脂质体介导的cripto反义寡核苷酸抑制结肠癌细胞端粒酶活性

目的：探讨cripto反义寡核苷酸（ASODN）对结肠癌细胞端粒酶活性的影响。 方法: 应用脂质体瞬时转染法介导cripto 反义寡核苷酸，处理人结肠癌细胞系后，分别采用实时定量PCR检测cripto mRNA表达，用TRAP检测端粒酶活性，采用软琼脂集落培养试验检测结肠癌细胞的生长。 结果: Cripto ASODN可有效抑制结肠癌细胞集落生长，且与浓度相关。结肠癌细胞经Cripto ASODN转染后，端粒酶活性明显受到抑制，呈作用浓度和时间依赖性。并与ASODN的浓度和处理时间有关。 结论: cripto基因可能参与对结肠癌细胞端粒酶活性的调控。     

亚砷酸对CNE1细胞株增殖和端粒酶hTRT的影响

目的　观察亚砷酸对人鼻咽癌CNE1细胞株增殖的抑制作用及端粒酶逆转录酶 (hTRT)的影响。方法　人鼻咽癌细胞株CNE1用不同浓度亚砷酸处理 ,MTT法观察不同浓度亚砷酸作用不同时间后对CNE1细胞生长的影响 ,流式细胞仪检测不同条件作用后CNE1细胞增殖指数和凋亡指数及周期分布改变 ,免疫组化法检测增殖细胞核抗原 (PCNA)及端粒酶hTRT表达情况。结果　亚砷酸抑制人鼻咽癌细胞株CNE1细胞的增殖 ,其作用随着亚砷酸浓度增加和作用时间延长而增加 ;一定浓度亚砷酸作用于CNE1细胞后 ,其S期细胞比例及增殖指数随着时间的延长而逐渐减少 ,但凋亡指数逐渐增多。随着亚砷酸浓度增加 ,PCNA及hTRT阳性细胞平均灰度值逐渐下降 (P <0 0 5 )。结论　亚砷酸可抑制人鼻咽癌细胞株CNE1的生长 ,除了诱导凋亡以外 ,调控DNA合成及细胞增殖的PCNA蛋白合成降低 ,抑制端粒酶的活性可能是亚砷酸的部分抗肿瘤作用机制。     

三氧化二砷对K562细胞端粒酶活性及P53蛋白表达水平的调节

目的：研究三氧化二砷(As2O3)诱导急性白血病K562细胞的凋亡机制。方法：以不同浓度的As2O3作用于体外培养的K562细胞，检测细胞生长抑制率，观察细胞凋亡时的形态学变化，对细胞端粒酶活性及P53蛋白的表达水平进行检测。结果：As2O3可显著地降低K562细胞端粒酶活性，升高P53蛋白的表达水平，抑制细胞的生长，诱导细胞发生凋亡。并呈现出明显的量-效与时-效关系。结论：As2O3能抑制K562细胞的生长并诱导细胞发生凋亡，降低细胞端粒酶活性及升高P53蛋白的表达水平是其重要作用机制之一。     

RNAi沉默Cripto基因对结肠癌细胞血管内皮生长因子的抑制

目的：研究Cripto基因对结肠癌细胞血管内皮生长因子的影响。方法:设计合成针对Cripto基因的小干扰RNA(small interfering RNA，siRNA)，转染结肠癌LS-174T细胞。细胞分4组：空载对照组（对照组）和不同浓度（3.125、6.25和12.5 nmol/L）的siRNA组。分别于转染24、48、72 h后收集细胞，以实时定量PCR检测结肠癌细胞Cripto mRNA，分别采用Northern blotting和免疫荧光标记法检测癌细胞VEGF mRNA和蛋白表达。分别收集转染72 h的12.5 nmol/L组和对照组细胞，接种于裸鼠背部。30 d后处死裸鼠，收集裸鼠肿瘤组织，对组织VEGF进行免疫组化染色，计算染色平均强度。结果:实时定量PCR检测显示，转染组Cripto mRNA被有效地抑制，且呈浓度和时间依赖性。转染组细胞VEGF 蛋白和mRNA明显低于对照组。裸鼠肿瘤免疫组化分析显示，转染组VEGF平均染色强度明显低于对照组。结论:Cripto基因参与了结肠癌组织血管生成的调控。采用siRNA下调Cripto基因表达，可抑制结肠癌组织血管生成。     

