MTS1基因β启动子转录活性研究

目的 研究IMTSl基因β启动子在急性T淋巴细胞白血病细胞株中的转录激活情况，鉴定β启动子转录活性的功能片段区。方法 用DNA重组方法，构建MTS1基因口启动子3′端转录起始点相同，而5′端序列不同的7种pGL3重组质粒。用脂质体介导的基因瞬时转染法，将构建的重组质粒分别转染MTSl基因双等位缺失的Jurkat细胞株，检测pGL3重组质粒中荧光素酶报告基因的表达，观察β启动子在Jurkat细胞中的激活情况及其基础转录活性片段区。结果 成功构建了MTSI基因β启动子7种不同片段的重组质粒，它们在Jurkat细胞中均有转录活性，其中0．38kb Sac Ⅱ-Sac Ⅰ酶切片段是MTSl基因β启动子转录活性的基础片段。结论 IMTSlβ启动子可在Jurkat细胞中被激活。     

生脉注射液对蛋白激酶C调控LPS诱导iNOS基因表达的影响

目的：探讨生脉注射液对蛋白激酶C调控LPS诱导单核／巨噬细胞iNOS基因表达的影响。方法：用蛋白激酶C（protein kinase C,PKC)活性测定法研究LPS对PKC的激活作用及Griess还原法测定NO的生成；用基因重组技术构建iNOS荧光素酶报告基因载体；用基因转染及报告基因检测等方法研究生脉注射液调控LPS刺激RAW264．7细胞对iNOS启动子活性的诱导作用及其与PKC的关系。结果：生脉注射液可负调节LPS刺激RAW264．7细胞引起的PKC磷酸化激活和膜移位，并可延长PKC抑制剂H－7下调NO作用时间；荧光素酶表示基因实验结果显示，生脉注射液可抑制iNOS启动子的转录活性，并可增强H－7和钙离子通道阻滞剂Verapamil作用。结论：生脉注射液抑制LPS刺激RAW264．7细胞所致的NO生成增加，其机制之一可能是通过抑制PKC激活和细胞钙增加而影响iNOS启动子的转录活性。     

牛珀至宝微丸对蛋白激酶C调控内毒素诱导iNOS基因表达的影响

目的 :探讨牛珀至宝微丸对蛋白激酶 C(PKC)调控 (L PS)诱导单核巨噬细胞诱导型一氧化氮合酶(i NOS)基因表达的影响。方法 :用 PKC活性测定法观察 L PS对 PKC的激活作用 ,用 Griess还原法测定一氧化氮 (NO)的生成 ,用基因重组技术构建 i NOS荧光素酶报告基因载体 ,用基因转染及报告基因检测等方法研究牛珀至宝微丸调控 L PS刺激 RAW2 6 4 .7细胞对 i NOS启动子活性的诱导作用及其与 PKC的关系。结果 :牛珀至宝微丸可负调节 L PS刺激 RAW2 6 4 .7细胞引起的 PKC磷酸化激活和膜移位 ,并可延长 PKC抑制剂 H 7下调 NO作用时间。荧光素酶报告基因实验结果显示 ,牛珀至宝微丸可抑制 L PS刺激诱导的 i NOS启动子的转录活性 ,并可增强 H 7和钙离子通道阻滞剂维拉帕米的作用。结论 :牛珀至宝微丸抑制 L PS刺激 RAW2 6 4 .7细胞所致的 NO生成增加 ,其机制之一是量效与时间两方面抑制 PKC激活和细胞内钙增加 ,从而影响 i NOS启动子的转录活性     

高迁移率族蛋白B1在白细胞介素-2转录表达信号调控中的作用

目的 探讨高迁移率族蚩白B1(HMGB1)在白细胞介素-2(IL-2)转录表达信号调控机制中的可能作用.方法首先将HMGB1和NFAT2质粒共同转染Hela细胞,同时转染IL-2报告基因,逐步增加HMGB1的转染剂量,检测IL-2报告基因的表达活性.观察HMGB1质粒用量埘IL-2报告基凶活性的影响;然后应用sRNAi质粒对内源性及外源性HMGB1表达进行特异性抑制,观察对IL-2报告基因活性的影响,以期从反而论证HMGB1可促进IL-2转录表达.结果 在Hela细胞中,随着HMGB1质粒转染剂量增加,IL-2报告基因活性增加了2.12倍(P<0.01).应用sRNAi抑制293T细胞中外源性以及Hela细胞中内源性HMGB1表达水平后,IL-2报告基因活性分别下降1.7倍和4.76倍(P<0.05或P<0.01).结论 HMGB1在NFAT2介导IL-2报告基冈转录表达的信号调控过程中发挥重要作用.     

