转录调节因子Ets-1在大鼠血管平滑肌细胞增殖与凋亡中的作用

目的： 研究转录调节因子Ets-1对大鼠血管平滑肌细胞增殖与凋亡的影响及其可能的机制。 方法： 用定量细胞DNA片段的方法观察大鼠血管平滑肌细胞增殖与凋亡。用Western印迹法检测磷酸化视网膜母细胞瘤（RB-P）蛋白表达。 结果： Ets-1抑制大鼠血管平滑肌细胞凋亡。反义P21WAF1/CIP1能够阻断Ets-1的抗凋亡作用，并抑制Ets-1诱导的平滑肌细胞增殖。Ets-1能够上调RB-P蛋白表达，反义P21WAF1/CIP1可以阻断Ets-1诱导的RB-P蛋白表达。 结论： 在大鼠血管平滑肌细胞中，Ets-1通过P21WAF1/CIP1旁路发挥抑制凋亡和促进增殖作用。     

乙型肝炎病毒I97L变异核壳蛋白对诱导HepG2细胞凋亡的影响

目的研究197L变异核壳蛋白对诱导HepG2细胞凋亡的影响。方法构建EGFP-野毒株核壳蛋白、197L变异核壳蛋白融合表达载体（pEGFP—WT和pEGFP—L97），酶切和测序鉴定；将pEGFP—WT、pEGFP—L97和对照质粒分别转染HepG2细胞．筛选阳性细胞株；荧光显微镜观察阳性克隆细胞荧光蛋白表达，Western—blot检测核壳蛋白表达：以TNF-α、Act-D诱导HepG2细胞株凋亡，0、16、32和48h采用流式细胞术检测细胞凋亡，32h时采用共聚焦检测细胞凋亡比例。结果成功建立了融合表达蛋白表达载体；荧光显微镜显示各细胞克隆有较好的荧光蛋白表达，Western-blot检测各细胞系核壳蛋白表达没有差异；16、32、48h时，pEGFP-WT、pEGFP-L97表达细胞株凋亡率均明显低于pEGFP-C1细胞株（P〈0．05）；32h和48h时，pEGFP-L97细胞株凋亡率明显高于pEGFP-WT细胞株（P〈0．05）；32h时激光共聚焦检测结果与流式细胞术检测结果一致。结论乙型肝炎病毒197L变异核壳蛋白对诱导HepG2细胞凋亡的影响与野毒株核壳蛋白不同，与野毒株核壳蛋白相比，197L变异核壳蛋白对凋亡因素可能更敏感。     

人PFP、GrB共表达诱导喉癌Hep-2细胞凋亡的研究

目的 探讨人穿孔素(PFP)、颗粒酶B(GrB)共表达是否可以诱导人的喉癌细胞系Hep-2的凋亡及其作用的机理.方法 利用脂质体2000将PFP、GrB共表达载体pVAX1-PIG(即pVAX1-PFP-IRES-GrB)转染人喉癌Hep-2细胞,采用荧光染料Hoechst33342法、流式细胞仪(FCM)检测、透射电镜观察Hep-2细胞的凋亡情况.以激光共聚焦显微镜检测重组载体转染后的Hep-2细胞内[Ca2+];浓度的变化,并探讨其作用机理.结果 pVAX1-PIG转染组的Hep-2细胞大量凋亡且其凋亡率显著高于对照组(P＜0.05),透射电镜研究显示单个Hep-2细胞内亦出现凋亡的特征.Hep-2细胞内[Ca2+];的浓度发生了变化,且由FI值增大可知细胞胞浆内[Ca2+];的浓度升高.结论 PFP、GrB共表达能够诱导人Hep-2细胞的凋亡,且凋亡的发生与细胞胞浆内[Ca2+];的浓度升高有关.     

Characterization and use of a rabbit-anti-mouse VPAC1 antibody by flow cytometry

Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.     

人骨肉瘤耐阿霉素细胞模型的建立及其生物学特性

背景：多药耐药是骨肉瘤化疗失败的重要原因,目前其耐药机制不明。 目的：诱导建立耐阿霉素的人骨肉瘤细胞株并观察多药耐药蛋白1、多药耐药相关蛋白1和肺耐药蛋白的表达。 方法：采用逐步递增阿霉素浓度间歇作用的方法诱导143B/WT细胞株建立143B/阿霉素耐药细胞株。 结果与结论：经阿霉素诱导45 d建立了143B/阿霉素细胞株,其对阿霉素高度耐药,对顺铂、甲氨蝶呤、异环磷酰胺、长春新碱和紫杉醇亦产生不同程度交叉耐药;流式细胞仪检测显示与143B野生型细胞相比,143B/阿霉素细胞周期中G1和S期所占比例增加,而G2/M期所占比例明显减少;罗丹明外排实验显示,143B/阿霉素细胞药物外排能力显著高于143B/WT细胞(P < 0.01);流式细胞仪和激光共聚焦显微镜观察发现,143B/阿霉素细胞阿霉素相关性细胞凋亡率显著低于143B/WT(P < 0.01);Western blot检测显示143B/阿霉素细胞多药耐药蛋白1表达水平较143B/WT显著升高(P < 0.01),二者多药耐药相关蛋白1和肺耐药蛋白表达差异无显著性意义(P > 0.05)。提示143B/阿霉素细胞多药耐药的产生与多药耐药蛋白1表达升高相关。     

