原发性肝癌中SHH信号通路的研究

目的:探讨SHH信号通路在原发性肝癌中的表达及变化。方法:RT-PCR反应检测肝癌细胞中SHH的mRNA表达结果,MTT法测定重组SHH-N及cyclopamine对肝癌细胞生长率的影响,western blot法测定重组SHH-N及cyclopamine对肝癌细胞中MMPs及PI3K/Akt的影响。结论:SHH信号通路对PI3K/Akt信号通路具有调控作用,激活SHH信号通路可上调MMP-2、MMP-9蛋白表达,MMP-2、MMP-9和Akt磷酸化是SHH信号通路影响肝癌细胞发展的重要机制。     

不同细胞周期肝癌细胞Integrin β1的表达

探讨人肝癌细胞（SMMC-7721）的同步化方法及粘附分子integrinβ1在不同周期肝癌细胞上的表达。采用胸腺嘧啶脱氧核苷、秋水仙碱顺序阻断和胸腺嘧啶脱氧核苷双阻断后释放培养的方法分别获得G1期和S期的肝癌细胞,用流式细胞仪进行周期分析;采用抗体阻断技术和流式细胞仪检测肝癌细胞上integrinβ1的表达。结果发现未同步化肝癌细胞integrinβ1表达的荧光强度为95.70;G1期和S期细胞的同步率分别为74.09％和98.29％,integrinβ1表达的荧光强度相应为76.90和94.09。得知药物胸腺嘧啶脱氧核苷和秋水仙碱能较好地将肝癌细胞同步于G1期和S期;粘附分子integrinβ1在SMMC-7721肝癌细胞上高表达,但表达水平呈现周期差异。     

免疫磁珠的制备及其在捕获乳腺癌患者白细胞中的应用

应用生物素-亲和素系统制备免疫磁珠,考察了不同条件对制备免疫磁珠的影响.结果表明:当反应温度为4℃、亲和素浓度为0.2mg/mL、PBS pH=5.8、反应时间为2h时,磁性微球的亲和素载量为25~55mg/g;BNHS与抗体质量比1∶7时,生物素与抗体的结合率最高;亲和素与生物素化抗体摩尔比在1∶3时,亲和素与生物素的偶联率最高.抗CD45免疫磁性微球的白细胞去除率高达78%,磁珠回收率达98%,可用于进一步捕获乳腺癌外周血循环肿瘤细胞.     

CD147与恶性肿瘤关系的研究进展

CD147是广泛表达于人体多个组织的跨膜糖蛋白,属于免疫球蛋白超家族。CD147在不同组织中呈现不同水平的表达,此外其在不同种属间的表达亦存在很大差异,有研究表明CD147在多种生理及病理过程起重要作用。经检测CD147在多种恶性肿瘤中均呈现高水平表达,在肿瘤的发生发展中,CD147通过诱导基质金属蛋白（MMPs）的产生,实现对肿瘤生长、浸润及转移的促进作用。     

人亲环素基因的克隆、表达及应用研究

亲环素(CyP)具有多种生物学功能,它是CsA的胞内受体,能和CsA特异结合;它又是人类免疫缺陷病毒(HIV)在细胞内复制的必要因子.研究首先从动物组织中提取、纯化CyP蛋白,并且克隆了CyPA基因,在Ecoli中获得高效表达.进而又克隆、表达了CyPB基因,进行了生物学活性测定,制备了CyP蛋白单克隆抗体和多抗,建立了多项检测方法.研制的CyP自身抗体及CsA血药浓度两种检测药盒已在临床应用,已取得良好的效益.     

大鼠肝细胞膜胰岛素受体与胰岛素的相互作用

采用125I-胰岛素标记法研究大鼠肝细胞膜中胰岛素受体与胰岛素的结合性质,用31P-核磁共振法(31P-NMR)研究大鼠肝细胞膜中胰岛素刺激下的胰岛素受体体外自磷酸化,实验结果表明大鼠肝细胞膜胰岛素受体与胰岛素结合的最佳酸度大约为pH=7.5,并且表明大鼠肝细胞膜上的胰岛素受体为高亲和、低容量和低亲和、高容量两型受体.非放射性的31P-NMR波谱法检测到胰岛素刺激下的大鼠肝细胞膜胰岛素受体的体外自磷酸说明所提取纯化的胰岛素受体具有生物活性.     

