發(fā)布時(shí)間：2018-04-22 08:27

  本文選題：HLA-A2 + HLA亞型�。� 參考：《華中科技大學(xué)》2009年博士論文

【摘要】： 中國人群常見HLA-A2亞型間抗原特異性差異的實(shí)驗(yàn)研究研究生：陸盛軍導(dǎo)師：吳雄文教授 MHC分子(鼠為H-2，人為HLA)的功能是將抗原肽提呈給T細(xì)胞識別，誘導(dǎo)抗原特異性T細(xì)胞發(fā)生增殖活化，進(jìn)而產(chǎn)生免疫效應(yīng)。T細(xì)胞抗原受體(TCR)識別的配體是抗原肽／MHC復(fù)合物(pMHC)。不同型別MHC分子之間的抗原特異性差異已經(jīng)廣為人知，例如應(yīng)用同種異體移植的體外模型—混合淋巴細(xì)胞培養(yǎng)，能夠觀察到不同型別MHC分子誘導(dǎo)淋巴細(xì)胞增殖。但是，人們尚未十分明確相同血清型的各亞型之間在抗原特異性方面是否存在差異，闡明這個(gè)問題有利于多肽疫苗設(shè)計(jì)和隨機(jī)移植供者選擇。 HLA-A2是HLA-A座位上最常見的型別，在漢族人中HLA-A2陽性者占50％左右，其亞型在中國人群中分布頻率較高的有HLA-A~*0201、HLA-A~*0203、HLA-A~*0206、HLA-A~*0207。雖然，這些亞型之間僅在抗原肽結(jié)合槽部位存在極少數(shù)幾個(gè)氨基酸的差異，仍有研究提示，不同HLA-A2亞型之間由于抗原肽結(jié)合槽部位氨基酸的差異，使得它們在抗原提呈和T細(xì)胞識別方面有一定的差異。但是由于HLA具有高度的多態(tài)性和單倍型遺傳的特點(diǎn)，很難找到僅HLA-A2亞型存在差異的樣本，目前尚缺乏HLA-A2亞型間抗原特異性存在差異的實(shí)驗(yàn)證據(jù)。 我們在前期工作中獲得了由HLA-A~*0201胞外段和人IgGl的Fc段構(gòu)成的融合蛋白(HLA-A~*0201／IgG二聚體)，借助該二聚體的Fc段與單核細(xì)胞表面Fc段受體(FcR)高親和力結(jié)合，將此HLA-A~*0201二聚體分子結(jié)合于HLA-A2陰性(HLA-A2-ve)的單核細(xì)胞表面，后者作為刺激細(xì)胞與同一個(gè)體的淋巴細(xì)胞進(jìn)行共培養(yǎng)，結(jié)果顯示表面結(jié)合HLA-A~*0201／IgG二聚體的HLA-A2-ve單核細(xì)胞能夠刺激自身淋巴細(xì)胞發(fā)生增殖，并產(chǎn)生相應(yīng)的特異性CTLs。很顯然，該共培養(yǎng)體系中淋巴細(xì)胞的增殖反映了單個(gè)表位的HLA-A2與非HLA-A2之間的抗原特異性差異，即上述共培養(yǎng)體系能用于單一pMHC分子的抗原特異性的實(shí)驗(yàn)觀察。 為了對HLA-A2常見亞型間的抗原特異性差異進(jìn)行實(shí)驗(yàn)研究，我們對原有的共培養(yǎng)體系進(jìn)行改進(jìn)，改進(jìn)之處在于選用HLA-A2陽性但非HLA-A~*0201型別的單核細(xì)胞和淋巴細(xì)胞，刺激細(xì)胞和效應(yīng)細(xì)胞之間僅有HLA-A2的亞型不同，而不是HLA-A2與非HLA-A2的差別。將表面結(jié)合HLA-A~*0201／IgG二聚體的單核細(xì)胞作為刺激細(xì)胞，與自身淋巴細(xì)胞共培養(yǎng)，通過檢測共培養(yǎng)體系中淋巴細(xì)胞的增殖強(qiáng)度，反映HLA-A*0201與特定HLA-A2亞型之間抗原特異性的差異。 鑒于HLA-A2亞型之間抗原特異性的差異較小，為了控制個(gè)體間淋巴細(xì)胞增殖活性的差異對實(shí)驗(yàn)結(jié)果的影響，我們采用“增殖系數(shù)”來衡量HLA-A2亞型之間抗原特異性的差異，而非直接采用增殖指數(shù)或增殖細(xì)胞頻率。具體做法是：試驗(yàn)個(gè)體(HLA-A2+ve)的單核細(xì)胞表面結(jié)合HLA-A~*0201／IgG二聚體后與自身淋巴細(xì)胞共培養(yǎng)后的增殖細(xì)胞頻率為試驗(yàn)組增殖細(xì)胞頻率，該試驗(yàn)個(gè)體(HLA-A2+ve)淋巴細(xì)胞與HLA-A2-ve的單核細(xì)胞混合培養(yǎng)的增殖細(xì)胞頻率作為陽性參照，而該HLA-A2+ve個(gè)體的淋巴細(xì)胞與自身單核細(xì)胞共培養(yǎng)所測得的增殖細(xì)胞頻率作為背景參照，采用公式((試驗(yàn)組增殖細(xì)胞頻率-背景參照的增殖細(xì)胞頻率)／(陽性參照的增殖細(xì)胞頻率-背景參照的增殖細(xì)胞頻率))對原始檢測值進(jìn)行校正，獲得的校正值稱為增殖系數(shù)。 