主要组织相容性复合体Ⅰ类分子相关A基因（MICA）抗体与临床肝移植排斥的相关性实验研究
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摘要
目的:本研究旨在通过对主要组织相容性复合体I类分子相关A基因(MICA)的多态性检测,探讨MICA抗体在肝移植排斥反应过程中的作用机制。本研究包括:
1)通过对主要组织相容性复合体I类分子相关A基因(MICA)转染的细胞系检测肝移植病人的血清,运用流式细胞仪检测是否产生针对MICA的抗体来探讨MICA抗体在肝移植免疫排斥反应中的影响机制。
2)运用PCR方法检测22例发生肝移植排斥反应供、受体病人血清中的MICA的等位基因型,确定MICA*008等位基因在肝移植排斥反应中的作用机制。
3)运用免疫组织化学方法进一步证实MICA抗原与肝移植排斥反应的关系。
方法:
课题第一部分:
1)转染MICA的人类B淋巴母细胞细胞系(721.221)通过细胞培养后,同84例接受肝移植手术的病人的血清进行流式细胞仪检测,实验分为五组,第一组:MICA转染细胞(5×10~5个)+羊抗鼠抗体2微升(二抗,荧光抗体);第二组:MICA转染细胞(5×10~5个)+抗MICA抗体(一抗,荧光抗体)+羊抗鼠抗体2微升(二抗,荧光抗体);第三组:MICA转染细胞(5×10~5个)+AB血清(阴性对照)+羊抗人(IgG+IgM)10微升(二抗);第四组:MICA转染细胞(5×10~5个)+阳性对照病人血清+羊抗人(IgG+IgM)10微升(二抗);第五组:MICA转染细胞(5×10~5个)+抗MICA抗体(一抗,荧光抗体)+产生排斥反应的病人的血清+羊抗人(IgG+IgM)10微升(二抗)。各组再用抗MICA单克隆抗体封闭,以检测血清中的抗体是否是MICA特异性抗体。
2)MICA转染细胞、胆管上皮细胞(BEC)、肝窦内皮细胞(LSEC)和肝细胞与5μl的抗MICA抗体结合,加入萤光抗体羊抗鼠IgG,分别行流式细胞仪检测,MICA的未转染细胞和MICA转染细胞分别作为阴性和阳性对照。
课题第二部分:
1)将22例发生肝移植排斥反应病人供体、受体的血清分别提取DNA并以分光光度计进行检测,采用PCR方法和标有MICA等位基因序列的引物进行电泳检测,分析记录电泳图,并与MICA等位基因图谱相对照得出各自的MICA等位基因型。
2)从健康肝脏中分别提取胆管上皮细胞(BEC)、肝窦内皮细胞(LSEC)和肝细胞,细胞经过培养后,提取RNA及cDNA合成,以葡萄糖-6-磷酸盐脱氢酶(G6PD)被当作内生的正常对照,MICA的第一代细胞系和MICA转染细胞分别作为阴性和阳性对照组,分别行RT-PCR检测和免疫印记检测。
课题第三部分:将已经病理检查确诊的13例肝移植并发生排斥反应病人的肝穿刺标本、15例没有发生排斥反应的肝移植病人的肝穿刺标本和11例患有结肠癌病人的结肠标本行免疫组织化学检查。其中没有发生排斥反应病人的标本设为阳性对照,患有结肠癌病人的结肠标本作为阴性对照。
结果:
1)总共22例(22/84)(26%)术前或/和术后病人产生MICA抗体,其中8例(8/84)(9.5%)为术前病人,14例(14/84)(17%)为术后病人,84例病人中,共有43例(43/84)发生排斥反应,在有MICA抗体组中,排斥发生率为63%(14/22),在没有MICA抗体组中,排斥发生率为47%(29/62),二者相比没有统计学差异。在非转染细胞组(721.221)没有发现MICA表达,而在转染组提示有高表达;用抗MICA单克隆抗体封闭后,血清与MICA转染的细胞系的结合被抑制,提示在这些病人血清中检测到的抗体是MICA特异性抗体。在MICA抗体阳性组和阴性组中,无论发生排斥反应及病人的存活与否,两组之间都没有临床联系。
2)流式细胞仪分析显示,MICA的表达在MICA转染细胞的表面上;而做为阴性对照,胆管上皮细胞、肝窦内皮细胞或肝细胞并没有发生任何变化,提示MICA的表达并不在这些细胞表面上。
3)在肝移植供、受体中出现频率最高的MICA等位基因是MICA*008,在9例病人供体和受体的抗体当中检测到了MICA*008,其中,4例病人的供体和受体检测出MICA*008完全相配;发现在4例病人中,供体不匹配的MICA等位基因是MICA*008;5例病人中,供体不匹配的MICA等位基因不是MICA*002就是MICA*010。在14/22(63%)例发生排斥反应的病人中可以检测到MICA*008细胞系抗体,而在29/62(47%)例病人中没有检测到MICA*008细胞系抗体,二者之间没有显著性差异(P>0.05)。
4)提取的新鲜肝脏细胞和抗MICA单克隆抗体经过免疫印迹法检测显示,62kDa条带与MICA多肽的相一致,这一结果仅见于MICA转染细胞而不是胆管上皮细胞、肝窦内皮细胞和肝细胞。
5)在那些发生排斥反应和没有发生排斥反应的肝移植病人标本中仅发现轻度的MICA抗原染色,而那些患有结肠癌并当作阴性对照组的病人,免疫组化显色后显示在上皮细胞有很强和散在的细胞质染色。
结论:
1)MICA的表达仅在MICA转染细胞的表面上,而在胆管上皮细胞、肝窦内皮细胞或肝细胞并没有发生表达,提示MICA的表达并不在这些细胞表面上。
2)MICA抗原仅在肝移植病人及发生排斥反应的肝移植病人的肝组织中发生微弱的表达,但却在结肠癌病人的结肠上皮组织中发生强烈表达。
3)统计结果显示,在肝移植预留抗体中没有发现MICA等位基因的表达,与肾移植相比较,MICA抗原不是引起肝移植排斥反应的目标抗原
Objective:
1)This research was on the polymorphism detection of Major Histrocompitbility complex ClassⅠreleted gene A (MICA) and determine the mechanism of MICA antibodies in liver transplantation rejection. The total experiment includes following sections: 1) Inspect the serum of patients who underwent liver transplantation with MICA tansfected cell line, detect patients serum with flow cytomertry for finding whether it had MICA antibodies or not, then determine the mechanism of MICA antibodies in liver transplantation rejection.
2)Using PCR method to detect the MICA alleles in serum of 22 cases in both donors and recipients who underwent liver transplantation rejection, and identify the mechanism of the MICA*008 allele in liver transplantation rejection.
3)Using the method of IHC to further verifying the methanism between MICA antigens and liver transplantation rejection.
Method:
First part of experement:
1)Test the transfected human B lymphoblastic cell line (721. 221) after cell culturing with serum of 84 cases who underwent liver transplantation, all the experiment were analysized with flow cytomertry. The experiment was identified as following groups, Group one: MICA transfected cells (the amount of cells are half million per sample) +goat anti mouse antibody, 2 nl (second antibody, also fluorescent antibody); Group two: MICA transfected cells (the amount of cells are half million per sample) +anti-MICA antibody(first antibody, also fluorescent antibody)+ goat anti mouse antibody, 2 nl(second antibody, also fluorescent antibody); Group three: MICA transfected cells ( the amount of cells are half million per sample) + the AB type sera for none- history of blood transfusion patients were considered as the negative control+goat anti human (IgG+IgM) 10nl (second antibody); Group four: MICA transfected cells (the amount of cells are half million per sample) + the serum of the patients who accepted blood transfusion several time or underwent organ transplantation and produced alloantibody, were classified as the positive control+goat anti human (IgG+IgM) 10nl (second antibody); Group five: MICA transfected cells (the amount of cells are half million per sample) + anti-MICA antibody (first antibody, also fluorescent antibody) +the serum of the patients who had the record of liver transplantation rejection+goat anti human (IgG+IgM) 10nl (second antibody); MICA monoclonal antibody was used to block in case of finding whether there are any MICA special antibody in serum of patients in all groups.
