ENDOGENOUS EXPRESSION AND HLA STABILIZATION ASSAY OF PLASMODIUM FALCIPARUM CTL EPITOPEMINIGENE IN HUMAN HLA－A2. 1 AND HLA－BS1 CELLS

Objective. To evaluate the Plasmodium falciparum CTL epitope vaccines in HLA class I allele specific human cell lines that have high frequency among Chinese population. Methods. Synthesized oligonucleotides encoding for P.f. CTL epitope genes, constructed eukaryotic expression plasmids, transfected the minigenes into HLA class I allele specific human cell lines and identified endogenous expressing of the minigenes by RT-PCR and HLA stabilization assay. Results. Two mini-genes encoding Plasmedium falciparum CTL epitopes were designed and cloned, respectively, into an eukaryotic expressing vector to form TR26 which was restricted to HLA-B51,SH6 which was restrictedto HLA-A2.1, and TS, which had the two aforementioned mini-genes fused in tandem. All of these CTL epitope genes were transfected and endogenously expressed in respective cell lines containing appropriate HLA molecules.The obviously increased expressions of HLA class I molecules were detected in the transfected cell lines. It was demonstrated that the two discrete Plasmedium falciparum epitope genes were effectively processed and presented, and the close proximity of the two epitope genes in one chain as in mini-gene TS did not interfere with the process-ing and presenting of each epitope gene in corresponding cell line. Conclusion. A successful expression and presentation of multiple CTL epitope mini-gene in MHC class I allele specific human cell lines were demonstrated by an in vitro assay, which could be corresponding to the vaccina-tion of CTL vaccines in people with different MHC I molecules. This work also suggested the possibility of constructing a multiple CI~ epitope plasmodium falciparum DNA vaccine that could cover most of Chinese population.     

恶性疟细胞毒性T淋巴细胞单表位疫苗抗原递呈细胞模型的建立

目的 构建恶性疟细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）单表位疫苗及建立抗原递呈细胞模型。方法 采用中国人群常见的HLAⅠ类分子A11限制的恶性疟（CTL）抗原表位(VTCGNGIQVR),合成其DNA序列并克隆于真核表达载体,构建CTL单表位DNA疫苗。将上述克隆在HLA－A11表型细胞株中进行表达,流式细胞仪检测细胞表面HLAⅠ类分子表达水平。结果 上述CTL单表位DNA疫苗编码的短肽在细胞内的表达明显促进HLA－A11分子在细胞表面的表达,用流式细胞仪测定平均荧光强度可量化表达水平（P＜0.05）。结论 成功构建CTL单表位短肽表达载体,模拟体内环境建立了抗原递呈细胞模型,提示CTL表位在该细胞模型内被内源性加工和递呈,以该表位为基础的疫苗可以为HLA－A11遗传背景的人群提供免疫防护。     

KBv200细胞株耐药逆转前后HLA、B7抗原的表达

目的 探讨肿瘤多药抗性细胞的免疫逃避机制。方法 利用特异性切割mdr1的核酶为工具,以表达Mdr1的耐药细胞株KBv200为靶细胞,采用脂质体转染技术,将含核酶的质粒pHβApr-1neo/5mR3及空载体pHβApr-1neo导入KBv200及其亲本KB细胞内,运用Northern-blotting、免疫组化方法观察核酶对mdr1 mRNA及P-gp的影响,运用流式细胞仪技术检测各种不同细胞亚株HLA-Ⅰ、HLA-Ⅱ、B7-1、B7-2的表达。 结果 含核酶的质粒pHβApr-1neo/5mR3及空载体pHβApr-1neo可以在KB、KBv200细胞中稳定表达,核酶可以特异性地切割mdr1,导致KBv200/5mR3的mdr1 mRNA含量下降,P 糖蛋白（P-gp）表达减低,各种不同细胞亚株均表达较强的HLA-Ⅰ类抗原,而HLA-Ⅱ、B7-1、B7-2的表达较低。各亚株HLA-Ⅰ表达无明显差异,但HLA-Ⅱ、B7-1、B7-2的表达变化较大,KB的HLA-Ⅱ、B7-1、B7-2的表达较KBv200强,经化疗药物作用后KB的HLA-Ⅱ、B7-2进一步表达增强,核酶逆转后,KBv200/5mR3 HLA-Ⅱ、B7-1、B7-2的表达趋于接近KB水平。 结论 mdr1-核酶在细胞内具有一定的逆转肿瘤多药抗性的生物学效应;多药耐药细胞和敏感株细胞有着不同的免疫逃避特点,与敏感株相比,KBv200较易逃避机体的免疫反应。     

Enhanced expression of HLA class I molecules in human hepatocellular carcinoma cells transduced with gamma-interferon gene.