MTS1基因ARF启动子基础转录调控区转录活性研究

目的　研究MTS1基因ARF启动子基础转录调控区的转录激活及其与E2 F1转录因子之间的关系。方法　以含ARF启动子基础转录调控区E2 F1结合位点野生型序列的W6重组质粒为模板 ,设计该片段中E2 F1A、B、C结合位点序列突变或缺失的引物 ,用聚合酶链反应构建该区域中E2 F1A、B、C结合位点序列突变或缺失的ZE、ZF、ZG重组质粒。再将W6、ZE、ZF、ZG重组质粒转染进Jurkat细胞 ,检测其荧光素酶报告基因的表达。结果　构建的ZE、ZF、ZG重组质粒经SacⅠ或NaeⅠ酶切鉴定和DNA序列分析得到证实。与E2 F1位点野生型重组质粒W6比较 ,突变型重组质粒ZE、ZF、ZG在Jurkat细胞中荧光素酶报告基因的表达量减少 ,以E2 F1A位点突变的重组质粒减少明显。结论　构建ARF启动子E2 F1A、B、C结合位点序列突变的重组质粒成功 ;MTS1基因ARF启动子基础转录调控区的转录激活可能与E2 F1转录因子的作用有关。     

抗CD_3抗体(HIT3a)基因突变及其表达研究

目的　提高可溶性抗CD3 scFv片段的表达量 ,并测定其生物学活性。方法　用PCR法致抗CD3 scFv基因突变 ;用DNA限制性酶切指纹图谱以及Westernblot法筛选突变克隆 ;用间接免疫荧光法测定抗体的特异性结合活性 ;用12 5I标记抗体 ,进行竞争抑制试验 ;采用51Cr释放试验 ,进行抗CD3scFv片段介导的T淋巴细胞细胞毒性测定。结果　DNA序列测定结果表明 ,抗CD3 scFv抗体突变克隆菌株m2为重链第六位氨基酸突变 ,即E(GAG)→Q(CAG)。突变后的可溶性抗CD3 scFv片段表达量(1μg/ml)比突变前 (0 .0 1μg/ml)高 10 0倍。突变前、后的抗CD3 scFv片段与Jurkat细胞 (CD3 )的结合特异性未改变。m2能竞争性封闭亲代鼠源性抗体HIT3a与CD3 阳性的Jurkat细胞的结合位点。体外杀伤实验结果显示 ,由m2与IL 2共刺激产生的CD3 AK细胞的细胞毒活性比单用IL 2刺激产生的LAK细胞强。结论　通过对抗CD3 scFv抗体基因进行定点突变 ,实现了该抗体片段的高表达 ;m2能与CD3 的Jurkat细胞结合 ;也能激活人外周血中的T淋巴细胞产生CD3 AK细胞毒活性     

小鼠adam10基因启动子克隆和双荧光素酶报告基因系统构建及鉴定

本研究旨在克隆小鼠adam10基因启动子,构建以adam10基因启动子为启动序列的双荧光素酶报告基因系统并分析其活性,为研究adam10基因转录调控提供有效工具。以BALB/c小鼠脑组织为模板,采用PCR方法克隆小鼠adam10基因启动子序列至pCR-Blunt载体,酶切后连接至荧光素酶报告质粒pGL4.10启动子区域,构建重组荧光素酶报告质粒pGL4.10-adam10,采用阳离子脂质体法与阳性对照质粒pGL4.74共转染真核细胞293FT,以离子霉素刺激为刺激组、DMSO为未加干预组,同时以不含启动子的pGL4.10与阳性对照质粒pGL4.74共转染组为阴性对照组,化学发光仪检测荧光强度及比例。结果表明,酶切及测序验证克隆的adam10启动子序列正确且无突变,构建的重组荧光素酶报告质粒pGL4.10-adam10与阳性对照质粒pGL4.74载体成功共转染293FT细胞,pGL4.10-adam10转染细胞后具有转录活性。离子霉素刺激组活性明显高于DMSO组,荧光比值约为DMSO组2倍(P<0.05),阴性对照组不具备转录活性。结论:成功克隆了adam10启动子并构建了重组荧光素酶报告质粒pGL4.10-adam10,离子霉素可增强adam10基因启动子转录活性。     