三氧化二砷抑制人神经胶质瘤细胞生长及其机制

目的：探讨三氧化二砷（As2O3）对人神经胶质瘤细胞株U251的生长抑制作用及对基因表达和钙含量的影响。 方法： MTT法观察As2O3对U251细胞的生长抑制作用；流式细胞仪测定不同浓度As2O3处理后U251细胞周期、增殖细胞核抗原（PCNA）、凋亡相关蛋白Fas和Bcl-2阳性细胞百分率及细胞内钙离子（IECa2+）含量变化。Annexin V-FITC+PI双参数检测细胞凋亡。 结果： (1) As2O3可显著抑制U251细胞生长，且剂量-效应和时间-效应关系显著（P<0.05），其24 h、48 h和72 h的IC50值分别为14.28 μmol/L、13.63 μmol/L和2.34 μmol/L。（2）As2O3能显著增加Fas蛋白表达和IECa2+含量（P<0.01），下调PCNA蛋白表达（P<0.01），并使细胞周期阻滞在G2 M期，但对Bcl-2表达无影响（P>0.05）。（3）U251细胞经As2O3处理后可出现凋亡。 结论: As2O3在体外可显著抑制人神经胶质瘤U251细胞生长并引起凋亡，其机制可能与上调Fas表达和IECa2+含量、降低PCNA蛋白表达及阻滞细胞周期有关。     

一定浓度下三氧化二砷对T细胞淋巴瘤Hut-78细胞株增殖的影响

背景：近年来有将三氧化二砷试用于T细胞肿瘤的有效报道,但未见其治疗机制的相关研究。 目的：检测三氧化二砷对人皮肤T细胞淋巴瘤Hut-78细胞的抑制增殖、诱导凋亡及细胞周期的影响。 方法：分别采用MTT、PI单标记流式细胞术和TUNEL法检测2,5,10 μmol/L的三氧化二砷干预24,48,72 h, 对人皮肤T细胞淋巴瘤Hut-78细胞株抑制增殖、诱导凋亡和细胞周期的影响,及其凋亡相关基因bcl-2及血管内皮细胞生长因子基因表达的变化。 结果与结论：在一定的浓度范围内,三氧化二砷对Hut-78细胞存在增殖抑制作用,其增殖抑制作用在一定程度上可能与下调血管内皮细胞生长因子基因的表达有关,同时还存在一定的细胞毒作用;三氧化二砷可诱导Hut-78细胞发生凋亡,凋亡机制主要发生在G2~M期,其凋亡作用可能与下调bcl-2基因表达有关。三氧化二砷对Hut-78细胞的增殖抑制及诱导凋亡作用呈时间剂量依赖性。     

Anticancer effect of arsenic trioxide on cholangiocarcinoma: in vitro experiments and in vivo xenograft mouse model

The purpose of our study was to investigate anticancer activity of arsenic trioxide (As₂O₃) on cholangiocarcinoma through in vitro and in vivo experiments using human cholangiocarcinoma cancer cells (CC-t6 cells) and a nude mouse model. The effect of As₂O₃ on CC-t6 cell survival was determined in vitro using MTT assay. Analysis of cell cycle phase distribution and quantification of apoptosis/necrosis, which were achieved by flow cytometry, were performed in order to understand the mechanism of As₂O_3. In vivo experiment was performed to assess the effectiveness of local injection of As₂O₃ on tumor inhibition by comparing the following three groups each consisting of five nude mouse xenograft models: high dose As₂O₃ (5 mg/kg), low dose As₂O₃ (1 mg/kg), and saline. In MTT assay, As₂O₃ inhibited the growth of CC-t6 cells more effectively than cisplatin or adriamycin at concentrations between 1 and 100 μM for most time points between 24 and 72 h (p < 0.05). With increased concentration of As₂O₃, there was dose-dependent increase in G ₀/G ₁ phase and dose-dependent decrease in S phase. As₂O₃-mediated inhibition of cell viability was achieved via induction of apoptosis and necrosis in a dose-dependent manner. Injection of As₂O₃ into CC-t6-induced tumors in nude mice inhibited the growth of subcutaneous tumor xenografts. As₂O₃ treatment dose-dependently inhibited the proliferation of CC-t6 cells via G ₀/G ₁ phase arrest and retarded tumor growth in nude mice, suggesting that As₂O₃ may be effective in the treatment of cholangiocarcinoma.     

