miR-21 targets Fas ligand-mediated apoptosis in breast cancer cell line MCF-7

Over-expression of Fas ligand （FasL） on tumor cell surface can induce the apoptosis of spe- cific activated tumor infiltrating lymphocytes （TILs） via the Fas/FasL pathway, leading to the formation of a site of immune privilege surrounding the tumor mass for escaping immune surveillance and pro- moting tumor proliferation, invasion and metastasis. The blocking effect of miR-21 on FasL-mediated apoptosis in breast cancers was investigated in this study. The expression levels of miR-21 and FasL in human breast carcinoma cell lines were detected by using RT-PCR and Western blotting. FasL as a tar- get gene of miR-21 was identified by Luciferase assay. The apoptosis of Jurkat T lymphocytes induced by MCF-7 cells was determined by flow cytometry. It was found that in four human breast cancer cell lines, FasL expression level in MCF-7 cells was the highest, while miR-21 was down-regulated the most notably. After miR-21 expression in MCF-7 cells was up-regulated, FasL was identified as a target gene of miR-21. When the effector/target （E/T） ratio of MCF-7 cells and Jurkat cells was 10：1, 5：1 and 1：1, the inhibitory rate of apoptosis of Jurkat T lymphocytes induced by MCF-7 cells was 95.81%, 93.16% and 91.94%, respectively. It is suggested that in breast cancers miR-21 expression is negatively associ- ated with FasL expression, and FasL is a target gene of miR-21, miR-21 targeting and regulating FasL-mediated apoptosis will bring us the possibility of a new tumor immunotherapy via breaking tu- mor immune privilege.     

Expression of HLA-DR antigen in hepatocellular carcinoma and its up-regulation by interferon

Objective: To observe the expression of human leukocytic antigen DR (HLA-DR) in primary hepatocellular carcinoma (HCC) and its up-regulation by interferon (IFN). Methods: The expression of HLA-DR in 46 specimens of human HCC tissues, 4 human HCC cell lines (SMMC-7721, HCC-9204, BEL-7402 and HHCC) and a human hepatocyte cell line QZG was respectively detected by immunohistochemical ABC staining and flow cytometry. The expression of HLA-DR in the 5 cell lines was detected by ELISA before and after the cells were treated with IFN-γ or IFN-α. Results:Eighteen out of 46 HCC tissues (39.1%) expressed HLA-DR, whereas all the normal liver tissues immediately adjacent to HCC tissues were HLA-DR-negative. No obvious HLA-DR-positive staining was found in all the 5 cell lines. The expression of HLA-DR was up-regulated in all the 5 cell lines after IFN-γ or IFN-α treatment. The up-regulation of HI A-DR in QZG cells was less obvious than that in HCC cell lines. The effect of IFN-γ was more significant than that of IFN-α. Conclusion: HCC tissues can express HLA-DR to some extent, but HCC cell lines do not express detectable HLA-DR. IFN can up-regulate HLA-DR expression in HCC cells.     

Five novel monoclonal antibodies to thymic epithelial cell surface antigens in rats

Objective To establish monoclonal antibodies (mAbs) against thymic epithelial cells and study the function of epithelial cells during T-cell differentiation in the thymus. Methods Hybridomas secreting mAbs against thymic epithelial cells were derived by immunization of Balb/c mice with two thymic epithelial cell lines, TaD3 and FTE. The distribution of antigens recognized by these mAbs was detected by immunochemical staining and cytofluorographic analysis, and the molecular weight of the antigens by immunoblotting. Results Five specific monoclonal antibodies (mAb) were obtained. On the basis of their distribution in the thymus determined by immunochemical staining, mAb RE-4D8 was regarded as clusters of thymic epithelium staining (CTES) type ⅡA; mAb RE-12B2, which showed a unique distribution pattern only in the medulla, was CTES type Ⅴ; mAb RE-5C6 was CTES type Ⅳ; mAb RE-6D6 might be CTES type ⅡB; and mAb RE-1D4 was classified as type Ⅴ. The molecular weight (MW) of antigen RE-4D8, RE-6D6 and RE-12B2 were 120 kDa, 220 kDa and 35 kDa, respectively. Antigen RE-1D4 is a novel marker of cortical epithelium, several established thymic epithelial cell lines were classified and their original intrathymic locations were determined by these mAbs. Thymic cell lines, TuD3 and FTE were cortical phenotypes whereas TaD3 had a medullar phenotype.Conclusions These mAbs clearly demonstrate the heterogeneity of the thymic epithelium; they could detect antigens not only in the cytoplasm but also on the surface of thymic epithelial cells. Our data suggest that these newly established mAbs may help elucidate the interaction between thymocytes and epithelial cells during T cell maturation.     