2) MICA transfected cells?BEC, LSEC and hepatocyte were conjugated with 5nl anti-MICA antibody, added goat anti mouse antibody (fluorescent antibody), and check them with flow cytomertry respectively. MICA none-transfected cells and transfected cells were considered as negative and positive control respectively.
Second part of experiment:
1) The DNA of serum for 22 cases of donors and recepients who had record of liver transplantation rejection were tested with spectrophotometer respectively for the case of finding whether there were enoght amount of DNA meet the requirement for PCR detection. Then tested them with MICA special primer through PCR method, the electrophoretogram was analysed and record, and every MICA allels genotype were obtained from contrast with MICA allels genotype map as control.
2) BEC, LSEC and hepatocyte were taken out from human health liver respectively, cells were cultured for a few days, RNA was taken out from them and synthesis for cDNA. G6PD was considered as endogenetic normal control, MICA first generation cell line and MICA transfected cells were regarded as negative and positive control respectively, tested them with RT-PCR and Western-blot then.
Third part of experiment:
13 cases who had record of liver transplantation rejection and 15 cases who underwent liver transplantation but no episode of rejection were taken liver biopsy at certain time point respectively. Those who had no record of rejection were applied as positive control. Again those who suffered colon cancer were taken colon section and applied as negative control. All samples were tested with IHC method.
Results:
1) A total of 22/84 (26%) patients with MICA antibodies were observed either pre and/or post-transplantation. 8/84 (9.5%) patients had MICA antibodies in the pre- and 14/84 (17%) in the post-transplantation period. Overall 43/84 (51%) patients had rejections. No correlation between rejection frequencies (14/22, 63%) or other clinical parameters was observed in patients with MICA antibodies as compared to those without MICA antibodies (29/62, 47% p=ns). There were no MICA expression in none-MICA transfected cells, however, there were highly MICA expression in MICA tranfected cells. After blocked with MICA monoclonal antibody, the conjugation between serum and MICA tranfected cells was inhibitted, suggests these detected antibodies in rerum of patients were MICA special antibodies. In MICA antibody negative and positive groups, regardless of whether there were rejection or not and patients’survival or not, there were not any clinical relation between two groups.
2)From the results of the folw cytomertry, there were MICA expression on the surface of MICA transfected cells, however, as the negative control, there were no any expression on the surface of BEC, LSEC and hepatocyte, suggests that the MICA were not expressed on the surface of these cells.
3) The most frequenct MICA allels occurring in both donors and recipients were MICA*008. In 9 patient’s serum both donors and recipients the MICA*008 allel could be detected, 4 of them were completely the same in MICA*008 typing; In 4 cases, donors were not cross-match, but their alleles are MICA*008. In 5 cases, the MICA alleles were 002 or 010 in donors without cross-match typing. In 14/22 (63%) patients who have suffered rejection, MICA*008 could be detected, but in 29/62 (47%) patients, MICA*008 could not be detected, there is no significant differentiation between them (P>0.05).
4) Testing the fresh liver cells and anti-MICA monoclonal antibody with Western-blot, shown that 62kDa band which observed was corresponging with MICA multipeptide. These results were solely detected in MICA transfected cell, not in liver cells, such as BEC, LSEC and hepatocyte.
5) MICA antigens can be slightly expressed in liver biopsy sections of who underwent liver transplantation and the patients who had rejection records at cerntain time point. However, there were quite strong and diffusing MICA expression was observed in section of those who suffered colon cancer.
Conclusion:
1) MICA antibody can be solely expressed on the surface of MICA tranfected cell and no expression can be found on the surface of other liver cells, such as BEC, LSEC and hepatocyte.
2) MICA antigen can be slightly expressed in the hepatic tissues of those who underwent liver transplantation and/or had records of rejection, however, strongly expressed in the tissues of those who suffered colon cancer.