Inhibition of T cell activity in vivo: a test model for quantitative evaluation.

A test model is presented which, in comparison with the conventional models of skin transplantation or graft-versus-host (GvH) reaction in mice, permits a more sensitive quantitative evaluation of T cell inhibition in vivo. Prospective donors (type AA) are immunized with prospective recipient material (type AB); the resulting T cell reaction of A versus B is inhibited by consecutive treatment. Extent of inhibition can be evaluated after transfer of the pretreated AA material onto AB recipients by calculation of remaining GvH reactivity, if compared to adequate control tranfers. In this model the target animal for T cell reactivity (the AB recipient) remains untouched from immunosuppressive regimen.     

Inhibition of in vitro and in vivo T cell responses by recombinant human Tim-1 extracellular domain proteins

Members of the T cell, Ig domain and mucin domain (Tim) family of proteins have recently been implicated in the control of T cell-mediated immune responses. Tim-1 (HUGO designation HAVCR1) polymorphisms have been linked to the regulation of atopy in mice and humans, suggestive of a role in immune regulation. Tim-1 is expressed upon activation of T cells. In concert with the increased expression of Tim-1, a binding partner for the extracellular domain of Tim-1 (eTim-1) was induced on activated T cells, and mRNA expression data was consistent with the binding partner being Tim-4. We found that co-immobilized recombinant eTim-1 was able to inhibit T cell activation mediated by CD3 + CD28 mAb. eTim-1 mediated its inhibitory effects on proliferation by arresting cell cycle at G(0)/G(1) phase through regulation of cell cycle proteins. In vivo, administration of eTim-1 proteins led to a decrease in both ear (contact hypersensitivity to oxazolone) and joint (methylated BSA antigen-induced arthritis) swelling. The inhibitory activity of eTim-1 in the T(h)1-dependent models was evidence that eTim-1 is able to modulate T cell responses. Manipulation of the Tim-1 interaction with its binding partner on T cells may therefore provide a novel target for therapeutic intervention in T cell-mediated diseases.     

Inhibition of accelerated rejection mediated by alloreactive CD4+ memory T cells and prolonged allograft survival by arsenic trioxide

The aim of this study was to evaluate and determine the potential mechanisms of As₂O₃ in accelerated rejection mediated by alloreactive CD4⁺ memory T cells. Vascularized heterotopic cardiac transplantation from C57BL/6 mice to nude mice (pre-transferred CD4⁺ memory T cells) was performed on Day 0, and As₂O₃ was administered to recipient mice from Day 0 to 10. As a result, As₂O₃ could reduce the proliferation of allo-primed CD4⁺ memory T cells in vitro in MLR and the baseline rate of proliferation was restored by the addition of exogenous IL-2. In vivo, compared with the control[+] group, the mean survival time of cardiac allografts in the As₂O₃ group was prolonged from 5.8?±?0.7 to 14.2?±?2.5 days. Five days after transplantation, the relative gene expression of IL-2, IFN-γ and Foxp3 was reduced in the grafts by As₂O₃ treatment, but the expression of IL-10 and TGF-β was increased. Correspondingly, the proportions of CD4⁺ T cells, CD4⁺ memory T cells and regulatory T cells (Tregs), both in recipient spleens and lymph nodes, were lowered. These results indicate the potential of As₂O₃ as a novel immunosuppressant targeting CD4⁺ memory T cells.     