Activation of CD40 by soluble recombinant human CD40 ligand inhibits human glioma cells proliferation via nuclear factor-κB signaling pathway

As CD40 transduces activation signals involved in inflammatory and immune disorders,we explored the expression and response to CD40 engagement in human glioma cell lines in this study.The CD40 expression in BT-325 and U251 cells was flow cytometrically detected.The cells were incubated with srhCD40L for 72 h to assess its effects on cell growth in vitro.TNF-α expression was quantified by real-time PCR,and protein expression was analyzed by ELISA.The I-κb mRNA was detected by RT-PCR.I-κB expression decreased after stimulation with 1 μg/mL srhCD40L,but it was upregulated after the cells were pretreated with CD40 antibody.srhCD40L significantly inhibited the proliferation of the CD40+ human glioma cells.The stimulation of CD40+ glioma cells with soluble CD40L (CD154) up-regulated the expression of TNF-α at both mRNA and protein levels.We are led to conclude that CD40L/CD40 could inhibit human glioma cells through I-κb signaling pathway.Interferon-γ can augment CD40 expression and the inhibitory effect of CD40 ligand on cell growth in vitro.These results suggest that srhCD40L may benefit the therapy strategy of glioma.     

miRNA-155 Modulates the Malignant Biological Characteristics of NK/T-Cell Lymphoma Cells by Targeting FOXO3a Gene

This study investigated the effects of miRNA-155 on malignant biological characteristics of NK/T-cell lymphoma cell lines and the possible mechanism. The expression of miRNA-155 was detected in lymphoma cell lines from different sources （SNK-6, YTS, Jurkat and DOHH2） by real-time PCR. Lentiviral vectors （pLL3.7） that could overexpress or downexpress miRNA-155 were constructed. Recombinant lentiviral particles were prepared and purified, and their titers determined. The expression of miRNA-155 in the infected SNK-6 cells and the cell proliferation were detected by PCR and CCK-8, respectively. Flow cytometry was used to determine the apoptosis of infected SNK-6 cells. The target of miRNA155 was predicted from Targetscan website. The effect of miRNA155 on FOXO3a expression was examined by Western blotting. The results showed that among the human NK/T-cell lymphoma cell lines SNK-6, YTS, Jurkat and DOHH2, the expression of miRNA-155 was highest in SNK-6. The infection efficiency of the recombinant lentivirns in SNK-6 was more than 70% at multiplicity of infection （MOI） of 100. The expression of miRNA-155 was significantly increased in SNK-6 cells infected by lentivirus vectors with high expression of miRNA-155 （4 times higher than the control group）, and profoundly decreased in those infected with lentivirnses with low expression of miRNA-155. The proliferation of letivirns-infected SNK-6 cells was decreased as the expression of miRNA-155 reduced. The apoptosis rate was increased with the reduction in the expression of miRNA-155. FOXO3a was found to be a possible target of miRNA155, as suggested by Targetscan website. Western blotting showed that the expression of FOXO3a was significantly elevated in SNK-6 cells with miRNA-155 inhibition. It was concluded that reduction in miRNA-155 expression can inhibit the proliferation of SNK-6 lymphoma cells and promote their apoptosis, which may be associated with regulation of FOXO3a gene.     

