In order to investigate the effect of T-bet on malignant cells, we selected SGC-7901, a kind of human gastric carcinoma cell line, and used gene clone technique and adeno-associated virus (AAV) packing technology, thus obtaining a recombinant rAAV-eGFP-T-bet and T-bet gene-transfected SGC-7901 cells. Then the function of T-bet gene-infected SGC-7901 cells was researched by detecting the levels of IFN-gamma and T-bet production. The results showed: (1) It was verified that rAAV-T-bet's packing was completed; (2) After SGC-7901 cells was transfected by rAAV-eGFP-T-bet, a green fluorescence was found in about 30%-40% SGC-7901s, and the gene of 1670 bp (T-bet) and 388 bp (IFN-gamma) were generated from SGC-7901s cells; (3) The proteins of IFN-gamma and T-bet secreted by SGC-7901 cells were also detected. These reveal that SGC-7901 cell is efficiently infected by rAAV encoding T-bet, which can induce transfected cells to secret IFN-gamma. It may be useful in the researches on cancer immune therapy of transfecting T-bet gene.

目的:构建含小鼠GITRL基因原核表达载体,经转化E.coli以表达GITRL融合蛋白,纯化蛋白并制备相应的抗血清.方法:将GITRL-pMD18-T重组质粒经EcoRⅠ和SalⅠ双酶切回收目的基因,与原核表达载体PET32a连接,应用电转法转化BL21(DE3)菌.阳性克隆经鉴定后用IPTG诱导GITRL蛋白表达,经Ni+-NTA层析柱纯化融合蛋白,通过SDS-PAGE、Western blot进行特异性鉴定,CD4+CD25+T细胞免疫抑制试验验证其生物学功能.采用弗氏完全佐剂常规免疫方案,制备抗GITRL抗血清,抗血清的效价和特异性测定采用双向琼脂免疫扩散法.结果:经酶切鉴定和测序分析证实原核表达质粒构建正确,SDS-PAGE和Western blot结果显示,在40 kD处呈现单一蛋白条带,该蛋白具有阻断CD4+CD25+T细胞免疫抑制功能的作用,双向免疫扩散结果显示兔抗小鼠GITRL抗血清效价为1∶16.结论:成功构建GITRL原核表达质粒,制备和纯化的GITRL融合蛋白具有较好的纯度和生物学功能,制备并获得特异性抗血清,可用于GITRL融合蛋白生物学活性的进一步研究.

T-bet, a Th1-specific transcription factor, can promote the production of IFN-γ. IFN-γ is the principal Th1 effector cytokine and it has a crucial role in Th1 differentiation, which can drive the differentiation of naïve CD4+T cells into T-helper 1 (Th1) cells. In our study, a human T-bet gene was fused with a gene fragment encoding HIV-1 protein transduction domain in a bacterial expression vector to produce a Tat/T-bet fusion protein. The expressed and purified Tat/T-bet proteins were transduced efficiently into THP-1 cells in a time- and dose-dependent manner; when Tat/T-bet pretreated THP-1 cells were co-cultured with CD4+T cells, the IFN-γ level increased higher to about 7 pg/ml, 10-folds as compared with the normal level when tested at 48 hours. The results demonstrated that the Tat/T-bet fusion protein can be efficiently transduced into antigen-presenting cells (APCs) like THP-1 cells and then regulated Th1/Th2 balance, which may act as a potential tool for gene therapy.