Inhibition of ‘in vitro’ tumor cell growth by aromatic polyamidines exhibiting antiproteinase activity

Aromatic polyamidines containing two, three or four benzamidine residues inhibit proteinase activity and proliferation of different human tumor cell lines, including leukemic (K562, HEL), melanoma (Colo 38) and B-lymphoid (WI-L2) cell lines. In addition, the benzamidine derivatives analysed in the present study inhibit cell growth of the Chinese hamster FHO6T1-1 cell line, obtained after transfection of primary lung cells with the activated human T24-Ha-ras-1 oncogene.After treatment of FHO6T1-1 cells with benzamidine derivatives, a sharp decrease of the content of Ha-ras-1 mRNA was found, but not of transferrin receptor mRNA.We found that inhibition of cell proliferation by tetra-benzamidine derivatives is not restricted to tumor cells, but concerns also non-tumorigenic cell lines as well as normal primary fibroblasts. Therefore, our analysis was extended to di- and tri-benzamidine derivatives, which could be proposed as useful substrates in the synthesis of drug-conjugated monoclonal antibodies or growth factors. The data obtained demonstrate that these latter compounds and their halo-derivatives also exhibit strong antiproliferative effects onin vitro cultured cells.     

外源性骨桥蛋白对人大肠癌LS-174T细胞增殖和侵袭能力的影响

目的观察人重组骨桥蛋白(recombinated human osteopontin,rhOPN)对大肠癌LS-174T细胞增殖、侵袭及其相关基因表达的影响,探讨骨桥蛋白在大肠癌演进中的作用。方法添加不同浓度rhOPN于人大肠癌LS-174T细胞培养液中,采用四甲基偶氮唑蓝(MTT)试验检测细胞的增殖能力,重组人工基底膜实验评价细胞侵袭能力,并运用实时荧光定量RT-PCR检测大肠癌细胞中尿激酶型纤溶酶原激活物(uPA)、基质金属蛋白酶-2(MMP-2)和血管内皮生长因子(VEGF)等mRNA表达水平。结果LS-174T细胞经不同浓度rhOPN作用24h后,实验组的MTT值显著高于对照组(P<0.01),并呈剂量依赖关系。Transwell上室加入5nmol/L、30nmol/L的rhOPN后,实验组LS-174T细胞穿膜数量明显多于对照组(P<0.05)。经5nmol/L、30nmol/L的rhOPN处理24h后,实验组LS-174T细胞的uPA mRNA及VEGF mRNA的表达水平明显高于对照组(P<0.05),而MMP-2 mRNA表达水平与对照组无显著性差别(P>0.05)。结论rhOPN促进人大肠癌LS-174T细胞的增殖,并提高其侵袭能力,可能与uPA和VEGF基因表达上调有关。     

三氧化二砷抑制大鼠脑胶质瘤增殖

目的:观察三氧化二砷(As2O3)瘤内给药对大鼠脑胶质瘤的疗效,以探讨胶质瘤的有效化疗手段。方法:实验用65只(180±10)g SD大鼠,5只做正常对照,在60只大鼠纹状体部埋植导管,其中30只通过导管接种9L胶质瘤细胞株,建立在体脑胶质瘤模型。15只注入等量D-Hanks平衡盐,作为阴性对照;另15只为安查组,进行药物安全性研究。于植入瘤细胞第9 d,30只模型鼠随机分为肿瘤组与治疗组(各15只)。治疗组及安查组通过导管将As2O3(10μmol/L)注入瘤内。阴性对照组和肿瘤组注等量生理盐水。(1)阴性对照组、肿瘤组、安查组和治疗组于注射As2O38 d后各随机处死5只,迅速剥离脑组织,测量肿瘤体积,HE染色,观察组织形态变化,免疫组化检测PCNA含量。(2)阴性对照组、肿瘤组、安查组和治疗组各剩余的10只大鼠,逐日称量体重,观察其行为学变化,待其自然死亡后计算生存时间,并取脑,验证肿瘤的存在。结果:治疗组肿瘤明显小于肿瘤组,大鼠体重下降较肿瘤组为轻。肿瘤组平均生存期为25.8天,治疗组平均生存时间40天。但治疗组有两只未计入平均,一只生存178天,另一只在研究结束时被处死(达14个月),且处死后未发现肿瘤。结论:As2O3可通过减缓脑胶质瘤的增殖,缓解大鼠体重下降,延长模型动物生存时间,对大鼠脑胶质瘤具有明显治疗作用。     