我們選擇了不同HLA-A2亞型型別組(包括HLA-A~*0201型別組與其它HLA-A2常見亞型型別組)的外周血樣本進(jìn)行了上述實(shí)驗(yàn)，將自身肽Tyr368-376(NH2-Y MDGTMSQV-COOH)加載的HLA-A~*0201／IgG二聚體結(jié)合到單核細(xì)胞表面，與自身淋巴細(xì)胞共培養(yǎng)后檢測淋巴細(xì)胞增殖系數(shù)。通過檢測不同HLA-A2亞型型別組淋巴細(xì)胞對表面結(jié)合自身肽／HLA-A~*0201二聚體的自身單核細(xì)胞的增殖反應(yīng)強(qiáng)度(用增殖系數(shù)表示)，觀察到HLA-A~*0201型別組與HLA-A~*0203型別組的增殖系數(shù)之間有統(tǒng)計(jì)學(xué)差異，其增殖系數(shù)分別為0．012±0．109，0．124±0．067(P≤0．05)；HLA-A~*0201型別組與HLA-A~*0206型別組的增殖系數(shù)之間可觀察到顯著統(tǒng)計(jì)學(xué)差異，，其增殖系數(shù)分別為0．012±0．109，0．225±0．184(P≤0．01)；HLA-A~*0201型別組與HLA-A~*0207型別組的增殖系數(shù)之間未觀察到統(tǒng)計(jì)學(xué)差異，其增殖系數(shù)分別為0．012±0．109，0．050±0．109(P＞0．05)。我們的實(shí)驗(yàn)結(jié)果提示，HLA-A~*0201與HLA-A~*0206之間以及HLA-A~*0201與HLA-A~*0203之間存在抗原特異性差異，而HLA-A~*0201與HLA-A~*0207之間未觀察到抗原特異性差異。 本研究的意義： 1．首次將單核細(xì)胞表面結(jié)合pMHC分子作為刺激細(xì)胞的培養(yǎng)體系用于亞型特異性差異的實(shí)驗(yàn)研究。本文研究的是HLA-A2主要亞型分子的抗原特異性差異，問題涉及的HLA分子范圍雖局限，但是為研究其它MHC分子亞型間的抗原特異性差異提供了一個(gè)新的實(shí)驗(yàn)研究策略。 2．用實(shí)驗(yàn)方法展示出HLA-A2某些亞型分子之間存在的抗原特異性差異。本文首次用實(shí)驗(yàn)方法顯示出HLA-A~*0201與HLA-A~*0206之間以及HLA-A~*0201與HLA-A~*0203之間存在抗原特異性差異；但HLA-A~*0201與HLA-A~*0207之間未觀察到抗原特異性差異。 本研究的不足之處： 本研究僅使用了HLA-A*0201這一個(gè)HLA-A2亞型型別的二聚體進(jìn)行實(shí)驗(yàn)，所得到的實(shí)驗(yàn)結(jié)果只能顯示HLA-A~*0201與其它非HLA-A*0201型別之間的差異，而非HLA-A~*0201型別的HLA-A2亞型相互之間的差異無法顯示出來。 今后研究設(shè)想： 為進(jìn)一步證實(shí)HLA-A~*0201分別與HLA-A~*0203和HLA-A~*0206之間的抗原特異性差異，我們認(rèn)為有希望從以下幾個(gè)方面改善和提高本論文： 1．構(gòu)建獲得HLA-A~*0203、HLA-A~*0206分子與IgG1的CH1-Fc片段的融合蛋白，借助新構(gòu)建和已構(gòu)建的分子，利用共培養(yǎng)體系。 2．盡量采用高親和力自身肽，采取多種自身肽的混合表位以提高刺激強(qiáng)度的策略，使個(gè)體共培養(yǎng)體系類的增殖更明顯。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Peptide-specific,allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2[J];Science in China(Series C:Life Sciences);2007年02期

本文編號：1786370