Hepatoma cells up-regulate expression of programmed cell death-1 on T cells

AIM： To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 （PD-1）, and the function of PD-1 on T cells. METHODS： HepG2 or HepG2.2.1.5 cells were cocultured with a lymphoma cell line-Jurkat cells. PD-1 expression was detected by flow cytometry. IL：2, INF-γ and IL-10 in culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. Cytotoxic action of T cells was determined by MIF reduction assay-direct mononuclear cell cytotoxicity assay. RESULTS： The PD-1 expression on Jurkat cells increased by 16.17% ± 2.5% and 17.43% ± 2.2% after HepG2 or HepG2.2.1.5 cells were co-cultured for 48 h. The levels of IL-2, INF-γ and IL-10 in the culture supernatant were 202.9 ＋ 53.0 pg/mL, 88.6 ± 4.6 pg/mL and 63.7± 13.4 pg/mL respectively, which were significantly higher than those （102.9 ± 53 pg/mL, 39.3 ± 4.2 pg/mL, and 34.6 =E13.7 pg/mL） in the control group （P 〈 0.05）. The OD value for MTT assay in the blocking group （0.29 ± 0.06） was significantly higher than that （0.19 ± 0.09） in the control group （P 〈 0.05）. CONCLUSION： PD-1 expression on Jurkat cells is upregulated by hepatoma cells, cytokines and cytotoxic action are elevated after PD-1/PD-L1 is blocked.     

Melanoma differentiation-associated gene-7, MDA-7/IL-24, selectively induces growth suppression, apoptosis in human hepatocellular carcinoma cell line HepG2 by replication-incompetent adenovirus vector

AIM: To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatoceliular carcinoma (HCC) cell line HepG2 and normal liver cell line L02. METHODS: We constructed the recombinant replication-incompetent Ad.mda-7 virus vector and infected it into the human HCC cell line HepG2 and normal liver cell line L02. RT-PCR was performed to detect the mRNA expressing in cells. by ELISA was used to detect MDA-7/ IL-24 protein expression in the culture supernatant. The effect of apoptosis induced by Ad.mda-7 was confirmed by Hoechst staining and flow cytometry assay with An-nexin-V and PI staining. MTT assay was used to determine growth inhibition of HepG2 cells, and cell-cycle and hypodiploidy analyses were performed by flow cytometry. RESULTS: Recombinant replication-defective virus expressing MDA-7/IL-24 was constructed successfully. RT-PCR showed that the Ad.mda-7 could mediate the expression of the exogenous gene MDA-7/IL-24 into HepG2 and L02. The concentration of MDA-7/IL-24 protein in supernatant was 130 pg/mL and 110 pg/mL in Ad.mda-7-infected L02 and HepG2 cells, respectively. Ad.mda-7 infection obviously induced apoptosis (from 2.60±0.72% to 33.6±13.2%, P= 0.00012) and growth suppression in HepG2 (inhibition ratio IR = 68%) and an increase in the percentage of specific cancer cell types at the G2/M phase of the cell cycle (from 6.44% to 32.29%, P<0.01), but not in L02 cells. CONCLUSION: These results confirm selectively induction of apoptosis and growth suppression by the mda-7/ IL-24 gene with replication-incompetent adenovirus vector in human hepatocellular carcinoma cell line HepG2.     

Apoptosis and its pathway in X gene-transfected HepG2 cells

AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells. METHODS: The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in-three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group. RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910±0.003164) than in HepG2(0.14410±0.004927) and HepG2/pcDNA3 cells (0.12150±0.007159) (P<0.05 and P<0.01). Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells (980/2 000) than HepG2 (420/2 000), HepG2/pcDNA3 cells (520/2 000) (P<0.05 and P<0.01). Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/ pcDNA3 cells (P<0.05 and P<0.01). Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells (P<0.05 and P<0.01). Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells (P<0.05 and P<0.01). CONCLUSION: HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.     