橙皮素对血管内皮细胞NO分泌功能的影响

考察了橙皮素对人脐静脉内皮细胞(HUVECs)NO分泌功能的影响,探讨其作用机制.为此采用DAN法测定HUVECs分泌NO的水平,用人乳腺癌MCF-7细胞(雌激素受体阳性)促增殖实验和报告基因模型实验评价橙皮素的雌激素样活性.结果显示在内源雌激素水平较低的条件下,橙皮素在12.5～100 μmoL/L浓度范围内,能够促进HUVECs分泌NO,并呈剂量依赖性.此作用能够被雌激素受体拮抗剂ICI182,780和放线菌素D阻断.橙皮素与E2同时作用时,HUVECs分泌NO水平较两者单独作用时显著下降.而在内源雌激素水平较高的条件下,橙皮素抑制HUVECs分泌NO.橙皮素能够促进MCF-7细胞增殖,并且能够被ICI182,780完全阻断,同时,橙皮素能够部分拮抗E2对MCF-7细胞的促增殖作用.另外,橙皮素能够诱导ERα控制的报告基因表达.从而可认为橙皮素属于雌激素受体部分激动剂,对血管内皮细胞NO分泌功能具有调节作用,其机理涉及雌激素受体信号途径和基因转录调节.     

HAb18G/CD147真核荧光蛋白的表达及鉴定

构建肝癌相关抗原HAb18G／CD147全长及缺失片段E51（第22—50位氨基酸缺失）的真核荧光蛋白表达载体,进一步研究该基因在肝细胞肝癌发生中的作用。通过PCR及Overlapping PCR的方法获得目的基因．与荧光载体pEGFP—N1双酶切、连接、转化E．coli JM109．构建含有肝癌相关抗原HAb18G／CD147全长及E51的真核荧光蛋白表达载体．并经限制性酶切及序列分析证明插入是否正确。用阳离子脂质体介导转染COS-7细胞,进行瞬时表达;荧光显微镜观察EGFP表达,通过流式细胞术检测蛋白的表达情况,明胶酶谱法鉴定其功能。成功地构建了真核荧光蛋白表达载体pEGFP—N1／HAb18G、pEGFP—N1/E51,并经限制性酶切及序列分析证明外源基因插入正确;流式细胞术鉴定该蛋白表达正确;功能实验证实缺失片段E51不具有诱导成纤维细胞分泌MMPs的功能．结果显示HAb18G／CD147基因第22—50位氨基酸与其刺激成纤细胞分泌MMP和功能有密切关系。实验结果为HAb18G蛋白分子的生物学功能研究奠定了基础。     

趋化因子IL-8和受体CXCR1,CXCR2在胃肠和肝肿瘤中的表达

为研究趋化因子IL-8和受体CXCR1、CXCR2在胃肠和肝肿瘤中的作用,应用免疫组织化学和RT-PCR的方法检测了趋化因子IL-8和受体CXCR1、CXCR2在食道癌、胃癌、结直肠癌和肝癌中的表达。在胃肠肿瘤中,CXCR1在癌细胞的细胞核膜和细胞质中表达,CXCR2在癌细胞的细胞质中表达。在肝癌中,CXCR1和CXCR2在癌巢中有表达而在邻近的肝组织中无表达。应用RT-PCR的方法检测了受体CXCR1、CXCR2的转录,其结果证实了免疫组织化学的结果。在胃肠和肝肿瘤中检测到了IL-8的表达。进一步分析表明在胃肠和肝肿瘤中IL-8与CXCR1/CXCR2共表达(p=0.0448)。实验结果说明趋化因子IL-8和受体CXCR1、CXCR2可能在胃肠和肝肿瘤的发生和发展中起作用。     

基于裸鼠模型研究EGFL9对肝癌细胞增殖、转移能力的调控作用

目的:旨在通过裸鼠模型分析EGFL9对肝癌细胞增殖、转移能力的影响。方法:通过慢病毒介导的小干扰RNA技术构建EGFL9沉默的JHH-7肝癌细胞系。采用MTT生长曲线分析EGFL9对JHH-7肝癌细胞增殖能力的影响。采用裸鼠皮下成瘤实验和尾静脉注射转移实验观察EGFL9基因在动物活体内对JHH-7肝癌细胞增殖、转移能力的影响。结果:荧光显微镜观察结果显示JHH-7^shEGFL9组和JHH-7^shCtrl组肝癌细胞慢病毒感染效率高于80%,qRT-PCR检测结果显示JHH-7^shEGFL9组EGFL9干扰效率达98.3%,提示EGFL9沉默的JHH-7人肝癌细胞系构建成功。MTT生长曲线结果提示，JHH-7^shEGFL9组肝癌细胞的增殖能力明显低于JHH-7^shCtrl组(P<0.05)。裸鼠皮下成瘤实验显示，JHH-7^shEGFL9组裸鼠皮下肿瘤的质量[(0.246±0.104)g vs(0.710±0.221)g]和瘤体体积均显著小于JHH-7     

Antibody and HIV-1 gp120 recognition of CD4 undermines the concept of mimicry between antibodies and receptors.

