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.

     

Expression of CⅡTA Gene in Five Human Malignant Hematological Cell Lines and Its Significance

The role of MHC class Ⅱ transactivator (CⅡTA) in constitutive or IFN-γ inducib|e expression of HLA molecules in human malignant hematological cell lines was investigated. The expression of HLA molecules and CⅡTA protein was detected by Western blot, immunohistochemistry and flow cytometry. The expression of CⅡTA 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 CⅡTA quite well, andthe expression of HLA Ⅰ＋Ⅱ was increased in the tumor cells with constitutive or inducible expression of CⅡTA after induced by IFN-γ. The tumor cells which did not express CⅡTA after in-duced 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 CⅡTA, indicating CⅡTA might take part in the regu|ation of HLA Ⅰ＋Ⅱ expression in the tumor cells, which might p|ay an important role in tumor immunologic escape.     

COMPARISON OF HLA CLASS I ANIIGEN BY SEROLOGY AND PCR- SSP TYPING

Objeetive A dcuble blind study was canied cut to evaluate the relative performance and reliability of PCR-SSP assay compared to standard serollgical typing in identifying HLA class I alleles in Chinses population, Methods A total of 525 consecutive samples were entered into the study-HLA-A and B antigens were typed by serology with two - stage microlympbocytotoxicity and by PCR arnplification with sequence - specific primers, Reliability, reproducibility and clinical practicability wine compared acocrding to typing results by both methods. Results All sarnples were successfully typed by both methods THe serological discrepancy far HLA - A was 8.95 %, consisting of 21 antigens being iocorrectly interpreced and 26 of serologic blankx tuming out to be second definable alleles by SSP typing, Misassignments of HLA-B by serology were 12.19%. In 4.8% (25/525)a serologic blank tumed out to be a definable allele by SSP, while in 7,4 % (39/525) an allele was incorrectly interpreted by serology. The results of this study study showed that PCR- SSP technique could proved ta be equivalent to or surpass deal serology, suitable for routine clinical typing of patients and donors for transplantation. Conclusion PCR-SSP typing for HLA-A and B araigens could offer the advantages 4 hettre reagent availability and greater precision than setology in Chinese population, It is necessary to adopt the DNA typing for HLA class I in whcm serology often fails with“blank”,“difficult“, or cross-reactive antigens.     

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo

OBJECTIVE： Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine（TCM） drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus（r AAV） vectors has not been attempted. METHODS： We synthesized the c DNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged c DNAs were subcloned into a r AAV plasmid vector. The protein expression was confi rmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into r AAV vectors, under the control of a liver cancer-specifi c promoter. The liver tumor growth was then monitored following intratumor administration of the r AAV vectors.RESULTS： The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin（TCS） gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of r AAV vectors containing the TCS gene signifi cantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model.CONCLUSION： Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.     

Protective effect of O6-methylguanine-DNA-methyltransferase on mammalian cells

Background O^6-methylguanine-DNA-methyltransferase （MGMT） is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells. Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells （PBMCs） via liposome, then assayed for gene expression at RNA and protein levels. MIF assay was used to check the drug resistance of cells transfected with MGMT gene. Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively. Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC50 values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC. Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.     

Effects of Exogenous p16^ink4a Gene on Biological Behaviors of Human Lung Cancer Cells

The effects of exogenous p16ink4a gene on biological behaviors of human lung cancer cell line with homozygous deletion of p16ink4a gene were investigated. Exogenous p16ink4a gene was transfected by lipofectin into human lung cell line A549, in which p16ink4a gene was homozygously deleted. The expression of p16ink4a mRNA and protein was detected by RT-PCR and immunocyto-chemistry, respectively. The changes in the behaviors of the transfected cell lines in vitro and in vivo were observed. In the transfected cell line A549, the exogenous p16ink4a gene could be stably ex-pressed. The growth of A549 cells transfected with p16ink4a gene was obviously slowed down. Flow cytometry revealed that transfection of the exogenous p16ink4a gene resulted in A549 cell lines arrest in G1 phase of cell cycle. The tumorigenicity of these transfected cells in nude mice could be inhib-ited, and the tumor growth of nude mice was significantly suppressed. It was concluded that exoge-nous p16ink4a gene may be stably expressed in human lung cancer cell line A549. The expression of the introduced p16ink4a could block lung cancer cells to entry into S phase of cell cycle and inhibit tumor malignant growth both in vitro and in vivo.     

HIV-1 DNA vaccine with adjuvant cytokines induces specific immune responses against HIV-1 infection in mice

There is mounting evidence that the induction ofstrong mucosal and cell-mediated immuneresponses is key element to consider in constructing efficacious HIV-1 vaccine. Therapeutic vaccines that induce high levels of CTL specific to HIV are currently being developed worldwide. To induce strong and stable cell-mediated immune responses against HIV infection, immunoregulatory molecules including cytokines and chemokines have been used in the immunization protocols, and some of them were found …     

RFLP OF HLA CLASS I GENES IN CHINESE POPULATION AND ITS CORRELATION WITH SEROLOGICALLY DEFINED ANTIGEN SPECIFICITIES

The restriction fragnent length polymorphisms (RFLPs) of HLA class I genes in 104 unrelated healthy Chinese individuals were analyzed with Southern blot assay. The DNAs from peripheral blood leucocytes were digested with EcoRI, EcoRV and XbaI respectively, and hybridized with ~(32)P-labeled HLA class I probe ( 1.4Kb, B7 cDNA ). The results showed that 3 EcoRI fragments (13.7, 8.1 and 5.2 Kb ), 3 EcoR V fragments (11.3, 7.8 and 4.1 Kb) and 7 Xbal fragmnents (21.9, 19.2, 16.3, 6.0, 3.8, 1.8 and 1.5 Kb) were polymorphic. Ten of the fragments were found to be correlated significantly with the serologically de fined antigen specificities. The significance of this kind of correlation is discussed.     

ENDOGENOUS EXPRESSION AND HLA STABILIZATION ASSAY OF PLASMODIUM FALCIPARUM CTL EPITOPE MINIGENE IN HUMAN HLA- A2.1 AND HLA- B51 CELLS

INTRODUCTION Malaria is responsible for 300～ 500 million morbidity per year in the endemic tropical and subtropical areas, and Plasmodium falciparum, the causative agent of the most virulent form of human malaria infections, causes about 3 million deaths. The increasing incidence of resistance to most antimalarial drugs has stimulated researches aimed at controlling this disease by vaccination. Many studies have demonstrated the critical role played by CD8＋ cytotoxic T lymphocytes (C…     

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。     

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     

利用RT—PCR的方法检测恶性疟原虫海南株FCC1／HN CT基因在红细胞内期的表达及CTP基因真核表达载体的构建

目的 利用反转录PCR（RT-PCR）的方法检测CTP基因是否在恶性疟原虫红内期表达。并构建CTP因的真核表达载体,以便进一步研究其功能。方法 按常规方法体外培养红内期恶性疟原虫,用Trizol试剂提取红内期疟原虫总RNA,通过RT-PCR方法扩增恶性疟原虫FCC1/HN株CTP编码基因并构建CTP基因的真核表达载体。结果获得了恶性疟原虫CTPcDNA的全编码区序列并将其克隆入真核表达载体pcDNA3。结论 CTP基因在恶性疟原虫FCC1/HN株红细胞内期表达并成功地构建了CTP基因的重组表达质粒。     

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