乙型肝炎病毒及其抗原成分对干扰素信号传导途径分子和抗病毒蛋白表达的影响

目的 了解HBV及其抗原成分对干扰素(IFN)αJanus激酶-信号传导和转录激活子(JAK-STAT)信号传导途径分子和抗病毒蛋白表达可能存在的影响.方法以人肝胚瘤细胞株HepG2细胞为研究对象,分别经质粒转染(以能够表达完整HBV病毒颗粒或HBsAg、HBcAg的质粒pSM2、pHBS2-S和pHBc-EGFP)、病毒感染(以HepG2.2.15细胞培养上清液感染HepG2细胞,其含有完整HBV病毒颗粒和HBV抗原)以及与HBV抗原直接接触刺激等方式处理不同组HepG2细胞,以Northern blot和RT-PCR等方法分析各处理组HepG2细胞的IFN α应答情况,如检测抗病毒蛋白[如粘病毒抵抗蛋白A(MxA)、2'-5'寡腺苷酸合成酶(2'-5'OAS)、9-27等]和JAKSTAT信号传导途径分子(如STAT1)的表达.对数据进行t检验.结果 转染pSM2、pHBS2-S和pHBc-EGFP质粒后,HepG2细胞能够表达完整的HBV颗粒或HBV抗原,且随着转染时间的延长,HBV颗粒或抗原表达量逐步增多,转染48、96h的细胞培养上清液中HBsAg的S/CO值为0.81±0.11和2.35±0.33(t=10.84,P<0.05),HBeAg的S/CO值为0.69±0.06和1.79±0.13(t=18.82,P<0.05).Northern blot分析提示HepG2细胞能够表达IFN α抗病毒蛋白MxA、2',5'OAS、9-27等,但质粒转染、病毒感染和HBV抗原直接接触刺激的HepG2细胞,IFN α抗病毒蛋白MxA、2',5'OAS、9-27等的表达量明显减低,且随着转染时间的延长而进一步减低;此外,STAT1的表达也随着HBV颗粒或HBV抗原的表达而受到抑制.结论在体外细胞模型中,HBV及其抗原成分影响IFN αJAK-STAT信号传导途径分子和抗病毒蛋白的表达;HBV具有拮抗或反作用于IFN α抗病毒活性的机制.

Abstract：

Objective To investigate the possible influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and the antiviral proteins of IFN α.Methods The HepG2 cells were transfected with pSM2.pHBS2-S and pHBc-EGFP plasmids which express HBV whole particles or S-antigen,Pre-S antigen and core antigens.The infectious supernatant from HepG2.2.15cells and the pured HBV proteins which contained tIle S.Pre-S antigens were used to treat the HepG2 cells.Northern blot and RT-PCR were applied to analyse the expresssions of the antiviral proteins MxA,2'-5'OAS.9-27 and the JAK-STAT signal transduction pathway molecules STAT1 in HepG2 cells responded to the IFN αtreatment.Results The HepG2 cells transfected with pSM2,pHBS2-S and pHBc-EGFP plasmids could express whole HBV particles and HBsAg,Pre-S antigen and HBcAg. The quantitation of expressed HBV particles and antigens increased significantly during the course of transfection. Northern blot hybridization analysis indicated that the HepG2 cells expressed IFN α antiviral proteins MxA,2' -5' OAS and 9-27.When transfected with pHBV-dimer,pHBS2-S,pHBc-EGFP plasmids,the IFN:A antiviral proteins MxA,2' -5' OAS and 9-27 in transfected cells were reduced greatly as compared to the un-transfected HepG2 cells,and the expressed antiviral proteins decreased sharply with the development of transfection time. Furthermore,the expression of IFN α JAK-STAT signal transduction pathway molecule STAT1 was also inhibited with the expression of HBV particles and HBV antigens in transfected HepG2 cells. Conclusions The HBV and its antigens influence the expressions of IFN α JAK-STAT signal transduction pathway molecules and antiviral proteins in the hepatocellular models in vitro. It is idicated that HBV might possess the activity to antagonise or counteract the IFN α antiviral action.     