Inhibitory Effects of Mild Hyperthermia plus Docetaxel Therapy on ER（＋/-） Breast Cancer Cells and Action Mechanisms

Summary： The purpose of this study was to verify that a combination of mild hyperthermia and do- cetaxel chemotherapy produces synergistic antitumor effects and to explore the action mechanisms of this treatment approach. The effects of docetaxel on the proliferation of cells from the estrogen receptor （ER）-positive human breast cancer cell line MCF-7 and the ER-negative human breast cancer cell line MDA-MB-453 were examined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, and effective experimental concentrations of docetaxel were determined. The effects of mild hy- perthermia plus docetaxel therapy on apoptosis rate in the MCF-7 and MDA-MB-453 human breast cancer cell lines were analyzed by using flow cytometry with Annexin-V fluorescein isothiocyanate （FITC）/propidium iodide （PI） staining. The effects of these combined treatments on cell cycle progres- sion in the MCF-7 and MDA-MB-453 human breast cancer cell lines were examined by using flow cy- tometry. The effects of these combined treatments on the expression of apoptosis-related proteins and proteins in the mitogen-activated protein kinase （MAPK） pathways were analyzed by using Western blotting. The effects of these combined treatments on the expression of the heat shock protein 70 （HSP70） and the multi-drug resistance （MDR） gene product P-glycoprotein （Pgp） were examined by using Western blotting. The results showed that the half-maximal inhibitory concentration （IC50） of do- cetaxel for MCF-7 and MDA-MB-453 cells was 19.57±1.12 and 21.64±2.31 gmol/L respectively. Mild hyperthermia with docetaxel therapy could increase apoptosis rate in the MCF-7 and MDA-MB-453 cells. Apoptosis rate in MCF-7 and MDA-MB-453 cells was increased from （23.66±3.59）% and （18.51±3.17）% in docetaxel treatment group to （47.12±6.73）% and （55.16±7.42）% in mild hyperthermia plus docetaxel group, indicating that the mild hyperthermia and docetaxel therapeutic approaches exhib- ited significant synergistic antitumor effects. Treatments of mild hyperthermia plus docetaxel induced G2/M cell cycle arrest in the MCF-7 and MDA-MB-453 cells. Western blotting demonstrated that pro- teins in the MAPK pathway were expressed at higher levels in docetaxel-treated cells following mild hypothermia than those in cells treated with docetaxel alone. As compared with blank control group, cells from the mild hyperthermia plus docetaxel group exhibited significantly decreased B-cell lym- phoma 2 （Bcl-2） protein expression but slightly increased Bcl-2-associated X protein （Bax） expression. Western blotting results revealed that HSP70 and Pgp expression levels were significantly increased following mild hypothermia. It was concluded that treatments of mild hyperthermia plus docetaxel in- hibited the proliferation of human breast cancer cells, promoted apoptosis of breast cancer cells, and produced synergistic antitumor effects.     

Enhanced Chemotherapy Sensitivity of Human Colon Cancer Cells to 5-Fluorouracil by siRNA Recombinant Expression Vector Targeting Survivin Gene

Objective To investigate the effects of small interfering RNA （siRNA） recombinant expression vector targeting survivin gene on chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil. Methods siRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cell lines LOVO. After 48 hours of transfection, cells were harvested for analysis of survivin mRNA and protein expressions using RT-PCR and Western blot. In addition, after human colon cancer cell lines were treated with Survivin siRNA and/or 5-fluorouracil, MTT assay and flow cytometry were used to analyze cell proliferation and apoptosis. Results Restriction endonuclease analysis confirmed that siRNA recombinant expression vector targeting survivin gene was successfully constructed. Inhibitory ratios of survivin mRNA and protein expressions by Survivin siRNA were 36.33% and 44.65%, respectively. Survivin siRNA combined with 5-fluorouracil significantly increased the cell proliferation inhibitory ratio and apoptosis ratio compared with 5-fluorouracil treatin~ alone （P〈0.05）. Conclusion The siRNA recombinant expression vector targeting survivin gene can inhibit the expression of survivin gene, and enhance chemotherapy sensitivity of human colon cancer cells to 5- fluorouracil.     

