寻找慢性移植肾失功患者血清标志物及对候选标志物Galectin-7的初步研究
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摘要
目的：慢性移植物失功(chronic allograft dysfunction，CGD)是影响移植肾长期存活的最重要的因素，但是它准确的诊断、发病机制和治疗措施还不十分明确。肾移植后诊断慢性移植物失功最可靠的方法是肾穿刺活检，但这种侵入性的诊断方法存在一些并发症，严重的可造成患者死亡。本研究的目的是利用蛋白组学二维差异性凝胶电泳(two-dimensional differential in-gel electrophoresis，2-D DIGE)和反相高效液相色谱法(reversed phase high-performance liquid chromatography，RP-HPLC)联合电喷射离子化质谱(electrospray ionization mass spectrometry mass spectrometry，ESI／MS)技术寻找慢性移植肾失功患者血清生物标志物，并对找到的血清候选标志物半乳糖苷凝集素-7(Galectin-7)在慢性移植物失功患者移植肾组织的表达以及其对免疫功能的影响进行了进一步的研究。
方法：：1、血清样本分成4组：慢性移植物失功组(CGD)、移植后长期肾功能稳定组(long-term stable renal function，SRF)、急性排斥组(acute rejection，AR)和健康对照组(normal volunteer，N)。血清样本经多重亲和吸附柱去除高丰度蛋白处理后，使用2-D DIGE技术找到差异蛋白点。切下这些差异蛋白点，胰蛋白酶消化，经RP-HPLC-ESI／MS分析鉴定，在另一独立样本中对鉴定出的差异蛋白Galectin-7和Clusterin进行了ELISA验证；2、利用免疫组化的方法对慢性移植物失功(CGD)，移植后长期肾功能稳定(SRF)和急性排斥(AR)患者移植肾活检标本Galectin-7的表达进行了检测；3、在体外利用不同浓度(0．2μg／mL，0．5μg／mL，1μg／mL，2μg／mL)的重组Galectin-7蛋白在有或无抗CD3和CD28抗体存在时与纯化的小鼠T淋巴细胞共同培养5天，流式细胞仪检测T淋巴细胞及各亚群的增殖情况。在体外利用另一浓度梯度(2ng／mL，4ng／mL，8ng／mL，20ng／mL，200ng／mL)的重组Galectin-7蛋白与纯化的小鼠T淋巴细胞共同培养4小时和24小时，流式细胞仪检测T淋巴细胞的早期凋亡和中晚期凋亡的情况。
结果：1、慢性移植物失功(CGD)组与其它三组比较，血清中的差异表达蛋白有39个，质谱鉴定出22个差异表达蛋白，包括载脂蛋白A-I前体、补体C4-A前体、补体H因子相关蛋白1前体、补体C9前体、Galectin-7、Clusterin前体、homerin、β2糖蛋白1前体等。ELISA结果提示，慢性移植物失功(CGD)患者血清中Galectin-7的表达较移植后长期肾功能稳定(SRF)患者和健康志愿者(N)有明显增高，慢性移植物失功(CGD)患者血清中Clusterin的表达较移植后长期肾功能稳定(SRF)患者有明显增高。2、在慢性移植物失功(CGD)患者移植肾活检标本中发现萎缩的肾小管上皮细胞的胞浆有Galectin-7的高表达，并且慢性移植物失功(CGD)组肾活检标本每个高倍视野下Galectin-7阳性细胞数较移植后长期肾功能稳定(SRF)组和急性排斥(AR)组明显增多；3、体外研究发现，重组Galectin-7蛋白(0．2μg／mL，0．5μg／mL，1μg／mL，2μg／mL)能明显诱导小鼠T淋巴细胞的增殖，在与抗CD3和抗CD28抗体联用时能协同促进T淋巴细胞增殖反应，增殖的细胞主要为CD8+T淋巴细胞；重组Galectin-7蛋白(2ng／mL，4ng／mL，8ng／mL，20ng／mL，200ng／mL)在与小鼠T淋巴培养4小时和24小时后发现Galectin-7对T淋巴细胞的早期凋亡和中晚期凋亡均无影响。
结论：移植肾慢性移植物失功患者血清中有一组表达差异的蛋白质，可区别于移植后长期肾功能稳定、急性排斥者和健康对照者，这些血清差异蛋白具有不同的生物功能，本研究的探索为大样本、多中心的验证奠定了基础。Galectin-7和Clusterin可能是慢性移植物失功(CGD)的血清标志物。Galectin-7在慢性移植物失功(CGD)患者血清和移植肾活检组织中高表达；重组Galectin-7蛋白在体外具有诱导和促进T淋巴细胞增殖的作用，提示Galectin-7可能在慢性移植物失功(CGD)的发病机制中发挥重要作用，Galectin-7可能成为慢性移植物失功(CGD)治疗的新靶点。
Objective:Most late renal allograft failure is attributed to chronic allograft dysfunction(CGD),but its precise diagnosis,pathogenesis and treatment are largely unknown.Renalbiopsy remains the most reliable method for detecting CGD post-transplantation,but it isassociated with significant patient morbidity and mortality.The purpose of this study was toidentify biomarkers of chronic renal allograft dysfunction in human serum usingtwo-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phasehigh-performance liquid chromatography (RP-HPLC) followed by electrospray ionizationmass spectrometry (ESI-MS).The expression of Galectin-7,a candidate biomarker,inallograft tissue and the effect of Galectin-7 on immunological function were further studied.
