新的T细胞活化标志ZCH-2B8a抗原的分子特性及功能研究

发布时间：2019-07-02 14:35

【摘要】： 目的： ZCH-2B8a单克隆抗体由本研究室采用经典的杂交瘤技术制备而成，，经HLDA8协作组鉴定后认为属国际上尚未认识的造血细胞膜新的分化抗原或新的CD分子，前期研究表明该抗原(Ag)可能为一活化分子。因此有必要对该Ag进一步研究以明确该其蛋白序列及其表达谱、是否为一新的活化分子及它的功能，开发其潜在的应用价值。方法：本研究主要包括以下四部分：(1)制备2B8a FITC直标抗体：采用SPASepharose亲和层析法从腹水中纯化2B8a抗体；聚丙烯酰胺凝胶电泳法(SDS-PAGE)鉴定抗体纯度及分子量；采用改良Marshall法制备2B8a FITC直标抗体，并通过流式细胞术鉴定。(2)检测2B8a Ag的分布情况：采用流式细胞术检测2B8a Ag在外周组织、正常外周血、白血病细胞、细胞株上的分布；采用激光共聚焦显微镜观察2B8a Ag在细胞上的定位。(3)2B8a Ag的功能研究：通过流式细胞术检测PHA刺激后T细胞活化过程中2B8a的表达规律；检测再生障碍性贫血等病人外周血标本2B8a Ag的表达情况及与其他活化标志的相关性；利用流式细胞微球技术(CBA)检测2B8a Ab对活化T细胞分泌细胞因子的影响；通过2B8a抗体封闭Raji细胞的Ag表位检测它与骨髓基质细胞粘附能力的变化；通过木瓜酶酶切法结合流式细胞术检测2B8a Ag介导抗体内化的能力；通过补体依赖的细胞毒实验(CDC)了解2B8a Ab与Ag特异性结合及激活补体的能力。(4)2B8a Ag蛋白的鉴定：将Raji细胞蛋白裂解液经免疫共沉淀法纯化后行SDS-PAGE检测，胶上的Ag蛋白条带送肽质量指纹图及串联质谱分析；Western blot法鉴定2B8a Ag蛋白的分子量。结果：经SDS-PAGE鉴定，2B8a抗体的重链和轻链分子量分别为52 kDa和9 kDa。2B8a直标法(2B8a FITC)和间标法(2B8a+GAM-FITC)与Raji细胞阳性反应率分别为98．48%和98．35%，与Molt-3细胞阳性反应率分别为9．35%和10．80%。2B8a Ag在正常外周血的T细胞表面不表达(5．4±3．8%)，B细胞表面弱表达(29．9±14．8%)，而中性粒细胞(92．2±8．7%)和单核细胞(81．9±19．3%)表面表达较强。在B系急性淋巴细胞白血病细胞表面，该Ag的表达较对照组显著增强(中位值99．0%vs．43．9%)。该Ag在所检测B系、T系、髓系的外周血细胞和肿瘤细胞株的胞浆内呈高表达。2B8a Ag在活化T细胞上的表达规律与CD69、CD25和HLA-DR不同，在T细胞受PHA刺激后12h内即可表达于胞膜上，72～96h达高峰。再生障碍性贫血(AA)等免疫相关疾病的T细胞表面，该Ag的表达明显上调(AA：CD4~+T细胞阳性率90．9%，CD8~+T细胞阳性率72．7%)。采用2B8a抗体封闭抗原表位后并不能减少Raji细胞与骨髓基质细胞的粘附(2h、4h、6h和12h时2B8a组与对照组的粘附率比分别为0．87、1．02、1．02和0．96，均为P＞0．05)，也并不影响T细胞活化后细胞因子的分泌水平和活化状态(PHA组与PHA+2B8a组IL-2中位值：425pg/ml vs．442pg/ml，P=0．465)。与3A4抗体在37℃时才能逐渐内化不同，2B8a抗体在4℃和37℃均可以快速“内化”(2B8a FITC与Raji细胞4℃和37℃孵育5min后再行木瓜酶酶切，流式检测阳性反应率分别达到76．7±2．5%和89．2±1．5%)。2B8a抗体能激活补体，介导CDC作用杀伤Raji细胞(按0．1μg抗体：10μl补体：5×10~4细胞的比例，Raji细胞死亡率88．6±7．9%)，而对Molt-3细胞基本无杀伤作用(死亡率3．6±0．6%)。将Raji细胞裂解液经免疫共沉淀纯化2B8a Ag后，Western blot鉴定证实该Ag的分子量为50 kDa左右。质谱分析未能明确得到唯一、可靠的蛋白序列，通过蛋白数据库比对及功能研究，冠蛋白的可能性较大，但它在表达谱上与2B8a Ag仍有较大不同。结论： (1)本研究自行研制的2B8a FITC荧光抗体具有良好的敏感性和特异性； (2)2B8a Ag在淋巴结、扁桃体、肝脏及胰腺组织中可检测到；在外周血，它主要分布在活化T细胞、B细胞、单核细胞、嗜中性粒细胞膜和细胞浆，而在静息T细胞膜上不表达，但在其胞浆中表达也很强； (3)2B8a Ag是一个早期稳定的T细胞活化标志；在再生障碍性贫血等免疫相关疾病中表达上调； (4)2B8a Ag可以介导抗体迅速内化； (5)2B8a Ab能激活补体，发挥明显的CDC作用； (6)2B8a Ag不能介导细胞间的粘附；对活化T细胞的细胞因子分泌无明显影响； (7)质谱分析未能在现有的蛋白质数据库中检索到与2B8a Ag相匹配的蛋白，该抗原可能为一新的蛋白分子。
[Abstract]:Purpose: The ZCH-2B8a monoclonal antibody is prepared by using the classical hybridoma technique in the laboratory, and it is considered to be a new differentiation antigen or a new CD molecule of the hematopoietic cell membrane which has not been recognized in the world after being identified by the HLDA8 cooperative group. The previous study indicates that the antigen (Ag) may be a living cell. It is therefore necessary to further study the Ag to define the protein sequence and its expression profile, whether it is a new activating molecule and its function, and to develop its potential value Methods: This study mainly includes the following four parts: (1) Preparation of 2B8a FITC direct-labeled antibody: purifying the 2B8a antibody from ascitic fluid by using the SPASephron affinity chromatography, and identifying the purity and molecular weight of the antibody by means of SDS-PAGE; and using the modified Marshall method to prepare 2. B8a FITC direct-labeled antibody And the distribution of 2B8a Ag was detected by flow cytometry. The distribution of 2B8a Ag in peripheral tissues, normal peripheral blood, leukemia cells and cell lines was detected by flow cytometry. (3) The function of 2B8a Ag: The expression of 2B8a in peripheral blood of patients with aplastic anemia was detected by flow cytometry, and the expression of 2B8a Ag in peripheral blood of patients with