Expression of EPO Receptor in Pancreatic Cells and Its Effect on Cell Apoptosis

In order to explore the expression of erythropoietin receptor （EPOR） in pancreatic cell line NIT-1 and its effect on cell apoptosis after binding with erythropoietin （EPO）, NIT-1 cells were cultured and expanded. The expression of EPOR was detected using electrophoresis. NIT-1 apoptosis was induced by cytokines and their effects on cell apoptosis and cell insulin secretion were assayed after binding of EPO to EPOR. The results showed that EPOR was expressed in NIT-1 cells. Recombinant human EPO （rHuEPO） had no effect on cell apoptosis but significantly inhibited apoptosis induced by cytokines, rHuEPO had no effect on cell insulin secretion but significantly improved insulin secretion inhibited by cytokines. From these findings, it was concluded that EPOR was expressed in NIT- 1 cells and EPO could protect NIT- 1 cells from apoptosis induced by cytokines.     

死亡受体5在肿瘤细胞系表面的表达

目的利用抗DR5单克隆抗体(mAb),检测死亡受体5(DeathRecepter5)在肿瘤细胞系表面的表达。方法sDR5免疫BALB/c小鼠,小鼠脾细胞与SP2/0-Ag14细胞在50%PEG条件下融合,获得能够分泌抗DR5单克隆抗体的杂交瘤细胞株,利用杂交瘤细胞株分泌的抗DR5特异性单克隆抗体,采用流式细胞仪技术测定抗DR5mAb结合至细胞表面的量来分析死亡受体在肿瘤细胞系的表达情况。结果不同肿瘤细胞表面DR5的表达水平分别为Jurkat细胞91.61%、NS-10.66%。结论抗DR5mAb能够特异性与DR5结合。DR5在Jurkat细胞系高水平表达,NS-1细胞系几乎不表达。该特异性mAb在研究TRAIL受体的生物功能上是一种有用的工具,对研究DR5在组织和细胞系表达十分有价值。     

白血病细胞系中WT1基因启动子区域DNA甲基化的研究

目的应用基于聚合酶链反应(PCR)的实验方法,研究白血病细胞系中WT1基因启动子区域的DNA甲基化水平及其与WT1基因表达的关系.方法采用RT-PCR技术及甲基化特异性PCR(methylation-specific PCR,MSP)技术,检测HL-60、Jurkat及KG-1等白血病细胞系中WT1基因mRNA表达水平及其启动子区域的DNA甲基化状态;以5-杂氮脱氧胞嘧啶(5-aza-CdR)对启动子区域DNA高甲基化的U937细胞系进行去甲基化处理后,观察WT1基因表达水平的改变.结果 HL-60、K562、KG-1、NB4及SHI-1细胞系中WT1水平高表达,而Jurkat和U937细胞表达水平极低,同时检测到这两个细胞系存在WT1基因启动子区域DNA高甲基化;经去甲基化处理后,U937细胞系的WT1基因表达水平较未处理者升高.结论 WT1基因启动子区域DNA高甲基化是抑制其表达的机制之一.     

B7H3分子在人髓性白血病细胞株U937和K562上的表达及生物学意义

目的:研究共刺激分子B7H3分子在人髓性白血病细胞株U937和K562上的表达及其生物学意义?方法:应用流式细胞术及RT-PCR法检测B7H3分子在人髓性白血病细胞株U937和K562上的表达;并用鼠抗人B7H3单克隆抗体(mAb)作用于U937和K562细胞株,细胞计数法检测鼠抗人mAb B7H3对U937和K562细胞株生长的影响?结果:流式细胞术分析显示U937和K562细胞株均表达B7H3膜蛋白,RT-PCR检测到U937和K562细胞有B7H3 mRNA产物;鼠抗人mAb B7H3对U937和K562细胞株有抑制生长的作用?结论:U937和K562细胞株组成性表达B7H3分子;鼠抗人mAb B7H3与U937和K562细胞表面的B7H3分子交联后可以抑制白血病细胞的生长?     

