中国人群常见HLA-A2亚型间抗原特异性差异的实验研究

发布时间：2018-04-22 08:27

本文选题：HLA-A2 + HLA亚型　；参考：《华中科技大学》2009年博士论文

【摘要】： 中国人群常见HLA-A2亚型间抗原特异性差异的实验研究研究生：陆盛军导师：吴雄文教授 MHC分子(鼠为H-2，人为HLA)的功能是将抗原肽提呈给T细胞识别，诱导抗原特异性T细胞发生增殖活化，进而产生免疫效应。T细胞抗原受体(TCR)识别的配体是抗原肽／MHC复合物(pMHC)。不同型别MHC分子之间的抗原特异性差异已经广为人知，例如应用同种异体移植的体外模型—混合淋巴细胞培养，能够观察到不同型别MHC分子诱导淋巴细胞增殖。但是，人们尚未十分明确相同血清型的各亚型之间在抗原特异性方面是否存在差异，阐明这个问题有利于多肽疫苗设计和随机移植供者选择。 HLA-A2是HLA-A座位上最常见的型别，在汉族人中HLA-A2阳性者占50％左右，其亚型在中国人群中分布频率较高的有HLA-A~*0201、HLA-A~*0203、HLA-A~*0206、HLA-A~*0207。虽然，这些亚型之间仅在抗原肽结合槽部位存在极少数几个氨基酸的差异，仍有研究提示，不同HLA-A2亚型之间由于抗原肽结合槽部位氨基酸的差异，使得它们在抗原提呈和T细胞识别方面有一定的差异。但是由于HLA具有高度的多态性和单倍型遗传的特点，很难找到仅HLA-A2亚型存在差异的样本，目前尚缺乏HLA-A2亚型间抗原特异性存在差异的实验证据。我们在前期工作中获得了由HLA-A~*0201胞外段和人IgGl的Fc段构成的融合蛋白(HLA-A~*0201／IgG二聚体)，借助该二聚体的Fc段与单核细胞表面Fc段受体(FcR)高亲和力结合，将此HLA-A~*0201二聚体分子结合于HLA-A2阴性(HLA-A2-ve)的单核细胞表面，后者作为刺激细胞与同一个体的淋巴细胞进行共培养，结果显示表面结合HLA-A~*0201／IgG二聚体的HLA-A2-ve单核细胞能够刺激自身淋巴细胞发生增殖，并产生相应的特异性CTLs。很显然，该共培养体系中淋巴细胞的增殖反映了单个表位的HLA-A2与非HLA-A2之间的抗原特异性差异，即上述共培养体系能用于单一pMHC分子的抗原特异性的实验观察。为了对HLA-A2常见亚型间的抗原特异性差异进行实验研究，我们对原有的共培养体系进行改进，改进之处在于选用HLA-A2阳性但非HLA-A~*0201型别的单核细胞和淋巴细胞，刺激细胞和效应细胞之间仅有HLA-A2的亚型不同，而不是HLA-A2与非HLA-A2的差别。将表面结合HLA-A~*0201／IgG二聚体的单核细胞作为刺激细胞，与自身淋巴细胞共培养，通过检测共培养体系中淋巴细胞的增殖强度，反映HLA-A*0201与特定HLA-A2亚型之间抗原特异性的差异。鉴于HLA-A2亚型之间抗原特异性的差异较小，为了控制个体间淋巴细胞增殖活性的差异对实验结果的影响，我们采用“增殖系数”来衡量HLA-A2亚型之间抗原特异性的差异，而非直接采用增殖指数或增殖细胞频率。具体做法是：试验个体(HLA-A2+ve)的单核细胞表面结合HLA-A~*0201／IgG二聚体后与自身淋巴细胞共培养后的增殖细胞频率为试验组增殖细胞频率，该试验个体(HLA-A2+ve)淋巴细胞与HLA-A2-ve的单核细胞混合培养的增殖细胞频率作为阳性参照，而该HLA-A2+ve个体的淋巴细胞与自身单核细胞共培养所测得的增殖细胞频率作为背景参照，采用公式((试验组增殖细胞频率-背景参照的增殖细胞频率)／(阳性参照的增殖细胞频率-背景参照的增殖细胞频率))对原始检测值进行校正，获得的校正值称为增殖系数。我们选择了不同HLA-A2亚型型别组(包括HLA-A~*0201型别组与其它HLA-A2常见亚型型别组)的外周血样本进行了上述实验，将自身肽Tyr368-376(NH2-Y MDGTMSQV-COOH)加载的HLA-A~*0201／IgG二聚体结合到单核细胞表面，与自身淋巴细胞共培养后检测淋巴细胞增殖系数。通过检测不同HLA-A2亚型型别组淋巴细胞对表面结合自身肽／HLA-A~*0201二聚体的自身单核细胞的增殖反应强度(用增殖系数表示)，观察到HLA-A~*0201型别组与HLA-A~*0203型别组的增殖系数之间有统计学差异，其增殖系数分别为0．012±0．109，0．124±0．067(P≤0．05)；HLA-A~*0201型别组与HLA-A~*0206型别组的增殖系数之间可观察到显著统计学差异，，其增殖系数分别为0．012±0．109，0．225±0．184(P≤0．01)；HLA-A~*0201型别组与HLA-A~*0207型别组的增殖系数之间未观察到统计学差异，其增殖系数分别为0．012±0．109，0．050±0．109(P＞0．05)。我们的实验结果提示，HLA-A~*0201与HLA-A~*0206之间以及HLA-A~*0201与HLA-A~*0203之间存在抗原特异性差异，而HLA-A~*0201与HLA-A~*0207之间未观察到抗原特异性差异。本研究的意义： 1．首次将单核细胞表面结合pMHC分子作为刺激细胞的培养体系用于亚型特异性差异的实验研究。本文研究的是HLA-A2主要亚型分子的抗原特异性差异，问题涉及的HLA分子范围虽局限，但是为研究其它MHC分子亚型间的抗原特异性差异提供了一个新的实验研究策略。 2．用实验方法展示出HLA-A2某些亚型分子之间存在的抗原特异性差异。本文首次用实验方法显示出HLA-A~*0201与HLA-A~*0206之间以及HLA-A~*0201与HLA-A~*0203之间存在抗原特异性差异；但HLA-A~*0201与HLA-A~*0207之间未观察到抗原特异性差异。本研究的不足之处：本研究仅使用了HLA-A*0201这一个HLA-A2亚型型别的二聚体进行实验，所得到的实验结果只能显示HLA-A~*0201与其它非HLA-A*0201型别之间的差异，而非HLA-A~*0201型别的HLA-A2亚型相互之间的差异无法显示出来。今后研究设想：为进一步证实HLA-A~*0201分别与HLA-A~*0203和HLA-A~*0206之间的抗原特异性差异，我们认为有希望从以下几个方面改善和提高本论文： 1．构建获得HLA-A~*0203、HLA-A~*0206分子与IgG1的CH1-Fc片段的融合蛋白，借助新构建和已构建的分子，利用共培养体系。 2．尽量采用高亲和力自身肽，采取多种自身肽的混合表位以提高刺激强度的策略，使个体共培养体系类的增殖更明显。