B7-1与B7-2对调节人IL-2基因的转录因子NF-κB和AP-1的相同作用

为了解B7共刺激对细胞因子,特别是对IL-2 mRNA及转录因子NF-κB出和AP-1的影响,探讨B7介导的IL-2调节的分子机制,在异基因混合淋巴细胞反应(MLR)体系中分别或联合加入抗B7-1、抗B7-2单克隆抗体和CTLA-4 Ig以阻断B7/CD28信号传导,通过竞争性PCR定量检测其对IL-2和IL-4 mRNA的影响,并初步测定IFN-γ mRNA的改变,同时用转染MHCⅡ类分子及联合转染等量B7-1或B7-2的NIH3T3转基因细胞tDR7,tDR7/B7-1和tDR7/B7-2刺激CD28+T细胞,通过DNA-蛋白结合实验观察B7对IL-2转录因子NF-出和AP-1的影响.结果表明:抗B7-2单抗和CTLA-4 Ig可明显抑制B7介导的IL-2和IL-4 mRNA合成,而抗B7-1单抗仅有轻度抑制作用,2种或3种抗体联合应用时抑制作用相加.MLR 1-6小时,单独tDR7即可诱导NF-κB的表达,联合转染B7早期对其结合活力无明显影响,6小时后tDR7诱导作用减弱,B7却可显著延长tDR7的诱导作用至72小时.tDR7早期同样可诱导AP-1的表达,联合转染B7分子在24小时内对其有一定的抑制作用,而在反应后期可延长tDR7对AP-1的上调作用,B7-1与B7-2间作用未见明显不同.结论:B7通过减少IL-2 mRNA降解和影响基因转录而上调IL-2分泌,并可同时影响多种细胞因子分泌;在转录水平B7-1与B7-2作用未见明显不同,提示两者的功能差异可能与细胞表达和时间动力学不同有关.详细了解B7介导的T细胞免疫耐受的分子机制有助于设计更好的临床方案预防移植排斥和GVHD.     

流式微珠阵列法检测IFN-γ诱导的Jurkat细胞Th1/Th2细胞因子表达

本研究旨在探讨流式微珠阵列法检测Th1／Th2细胞因子表达及IFN-1应用于T淋巴细胞白血病治疗的临床价值。以不同浓度IFN-γ诱导培养Jurkat细胞48小时，用流式微珠阵列法检测培养上清液中IL-2、IL-6、TNF-α和IFN-γ表达，并采用流式细胞术检测膜上IL-2受体（CD25）的表达。结果表明：IFN-γ诱导Jurkat细胞IL-2和TNF-α的表达呈浓度依赖性升高，未检测到IL-6表达，CD25表达明显升高。结论：Jurkat细胞能被IFN-γ诱导高表达Th1细胞因子和CD25，流式微珠阵列法能准确客观地反映Th1／Th2细胞因子表达的动态变化。     

抗CD3抗体（HIT3a）基因突变及其表达研究

目的 提高可溶性抗CD3scFv片段的表达量,并测定其生物学活性。方法 用PCR法致抗CD3scFv基因突变;用DNA限制性酶切指纹图谱以及Western blot法筛选突变克隆;用间接免疫荧光法测定抗体的特异性结合活性;用^125I标记抗体,进行竞争抑制试验;采用^51Cr释放试验,进行抗CD3scFv片段介导的T淋巴细胞细胞毒性测定。结果 DNA序列测定结果表明,抗CD3scFv抗体突变克隆菌株m2为重链第六位氨基酸突变,即E（GAG）→Q(CAG)。突变后的可溶性抗CD3scFv片段表达量（1μg/ml）比突变前（0．01μg/ml）高100 。突变前、后的抗CD3scFv片段与Jurkat细胞（CD3^ ）的结合特异性改变。m2能竞争性封闭亲代鼠源性抗体HIT3a与CD3阳性的Jurkat细胞的结合位点。体外杀伤实验结果显示,由m2与IL－2共刺激产生的CD3AK细胞的细胞毒活性比单用IL－2刺激产生的LAK细胞强。结论 通过对抗CD3scFv抗体基因进行定点突变,实现了该抗体片段的高表达;m2能与CD3^ 的Jurkat细胞结合;也能激活人外周血中的T淋巴细胞产生CD3AK细胞毒活性。     

Expression and release of IL-2 receptor and production of IL-2 by activated T lymphocyte subsets.