OBJECTIVE To investigate the expression of exogenous gamma-interferon gene in human hepatocellular carcinoma cells following retroviral transduction and the effect on the expression of surface HLA class I molecules.

METHODS Retroviral vector pLXSN was used to introduce human gamma-interferon (IFN-gamma) gene into four different human hepatocellular carcinoma cell lines (HCC). The G418-resistant colonies were isolated and cloned. The integration and expression of IFN-gamma gene were determined by PCR and RT-PCR analysis. A bioassay method was used to test the amount of IFN-gamma secreted by gene modified HCC cells. The expression of HLA class I molecules in HCC cells were analyzed by flow cytometry using indirect fluorescence staining.

RESULTS Four different HCC cell lines were successfully transduced with human IFN-gamma gene using retroviral vector. The integration and expression of IFN-gamma gene were shown only in the transduced cells. All four genetically modified HCC cells can secrete varied amount of IFN-gamma and demonstrate a significant up-regulation of surface HLA class I antigens. One specific HLA class I antigen, HLA-A2, has almost the same degree of increase as that of the total HLA class I molecules after transduction with IFN-gamma gene.

CONCLUSIONS Gene modification with IFN-gamma gene can significantly enhance the expression of HLA class I molecules in HCC cells and may increase its immunogenicity. These gene modified tumor vaccines can be helpful in tumor biotherapy.

     

人类白细胞抗原Ⅱ类基因多态性与胃肠道疾病相关性的研究进展

人类白细胞抗原(HLA)是目前发现的与疾病关联最为密切的基因复合体,决定疾病易感性和个体差异。胃肠道疾病(如消化性溃疡)是人类最为常见的疾病。HLA-Ⅱ类基因多态性可以改变HLA分子、抗原和T细胞之间相互作用的特性,并因此控制对外来抗原(或自身抗原)的免疫应答,致人类疾病的易感性和临床表现不同。现就HLA-Ⅱ类基因多态性与胃肠道疾病的相关性研究予以综述。     

T辅助细胞在疫苗研制中的作用

发展感染性疾病疫苗之关键挑战在于利用确定的抗原以刺激产生能引起保护作用的合适的免疫反应。肽类疫苗的运用得到了极大的关注，其意义在于，已知不同的多表位构成单一结构以诱导出所希望的免疫反应所表现出的灵活性。这一般比利用减毒的活疫苗要安全并且相对而言比制造亚单位疫苗要容易。然而，多肽疫苗的发展面临巨大挑战。这一方法在诱导遗传背景复杂的人群免疫反应方面受到限制，这与主要组织相溶性复合物(MHC)多态性有关。因同样的理由，肽类免疫应答常因缺乏适当的辅助T淋巴细胞(HIL)而引导出不充分的细胞毒素T淋巴细胞(CTL)和抗体反应。另一个运用线性肽链结构的可能缺点是：为了引导出合适抗体反应，表面免疫球蛋白受体簇对于激活静息的B细胞就成为必须因素。由WHC多肽性引起的问题可由运用不加区别的T细胞表位来解决。从麻疹病毒F蛋白(氨基酸288到302)中得到的不加区别的T细胞表位和鼠的确定结合在多种MHC分子上的辅助T细胞表位(v1EB，aa191-209)已被定性并且被用于能极大激发免疫应答的结构中，以克服单一限制型免疫应答的缺陷。合成的，非自然Pan DR表位(PADRE)具有退化的结合几种通常HLA—DR的能力，能以绝对效价和抗体反应质量两种形式来增强激发短肽链的免疫应答。另外，一些所谓的从流感病毒血凝素(HA)来的“不加区别的”T细胞表位，恶性疟疟原虫红细胞前期抗原和分枝杆菌蛋白被报道能激发广泛的免疫应答。为了不加区别地结合于几种同型和同种异型的MHCⅡ类分子，这些肽类应显示出部重叠MHC结合形式或应利用保存于配体中的固定位点和应缺失等位基因特异性固定残基，以防止结合于其它Ⅱ类分子。了解MHCⅡ类分子对肽链的不加区别及特异性识别的生物物理学基础将为在疫苗设计中突破遗传限制的策略提供分子水平的依据。     