[Abstract]:An experimental study on the differences in antigen specificity between HLA-A2 subtypes in Chinese population: Professor Lu Sheng Jun: Professor Wu Xiongwen
The function of MHC molecule (rat H-2, human HLA) is to present antigen peptide to T cell recognition, induce antigen specific T cells to proliferate and activate, and then produce immune effect.T cell antigen receptor (TCR) recognition ligand is antigen peptide / MHC complex (pMHC). The difference of antigen specificity among different types of MHC molecules is well known, for example In vitro model of allograft - mixed lymphocyte culture, different types of MHC molecules can be observed to induce lymphocyte proliferation. However, it is not clear whether there is any difference in antigen specificity between the various subtypes of the same serotypes, and that this problem is beneficial to the design of polypeptide vaccine and the randomness of transplantation. The choice of the person.
HLA-A2 is the most common type of HLA-A, which accounts for about 50% of the HLA-A2 positive in the Han people. Its subtypes are highly distributed in Chinese people, including HLA-A~*0201, HLA-A~*0203, HLA-A~*0206, HLA-A~*0207., although these subtypes exist only in a very few amino acids in the antigen peptide binding slot. The difference in the amino acid of the antigen peptide binding slot between different HLA-A2 subtypes makes them different in antigen presentation and T cell recognition. However, because HLA has high polymorphism and haplotype inheritance, it is difficult to find a sample with different HLA-A2 subtypes, and there is still a lack of HLA-A2 subtypes. Experimental evidence of differences in the opposite sex.
In our previous work, we obtained the fusion protein (HLA-A~*0201 / IgG two polymer) composed of the HLA-A~*0201 segment and the Fc segment of human IgGl. By combining the Fc segment of the polymer with the high affinity of the Fc segment receptor (FcR) on the surface of monocyte, the HLA-A~*0201 two polymer molecules were bonded to the monocyte surface of HLA-A2 negative (HLA-A2-ve), and the latter was the latter. As a co culture of the lymphocytes of the same individual, the results showed that the HLA-A2-ve monocytes with HLA-A~*0201 / IgG two polymer can stimulate the proliferation of their own lymphocytes and produce the corresponding specific CTLs.. The proliferation of the lymphoblastic cells in the co culture system reflects the HLA-A2 of the single epitope and the HLA-A2 of the single epitope. The antigenic difference between non HLA-A2 is that the above co culture system can be used for antigen specific observation of single pMHC molecule.