Inhibition of tumor growth induced by histamine: In vivo and in vitro studies

An experimental mammary carcinoma was induced in rats by the i.p. administration of N-nitroso N-methylurea (NMU) in 3 doses of 50 mg/kg. In order to study the role of histamine (Hi) and its receptors in tumor growth,in vivo andin vitro treatments were carried out. Different groups of animals received a daily s.c. injection of Hi 0.1 mg/kg, starting with the first dose of NMU. Hi significantly reduced tumor incidence and the number of tumors developed per rat. Thein vitro studies, using the clonogenic agar technique, showed no difference in colony formation between control, 0.01 and 0.1 M Hi treatment, while 1 and 10 M Hi concentrations induced inhibition of cell growth up to 60%. This effect was abolished by H₁ antagonists. Conversely, the action of the H₂ antagonists was a significant inhibition of colony formation. We may then conclude that in these experimental tumors, histamine exerts a regulatory function on cell growth by acting directly on specific membrane receptors.     

Regulation of in vitro and in vivo T cell activation by CD43

Accessory molecule interactions can be critical in determining the outcome of a T cell's encounter with antigen. Cell adhesion proteins may augment T cell responses by facilitating TCR engagement of the antigen-MHC complex, while co-stimulatory molecules may deliver distinct signals that modulate T cell responsiveness. CD43 (leukosialin, sialophorin) has been suggested to influence cell activation by steric hindrance based upon the large size and glycosylation of the protein, as well as the relative abundance of the protein on the cell surface. In this paper we examine both in vitro and in vivo T cell-dependent responses in CD43-deficient mice. We demonstrate that T cells from CD43-deficient mice are hyper-responsive following both in vivo and in vitro activation, and that this is observed in response to not only TCR-CD3-mediated stimulation, but also following receptor-independent activation. This data suggests that mechanisms other than non-specific steric hindrance are important in the regulation of T cell activation by CD43.      

Inhibition of in vitro and in vivo mast cell degranulation by Taenia crassiceps metacestodes in vitro incubation products

In vitro released products of T. crassiceps metacestodes (TcIP) harvested from the peritoneal cavity of NMRI mice were tested for inhibitory effects on the in vitro degranulation of peritoneal mast cells (MCs) of normal mice (NMRI) and rats (Wistar) and on the in vivo degranulation of rat (Wistar) skin MCs (PCA-assay). In vitro degranulation was elicited chemically (compound 48/80, polymyxin B or the bee venom peptide, mellitin). In vivo degranulation was triggered immunologically (anaphylactic systems ovalbumin/anti-ovalbumin or Fasciola hepatica crude fluke extract antigen/serum of fluke-infected rats (Wistar]. In vitro degranulation of murine peritoneal MCs or the in vitro histamine release of rat peritoneal MCs normally induced chemically was significantly inhibited when the MCs were preincubated with the TcIP or with serum of T. crassiceps-infected NMRI mice from day 35 post infection and thereafter. In vitro degranulation of peritoneal MCs of infected mice was strongly inhibited beginning on day 10 after infection. Also in vivo degranulation of the IgE-sensitized rat skin MCs was significantly reduced by intradermal injection of the TcIP before (6, 3 and 1 h) antigen challenge and by preinjection (1 h) of serum from infected mice (day 80 p.i.)-The inhibitory effect was also demonstrated after immunoadsorption of mouse serum proteins naturally contaminating the TcIP. Heating (100 degrees C/15 min), even in the presence of 0.25 M HCl, did not suppress the inhibitory activity.     