胡桃醌对白血病细胞增殖的抑制作用

目的: 探讨胡桃醌对白血病细胞增殖的抑制作用,阐明胡桃醌抗白血病的机制。方法: 体外培养人白血病K562、Jurkat、U937、U266及NB4细胞株,按不同处理因素分组：调零组,含有培养基、MTT和三联溶解液;空白对照组,含有各种人白血病细胞、药物溶解介质、培养液、MTT 和三联溶解液;实验组,分别采用不同浓度（终浓度为0.25、0.50、1.00、2.00、4.00、6.00、8.00和16.00 mg/L）的胡桃醌作用各种细胞株。MTT比色法检测胡桃醌对白血病细胞的增殖抑制作用。流式细胞术测定不同浓度胡桃醌对K562细胞株凋亡的影响。结果: 胡桃醌对白血病K562、Jurkat、U937、U266及NB4细胞株增殖均有较强的抑制作用,其半数抑制浓度(IC50)(48 h)分别为3.87、1.13、4.48、1.87和4.67 mg/L。其中胡桃醌对Jurkat和U266细胞的抑制效果最为明显（P<0.05）,胡桃醌对其他3个白血病细胞株的IC50值均<40 mg/L,且胡桃醌对这5种细胞株增殖的抑制作用呈时间和剂量依赖性。流式细胞术检测显示,胡桃醌可诱导K562细胞凋亡,1、2、4、8和16 mg/L胡桃醌作用6 h,K562细胞凋亡率分别为(4.54±0.6) %、(11.5 4±0.6) %、(28.7±0.3) % 、(45.0±1.2) %和(76.1±1.4) %。结论: 胡桃醌可抑制多种白血病细胞株增殖,其机制可能与促进白血病细胞凋亡相关。     

抗肿瘤坏死因子相关凋亡诱导配体受体死亡受体5嵌合抗体的构建及其真核表达

目的构建以人肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体死亡受体5(DR5)为抗原的鼠/人重组嵌合抗体。方法采用基因工程技术,扩增和克隆抗DR5嵌合抗体的轻、重链表达载体,共转染二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞(CHO-dhfr-),筛选稳定分泌表达抗DR5嵌合抗体的细胞株。采用ELISA和Western bloting法鉴定抗体的人源性和抗体-抗原结合活性,四唑盐/吩嗪硫酸甲酯比色法检测抗体的生物学活性。结果获得了稳定表达和分泌抗人DR5的嵌合抗体(anti-DR5mV-hH)的重组细胞;与人DR5蛋白具有高的特异性结合活性;能使体外培养的人髓性白血病Jurkat细胞和人结肠癌HCT116细胞的存活率分别下降到73.15%和77.30%。结论抗DR5嵌合抗体anti-DR5mV-hH具有抗肿瘤活性,为发展人源化抗体药物的研究奠定了基础。     

重组人可溶性DR5蛋白的分离纯化及其生物活性检测

目的:提纯毕赤酵母GS115菌株表达的重组人可溶性死亡受体5(sDR5)蛋白,并研究其对肿瘤坏死因子相关凋亡诱导配体(TRAIL)所诱导细胞的阻断效应。方法:大量扩增可溶性DR5酵母表达载体转化阳性的毕赤酵母GS115菌株,收集上清,利用Ni-NTA Agarose亲和层析柱纯化获得重组可溶性DR5,用SDS-PAGE检测纯化产物的纯度。用流式细胞仪检测纯化的可溶性DR5对TRAIL诱导Jurkat细胞凋亡作用的阻断效应。结果:转化阳性的毕赤酵母GS115菌株可以成功表达分子量为23kD的可溶性DR5蛋白,经过Ni-NTAAgarose亲和层析柱纯化,SDS-PAGE鉴定显示获得了高纯度的重组人可溶性DR5蛋白,培养上清产量达28.69mg/L;体外活性检测结果显示,纯化的可溶性DR5蛋白几乎完全可以阻断TRAIL诱导的Jurkat细胞凋亡作用。结论:经纯化后的重组人可溶性DR5蛋白可以有效阻断TRAIL诱导的细胞凋亡作用,显示DR5在TRAIL诱导Jurkat细胞凋亡作用中起着十分关键的作用。     