It has been proposed that antibodies can mimic the binding of a receptor to its ligand and that anti-idiotype antibodies raised against such antibodies can be used to identify the receptor. A large number of antibodies have been raised against CD4, the receptor on T cells for the envelope glycoprotein gp120 of the human immunodeficiency virus, and the site at which gp120 binds to CD4 has been delineated. It has therefore become possible to contrast the fine specificities of a natural ligand (gp120) and antibodies that interact with the receptor at the same site. Here we report that out of a panel of 225 anti-CD4 antibodies, only one showed fine binding specificity that was broadly like that of gp120, but the evidence was against this being an exact mimic. Thus the data indicate that the production of antibody mimics will occur very rarely or not at all and that the anti-idiotype approach is unlikely to be useful. This contention is supported by a review of the results of attempts to use this approach. Taking strict criteria for success, there is no example for which the anti-idiotype approach has led to the discovery of a previously undescribed receptor or other protein of interest.     

Evidence for a physical association of CD4 and the CD3:alpha:beta T-cell receptor

CD4 is a molecule expressed on the surface of T lymphocytes which recognize foreign protein antigens in the context of class II major histocompatibility complex (MHC) molecules. Recognition of antigen:class II MHC complexes by CD4+ T cells can be inhibited by anti-CD4 (ref. 3). Nevertheless, specific recognition of the antigen:Ia complex is clearly a function of the T-cell receptor, which is composed of CD3 and the variable polypeptides alpha and beta. Thus, it has been proposed that CD4 serves an accessory function in the interaction of CD4+ T cells and Ia-bearing antigen-presenting cells by binding to non-polymorphic portions of class II MHC molecules and stabilizing the cell interaction. Based on our observation that anti-CD4 could inhibit activation of a cloned line of CD4+ T cells by antibodies directed at a particular epitope on the variable region of the T-cell receptor, we have recently proposed that CD4 is actually part of the T-cell antigen recognition complex, physically associated with CD3:alpha:beta. But numerous studies showing that CD3 and CD4 are not stably associated on the T-cell surface would appear to contradict this model. Here we show that anti-T-cell-receptor antibodies can co-modulate expression of the T-cell receptor and CD4, and that the monovalent Fab fragment of such an anti-T-cell-receptor antibody can, in conjunction with bivalent anti-CD4 antibody, generate an activating signal for the T cell. These findings provide further evidence for a physical association of the T-cell receptor complex and CD4.     

CD21 is a ligand for CD23 and regulates IgE production.

The molecule CD23, a low-affinity receptor for IgE (Fc epsilon R2), is a type II transmembrane molecule expressed on many haemopoietic cell types. CD23 has pleiotropic roles in the control of lymphocyte behaviour, suggesting that CD23 may interact with another ligand in addition to IgE. To identify such a CD23 ligand, we expressed and purified full-length recombinant CD23, incorporated it into fluorescent liposomes and used these as a probe. We report here that fluorescent liposomes carrying CD23 interact specifically with the cell-surface protein CD21, identified as the receptor for Epstein-Barr virus and the complement receptor-2 on B cells, some T cells and follicular dendritic cells. In addition, fluorescent CD23-liposomes were shown to bind to hamster kidney cells (BHK-21) transfected with CD21 complementary DNA. The interaction between fluorescent CD23-liposomes and B cells or CD21-transfected BHK-21 cells was specifically inhibited by anti-CD21 and anti-CD23 monoclonal antibodies. Western blotting analysis revealed that 14C-labelled liposomes carrying CD23, in contrast to anti-CD21 antibodies, reacted with a subtype of CD21 molecules. Triggering of CD21 either with an anti-CD21 antibody or with recombinant soluble CD23 was shown to increase specifically interleukin-4-induced IgE production from blood mononuclear cells. These results demonstrate that the cell-surface protein CD21 is a ligand for CD23 and that the pairing of these molecules may participate in the control of IgE production.     