In order to study the antigen specificity difference between the common subtypes of HLA-A2, we improved the original co culture system. The improvement was that the HLA-A2 positive but non HLA-A~*0201 type monocytes and lymphocytes were selected, and the only HLA-A2 subtypes were different between the stimulant cells and the effector cells, not the HLA-A2 and the non HLA-A2. The mononuclear cells with HLA-A~*0201 / IgG two polymer were used as stimulating cells to co culture with their own lymphocytes. By detecting the proliferation of lymphocyte in the co culture system, the difference of antigen specificity between HLA-A*0201 and specific HLA-A2 subtypes was reflected.
In view of the small difference in antigen specificity between HLA-A2 subtypes, in order to control the effects of the difference in proliferation activity between individuals, we used the "multiplication factor" to measure the difference in antigen specificity between HLA-A2 subtypes instead of directly using the proliferating index or proliferating cell frequency. The frequency of proliferation cells after co culture of mononuclear cells with HLA-A~*0201 / IgG two polymer after co culture with HLA-A~*0201 / IgG two cells is the frequency of proliferating cells in the test group. The frequency of the proliferation cell frequency of the test individual (HLA-A2+ve) lymphocyte and HLA-A2-ve mononuclear cells is the positive reference, and the lymph nodes of the HLA-A2+ve individual The frequency of the proliferating cell measured by co culture of the cell and its own mononuclear cells was used as the background reference. The original detection values were corrected by the formula (the frequency of the proliferating cell of the experimental group - the background reference, the frequency of the proliferating cell with the background reference). The corrected value was called the proliferation line. Number.
We selected the peripheral blood samples from different HLA-A2 subtypes (including the HLA-A~*0201 type group and other HLA-A2 subtypes). We combined the HLA-A~*0201 / IgG two polymer loaded with the autopy Tyr368-376 (NH2-Y MDGTMSQV-COOH) to the monocyte surface and co culture with the lymphocyte to detect the lymph nodes. Cell proliferation coefficient. By detecting the proliferation response intensity of the lymphocytes of different HLA-A2 subtypes to the autogenous mononuclear cells of the autogenous peptide / HLA-A~*0201 two polymer (using the proliferation coefficient), the proliferation coefficient of the HLA-A~*0201 type group and the HLA-A~*0203 type group was observed to be statistically different. 0.012 + 0.109,0.124 + 0.067 (P < 0.05), the proliferation coefficient of HLA-A~*0201 type group and HLA-A~*0206 type group can be observed statistically significant difference, its proliferation coefficient is 0.012 + 0.109,0.225 + 0.184 (P < 0.01), and the proliferation coefficient of HLA-A~*0201 type group and HLA-A~*0207 type group is not observed. The proliferation coefficient was 0.012 + 0.109,0.050 + 0.109 (P > 0.05). Our experimental results suggest that there is a specific difference in antigen between HLA-A~*0201 and HLA-A~*0206 and between HLA-A~*0201 and HLA-A~*0203, but there is no specific difference in antigen specificity between HLA-A~*0201 and HLA-A~*0207.
The significance of this study is:
1. for the first time, an experimental study on the specific differences in subtypes of the mononuclear cell surface combined with pMHC molecules as a stimulating cell is used to study the specific differences in the antigen specificity of the main subtypes of HLA-A2. The scope of the problem involves the limitation of the HLA molecular scope, but it provides a study of the specific differences in the antigen of other MHC subtypes. A new experimental research strategy.
2. an experimental method was used to show the difference in antigen specificity between some subtypes of HLA-A2. In this paper, the antigen specific differences between HLA-A~*0201 and HLA-A~*0206 and between HLA-A~*0201 and HLA-A~*0203 were revealed for the first time, but the difference of antigen specificity was not observed between HLA-A~*0201 and HLA-A~*0207.
The shortcomings of this study are as follows:
In this study, only HLA-A*0201, a HLA-A2 subtype two polymer, was used to experiment. The results obtained only showed the difference between HLA-A~*0201 and other non HLA-A*0201 types, but the difference between the HLA-A2 subtypes of non HLA-A~*0201 types could not be shown.
Future research envisages:
In order to further confirm the difference in antigen specificity between HLA-A~*0201 and HLA-A~*0203 and HLA-A~*0206, we believe that we hope to improve and improve this paper in the following aspects:
1. we constructed the fusion protein of CH1-Fc fragment of HLA-A~*0203, HLA-A~*0206 and IgG1, and co cultured with the newly constructed and constructed molecule.
2. the high affinity self peptide is used as much as possible, and a variety of mixed epitopes of self peptides are adopted to improve the intensity of stimulation, so that the proliferation of the individual co culture system is more obvious.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

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