T lymphocyte subsets differ in expression of cell surface antigen and functional properties. Both CD4+ and CD8+ subsets express interleukin-2 receptor (IL-2R) following their activation in vitro. In the present investigation T lymphocyte subsets were activated by different mitogens and IL-2R expression was enumerated on these stimulated subsets. Peripheral blood mononucleated cells (PBMC) were stimulated with phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) and then stained with anti-CD4 or anti-CD8 antibodies conjugated with fluorescein isothiocyanate and anti-IL-2R monoclonal antibody conjugated with phycoerythrin using a direct immunofluorescence technique. The percentage of IL-2R positive lymphocytes was enumerated by flow cytometry. The results showed that mitogen activated lymphocytes expressed variable degrees of IL-2R which were significantly higher than the control. 53% of CD4+ lymphocytes and 28% of CD8+ expressed IL-2R following PHA stimulation in vitro. Similarly, 47% of CD4+ lymphocytes and 23% of CD8+ lymphocytes expressed IL-2R following PWM stimulation. The present study also revealed that the release of soluble IL-2R (sIL-2R) and IL-2 production in supernatant from cultured PBMC varied with different mitogen stimulation. Using the same concentration of PHA and PWM as used to study IL-2R expression, higher activity of sIL-2R was detected in PHA stimulated lymphocytes as compared to PWM treated lymphocytes. However, IL-2 production was more in culture medium from PMW treated PBMC. Thus, there was a significant correlation between the cellular and soluble IL-2R but the production of IL-2 from activated PBMC cells had no good correlation with either the cellular IL-2R expression or the release of sIL-2R.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3

Injection of anti-CD3 antibodies causes prompt expression of interleukin (IL)-4, IL-2, and interferon gamma (IFN-gamma) mRNA among spleen cells. The optimal dose of anti-CD3 for such induction was 1.33 microgram/animal; lymphokine mRNA was first observed at 30 min, peaked at 90 min, and was undetectable (for IL-4) or had declined markedly by 4 h. Cells harvested from spleens of mice injected with anti-CD3 90 min earlier secreted IL-4, IL-2, and IFN-gamma without further stimulation. By contrast, in vitro stimulation with anti-CD3 of spleen cell suspensions or splenic fragments from noninjected donors failed to cause prompt production of IL-4 and, even after 24 h of stimulation, the amount of IL-4 produced in such cells was substantially less than that secreted within 1 h by spleen cell suspensions or splenic fragments from mice injected with anti-CD3 90 min earlier. Production of IL-4 by spleen cells from anti-CD3-injected mice was not inhibited by pretreatment with anti-IL-4 antibody or with IFN-gamma or tumor growth factor beta nor enhanced by treatment with IL-4. By contrast, CTLA-4 immunoglobulin (Ig) treatment clearly diminished IL-4 production in response to in vivo anti-CD3, indicating that cellular interactions involving CD28 (or related molecules) were important in stimulation. Cell sorting analysis indicated that the cells that produced IL-4 in response to in vivo injection of anti-CD3 were highly enriched in CD4pos cells with the phenotype leukocyte cell adhesion molecule-1 (LECAM-1)dull, CD44bright, CD45RBdull, NK1.1pos. Indeed, the small population of CD4pos, NK1.1pos cells had the great majority of the IL-4- producing activity of this population. Injection with Staphylococcal enterotoxin B also caused prompt induction of IL-4 mRNA; the cells that were principally responsible for production also had the phenotype of CD4pos, NK1.1pos. These results suggest that possibility that this rare population of T cells may be capable of secreting IL-4 at the outset of immune responses and thus may act to regulate the pattern of priming of naive T cells, by providing a source of IL-4 to favor the development of T cell helper 2-like IL-4-producing cells.     

Plasmapheresis affects T helper type-1/T helper type-2 balance of circulating peripheral lymphocytes.

Plasmapheresis not only removes humoral factors, but may also modulate cellular immunity. We investigated whether plasmapheresis influenced T helper type-1/T helper type-2 (Th1/Th2) cytokine-producing-cell balance in 3 patients with neuroimmunological disease. The production of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and IL-4 in the culture supernatant of peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 was assayed. In 2 of 3 patients, plasmapheresis (immunoadsorption or plasma exchange) reduced Th1/Th2 cytokine ratio. The results may suggest that plasmapheresis induces a shift of Th1/Th2 balance in peripheral blood.     

Gene transfer and expression of a non-viral polycation-based vector in CD4+ cells.