Methods:Serum samples were divided into four groups:chronic allograft dysfunction(CGD),long-term stable renal function (SRF),acute rejection (AR) and normal volunteer (N).Firstly,the highest abundance proteins in serum samples were selectively removed usingMultiple Affinity Removal Column.Differential protein analysis was performed using 2-DDIGE.These spot features were excised,digested by trypsin,and analyzed byRP-HPLC-ESI/MS.Galectin-7 and Clusterin,two of these identified proteins was confirmedby ELISA analysis in the independent set of serum samples.The expression of Galectin-7 inrenal biopsy specimen from CGD,SRF and AR groups was detected using immunohistochemistry technology.Recombined Galectin-7 at different concentrations(0.2μg/mL,0.5μg/mL,1μg/mL,2μg/mL) and T lymphocytes of mouse had been coculturedwith or without anti-CD3 and anti-CD28 antibody for 5 days in vitro.The proliferation of Tlymphocytes and subpopulations was analyzed using FACS.Recombined Galectin-7 atanother concentration gradient (2ng/mL,4ng/mL,8ng/mL,20ng/mL,200ng/mL) had beencocultured with T lymphocytes of mouse for 4 hours and 24 hours in vitro.The earlyapoptosis and late apoptosis of T lymphocytes were analyzed using FACS.
Results:A total of 39 protein spots were found to be significantly up regulated or downregulated in serum from CGD group compared to other three groups.22 differentiallyexpressed proteins were identified in CGD serum,including apolipoprotein A-I precursor,complement C4-A precursor,complement factor H-related protein 1 precursor,component C9precursor,galectin-7,clusterin precursor,homerin,Beta-2-glycoprotein 1 precursor,et al.ELISA result showed that Galetin-7 was significantly up regulated in serum from CGD groupcompared to SRF group and to N group and Clusterin was significantly up regulated in serumfrom CGD group compared to SRF group.Galectin-7 was high expressed in endochylema ofatrophied tubular epithelial cells of renal biopsy specimen from CGD patients.The count ofGalectin-7 positive cells per high power field was significantly greater in renal biopsyspecimen from CGD group compared to SRF group and to AR group.In vitro recombinedGalectin-7(0.2μg/mL,0.5μg/mL,1μg/mL,2μg/mL) could significantly induce theproliferation of T lymphocytes of mouse.Recombined Galectin-7 could synergy to promotethe proliferation of T lymphocytes when it was used with anti-CD3 and anti-CD28 Antibody.CD8+T lymphocytes were the major proliferated T lymphocytes.RecombinedGalectin-7(2ng/mL,4ng/mL,8ng/mL,20ng/mL,200ng/mL) had no effect on the earlyapoptosis and late apoptosis of T lymphocytes of mouse after 4 hours or 24 hours' cultivationwith T lymphocytes.
