系统性红斑狼疮相关T细胞克隆的实验研究
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摘要
目的:通过研究系统性红斑狼疮患者T细胞受体可变区β链互补决定区3(TCRVβCDR3)的表达,寻找与病证相关的T细胞克隆。
方法:收集系统性红斑狼疮患者12例,根据痰瘀辨证标准进行分类,设立正常对照6例和类风湿关节炎对照5例。每例抽取5ml外周血,运用RT—PCR技术,扩增TCRVβ25个家族的CDR3区,通过长泳道测序胶电泳观察TCRVβCDR3谱型图,分析T细胞表达特征;对发生优势克隆增生的异常条带进行二次PCR,测序,进行比对分析。
结果:1.正常人外周血TCRβ链的25个V基因家族均有表达,条带的强弱呈现正态的高斯(Gaussian)分布。2.系统性红斑狼疮患者与正常人群相比,发生异常变化的TCRVβ家族明显增多,具有统计学意义(P＜0.05);而类风湿性关节炎患者与正常组相比,发生异常的家族虽较之有一定增多,但无统计学意义。3.痰瘀兼夹型系统性红斑狼疮组比正常人群异常TCRVβ家族数目明显增多(P＜0.01),与非痰瘀兼夹型SLE组相比有统计学意义(P＜0.01);而非痰瘀兼夹型系统性红斑狼疮患者其异常家族虽较之正常组增多,但与正常人群相比无意义。兼夹证型较之单一证型异常家族数目明显增多。4.编号为SLE5、SLE7、RA1的患者Vβ25CDR3区完全一致(CASSLRGSTEAFF),三者辨证均属于血瘀证;编号为SLE1和RA1的患者Vβ12CDR3区完全一致(CAISELREGDMSSGNTIYF),SLE1为痰瘀兼夹证,RA1为血瘀证;编号为SLE12、RA3的患者Vβ4CDR3区完全一致(CSVGGLAGETQYF),SLE12为痰瘀兼夹证,RA3为血瘀证;编号SLE9的患者与造血干细胞移植后GVHD和CMV相关的异常扩增的T淋巴细胞CDR3序列完全相同(CASSPGDEQFF),属于Vβ11家族,SLE9为非痰非瘀型缓解期患者。
结论1.TCRVβ谱系可作为系统性红斑狼疮一个微观辨证参考指标。2.不同自身免疫病有相同的序列,推测LREGDMS、LRGS、GGLAG、PGD是与自身免疫病发病相关的模体。3.同一证型可能存在着有相同序列的T细胞。CASSLRGSTEAFF、CAISELREGDMSSGNTIYF、CSVGGLAGETQYF可能与血瘀证具有一定的相关性,模体为LREGDMS、LRGS、GGLAG的T细胞可能是在自身免疫病中,与血瘀证发生相关的T细胞,其活化所介导的自身免疫反应可能使患者有表现为血瘀证的倾向。
Object:Through researching the change of variable region of SLE patients' T cell receptorβchain(TCRVβ),to search T cell clone which related with disease syndrome.
Method:Collected 12 cases of SLE patients,classified on the base of Phlegm and Blood stasis,set up normal contrast group and RA control group.Each case was draw-off 5ml of peripheral blood,deployed RT-PCR technique,amplified the district of TCRVβCDR3.Through silver staining of denaturing polyacrylamide gel electrophoresis to observe the spectral pattern picture of TCRVβand analyzed the characteristic of T cell clone's hyperplasy;carded out twice PCR on the hyperplastic abnormal strap,sequenced,compared and analyzed.
Result:1.25 V gene family of TCRβin health adult PB had expression,the powerfulness and weakness of strap presented Gaussian distribution.2.Compared SLE patient with health people,the abnormal change of TCRVβfamily clan increased obviously,it had statistical significance(P=0.013＜0.05);compared RA patient with health people,although abnormal family clan had invariably increase, it had no statistical significance.3.The amount of abnormal TCRVβfamily clan in Phlegm and Blood stasis pattern of SLE patient had obviously increase compared health people(P=0.000＜0.01),it had statistical significance compared non-Phlegm and Blood stasis pattern of SLE patient(P=0.009＜0.01);non-Phlegm and Blood stasis pattern of SLE patient's abnormal family clan had increase compared health people,it had no statistical significance,but it had significant statistical difference compared Phlegm and Blood stasis pattern.Complex pattern increased obviously than single pattern.4.Number SLE5,SLE7 and RA1 patients' Vβ25CDR.3 districts were completely coincident(CASSLRGSTEAFF).The three kinds of patients differentiated Blood stasis;number SLE1 and RA1 patients' Vβ12CDR3 districts were completely coincident(CAISELREGDMSSGNTIYF);SLE1 was Phlegm complex Blood stasis,RA1 was Blood stasis.Number SLE12 and RA3 patients' Vβ4CDR3 districts were completely coincident(CSVGGLAGETQYF), SLE12 was Phlegm complex Blood stasis,RA3 was Blood stasis.Number SLE9 patient was coincident with abnormal amplification of T cell CDR3 series related GVHD and CMV of HBSC PG(CASSPGDEQFF),it belong to VB11 family, SLE9 was non-Phlegm and non-Blood stasis paracmasis patient.
Conclusion:1.TCRVβpedigree can use as one of micro indexes to evaluate Phlegm and Blood stagnated SLE.2.Different autoallergic disease had identical series,it inferred LREGDMS,LRGS,GGLAG and PGD were motifs related with autoallergic disease antigen.3.Pattern had its material foundation,the same pattern might exist identical series,it personalized on molecular level.CASSLRGSTEAFF, CAISELREGDMSSGNTIYF and CSVGGLAGETQYF might have invariably dependability with Blood stasis pattern.Motifs LREGDMS、LRGS、GGLAG might the T cells those dependent with Blood stasis pattern in autoallergic disease.