柔红霉素、地塞米松、全反式维甲酸对白血病细胞系CYP3A5基因转录及活性的调控

目的 探讨化疗药对白血病细胞系CYP3A5基因转录及活性的调控。方法 采用RT-PCR、高压液相色谱法(HPLC)检测药物干预后白血病细胞系CYP3A5基因的转录及活性。结果 K562／A02细胞经柔红霉素作用后CYP3A5转录增强，HL-60／ADR细胞则经诱导后出现CYP3A5转录。Jurkat细胞经地塞米松作用24h后出现微弱的CYP3A5转录，48h后转录明显增强。NB4细胞经全反式维甲酸作用24～96h均诱导出现CYP3A5转录。K562细胞的CYP3A5活性显著高于HL-60、NB4和Jurkat细胞。柔红霉素作用24h后K562细胞的CYP3A5活性增高，48h后活性显著增高；而NB4和Jurkat细胞在柔红霉素作用24h后CYP3A5活性无改变。地塞米松作用Jurkat细胞、全反式维甲酸作用NB4细胞均能诱导CYP3A5活性显著增高，并呈时间依赖性。结论 柔红霉素、地塞米松、全反式维甲酸的应用可诱导某些白血病细胞株CYP3A5基因的转录及活性。     

CLONING PROMOTER OF HUMAN SATBl GENE AND EFFECT OF ATRA AND CoCl_2 ON ITS ACTIVITY

Objective To explore the structure and activity of SATBI promoter in different cells, ATRA and CoCl2 effect on its activity. Methods Using luciferase system to assay the promoter activity of human SATB1 gene, three luciferase reporter vectors were constructed which driven by different regions of 5' untranslated sequence from human SATB1 gene, called pGL3-SP2946-luc, pGL3-SP1718-luc and pGL3-SP751-luc, and transfeted into Jurkat T,K562, U937 and Hela cells transiently using lipofectinamine, the expression activity was detected at different dosage of ATRA and CoCl2 treatment for different time course. Results The reporter gene expression from SATB1 promoter were high activity in U937 cell, moderate in Jurkat T cell, low activity in K562 cell and showed no obvious activity in Hela cell, the reporter gene expression from pGL3-SP751-luc kept on the higher lever in Jurkat T,K562 and U937 cells than the other two vectors. We also found that the repressive effect of CoCl2 on SATBI' s mRNA expression and the relative luciferase expression from pGL3-SP751~luc in U937 cell was down-regulated obviously by ATRA and CoCl2 in the concentration- and time-dependent manners. Conclusion SATB1 promoter drives gene expression with cell-specificity and its core promoter region maybe exist in the - 751 ~ - 9bp of 5' untranslated region of human SATBI gene. Combined with the experiment result we found before that SATB1 was down-regulated by ATRA in U937, the results imply that STAB1 maybe is down-regulated by ATRA and CoCl2 through its promoter in the differentiation of myeloid cell line-U937.     

阿霉素对HL-60、K562细胞表面DR5表达的影响

目的:观察白血病细胞HL-60、K562细胞表面DR5表达水平及阿霉素对其DR5表达的影响。方法:利用抗DR5单克隆抗体,流式细胞仪测定HL-60、K562细胞表面DR5表达量及1×10-4g/L的阿霉素作用细胞10h,HL-60、K562细胞表面DR5表达水平。结果:HL-60、K562细胞表面DR5表达量分别为58.66%、14.92%;1×10-4g/L阿霉素作用10h,HL-60、K562细胞表面DR5表达分别增至86.89%、15.98%。结论:阿霉素提高HL-60、K562细胞表面DR5表达量。     

化疗药物增强重组可溶性TRAIL抗肿瘤作用

目的探讨化疗药物对重组可溶性TRAIL(rsTRAIL)抗肿瘤作用的影响及其分子机制.方法 13株不同肿瘤细胞经rsTRAIL处理后,采用MTS-PMS染色法和流式细胞仪技术测定细胞凋亡率,观察肿瘤细胞对rsTRAIL的敏感性;化疗药物与rsTRAIL联合作用于敏感性不同的肿瘤细胞检测细胞敏感性的变化;免疫印迹技术分析TRAIL受体DR5蛋白表达.结果 13株肿瘤细胞中约46.2%(6/13)的细胞株对rsTRAIL敏感;化疗药物CHX、CP和8-CA可显著提高肿瘤细胞株对rsTRAIL细胞毒作用的敏感性;8-CA和TRAIL联合作用可上调DR5的蛋白表达.结论化疗药物CHX、CP和8-CA可增强rsTRAIL的抗肿瘤作用.