脂质体转染细胞周期素B1反义脱氧寡核苷酸对HL60细胞增殖调控的影响

目的:探讨脂质体转染细胞周期素B1(cyclinB1)反义脱氧寡核苷酸(ASON)对HL60细胞增殖调控的作用。方法:用针对cyclinB1mRNA5’端编码区起始密码子(ATG/AUG)的ASON,通过脂质体导入HL60细胞共培养后,用流式细胞术(FCM)和RT-PCR分别检测cyclinB1蛋白和mRNA的表达水平,电镜和原位细胞凋亡检测法(POD)、FCM及DNA凝胶电泳法检测细胞凋亡。结果:CyclinB1ASON组与SON及空白对照组相比,ASON能特异地抑制cyclinB1蛋白及mRNA水平的表达,当ASON的浓度达到一定程度时,HL60细胞的增殖及集落形成率均明显受抑制,出现细胞凋亡,并且此作用随ASON浓度的升高而增强。结论:CyclinB1的特异ASON能封闭其蛋白及mRNA的表达水平,可剂量依赖性地抑制白血病细胞增殖,诱导细胞凋亡。     

人外周血淋巴细胞及单核细胞表达PD-L1和PD-L2的动力学研究

目的： 探讨PD-L1和PD-L2在正常人外周血静息和活化的B细胞、T细胞及单核细胞表面的表达规律。 方法： 利用荧光抗体染色和流式细胞术分析静息状态及经多克隆刺激剂脂多糖（LPS）和美洲商陆丝裂原（PWM）刺激6、24、48和72 h后表达PD-L1和PD-L2的B细胞和T细胞百分率，同时分析静息状态及经IFN-γ和LPS共同刺激24、48、72 和96 h后表达PD-L2的单核细胞百分率。 结果： 静息B细胞和T细胞表面不表达PD-L1，LPS和PWM刺激6 h后表达PD-L1的B细胞百分率均显著高于静息B细胞，24 h达到最高，分别为(46.26±10.71)%和(43.67±6.14)%，之后随时间延长而降低；表达PD-L1的T细胞百分率在LPS刺激下无显著变化，但PWM刺激6 h后百分率明显高于静息条件，24 h达到最高，为(25.42±9.23)%，之后下降。静息B细胞和T细胞不表达PD-L2，活化后PD-L2表达也无明显上调。另外，静息状态单核细胞表面不表达PD-L2，体外活化24 h后表达PD-L2的单核细胞百分率显著上调，48 h达到最高为(28.70±14.22)%，之后随时间而降低。 结论： 活化的淋巴细胞表达PD-L1，但不表达PD-L2，后者在活化的单核细胞表面表达，并且后者达到高峰的时间迟于前者。     

CTLA4-EGFP融合蛋白在K562细胞中的表达及其亚细胞定位研究

目的 :构建增强型绿色荧光蛋白 (EGFP)与CTLA4融合蛋白真核表达载体 ,分析其在K562细胞中的表达和亚细胞定位。方法 :以RT PCR方法克隆人CTLA4基因 ,构建CTLA4 EGFP融合蛋白的表达载体。以其转染K562细胞后 ,以流式细胞仪和激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果 :从人外周血细胞中克隆到CTLA4基因的cDNA。通过PCR方法在起始位点前加入Kozak序列并删除终止密码 ,成功地构建CTLA4 EGFP融合蛋白表达载体。以该质粒转染K562细胞 2 4h后 ,CTLA4和EGFP双阳性细胞的百分率为 16%。表达的融合蛋白主要分布于胞内 ,细胞膜上分布较少。相反 ,转染空载体的细胞仅表达EGFP ,且其在细胞内呈弥散样分布。以佛波醇酯加离子霉素刺激后 ,CTLA4 EGFP融合蛋白在细胞表面表达水平升高到 2 9% ,且分布于胞内的融合蛋白向细胞膜靠近并与质膜融合 ;而转染空载体的细胞内EGFP的分布无明显改变。结论 :成功地构建CTLA4 EGFP融合蛋白表达载体 ,并在K562细胞中得到表达。表达的融合蛋白与天然CTLA4在活化T细胞中的定位和转运特点相似     

A novel monoclonal antibody, C41, reveals IL-13Ralpha1 expression by murine germinal center B cells and follicular dendritic cells

Responsiveness to IL-13 involves at least two chains, IL-4Ralpha and IL-13Ralpha1. Although mouse B cells express IL-4Ralpha, little is known about their expression of IL-13Ralpha chains. To investigate this topic further, we have generated a monoclonal antibody (C41) specific for murine IL-13Ralpha1. Using C41, IL-13Ralpha1 expression was detected on germinal center (GC) B cells by flow cytometry and immunohistochemistry. In addition, IL-13Ralpha1 was observed on follicular dendritic cells, but not interdigitating dendritic cells in the T cell areas. Furthermore, resting B cells also expressed IL-13Ralpha1, and in the presence of IL-13 produced increased amounts of IgM in response to in vitro CD40 stimulation. However, C41 was unable to neutralize this bioactivity. The distribution of IL-13Ralpha1 on murine B cells and during GC reactions suggests a role for IL-13 during B cell differentiation.     