Background: CD4+CD25+ regulatory T (Treg) cells play a pivotal role in the maintenance of the homeostatic balance of the immune system. The current study was conducted to evaluate the prevalence of CD4+CD25+ Treg cells in cancer patients and to identify possible mechanisms contributing to Treg cell expansion. Materials and Methods: The frequencies of CD4+CD25+FOXP3+ T cells in the peripheral blood mononuclear cells (PBMCs) of 62 cancer patients and 25 healthy individuals were determined by flow cytometry. The purified CD4+CD25– T cells were stimulated with malignant pleural effusion (MPE) or tumor cell-derived supernatants (TCS) from cancer patients in vitro. MPE/TCS-exposed CD4+ CD25– T cells were also characterized in terms of phenotype (CD4, CD25, and FOXP3 expression) and function (i.e. suppression assays). Results: An increased population of CD4+ CD25+FOXP3+ T cells was detected in the peripheral blood of cancer patients, with no obvious difference between the adenocarcinoma group and the squamous carcinoma group. When stimulated with MPE or TCS, a fraction of natural CD4+CD25– T cells was converted into CD4+CD25+ FOXP3+ T cells which had the functional activity resembling natural CD4+CD25+ Treg cells with anergy and imm unosuppression. Conclusion: The percentages of CD4+CD25+ FOXP3+ Treg cells in PBMCs of cancer patients are significantly increased. Tumor-derived factors such as TGF-βmay contribute to CD4+CD25+ Treg cell expansion in cancer patients.

目的:利用载体pQE30在大肠埃希菌(M15)原核表达人T-bet基因全长序列,并对表达产物进行纯化、免疫动物和制备多克隆抗体.方法:利用PCR技术从克隆载体pGEM-T/T-bet获得人T-bet的全长编码序列,并将其亚克隆至原核表达载体pQE30,形成重组表达质粒pT-bet;酶切鉴定挑选阳性重组质粒转化大肠埃希菌JM109,测序鉴定后转化M15,经IPTG 37℃诱导4 h后,SDS-PAGE电泳、考马斯亮蓝染色判断以包涵体形式存在的带有6×His标签的融合蛋白(表达产物),用Ni2+-IMAC层析柱对融合蛋白进行纯化;将纯化的蛋白免疫BALB/c小鼠,制备人T-bet的多克隆抗体.结果:成功表达和纯化了人T-bet,并制备了多克隆抗体,ELISA和Western blot结果显示了血清抗体的高效价(1:100 000)和高度特异性.结论:成功构建了原核表达载体pT-bet,并在工程菌M15中获得大量表达,经纯化后获得高纯度的人T-bet蛋白,制备了效价和特异性良好的多克隆抗体,为进一步研究T-bet的生物学功能奠定了的基础.

目的:分析胃癌患者Th1和Th2两类细胞因子的基因表达与转录因子T-bet和GATA3表达的相关性,从细胞因子与转录因子角度考证胃癌患者Th1/Th2细胞分化趋势,探讨p53与T-bet的相关性,了解T-bet与肿瘤转移之间的关系.方法:利用荧光定量PCR技术检测55例胃癌患者及45例正常人外周血单个核细胞(PBMC)中T-bet、GATA3转录因子和IFN-γ、IL-4等细胞因子的水平,应用免疫组化法检测胃癌组织中p53的表达状况.结果:55例胃癌患者T-bet、GATA3、IFN-γ、IL-4的表达率依次为51%(28/55)、78%(43/55)、27%(15/55)、69%(38/55).定量PCR结果显示,病人组的T-bet和IFN-γ明显低于正常对照组(P＜0.01),而GATA3和IL-4则明显高于正常对照组(P＜0.01).T-bet与IFN-γ在病人体内的mRNA表达存在正相关性(P＜0.01),正常人则没有.GATA3与IL-4的表达在两组中均呈明显正相关(P＜0.01和P＜0.05),且在p53阳性的患者中,T-bet的表达率较低,而GATA3的表达率较高.结论:胃癌患者有明显Th2漂移现象,且p53的表达与T-bet的表达呈负相关.