3) From the statistical results, in pre-existed antibodies in drafted liver, no MICA allels were observed. Therefore, MICA may not be the reasoning and target MHC gene for liver transplantation rejection compared with kidney transplantation.
1)通过对主要组织相容性复合体I类分子相关A基因(MICA)转染的细胞系检测肝移植病人的血清,运用流式细胞仪检测是否产生针对MICA的抗体来探讨MICA抗体在肝移植免疫排斥反应中的影响机制。
2)运用PCR方法检测22例发生肝移植排斥反应供、受体病人血清中的MICA的等位基因型,确定MICA*008等位基因在肝移植排斥反应中的作用机制。
3)运用免疫组织化学方法进一步证实MICA抗原与肝移植排斥反应的关系。
方法:
课题第一部分:
1)转染MICA的人类B淋巴母细胞细胞系(721.221)通过细胞培养后,同84例接受肝移植手术的病人的血清进行流式细胞仪检测,实验分为五组,第一组:MICA转染细胞(5×10~5个)+羊抗鼠抗体2微升(二抗,荧光抗体);第二组:MICA转染细胞(5×10~5个)+抗MICA抗体(一抗,荧光抗体)+羊抗鼠抗体2微升(二抗,荧光抗体);第三组:MICA转染细胞(5×10~5个)+AB血清(阴性对照)+羊抗人(IgG+IgM)10微升(二抗);第四组:MICA转染细胞(5×10~5个)+阳性对照病人血清+羊抗人(IgG+IgM)10微升(二抗);第五组:MICA转染细胞(5×10~5个)+抗MICA抗体(一抗,荧光抗体)+产生排斥反应的病人的血清+羊抗人(IgG+IgM)10微升(二抗)。各组再用抗MICA单克隆抗体封闭,以检测血清中的抗体是否是MICA特异性抗体。
2)MICA转染细胞、胆管上皮细胞(BEC)、肝窦内皮细胞(LSEC)和肝细胞与5μl的抗MICA抗体结合,加入萤光抗体羊抗鼠IgG,分别行流式细胞仪检测,MICA的未转染细胞和MICA转染细胞分别作为阴性和阳性对照。
课题第二部分:
1)将22例发生肝移植排斥反应病人供体、受体的血清分别提取DNA并以分光光度计进行检测,采用PCR方法和标有MICA等位基因序列的引物进行电泳检测,分析记录电泳图,并与MICA等位基因图谱相对照得出各自的MICA等位基因型。
2)从健康肝脏中分别提取胆管上皮细胞(BEC)、肝窦内皮细胞(LSEC)和肝细胞,细胞经过培养后,提取RNA及cDNA合成,以葡萄糖-6-磷酸盐脱氢酶(G6PD)被当作内生的正常对照,MICA的第一代细胞系和MICA转染细胞分别作为阴性和阳性对照组,分别行RT-PCR检测和免疫印记检测。
课题第三部分:将已经病理检查确诊的13例肝移植并发生排斥反应病人的肝穿刺标本、15例没有发生排斥反应的肝移植病人的肝穿刺标本和11例患有结肠癌病人的结肠标本行免疫组织化学检查。其中没有发生排斥反应病人的标本设为阳性对照,患有结肠癌病人的结肠标本作为阴性对照。
结果:
1)总共22例(22/84)(26%)术前或/和术后病人产生MICA抗体,其中8例(8/84)(9.5%)为术前病人,14例(14/84)(17%)为术后病人,84例病人中,共有43例(43/84)发生排斥反应,在有MICA抗体组中,排斥发生率为63%(14/22),在没有MICA抗体组中,排斥发生率为47%(29/62),二者相比没有统计学差异。在非转染细胞组(721.221)没有发现MICA表达,而在转染组提示有高表达;用抗MICA单克隆抗体封闭后,血清与MICA转染的细胞系的结合被抑制,提示在这些病人血清中检测到的抗体是MICA特异性抗体。在MICA抗体阳性组和阴性组中,无论发生排斥反应及病人的存活与否,两组之间都没有临床联系。
2)流式细胞仪分析显示,MICA的表达在MICA转染细胞的表面上;而做为阴性对照,胆管上皮细胞、肝窦内皮细胞或肝细胞并没有发生任何变化,提示MICA的表达并不在这些细胞表面上。
3)在肝移植供、受体中出现频率最高的MICA等位基因是MICA*008,在9例病人供体和受体的抗体当中检测到了MICA*008,其中,4例病人的供体和受体检测出MICA*008完全相配;发现在4例病人中,供体不匹配的MICA等位基因是MICA*008;5例病人中,供体不匹配的MICA等位基因不是MICA*002就是MICA*010。在14/22(63%)例发生排斥反应的病人中可以检测到MICA*008细胞系抗体,而在29/62(47%)例病人中没有检测到MICA*008细胞系抗体,二者之间没有显著性差异(P>0.05)。
4)提取的新鲜肝脏细胞和抗MICA单克隆抗体经过免疫印迹法检测显示,62kDa条带与MICA多肽的相一致,这一结果仅见于MICA转染细胞而不是胆管上皮细胞、肝窦内皮细胞和肝细胞。
5)在那些发生排斥反应和没有发生排斥反应的肝移植病人标本中仅发现轻度的MICA抗原染色,而那些患有结肠癌并当作阴性对照组的病人,免疫组化显色后显示在上皮细胞有很强和散在的细胞质染色。
结论:
1)MICA的表达仅在MICA转染细胞的表面上,而在胆管上皮细胞、肝窦内皮细胞或肝细胞并没有发生表达,提示MICA的表达并不在这些细胞表面上。
2)MICA抗原仅在肝移植病人及发生排斥反应的肝移植病人的肝组织中发生微弱的表达,但却在结肠癌病人的结肠上皮组织中发生强烈表达。
3)统计结果显示,在肝移植预留抗体中没有发现MICA等位基因的表达,与肾移植相比较,MICA抗原不是引起肝移植排斥反应的目标抗原
Objective:
1)This research was on the polymorphism detection of Major Histrocompitbility complex ClassⅠreleted gene A (MICA) and determine the mechanism of MICA antibodies in liver transplantation rejection. The total experiment includes following sections: 1) Inspect the serum of patients who underwent liver transplantation with MICA tansfected cell line, detect patients serum with flow cytomertry for finding whether it had MICA antibodies or not, then determine the mechanism of MICA antibodies in liver transplantation rejection.
2)Using PCR method to detect the MICA alleles in serum of 22 cases in both donors and recipients who underwent liver transplantation rejection, and identify the mechanism of the MICA*008 allele in liver transplantation rejection.
3)Using the method of IHC to further verifying the methanism between MICA antigens and liver transplantation rejection.
Method:
First part of experement:
1)Test the transfected human B lymphoblastic cell line (721. 221) after cell culturing with serum of 84 cases who underwent liver transplantation, all the experiment were analysized with flow cytomertry. The experiment was identified as following groups, Group one: MICA transfected cells (the amount of cells are half million per sample) +goat anti mouse antibody, 2 nl (second antibody, also fluorescent antibody); Group two: MICA transfected cells (the amount of cells are half million per sample) +anti-MICA antibody(first antibody, also fluorescent antibody)+ goat anti mouse antibody, 2 nl(second antibody, also fluorescent antibody); Group three: MICA transfected cells ( the amount of cells are half million per sample) + the AB type sera for none- history of blood transfusion patients were considered as the negative control+goat anti human (IgG+IgM) 10nl (second antibody); Group four: MICA transfected cells (the amount of cells are half million per sample) + the serum of the patients who accepted blood transfusion several time or underwent organ transplantation and produced alloantibody, were classified as the positive control+goat anti human (IgG+IgM) 10nl (second antibody); Group five: MICA transfected cells (the amount of cells are half million per sample) + anti-MICA antibody (first antibody, also fluorescent antibody) +the serum of the patients who had the record of liver transplantation rejection+goat anti human (IgG+IgM) 10nl (second antibody); MICA monoclonal antibody was used to block in case of finding whether there are any MICA special antibody in serum of patients in all groups.