Conclusion:There are set of differentially expressed proteins from CGD groupcompared with AR,SRF and N groups.These differentially expressed proteins were involvedin different physiologic functions and this research provided the basis for further validation oflarger sample from multiple centers.Our data also showed that confirmed Galectin-7 andClusterin may be serum biomarkers of CGD patients.Galectin-7 was significantly up regulated in serum and renal biopsy specimen from CGD group.Recombined Galectin-7promoted the proliferation of T lymphocyte but not apoptosis in vitro which suggestsGalectin-7 may play an important role in mechanisms of CGD.Galectin-7 may become a newtarget for treatment of CGD.
方法：：1、血清样本分成4组：慢性移植物失功组(CGD)、移植后长期肾功能稳定组(long-term stable renal function，SRF)、急性排斥组(acute rejection，AR)和健康对照组(normal volunteer，N)。血清样本经多重亲和吸附柱去除高丰度蛋白处理后，使用2-D DIGE技术找到差异蛋白点。切下这些差异蛋白点，胰蛋白酶消化，经RP-HPLC-ESI／MS分析鉴定，在另一独立样本中对鉴定出的差异蛋白Galectin-7和Clusterin进行了ELISA验证；2、利用免疫组化的方法对慢性移植物失功(CGD)，移植后长期肾功能稳定(SRF)和急性排斥(AR)患者移植肾活检标本Galectin-7的表达进行了检测；3、在体外利用不同浓度(0．2μg／mL，0．5μg／mL，1μg／mL，2μg／mL)的重组Galectin-7蛋白在有或无抗CD3和CD28抗体存在时与纯化的小鼠T淋巴细胞共同培养5天，流式细胞仪检测T淋巴细胞及各亚群的增殖情况。在体外利用另一浓度梯度(2ng／mL，4ng／mL，8ng／mL，20ng／mL，200ng／mL)的重组Galectin-7蛋白与纯化的小鼠T淋巴细胞共同培养4小时和24小时，流式细胞仪检测T淋巴细胞的早期凋亡和中晚期凋亡的情况。
结果：1、慢性移植物失功(CGD)组与其它三组比较，血清中的差异表达蛋白有39个，质谱鉴定出22个差异表达蛋白，包括载脂蛋白A-I前体、补体C4-A前体、补体H因子相关蛋白1前体、补体C9前体、Galectin-7、Clusterin前体、homerin、β2糖蛋白1前体等。ELISA结果提示，慢性移植物失功(CGD)患者血清中Galectin-7的表达较移植后长期肾功能稳定(SRF)患者和健康志愿者(N)有明显增高，慢性移植物失功(CGD)患者血清中Clusterin的表达较移植后长期肾功能稳定(SRF)患者有明显增高。2、在慢性移植物失功(CGD)患者移植肾活检标本中发现萎缩的肾小管上皮细胞的胞浆有Galectin-7的高表达，并且慢性移植物失功(CGD)组肾活检标本每个高倍视野下Galectin-7阳性细胞数较移植后长期肾功能稳定(SRF)组和急性排斥(AR)组明显增多；3、体外研究发现，重组Galectin-7蛋白(0．2μg／mL，0．5μg／mL，1μg／mL，2μg／mL)能明显诱导小鼠T淋巴细胞的增殖，在与抗CD3和抗CD28抗体联用时能协同促进T淋巴细胞增殖反应，增殖的细胞主要为CD8+T淋巴细胞；重组Galectin-7蛋白(2ng／mL，4ng／mL，8ng／mL，20ng／mL，200ng／mL)在与小鼠T淋巴培养4小时和24小时后发现Galectin-7对T淋巴细胞的早期凋亡和中晚期凋亡均无影响。
结论：移植肾慢性移植物失功患者血清中有一组表达差异的蛋白质，可区别于移植后长期肾功能稳定、急性排斥者和健康对照者，这些血清差异蛋白具有不同的生物功能，本研究的探索为大样本、多中心的验证奠定了基础。Galectin-7和Clusterin可能是慢性移植物失功(CGD)的血清标志物。Galectin-7在慢性移植物失功(CGD)患者血清和移植肾活检组织中高表达；重组Galectin-7蛋白在体外具有诱导和促进T淋巴细胞增殖的作用，提示Galectin-7可能在慢性移植物失功(CGD)的发病机制中发挥重要作用，Galectin-7可能成为慢性移植物失功(CGD)治疗的新靶点。
Objective:Most late renal allograft failure is attributed to chronic allograft dysfunction(CGD),but its precise diagnosis,pathogenesis and treatment are largely unknown.Renalbiopsy remains the most reliable method for detecting CGD post-transplantation,but it isassociated with significant patient morbidity and mortality.The purpose of this study was toidentify biomarkers of chronic renal allograft dysfunction in human serum usingtwo-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phasehigh-performance liquid chromatography (RP-HPLC) followed by electrospray ionizationmass spectrometry (ESI-MS).The expression of Galectin-7,a candidate biomarker,inallograft tissue and the effect of Galectin-7 on immunological function were further studied.