Objective:To investigate the regulatory effects of synthetic oligodeoxynucleo tides containing CpG motif (CpG-ODN) on the expression of T-bet and the secretion of IFN-γ from peripheral blood mononuclear cells (PBMC) from infants with wheezing bronchitis.Methods:28 cases of wheezing bronchitis (W.B group) were enrolled, and 26 normal infants were served as control. PBMCs were isolated from peripheral blood over a Ficoll-Hypaque gradient reagent according to standard procedures and incubated with CpG-ODN (10μg/ml) at 37℃, 5%CO2 for 48 hours. The mRNA and protein expression levels of T-bet in PBMC were detected by both reverse transcription PCR (RT-PCR) and Western blot. IFN-γ levels in culture supernatant of PBMC were also tested.Results:The mRNA and protein levels of T-bet from PBMC in infants with wheezing bronchitis were lower than those of control, and were up-regulated after the stimulation of CpG-ODN at 10μg/ml. The IFN-γ levels secreted by PBMC were not affected by the treatment of control-ODN stimulating.Conclusion:The mRNA and protein levels of T-bet in PBMC are lower in infants suffered from wheezing bronchitis. It is suggested that the decreased expression of T-bet may play an important role in immunopathology of the disease. As an immunoregulator, CpG-ODN can up-regulate T-bet mRNA and protein in PBMC remarkably, despite of not driving PBMC to express more IFN-γ.

Objective To investigate the effects of T-bet gene in leukermic cell on the level of IFN-γ for seeking a way of improving the immune state of leukemia patient by immunomodulation. Methods pIRES-eGFP-hTbet recombinant plasmid was established, and then transfected into U937 cells by using electroporation methods. The human T-bet gene was amplified and identified by RT-PCR; the expression protein of T-bet was observed by Western blotting; the level of IFN-γ was detected by ELISA. Results After transfection, about 80% fluorescent U937 cells were detected. T-bet mRNA and protein were found in transfected T-bet U937 cells but not in negative and untransfected U937 cells. The level of IFN-γ protein was obviously high in T-bet -transfected U937 cells but was not obviously detected in negative and untransfected U937 cells. Conclusion Human T-bet transfected-U937 cell is successfully obtained and the cell secretes high level of IFN-γ. Our study may shed some light upon the clinical usage of T-bet gene or its expression product as a therapeutic reagent for induction of Th1-dominated response.

Objective:To confirm differentiation of Th1/Th2 on the levels of transcription and cytokines production in patients with gastric carcinoma,and to reveal the relationship between the expression of T-bet/GATA3 and Th1/Th2 type cytokines in peripheral blood mononuclear cells(PBMC).In addition, the relation of p53 and T-bet expression was discussed for understanding the role of T-bet in metastasis. Methods:The gene expression of Th1/Th2 type cytokines IFN-γ/IL-4,transcription factors T-bet/GATA3 were determined by RT-PCR and realtime RT-PCR from 55 patients with gastric cancer and 45 normal person. The expression of p53 was analyzed by immunohistochemistry. Results: Predominant expression of Th2 type cytokines was detected in 55 gastric cancer patients. The expression rates of T-bet,GATA3,IFN-γ and IL-4 was 51%(28/55),78%(43/55),27%(15/55)and 69%(38/55),respectively. The expression rates of T-bet and IFN-γ in patients were lower than control groups, but the results of GATA3 and IL-4 were opposite. The similar results were obtained by realtime PCR. There was a positive correlation between the expression of T-bet and IFN-γ in patient, but no relationship in normal person. The positive correlation between GATA3 and IL-4 was also found in patients and in normal person. The expression of p53 in patients was accompanied with lower expression rate of T-bet and higher expression of GATA3.Conclusions:Predominant expression of Th2 type cytokines may be related to lower expression of T-bet or higher expression of GATA3 in PBMC of gastric cancer patients. The negative correlation exists between p53 and T-bet.

Objective:To obtain recombinant protein of an soluble extracellular fragment of activation-inducible tumor necrosis factor ligand ( sAITRL), and to analyze its bioactivity.Methods:sAITRL gene was amplified by PCR from the full-length plasmid AITRL-pMD18-T and subcloned into the pQE30 vector. The recombinant plasmid was transformed into E.coli M15, and the expression of recombinant protein was induced by IPTG. After purified and refolded with Ni2+-IMAC column, identification of the recombinant protein by Western blot was performed. The binding ability of sAITRL protein to natural AITR was evaluated by FCM, and the bioactivity was estimated by proliferation assay in vitro.Results:The prokaryotic expression vector for sAITRL-pQE30 was constructed successfully. sAITRL protein with molecular mass about 15 kD was expressed in E.coli M15 after IPTG induction. This recombinant sAITRL,which was able to bind to natural AITR on surface of activated lymphocytes,significantly stimulated proliferation of lymphocytes at low doses, but had an opposite effect at higher concentrations.Conclusion:Recombinant sAITRL with bioactivity is expressed successfully. AITR-AITRL interaction appears to be crucial in regulation the ongoing immune responses.