热休克蛋白70在人食管癌EC9706细胞凋亡过程中的表达与定位变化

目的 探讨姜黄素对人食管癌EC9706细胞凋亡的诱导作用,热休克蛋白70(HSP70)在肿瘤细胞凋亡过程中在核基质上的变化及其与凋亡调控相关蛋白的关系.方法 用细胞计数和流式细胞仪检测姜黄素对人食管癌EC9706细胞的增殖抑制作用,以光学显微镜和透射电镜观察姜黄素诱导人食管癌EC9706细胞凋亡前后的细胞结构变化,琼脂糖凝胶电泳观察人食管癌EC9706细胞凋亡前后的DNA结构变化.双向凝胶电泳和质谱鉴定分析HSP70在核基质中的存在与变化;并以Western blotting进行确证;激光扫描共焦显微镜观察HSP70在EC9706细胞凋亡过程中的定位及其与Bax、Bcl-2等基因产物的共定位关系.结果 姜黄素能显著抑制人食管癌EC9706细胞增殖并诱导人食管癌EC9706细胞凋亡,双向凝胶电泳、质谱鉴定和结果 发现并证实,HSP70在姜黄素处理前后的EC9706细胞核基质蛋白中的存在及其表达下调变化.激光扫描共焦显微镜观察结果 显示,HSP70在EC9706细胞凋亡过程中与Bax、Bcl-2等基因产物具有共定位关系,且其共定位区域发生了变化.结论 姜黄素对人食管癌EC9706细胞具有显著的凋亡诱导作用;HSP70作为一种新发现的核基质蛋白,在姜黄素诱导人食管癌EC9706凋亡过程中的表达与分布发生了显著变化.HSP70与凋亡相关基因的关系对EC9706细胞凋亡具有重要影响.     

Regeneration and tolerance factor is expressed during T-lymphocyte activation and plays a role in apoptosis

Regeneration and tolerance factor (RTF) is a protein cloned from the thymus and expressed on B lymphocytes in normal pregnancy, B lymphocytic leukemia lines, and T and B lymphocytes in individuals with HIV infection. Findings, using the Jurkat T-cell model, revealed that RTF is upregulated after activation and anti-RTF antibody-induced apoptosis. In this article anti-RTF antibody-induced apoptosis of both unstimulated and activated T lymphocytes. RTF expression was examined in human PBMC or purified T lymphocytes after their in vitro activation. Kinetic studies indicated maximal RTF cell surface expression on activated T lymphocytes occurred between expression of the early activation antigen CD69 and the IL-2alpha receptor (CD25) by multiparameter flow cytometry. RTF receptor expression correlated with Fas (CD95) and CD25 receptor expression (r2 = 0.6 and 0.5, respectively). RTF surface expression was dependent on the stimuli used to activate T lymphocytes. T lymphocytes obtained maximal RTF expression when activated through the TCR signal complex using anti-CD3epsilon antibody alone when compared with T lymphocytes activated with costimulation provided by anti-CD28 antibody alone or with anti-CD28 and anti-CD3epsilon antibody. RTF is expressed under conditions of both activation and anergy. The RTFs increased concentration on the surface of anergic T cells may protect these cells from apoptosis because increased RTF concentrations inhibited anti-RTF induced apoptosis. These data further characterize the expression of RTF on activated T lymphocytes and the role of anti-RTF antibody in T-lymphocyte apoptosis.     

Reduced expression of arrestin beta 2 by graft monocytes during acute rejection of rat kidneys

During acute rejection, numerous pro-inflammatory and cytotoxic monocytes accumulate in the vasculature of experimental renal allografts. Arrestins (ARRBs) are cellular regulators of inflammation, but nothing is known about their expression during rejection. Intravascular mononuclear graft leukocytes were isolated 4 days after kidney transplantation. ARRB1 and ARRB2 mRNA expression was reduced in blood leukocytes from allografts undergoing acute rejection, whereas on the protein level only ARRB2 was changed. Flow cytometry and confocal microscopy revealed ARRB1 and ARRB2 expression by monocytes and T cells, with a selective decrease in ARRB2 expression in monocytes during acute rejection. I-κB directly interacted with ARRB2 and the levels of both proteins strongly correlated. Concomitantly, the mRNA expression of NF-κB targeted genes increased. Our results suggest that activation of blood monocytes in renal isografts is dampened by high ARRB2 levels. During acute rejection, ARRB2 levels are reduced and classical monocyte activation is enabled via NF-κB activation.