方法:收集系统性红斑狼疮患者12例,根据痰瘀辨证标准进行分类,设立正常对照6例和类风湿关节炎对照5例。每例抽取5ml外周血,运用RT—PCR技术,扩增TCRVβ25个家族的CDR3区,通过长泳道测序胶电泳观察TCRVβCDR3谱型图,分析T细胞表达特征;对发生优势克隆增生的异常条带进行二次PCR,测序,进行比对分析。
结果:1.正常人外周血TCRβ链的25个V基因家族均有表达,条带的强弱呈现正态的高斯(Gaussian)分布。2.系统性红斑狼疮患者与正常人群相比,发生异常变化的TCRVβ家族明显增多,具有统计学意义(P＜0.05);而类风湿性关节炎患者与正常组相比,发生异常的家族虽较之有一定增多,但无统计学意义。3.痰瘀兼夹型系统性红斑狼疮组比正常人群异常TCRVβ家族数目明显增多(P＜0.01),与非痰瘀兼夹型SLE组相比有统计学意义(P＜0.01);而非痰瘀兼夹型系统性红斑狼疮患者其异常家族虽较之正常组增多,但与正常人群相比无意义。兼夹证型较之单一证型异常家族数目明显增多。4.编号为SLE5、SLE7、RA1的患者Vβ25CDR3区完全一致(CASSLRGSTEAFF),三者辨证均属于血瘀证;编号为SLE1和RA1的患者Vβ12CDR3区完全一致(CAISELREGDMSSGNTIYF),SLE1为痰瘀兼夹证,RA1为血瘀证;编号为SLE12、RA3的患者Vβ4CDR3区完全一致(CSVGGLAGETQYF),SLE12为痰瘀兼夹证,RA3为血瘀证;编号SLE9的患者与造血干细胞移植后GVHD和CMV相关的异常扩增的T淋巴细胞CDR3序列完全相同(CASSPGDEQFF),属于Vβ11家族,SLE9为非痰非瘀型缓解期患者。
结论1.TCRVβ谱系可作为系统性红斑狼疮一个微观辨证参考指标。2.不同自身免疫病有相同的序列,推测LREGDMS、LRGS、GGLAG、PGD是与自身免疫病发病相关的模体。3.同一证型可能存在着有相同序列的T细胞。CASSLRGSTEAFF、CAISELREGDMSSGNTIYF、CSVGGLAGETQYF可能与血瘀证具有一定的相关性,模体为LREGDMS、LRGS、GGLAG的T细胞可能是在自身免疫病中,与血瘀证发生相关的T细胞,其活化所介导的自身免疫反应可能使患者有表现为血瘀证的倾向。
Object:Through researching the change of variable region of SLE patients' T cell receptorβchain(TCRVβ),to search T cell clone which related with disease syndrome.
Method:Collected 12 cases of SLE patients,classified on the base of Phlegm and Blood stasis,set up normal contrast group and RA control group.Each case was draw-off 5ml of peripheral blood,deployed RT-PCR technique,amplified the district of TCRVβCDR3.Through silver staining of denaturing polyacrylamide gel electrophoresis to observe the spectral pattern picture of TCRVβand analyzed the characteristic of T cell clone's hyperplasy;carded out twice PCR on the hyperplastic abnormal strap,sequenced,compared and analyzed.
Result:1.25 V gene family of TCRβin health adult PB had expression,the powerfulness and weakness of strap presented Gaussian distribution.2.Compared SLE patient with health people,the abnormal change of TCRVβfamily clan increased obviously,it had statistical significance(P=0.013＜0.05);compared RA patient with health people,although abnormal family clan had invariably increase, it had no statistical significance.3.The amount of abnormal TCRVβfamily clan in Phlegm and Blood stasis pattern of SLE patient had obviously increase compared health people(P=0.000＜0.01),it had statistical significance compared non-Phlegm and Blood stasis pattern of SLE patient(P=0.009＜0.01);non-Phlegm and Blood stasis pattern of SLE patient's abnormal family clan had increase compared health people,it had no statistical significance,but it had significant statistical difference compared Phlegm and Blood stasis pattern.Complex pattern increased obviously than single pattern.4.Number SLE5,SLE7 and RA1 patients' Vβ25CDR.3 districts were completely coincident(CASSLRGSTEAFF).The three kinds of patients differentiated Blood stasis;number SLE1 and RA1 patients' Vβ12CDR3 districts were completely coincident(CAISELREGDMSSGNTIYF);SLE1 was Phlegm complex Blood stasis,RA1 was Blood stasis.Number SLE12 and RA3 patients' Vβ4CDR3 districts were completely coincident(CSVGGLAGETQYF), SLE12 was Phlegm complex Blood stasis,RA3 was Blood stasis.Number SLE9 patient was coincident with abnormal amplification of T cell CDR3 series related GVHD and CMV of HBSC PG(CASSPGDEQFF),it belong to VB11 family, SLE9 was non-Phlegm and non-Blood stasis paracmasis patient.
Conclusion:1.TCRVβpedigree can use as one of micro indexes to evaluate Phlegm and Blood stagnated SLE.2.Different autoallergic disease had identical series,it inferred LREGDMS,LRGS,GGLAG and PGD were motifs related with autoallergic disease antigen.3.Pattern had its material foundation,the same pattern might exist identical series,it personalized on molecular level.CASSLRGSTEAFF, CAISELREGDMSSGNTIYF and CSVGGLAGETQYF might have invariably dependability with Blood stasis pattern.Motifs LREGDMS、LRGS、GGLAG might the T cells those dependent with Blood stasis pattern in autoallergic disease.
引文
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