylatoxin C3 a secrectory expression lentiviral system was also useful in study of the function and mechanisms of C3 a in other cells.
Objective To pave the way for investigation of the roles of anaphylatoxin C3 a in glomerular podocytes,in the present study,an anaphylatoxin C3 a secrectory expression lentiviral system and a podocytes strain highly expressing C3 a in a secretory manner was constructed. Methods Anaphylotoxin C3 a secretory expression unit was synthesized and cloned into a lentivirus expression vector. After the sequence was confirmed by direct sequencing,recombinant lentivirus was packaged in 293 T cells. Infection of HPC,a human glomerular podocytes cell line,with the recombinant lentivirus was carried out. Blasticidin resistant cell clones were screened out. Significantly increased expression and secretion of anaphylatoxin C3 a was proved by real-time PCR and enzyme linked immunosorbent assay. Results An anaphylotoxin C3 a secrectory expression lentiviral system was correctly established. A podocyte strain highly expressing anaphylotoxin C3 a in a secretory manner was obtained. Conclusion An anaphylatoxin C3 a secrectory expression lentiviral system and a podocytes strain highly expressing C3 a in a secretory manner were successfully constructed. The podocytes strain was very useful for study of the function of C3 a/C3 aR axis in podocytes. The anaphylatoxin C3 a secrectory expression lentiviral system was also useful in study of the function and mechanisms of C3 a in other cells.
引文
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[13]郑敬民,朱小东,张明超,等.过敏毒素受体(C3aR)在db/db糖尿病肾病小鼠肾脏中的表达及病理意义分析[J].生物化学与生物物理进展,2010,37(8):847-845.
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[15]Niebuhr M,Bumer W,Kietzmann M,et al.Participation of complement 3a receptor(C3aR)in the sensitization phase of Th2 mediated allergic contact dermatitis[J].Exp Dermatol,2012,21(1):52-56.
[16]Bao L,Wang Y,Haas M,et al.Distinct roles for C3a and C5a in complement-induced tubulointerstitial injury[J].Kidney Int,2011,80(5):524-534.
[17]Zhou W.The new face of anaphylatoxins in immune regulation[J].Immunobiology,2012,217(2):225-234.
[18]Carmona-Fontaine C,Theveneau E,Tzekou A,et al.Complement fragment C3a controls mutual cell attraction during collective cell migration[J].Dev Cell,2011,21(6):1026-1037.
[19]Yu M,Zou W,Peachey NS,et al.A novel role of complement in retinal degeneration[J].Invest Ophthalmol Vis Sci,2012,53(12):7684-7692.
[20]Tu Z,Bu H,Dennis JE,et al.Efficient osteoclast differentiation requires local complement activation[J].Blood,2010,116(22):4456-4463.
[21]Lim J,Iyer A,Suen JY,et al.C5aR and C3aR antagonists each inhibit diet-induced obesity,metabolic dysfunction,and adipocyte and macrophage signaling[J].FASEB J,2013,27(2):822-831.
[22]Xu XH,Peng HS,Sun MQ,et al.C-terminal peptide of anaphylatoxin C3a enhances hepatic function after steatotic liver transplantation:a study in a rat model[J].Transplant Proc,2010,42(3):737-740.
[2]Schramm EC,Clark SJ,Triebwasser MP,et al.Genetic variants in the complement system predisposing to age-related macular degeneration:a review[J].Mol Immunol,2014,61(2):118-125.
[3]Peterson SL,Anderson AJ.Complement and spinal cord injury:traditional and non-traditional aspects of complement cascade function in the injured spinal cord microenvironment[J].Exp Neurol,2014,258:35-47.
[4]Hertle E,Stehouwer CD,van Greevenbroek MM.The complement system in human cardiometabolic disease[J].Mol Immunol,2014,61(2):135-148.
[5]甘燕玲,杨至宜,孙朝晖,等.快速CRP检测对儿童上呼吸道感染的鉴别诊断价值[J].华南国防医学杂志,2011,25(3):264-265.
[6]陈金弟,王智华,李华良,等.抗脑抗体对精神分裂症自身免疫反应的探讨[J].东南国防医药,2004,6(3):171-172.
[7]任红旗,吴梅月,蔡青,等.肾淀粉样变性10例临床病理分析[J].东南国防医药,2011,13(4):348-349.
[8]Fearn A,Sheerin NS.Complement activation in progressive renal disease[J].World J Nephrol,2015,4(1):31-40.
[9]Thurman JM1.Complement in kidney disease:core curriculum2015[J].Am J Kidney Dis,2015,65(1):156-168.
[10]郑敬民,尹广,姚根宏,等.肾病患者肾组织补体活化与肥大细胞滑润的关系研究[J].医学研究生学报,2012,25(10):1040-1044.
[11]Klos A,Wende E,Wareham KJ,et al.International Union of Basic and Clinical Pharmacology[corrected].LXXXVII.Complement peptide C5a,C4a,and C3a receptors[J].Pharmacol Rev,2013,65(1):500-543.
[12]Braun MC,Reins RY,Li TB,et al.Renal expression of the C3a receptor and functional responses of primary human proximal tubular epithelial cells[J].J Immunol,2004,173(6):4190-4196.
[13]郑敬民,朱小东,张明超,等.过敏毒素受体(C3aR)在db/db糖尿病肾病小鼠肾脏中的表达及病理意义分析[J].生物化学与生物物理进展,2010,37(8):847-845.
[14]Mueller-Ortiz SL,Morales JE,Wetsel RA.The receptor for the complement C3a anaphylatoxin(C3aR)provides host protection against listeria monocytogenes-induced apoptosis[J].J Immunol,2014,193(3):1278-1289.
[15]Niebuhr M,Bumer W,Kietzmann M,et al.Participation of complement 3a receptor(C3aR)in the sensitization phase of Th2 mediated allergic contact dermatitis[J].Exp Dermatol,2012,21(1):52-56.
[16]Bao L,Wang Y,Haas M,et al.Distinct roles for C3a and C5a in complement-induced tubulointerstitial injury[J].Kidney Int,2011,80(5):524-534.
[17]Zhou W.The new face of anaphylatoxins in immune regulation[J].Immunobiology,2012,217(2):225-234.
[18]Carmona-Fontaine C,Theveneau E,Tzekou A,et al.Complement fragment C3a controls mutual cell attraction during collective cell migration[J].Dev Cell,2011,21(6):1026-1037.
[19]Yu M,Zou W,Peachey NS,et al.A novel role of complement in retinal degeneration[J].Invest Ophthalmol Vis Sci,2012,53(12):7684-7692.
[20]Tu Z,Bu H,Dennis JE,et al.Efficient osteoclast differentiation requires local complement activation[J].Blood,2010,116(22):4456-4463.
[21]Lim J,Iyer A,Suen JY,et al.C5aR and C3aR antagonists each inhibit diet-induced obesity,metabolic dysfunction,and adipocyte and macrophage signaling[J].FASEB J,2013,27(2):822-831.
[22]Xu XH,Peng HS,Sun MQ,et al.C-terminal peptide of anaphylatoxin C3a enhances hepatic function after steatotic liver transplantation:a study in a rat model[J].Transplant Proc,2010,42(3):737-740.