2) MICA transfected cells?BEC, LSEC and hepatocyte were conjugated with 5nl anti-MICA antibody, added goat anti mouse antibody (fluorescent antibody), and check them with flow cytomertry respectively. MICA none-transfected cells and transfected cells were considered as negative and positive control respectively.
Second part of experiment:
1) The DNA of serum for 22 cases of donors and recepients who had record of liver transplantation rejection were tested with spectrophotometer respectively for the case of finding whether there were enoght amount of DNA meet the requirement for PCR detection. Then tested them with MICA special primer through PCR method, the electrophoretogram was analysed and record, and every MICA allels genotype were obtained from contrast with MICA allels genotype map as control.
2) BEC, LSEC and hepatocyte were taken out from human health liver respectively, cells were cultured for a few days, RNA was taken out from them and synthesis for cDNA. G6PD was considered as endogenetic normal control, MICA first generation cell line and MICA transfected cells were regarded as negative and positive control respectively, tested them with RT-PCR and Western-blot then.
Third part of experiment:
13 cases who had record of liver transplantation rejection and 15 cases who underwent liver transplantation but no episode of rejection were taken liver biopsy at certain time point respectively. Those who had no record of rejection were applied as positive control. Again those who suffered colon cancer were taken colon section and applied as negative control. All samples were tested with IHC method.
Results:
1) A total of 22/84 (26%) patients with MICA antibodies were observed either pre and/or post-transplantation. 8/84 (9.5%) patients had MICA antibodies in the pre- and 14/84 (17%) in the post-transplantation period. Overall 43/84 (51%) patients had rejections. No correlation between rejection frequencies (14/22, 63%) or other clinical parameters was observed in patients with MICA antibodies as compared to those without MICA antibodies (29/62, 47% p=ns). There were no MICA expression in none-MICA transfected cells, however, there were highly MICA expression in MICA tranfected cells. After blocked with MICA monoclonal antibody, the conjugation between serum and MICA tranfected cells was inhibitted, suggests these detected antibodies in rerum of patients were MICA special antibodies. In MICA antibody negative and positive groups, regardless of whether there were rejection or not and patients’survival or not, there were not any clinical relation between two groups.
2)From the results of the folw cytomertry, there were MICA expression on the surface of MICA transfected cells, however, as the negative control, there were no any expression on the surface of BEC, LSEC and hepatocyte, suggests that the MICA were not expressed on the surface of these cells.
3) The most frequenct MICA allels occurring in both donors and recipients were MICA*008. In 9 patient’s serum both donors and recipients the MICA*008 allel could be detected, 4 of them were completely the same in MICA*008 typing; In 4 cases, donors were not cross-match, but their alleles are MICA*008. In 5 cases, the MICA alleles were 002 or 010 in donors without cross-match typing. In 14/22 (63%) patients who have suffered rejection, MICA*008 could be detected, but in 29/62 (47%) patients, MICA*008 could not be detected, there is no significant differentiation between them (P>0.05).
4) Testing the fresh liver cells and anti-MICA monoclonal antibody with Western-blot, shown that 62kDa band which observed was corresponging with MICA multipeptide. These results were solely detected in MICA transfected cell, not in liver cells, such as BEC, LSEC and hepatocyte.
5) MICA antigens can be slightly expressed in liver biopsy sections of who underwent liver transplantation and the patients who had rejection records at cerntain time point. However, there were quite strong and diffusing MICA expression was observed in section of those who suffered colon cancer.
Conclusion:
1) MICA antibody can be solely expressed on the surface of MICA tranfected cell and no expression can be found on the surface of other liver cells, such as BEC, LSEC and hepatocyte.
2) MICA antigen can be slightly expressed in the hepatic tissues of those who underwent liver transplantation and/or had records of rejection, however, strongly expressed in the tissues of those who suffered colon cancer.
3) From the statistical results, in pre-existed antibodies in drafted liver, no MICA allels were observed. Therefore, MICA may not be the reasoning and target MHC gene for liver transplantation rejection compared with kidney transplantation.
引文
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