CD4-selective targeting of an antibody-polycation-DNA complex was investigated. The complex was synthesized with the anti-CD4 monoclonal antibody B-F5, polylysine268 (pLL) and either the pGL3 control vector containing the luciferase reporter gene or the pGeneGrip vector containing the green fluorescent protein (GFP) gene. B-F5-pLL-DNA complexes inhibited the binding of 125I-B-F5 to CD4+Jurkat cells, while complexes synthesised either without B-F5 or using a non-specific mouse IgG1 antibody had little or no effect. Expression of the luciferase reporter gene was achieved in Jurkat cells using the B-F5-pLL-pGL3 complex and was enhanced in the presence of PMA. Negligible luciferase activity was detected with the non-specific antibody complex in Jurkat cells or with the B-F5-pLL-pGL3 complex in the CD4- K-562 cells. Using complexes synthesised with the pGeneGrip vector, the transfection efficiency in Jurkat and K-562 cells was examined using confocal microscopy. More than 95% of Jurkat cells expressed GFP and the level of this expression was markedly enhanced by PMA. Negligible GFP expression was seen in K-562 cells or when B-F5 was replaced by a non-specific antibody. Using flow cytometry, fluorescein-labelled complex showed specific targeting to CD4+ cells in a mixed cell population from human peripheral blood. These studies demonstrate the selective transfection of CD4+ T-lymphoid cells using a polycation-based gene delivery system. The complex may provide a means of delivering anti-HIV gene therapies to CD4+ cells in vivo.     

A cysteine residue located in the transmembrane domain of CD44 is important in binding of CD44 to hyaluronic acid

In the transmembrane domain and cytoplasmic domain of human CD44 protein there are two cysteine residues. These two cysteines are conserved in all known mammalian CD44 proteins. The functions of these cysteine residues are not known. Site-specific mutagenesis was used to create CD44 mutant proteins lacking either one or both of these cysteine residues. Wild-type CD44 and mutant CD44 genes were transfected into CD44- Jurkat cells to establish stable transfectants. These transfectants were used to study whether these two cysteine residues are important in the binding of CD44(H) to fluorescein- conjugated hyaluronic acid (F-HA). Jurkat transfectant bearing wild- type CD44 did not bind F-HA, unless they were stimulated in vitro with immobilized anti-CD3 monoclonal antibody. Anti-CD3 antibody also stimulated the binding of F-HA in Jurkat CD44.C295A transfectant in which the cytoplasmic cysteine residue has been replaced with alanine. In contrast, anti-CD3 antibody failed to stimulate the binding of F-HA in Jurkat transfectant (CD44.C286A), in which the transmembrane domain cysteine 286 has been replaced with an alanine, and in Jurkat transfectant CD44.2C2A, in which both of the cysteine residues have been altered. Binding can also be induced with a monoclonal anti-CD44 antibody (F-44-10-2) in Jurkat wild-type CD44 and Jurkat CD44.C295A transfectants but not in CD44. C286A transfectant. These results provide evidence that the transmembrane domain of CD44, more specifically the cysteine residue in the transmembrane domain, is important for both activation-induced and anti-CD44 antibody-induced binding of soluble HA.     

类风湿关节炎患者外周血Th17细胞的检测及其临床意义

目的探讨Th17细胞与类风湿关节炎(RA)疾病的相关性。方法采用RT-PCR检测外周血单个核细胞(PBMCs)中Th17细胞的特异性转录因子视磺醛酸相关的孤儿核受体(RORγt)mRNA;酶联免疫吸附法(ELISA)检测20例健康体检者和22例RA患者外周血单个核细胞(PBMCs)刺激培养后,上清液中白细胞介素-17(IL-17)的水平。结果与健康对照组相比,RA患者PBMCs刺激后产生IL-17的量明显增高(P<0.01),RORγtmRNA也高于健康对照组(P<0.01)。结论 Th17细胞参与了RA发病及病情发展。     

Differential regulation of transforming growth factor beta and interleukin 2 genes in human T cells: demonstration by usage of novel competitor DNA constructs in the quantitative polymerase chain reaction

The regulation of mRNA encoding transforming growth factor beta (TGF-beta) and interleukin 2 (IL-2) in normal human T cells was explored using novel competitor DNA constructs in the quantitative polymerase chain reaction and accessory cell-independent T cell activation models. Our experimental design revealed the following: (a) TGF-beta mRNA and IL-2 mRNA are regulated differentially in normal human T cells, quiescent or signaled with the synergistic combinations of: sn-1,2-dioctanoylglycerol and ionomycin or anti-CD3 monoclonal antibody (mAb) and anti-CD2 mAb; (b) the steady-state level of TGF-beta mRNA in the stimulated T cells, in contrast to that of IL-2 mRNA, is increased by the immunosuppressant cyclosporine (CsA); and (c) the paradoxical effect of CsA on TGF-beta mRNA levels is also appreciable at the level of production of functionally active TGF-beta protein. Our findings, in addition to demonstrating the utility of the competitor DNA constructs for the precise quantification of immunoregulatory cytokines, suggest a novel and unifying mechanistic basis for the immunosuppression and some of the complications (e.g., renal fibrosis) associated with CsA usage.