aplastic anemia was detected. Correlations with other activation markers; the effect of 2B8a Ab on the secretion of cytokines in activated T cells by flow cytometry (CBA); the detection of the Ag epitope of Raji cells by the 2B8a antibody and the bone marrow Changes of the adhesion ability of the matrix cells; the ability of the 2B8a Ag-mediated antibody to be internalized by the enzyme-cutting of the papain and the flow cytometry;2 B8a Ab and Ag-specific by the complement-dependent cytotoxicity assay (CDC) The ability of binding and activating complement. (4) Identification of the 2B8a Ag protein: the Raji cell protein lysate was purified by the immunoprecipitation method, and the SDS-PAGE was detected by SDS-PAGE, and the Ag protein band on the gel was sent to the peptide mass fingerprint and the tandem mass spectrum analysis; and the Western blot method was used to identify 2. B8a The molecular weight of the Ag protein was determined by SDS-PAGE. The heavy chain and light chain molecular weight of the 2B8a antibody were 52 kDa and 9 kDa, respectively. The 2B8a direct standard method (2B8a FITC) and the m-standard method (2B8a + GAM-FITC) were positive for Raji cells. The reaction rates were 98.48% and 98.35%, respectively. The positive rate of reaction with Molt-3 cells was 9.35% and 10.80%, respectively. The expression of Ag in B-series acute lymphoblastic leukemia cells increased significantly compared with the control group. The expression of 2B8a Ag on activated T cells was different from that of CD69, CD25 and HLA-DR. The positive rate of Ag expression was up-regulated (AA: CD4 + T cell positive rate was 90.9). The rate of adhesion between Raji cells and bone marrow stromal cells (2 h,4 h,6 h, and 12 h) was 0.87, 1.02 respectively after the antigen epitope was closed by the 2B8a antibody and the adhesion of Raji cells to the bone marrow stromal cells (2 h,4 h,6 h and 12 h) was not reduced. 1.02 and 0.96 (both P> 0.05) and did not affect the level of secretion and activation of cytokines after T cell activation (PHA group and PHA + 2 B8a group IL-2 median value:425 pg/ ml v (s.442 pg/ ml, P = 0.465). 2 (1.5%). The B8a antibody can activate complement and mediate CDC to kill Raji cells (according to 0.1. mu.g of antibody:10. mu. l complement:5%10-4 cells, Raji cell death rate of 88.6% 7.9%), and Molt-3 cell group The cell lysate of Raji was purified by immunoprecipitation and purified by Western blot. the molecular weight of the Ag is confirmed to be about 50 kDa. The mass spectrum analysis fails to clearly obtain a unique and reliable protein sequence, It's in the table The results are as follows: (1) The study itself The 2B8a FITC fluorescent antibody has good sensitivity and specificity; (2) 2B8a Ag is in the lymph node, tonsil, liver and pancreas can be detected in peripheral blood, which is mainly distributed in activated T cells, B cells, monocytes, neutrophils, The cell membrane and the cell pulp were not expressed on the resting T cell membrane, but the expression was also strong in its cytoplasm; (3) 2B8a Ag was An early-stable T-cell activation marker; in regeneration Up-regulation of expression in immune-related diseases, such as anemia; (4) 2B8 a) Ag can mediate the rapid internalization of the antibody; (5) 2B8a Ab can activate complement and play a significant role in C (6) 2B8a Ag could not mediate the adhesion between cells;
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

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