大约有20多种人重组体蛋白药物已用于临床治疗,特别是细胞因子和抗细胞因子用途尤为广泛,重组体药物在患者体内的转运能够诱导免疫应答,削弱治疗效果,导致超敏反应和自身免疫方面的疾病.本文对不同细胞因子治疗引起的自身免疫性疾病进行综述.

Objective: To clone and analyze the cDNA encoding human T-bet expressed in T lymphocyte(T-bet) gene,a type of T-bet family member.Methods: The cDNA of T-bet was amplified from total RNA extracted from human peripheral blood mononuclear cell(PBMC) by RT-PCR and inserted into pGEM-Teasy vector,and the sequence of the DNA was analyzed.Results: The cDNA of T-bet has the length of 1 607 bp with an complete open reading frame,which encodes a product of 530 amino acid and shares 100% homology with the sequence of mRNA for human T-bet in GeneBank.Conclusion: The cDNA of T-bet was successfully cloned,which brought a foundation for further research on its biological function.

Objective To investigate the modulation function of transcription factor T-bet/GATA-3 in infants with wheezing bronchitis.Methods Thirty-two cases with wheezing bronchitis were collected while 30 normal infants were served as control.Lymphocytes were isolated from peripheral blood by Ficoll and incubated with phytohemagglutinin(PHA)(100 mg/L) at 37 ℃,5% CO_2 for 48 hours.Interferon-γ(IFN-γ) and interleukin 4(IL-4) levels in the supernatant were detected by enzyme linked immunosorbent assay(ELISA).The mRNA levels of T-bet and GATA-3 gene in lymphocytes were amplified by reverse transcription polymerase chain reaction(RT-PCR).Results In infants with wheezing bronchitis,the levels of IFNγ were lower than those of control(P0.05) and(IL-4) were higher than those of control(P0.01).In the matter of the fact,the T-bet did also reduce when GATA-3 relatively increased in this kind of dieseae.Two positive correlations were found between the level of IFN-γ and T-bet mRNA(r=0.57 P0.01) as well as IL-4 and GATA-3 mRNA(r=0.60 P0.01).Two negative correlation were also proved in the level of IFN-γ and IL-4(r=-0.38 P0.05),T-bet mRNA and GATA-3 mRNA(r=-0.46 P0.05).Conclusions In infants with wheezing bronchitis,there is a predominant differentiation of Th2 type cells accompanying with over-production of Th2 cytokine IL-4 and lower production of Th1 cytokine IFN-γ.From this research,it is suggested that the imbalance differentiation of Th cells is regulated by transcription factor T-bet and GATA-3,of which the less T-bet expression may be a key factor.

AIM To determine the rate of furazolidone resistance of Helicobacter pylori (H. pylori) isolated from gastric biopsy specimens and to explore the relationship between genetic mutations in porD and oorD genes of H. pylori and its resistance to the antibiotic. METHODS Gastric biopsy was performed in 83 adult patients aged 31-77 years with gastric complaints. H. pylori was isolated from biopsy specimens of 46 patients. E-test and 2-fold agar dilution method were used to determine the rate of H. pylori resistance to furazolidone. The genes porD and oorD from susceptible and resistant isolates were amplified by polymerase chain reaction (PCR), and their PCR products were sequenced. RESULTS Resistance to furazolidone was found in 8.7% of H. pylori isolates and 6 mutations were detected in porD and oorD genes of the resistant isolates. Three mutations--G353A, A356G, and C357T--occurred in porD and the other mutations--A041G, A122G, C349A(G)--occurred in oorD genes. CONCLUSIONS Changes in 6 amino acids may be associated with the resistance of H. pylori to furazolidone.