自噬对肝癌SMMC-7721细胞侵袭迁移能力的影响

为探讨自噬对照射过程中肝癌SMMC-7721细胞侵袭和迁移能力的影响及其潜在的作用机制,将肝癌SMMC-7721细胞分为6组,分别为阴性对照(NC)组、8 Gy X-线照射(IR)组、自噬激活剂雷帕霉素(Rapa)组、自噬抑制剂三甲基腺嘌呤(3-MA)组、照射+雷帕霉素(IR+Rapa)组和照射+三甲基腺嘌呤(IR+3-MA)组,利用划痕实验和Transwell实验检测自噬对肝癌SMMC-7721细胞迁移和侵袭的影响;用CCK8实验检测照射、雷帕霉素和3-MA对肝癌细胞增殖的影响;用蛋白免疫印迹(Western blot)检测N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和LC3B蛋白表达情况。研究结果显示,照射可增强肝癌SMMC-7721细胞侵袭迁移的能力(P<0.05),同时,照射可抑制细胞的增殖能力(P<0.05);雷帕霉素激活自噬可增强细胞的侵袭迁移能力(P<0.05),3-MA抑制自噬可抑制细胞侵袭迁移和细胞的增殖能力(P<0.05)。照射可上调N-cadherin、Vimentin表达水平(P<0.05),激活或抑制自噬可分...     

Activated CD8 binding to class I protein mediated by the T-cell receptor results in signalling

The CD8 glycoprotein of T cells bind nonpolymorphic regions of class I major histocompatibility complex proteins on target cells and these interactions promote antigen recognition and signalling by the T-cell receptor. Studies using artificial membranes indicated that effective CD8/class I interaction is critical for response by alloantigen-specific cytotoxic T lymphocytes when class I protein is the only ligand on the antigen-bearing surface. But significant CD8-mediated binding of cytotoxic T lymphocytes to non-antigenic class I protein could not be detected in the absence of the alloantigen. These apparently contradictory findings indicate that CD8 binding to class I protein might be activated through the T-cell receptor and the results reported here demonstrate that this is the case. Treatment of cytotoxic T lymphocytes with soluble anti-T-cell receptor antibody activates adhesion of the cytotoxic T lymphocytes to class I, but not class II proteins. The specificity of this binding implies that it is mediated by CD8 and blocking by anti-CD8 antibodies confirmed this. Furthermore, binding of CD8 to class I protein resulted in generation of an additional signal(s) necessary to initiate response at low T-cell receptor occupancy levels.     

Interleukin-4 mediates CD8 induction on human CD4+ T-cell clones

CD4 and CD8 antigens are simultaneously expressed on most of the cortical thymocytes, that weakly express the T-cell antigen receptor(TCR)/CD3 complex. Mature peripheral T cells, however, strongly express the TCR complex and are positive for either CD4 or CD8. Nevertheless, a small percentage of peripheral CD3+ T cells express CD4 and CD8 simultaneously. These mature, double positive cells could be intermediates between CD4+CD8+ thymocytes and mature, single positive T cells, or they may originate from single positive T cells that acquire either CD4 or CD8. Here we report that activation and culturing of cloned CD4+ T cells in interleukin-4 (IL-4), results in the acquisition of CD8 due to its de novo synthesis. The IL-4-induced co-expression of CD8 on CD4+ T cells is reversible, in that CD8 disappeared from double positive T-cell clones isolated in IL-4, when they were cultured in IL-2. CD8 induced by IL-4 can be functional as a monoclonal antibody to CD8 inhibited anti-CD3-mediated cytotoxicity by a double positive T-cell clone.     

Clinical and pathological features of miR-10b and RHOC gene expression in hepatocellular carcinoma

Aberrant expression of microRNAs （miRNAs） was reported frequently in different human cancers. The major role of miRNA is targeting 31-UTR of coding gene and causing translational repression or mRNA degradation. miR-10b overexpression was reported to promote breast cancer metastasis by up-regulating RHOC expression. But its expression in hepatocellular carcinoma （HCC） remains unclear. Our study indicated that the expression of miR-10b was different in HCC and adjacent tissue samples, and reduced expression of miR-10b in HCC was related tovein invasion. High-level expression of RHOC was also related to vein invasion in HCC. But no correlation was found between miR-10b and RHOC expression. These results suggest that miR-10b and RHOC are independent predictors of HCC invasion and metastasis.