CⅡTA基因在五种人类恶性血液病细胞株细胞中的表达及意义

目的:探讨5种血液病细胞株细胞的HLA-Ⅱ类抗原表达,以及对IFN-γ诱导HLA分子表达的反应性与MHCⅡ类分子反式激活因子 (CⅡTA) 表达的关系.方法:采用流式细胞术和免疫组化法检测肿瘤细胞HLA分子及CⅡTA 蛋白的表达,RT-PCR检测肿瘤细胞CⅡTA基因表达.混合淋巴细胞反应检测肿瘤细胞刺激外周血T细胞反应的能力.结果:肿瘤细胞HLAⅡ类分子表达与CⅡTA表达一致; 结构型或诱导型表达CⅡTA的肿瘤细胞,经IFN-γ作用后其HLAⅠ、Ⅱ类抗原表达增高;IFN -γ诱导后仍不表达CⅡTA的肿瘤细胞,其对IFN-γ促HLAⅡ表达的作用不反应.Jurkat诱导后刺激T细胞表达高水平的IL-2 mRNA.结论:某些恶性血液病细胞株细胞对IFN-γ不能诱导HLA分子表达与CⅡTA诱导型表达缺陷有关,表明CⅡTA参与调控肿瘤细胞 HLAⅠ、Ⅱ类抗原表达,可能在肿瘤免疫逃逸中起重要作用.     

丙型肝炎病毒NS3区C末端HLA-11限制性CTL表位的作用

目的 研究丙型肝炎病毒（ｈｅｐａｔｉｔｉｓ Ｃ ｖｉｒｕｓ,ＨＣＶ）非结构３区（ＮＳ３）Ｃ末端ＨＬＡ－１１限制的细胞Ｔ细胞（ＣＴＬ）表位在ＨＣＶ感染中的作用。方法 血清学方法检测３例患的ＨＬＡ分型,他们均存在ＨＬＡ－Ａ１１表型,以ＨＬＡ－１１结合基序为依据,预测了ＨＬＡ－１１限制的ＣＴＬ表位。２例慢性患５ａ内３个时间点和３ａ内２个时间点及转阴患的血清样本,用反转录ＰＣＲ扩增出ＨＣＶ基因片段,     

MART-1 HLA-A2限制性CTL表位的预测

目的 预测黑色素瘤分化抗原ＭＡＲＴ－１的ＨＬＡ－Ａ２限制性细胞毒性Ｔ淋巴细胞（ＣＴＬ）表位。方法 采用超基序与量化基序多项式方案相结合的办法,对目的抗原ＭＡＲＴ－１的ＨＬＡ－Ａ２限制性ＣＴＬ表位进行预测。结果 预测出了６个九肽表位。结论 两种方法预测结果的一致性较好,所预测出的６个ＭＡＲＴ－１的ＨＬＡ－Ａ２限制性ＣＴＬ表位经后续实验筛选,鉴定后,可望用于新型ＭＡＲＴ－１肿瘤治疗性多肽疫苗的设计研究     

Expression of C II TA gene in five human malignant hematological cell lines and its significance

Summary The role of MHC class II transactivator (C II TA) in constitutive or IFN-γ inducible expression of HLA molecules in human malignant hematological cell lines was investigated. The expression of HLA molecules and C II TA protein was detected by Western blot, immunohistochemistry and flow cytometry. The expression of C II TA gene was determined by RT-PCR. The capability of peripheral blood T cell reaction stimulated by tumor cells was monitored by mixed lymphocyte reaction. It was found that the HLA II-positive tumor cells expressed the C II TA quite well, and the expression of HLA I + II was increased in the tumor cells with constitutive or inducible expression of C II TA after induced by IFN-γ. The tumor cells which did not express C II TA after induced by IFN-γ were not response to the expression of HLA II promoted by IFN-γ. It suggests a correlation between the inability of some malignant hematological cell lines in response to IFN-γ for HLA expression and the deficiency in the inducible expression of C II TA, indicating C II TA might take part in the regulation of HLA I+II expression in the tumor cells, which might play an important role in tumor immunologic escape. YOU Yong, male, born in 1969, Doctor in Charge     

应用计算机预测T细胞抗原表位研究HCV免疫反应

目的：编写预测Ｔ细胞怕表位的软件ＧＵＯＴＩＦ,对１例丙型肝炎病毒（ＨＣＶ）健康带者进行人类白细胞怕（ＨＬＡ）分型及ＨＣＶＮＳ３区部分区段的一级结构测定,用ＧＵＯＴＩＦ预测其Ｔ细胞怕表位,。为合成肽研究针对ＨＣＶ的免疫反应提供指导。方法：在Ｉｎｔｅｒｎｅｔ上搜集各型主要组织相容性复合体（ＭＨＣ）分子的ｍｏｔｉｆ资料,以之为基础用ＶｉｓｕａｌＣ＋＋语言编写ＧＵＯＴＩＦ软件,用已报道的ＨＣＶＴ细胞怕表位     