Inhibition of macrophage phagocytosis by Bacteroides fragilis in vivo and in vitro.

A number of investigators have provided experimental evidence for synergistic effects in mixed infections with Escherichia coli and Bacteroides fragilis. In vitro studies have suggested that competition for serum opsonins and diminished subsequent phagocytosis by polymorphonuclear leukocytes might explain these effects. In the present study we evaluated the effect of B. fragilis on macrophage phagocytosis. It was shown that peritoneal macrophages from mice injected intravenously 6 to 12 h earlier with 10(8) CFU of encapsulated B. fragilis were markedly suppressed in their phagocytic ability. Injections of laboratory-passaged, less-encapsulated B. fragilis, other bacteria, or latex particles were either not suppressive of macrophage phagocytosis or less effective. When peritoneal macrophages were treated in vitro for 24 h with the same challenge organisms prior to assessing their phagocytic capacity, encapsulated B. fragilis also proved significantly more suppressive than challenges with other organisms or latex particles. We conclude that suppression of macrophage phagocytosis by B. fragilis seems to be an important mechanism contributing to synergistic effects described for mixed aerobic and anaerobic infections.     

阻抑eIF-4E对大肠腺癌LS-174T细胞乙酰肝素酶mRNA及其翻译表达水平的影响

目的:确定阻抑结肠癌LS-174T细胞中过量表达的真核细胞起始因子-4E(eukaryoticinitiationfactor-4E,eIF-4E)是否促进乙酰肝素酶(heparanase)mRNA的降解,并改变其翻译表达水平。方法:应用脂质体包裹与eIF-4EmRNA翻泽起始点互补的asODN,转染处理人大肠腺癌细胞LS-174T。使用Westernblot和RT-PCR方法分别检测eIF-4E被阻抑后其转录和翻译水平的改变。乙酰肝素酶mRNA在细胞内水平采用Northernblot定量检测,其蛋白表达水平改变采用Westernblot检测。结果:asODN经脂质体转染LS-174T细胞后,eIF-4E基因表达明显受到抑制,其蛋白表达产物也显著下降。伴随eIF-4E被阻抑表达,Northernblot结果显示乙酰肝素酶mRNA水平下降,且其蛋白翻译表达量也降低。结论:阻抑eIF-4E影响LS-174T细胞乙酰肝素酶mRNA稳定、促使其降解,并降低乙酰肝素酶表达。     

三氧化二砷体内化疗抑制大肠癌转移的分子生物学机制研究

目的研究三氧化二砷(As2O3)术前短期体内化学治疗对大肠癌细胞分子生物学作用机制,评价其体内化学治疗效果,为临床治疗大肠癌提供理论依据。方法选择消化内镜检查及病理活组织检查诊断为大肠腺癌且计划进行手术切除的患者30例,其中男性17例,女性13例;年龄38～73岁,平均年龄55.1岁。随机分为试验组和空白对照组,每组15例,保存患者术前行内镜诊断时所取得的癌组织。试验组:As2O3(亚砷酸)注射液10 mL加入5%葡萄糖注射液500 mL中,给予患者术前每天1次静脉滴注,连用3 d;空白对照组:不用药。两组均于第4日复查血常规和肝、肾功能后手术。取术后病理癌组织。将患者术前与术后所取标本进行如下处理:固定、包埋、苏木精-伊红染色,免疫组织化学法检测突变型P53蛋白、血管内皮生长因子(VEGF)、nm23蛋白表达情况。结果As2O3体内化学治疗可以使大肠癌细胞P53蛋白、VEGF蛋白的表达下调,差异具有显著统计学意义(P<0.01);As2O3体内化学治疗可以使大肠癌细胞nm23蛋白表达率增加,差异具有显著统计学意义(P<0.01)。结论As2O3短期体内化学治疗有抑制大肠癌细胞转移的作用。