Methods:Serum samples were divided into four groups:chronic allograft dysfunction(CGD),long-term stable renal function (SRF),acute rejection (AR) and normal volunteer (N).Firstly,the highest abundance proteins in serum samples were selectively removed usingMultiple Affinity Removal Column.Differential protein analysis was performed using 2-DDIGE.These spot features were excised,digested by trypsin,and analyzed byRP-HPLC-ESI/MS.Galectin-7 and Clusterin,two of these identified proteins was confirmedby ELISA analysis in the independent set of serum samples.The expression of Galectin-7 inrenal biopsy specimen from CGD,SRF and AR groups was detected using immunohistochemistry technology.Recombined Galectin-7 at different concentrations(0.2μg/mL,0.5μg/mL,1μg/mL,2μg/mL) and T lymphocytes of mouse had been coculturedwith or without anti-CD3 and anti-CD28 antibody for 5 days in vitro.The proliferation of Tlymphocytes and subpopulations was analyzed using FACS.Recombined Galectin-7 atanother concentration gradient (2ng/mL,4ng/mL,8ng/mL,20ng/mL,200ng/mL) had beencocultured with T lymphocytes of mouse for 4 hours and 24 hours in vitro.The earlyapoptosis and late apoptosis of T lymphocytes were analyzed using FACS.
Results:A total of 39 protein spots were found to be significantly up regulated or downregulated in serum from CGD group compared to other three groups.22 differentiallyexpressed proteins were identified in CGD serum,including apolipoprotein A-I precursor,complement C4-A precursor,complement factor H-related protein 1 precursor,component C9precursor,galectin-7,clusterin precursor,homerin,Beta-2-glycoprotein 1 precursor,et al.ELISA result showed that Galetin-7 was significantly up regulated in serum from CGD groupcompared to SRF group and to N group and Clusterin was significantly up regulated in serumfrom CGD group compared to SRF group.Galectin-7 was high expressed in endochylema ofatrophied tubular epithelial cells of renal biopsy specimen from CGD patients.The count ofGalectin-7 positive cells per high power field was significantly greater in renal biopsyspecimen from CGD group compared to SRF group and to AR group.In vitro recombinedGalectin-7(0.2μg/mL,0.5μg/mL,1μg/mL,2μg/mL) could significantly induce theproliferation of T lymphocytes of mouse.Recombined Galectin-7 could synergy to promotethe proliferation of T lymphocytes when it was used with anti-CD3 and anti-CD28 Antibody.CD8+T lymphocytes were the major proliferated T lymphocytes.RecombinedGalectin-7(2ng/mL,4ng/mL,8ng/mL,20ng/mL,200ng/mL) had no effect on the earlyapoptosis and late apoptosis of T lymphocytes of mouse after 4 hours or 24 hours' cultivationwith T lymphocytes.
Conclusion:There are set of differentially expressed proteins from CGD groupcompared with AR,SRF and N groups.These differentially expressed proteins were involvedin different physiologic functions and this research provided the basis for further validation oflarger sample from multiple centers.Our data also showed that confirmed Galectin-7 andClusterin may be serum biomarkers of CGD patients.Galectin-7 was significantly up regulated in serum and renal biopsy specimen from CGD group.Recombined Galectin-7promoted the proliferation of T lymphocyte but not apoptosis in vitro which suggestsGalectin-7 may play an important role in mechanisms of CGD.Galectin-7 may become a newtarget for treatment of CGD.
引文
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