HLA基因多态性与原发性高血压的相关性研究进展

人类白细胞抗原（HLA）由称人类主要组织相容性复合体（MHC）,它由一组具有高度多态性和连锁的不均衡性基因群体构成,其研究主要于器官移植、免疫应答、法医学、人类学和疾病相关性等领域。本文介绍了HLA的基因结构、多态性及HLA—Ⅱ类基因多态性与EH相关性等方面内容。     

Enhanced expression of HLA class Ⅰ molecules in human hepatocellular carcinoma cells transduced with γ-interferon gene

ＥｎｈａｎｃｅｄｅｘｐｒｅｓｉｏｎｏｆＨＬＡｃｌａｓⅠｍｏｌｅｃｕｌｅｓｉｎｈｕｍａｎｈｅｐａｔｏｃｅｌｕｌａｒｃａｒｃｉｎｏｍａｃｅｌｓｔｒａｎｓｄｕｃｅｄｗｉｔｈγｉｎｔｅｒｆｅｒｏｎｇｅｎｅＱｉａｎＳｈｕｂｉｎｇ钱书兵，ＺｈａｎｇＴｅｎｇｆｅ...     

MHC-Ⅱ类分子与hTSH受体共表达系统的建立

目的 :应用电击基因导入法 ,建立 MHC- 类分子与 h TSHR共表达体系 (h Ml2 ) ,为 GD发病机制的研究奠定基础。方法 :采用电击基因导入法将质粒 p CRTM3- h TSHR转染于 M12细胞 (H- 2 d) ,应用 G4 18筛选出阳性细胞 ,经有限稀释获得单克隆细胞株。用逆转录聚合酶链反应 (RT- PCR)法检测单克隆细胞内 h TSHR胞外段 m RNA的表达 ;用甲亢病人血清粗提 Ig G及 b TSH刺激后测定上清中 c AMP值以检测单克隆细胞株表达的 h TSHR是否具有天然 h TSHR功能。结果 :应用有限稀释法获得 14株单克隆细胞。RT- PCR结果显示 :3号、5号、6号、8号、11号细胞内有高水平的 h TSHR胞外段 m RNA的转录。应用甲亢病人血清粗提 Ig G及 b TSH刺激后 ,6号、8号细胞上清中c AMP值明显升高 ,与对照组之间存在显著差别 (P<0 .0 5 )。结论 :应用电击基因导入法成功地建立了 MHC- 类分子与 h TSH受体共表达体系。     

肾透明细胞癌和卵巢癌与HLAI类抗原关联性研究

目的 分析HLAI类抗原与肾透明细胞癌和卵巢癌的关联性。为确定与这些肿瘤的易感性和抵抗性有关的基因。并为寻找肿瘤特异性的抗原肽,开展肿瘤免疫诊断和治疗奠定基础。方法 实验采用微量淋巴细胞毒试验。分析82例肾透明细胞癌和21例卵巢癌患者外周血HLA-A抗原和-B抗原的抗原频率。结果 与肾透明细胞癌和卵巢癌关联的HLAI类完全不同,其中与肾透明细胞癌关联的位点大大多于卵巢癌的位点,有A9、A11、A28、BG15、B22、B27和B40;而与卵巢癌关联的HLAI类抗原位点只有A10、A36和B12,且卵巢癌关联A10抗原保持显著关联。结论 HLAI类抗原关联性研究显示,HLA-A10可能为卵巢癌提供抵抗性。     

肿瘤抗原MAGE-3 CTL预测表位的计算机分子模拟研究

目的 从理论上分析预测表位与ＨＬＡ－Ａ２分子的结合情况及其为ＨＬＡ－Ａ２限制性ＣＴＬ表位的可能性。方法 应用计算机分子模拟的方法,建立ＭＡＧＥ－３４个ＣＴＬ预测表位的ＨＬＡ－Ａ２结合三维结构和各多肽与ＨＬＡ－Ａ２分子所形成复合的三维结构。结果 ４个预测表位的三维结构完全符合ＨＬＡ－Ａ２限制性ＣＴＬ表位的结构要求,且都能与ＨＬＡ－Ａ２分子结合。结论 ４个ＣＴＬ预测表位为ＨＬＡ－Ａ２限制性ＣＴＬ表位的     

Expression of C Ⅱ TA Gene in Five Human Malignant Hematological Cell Lines and Its Significance

The role of MHC class Ⅱ transactivator (C Ⅱ TA) in constitutive or IFN-γ inducible expression of HLA molecules in human malignant hematological cell lines was investigated. The expression of HLA molecules and C Ⅱ TA protein was detected by Western blot, immunohistochemistry and flow cytometry. The expression of C Ⅱ TA gene was determined by RT-PCR. The capability of peripheral blood T cell reaction stimulated by tumor cells was monitored by mixed lymphocyte reaction. It was found that the HLA Ⅱ-positive tumor cells expressed the C Ⅱ TA quite well, and the expression of HLA Ⅰ + Ⅱ was increased in the tumor cells with constitutive or inducible expression of C Ⅱ TA after induced by IFN-γ. The tumor cells which did not express C Ⅱ TA after induced by IFN- γ were not response to the expression of HLA Ⅱ promoted by IFN- γ. It suggests a correlatior between the inability of some malignant hematological cell lines in response to IFN-γ for HLA expression and the deficiency in the inducible expression of C Ⅱ TA, indicating C Ⅱ TA might take part in the regulation of HLA Ⅰ + Ⅱ expression in the tumor cells, which might play an important role in tumor immunologic escape.     

肾移植术后新生HLA抗体和MICA抗体对移植肾功能的损伤作用

目的研究肾移植术后新生人类白细胞抗原(HLA) 抗体及主要组织相容性I类相关链A抗原（MICA）抗体的产生对移植肾功能的损伤作用。方法采用免疫磁珠流式细胞仪液相芯片技术检测96例肾移植患者HLA及MICA抗体。根据检测结果,将患者分为HLA抗体阳性组（HLA+）、MICA抗体阳性组（MICA+）、以及HLA、MICA抗体阴性组（HLA /MICA ）,观察并比较各组不良免疫事件的发生率。再根据有无急性排斥（AR）,将患者分为排斥组（AR+）和非排斥（AR ）组,观察HLA抗体、MICA抗体对移植肾功能的影响。结果HLA+组急性排 斥反应发生率高于HLA /MICA 组(43．5％ vs 11.7％,P＜0.05);MICA+组急性排斥反应发生率与HLA /MICA 组比较差异无统计学意义(15．3％ vs 11．7％,P＞0．05);AR+组中,HLA+患者术后1、3、6和12个月时血清肌酐水平高于HLA /MICA 患者, MICA+患者在术后1、3月时血肌酐水平高于抗体阴性患者;AR 组中,HLA+患者术后6、12个月血肌酐水平高于HLA /MICA 患者;MICA+组在术后6个月及12个月的尿蛋白定量均值高于150?mg,并随时间逐渐升高。结论HLA抗体对移植肾功能的损伤作用表现为血清肌酐的升高以及与急性排斥反应的发生有关;MICA抗体对移植肾功能的损伤主要表现为尿蛋白定量的升高。     

恶性疟原虫乳酸脱氢酶单克隆抗体的制备及其特性鉴定

为制备抗恶性疟原虫乳酸脱氢酶（LDHp）单克隆抗体（McAb）,并对其特异性进行鉴定,用纯化的LDHp重组抗原免疫BALB/c小鼠,采用杂交瘤技术制备McAb,筛选出分泌高滴度McAb的杂交瘤细胞株,测定其免疫球蛋白亚类及其效价,ELISA、Westen blotting试验分析其特导性。结果,制备出2A5和 1H10两株能稳定分泌抗LDHp　McAb的杂交瘤细胞株,两株单抗均为IgG2b,2A5和1H10培养上清的ELISA效价分别为1:512和1:256,腹水效价分别为1:25600和1:12800,两株单抗与间日疟、红细胞、弓形虫、日本血吸虫等抗原均不发生交叉反应,能识别恶性疟原虫的33Kda虫源蛋白。证明制备的抗LDHp杂交瘤细胞株能分泌高滴度和高特异性的单抗。     

脂质体包裹可溶性蛋白质抗原诱导特异性CTL的研究

目的：搪塞用可溶性蛋白质抗原诱导特异性ＣＴＬ的途径。方法：用冻融－超声法制备包裹可溶性鸡卵蛋白（ＯＶＡ）和ＣＡ－Ｈｂ３蛋白质抗原的阳离子脂质体，并借助脂南体将抗的导入Ｃ５７ＢＬ／６小鼠脾细胞胞浆内，以此细胞经尾静脉免疫Ｃ５７ＢＬ／６小鼠，取体内初致敏脾细胞，在体外以渗透方式导入ＯＶＡ或ＣＡ－Ｈｂ３抗原的脾细胞再刺激，诱导特异性ＣＴＬ，并进行Ｉ类途径阻断实验和ＭＨＣＩ类分子中和实验，结果：脂质体成功