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

AIM： To study the expression and phosphorylation of extracellular signal-regulated kinase （ERK） i and ERK2 in multidrug resistant （MDR） hepatocellular carcinoma （HCC） cells.
METHODS： MDR HCC cell lines, HepG2/adriamycin （ADM） and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein （P-gp） and multidrug resistant protein 1 （MRP1） expression levels. ERK1 and ERK2 mRNA expression lev-ls were measured by quantitative real-time PCR （QRTPCR）. Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.
RESULTS： MTT assay showed that HepG2/ADM andSMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells （8.92% ±0.22% vs 0.88% ± 0.05%, P 〈 0.001） and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells （7.37% ± 0.26% vs 1.74% ± 0.25%, P 〈 0.001）. However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.
CONCLUSION： ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells,     

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

AIM:To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action.METHODS:HepG2 cells were treated with different concentrations of cinobufacini.Cell viability was measured by methylthiazolyl tetrazolium(MTT) assay.Cell cycledistribution was analyzed by flow cytometry(FCM).Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope.Changes in morphology and ultrastructure of cells were detected by atomic force microscopy(AFM) at the nanoscale level.RESULTS:MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dosedependent manner.With the concentration of cinobufacini increasing from 0 to 0.10 mg/m L,the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1%(P 0.05).FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini.The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment,the regular reorganization of actin filaments in HepG2 cells become chaotic,while the nuclei were not damaged seriously.Additionally,high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini.It appeared as significant shrinkage and deep pores in the cell membrane,with larger particles and a rougher cell surface.CONCLUSION:Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity.     

Antiproliferation and apoptosis induction of paeonol in HepG2 cells

AIM To investigate the antiproliferative effect of paeonol (Pae) used alone or in combination with chemotherapeutic agents [cisplatin (CDDP), doxorubicin (DOX) and 5-fluorouracil (5-FU)] on human hepatoma cell line HepG2 and the possible mechanisms.METHODS The cytotoxic effect of drugs on HepG2 cells was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) assay.Morphologic changes were observed by acridine orange (AO) fluorescence staining. Cell cycle and apoptosis rate were detected by flow cytometry (FCM). Drug-drug interactions were analyzed by the coefficient of drug interaction (CDI).RESULTS Pae (7.81-250 mg/L) had an inhibitory effect on the proliferation of HepG2 cells in a dose-dependent manner, with the IC50 value of (104.77 7.28) mg/L. AO fluorescence staining and FCM assays showed that Pae induced apoptosis and arrested cell cycle at S phase in HepG2 cells. Further, different extent synergisms were observed when Pae (15.63, 31.25, 62.5 mg/L) was combined with CDDP (0.31-2.5 mg/L), DOX (0.16-1.25 mg/L), or 5-FU (12.5-100 mg/L) at appropriate concentrations. The IC50 value of the three drugs decreased dramatically when combined with Pae (P ＜0.01). Of the three different combinations, the sensitivity of cells to drugs was considerably different.CONCLUSION Pae had a significant growth-inhibitory effect on the human hepatoma cell line HepG2,which may be related to apoptosis induction and cell cycle arrest. It also can enhance the cytotoxicity of chemotherapeutic agents on HepG2 cells, and the S phase arrest induced by Pae may be one of the mechanisms of these interactions.     

丙型肝炎病毒核心蛋白对低氧诱导因子-1α和血管内皮生长因子表达的影响

目的 探讨HCV核心蛋白对低氧诱导因子1 α(HIF-1 α)和血管内皮生长因子(VEGF)表达的影响. 方法 将HCV核心蛋白基因的真核表达载体flag2B-core和HIF-1 αsiRNA转染Huh7.5.1细胞;采用RT-PCR和Western blot法分别检测HIF-1 α、VEGF在mRNA和蛋白水平的表达变化,采用酶联免疫吸附试验检测细胞上清液中VEGF的含量.对各组间数据进行t检验.结果 Huh7.5.1细胞转染flag2B-core后,HIF-1 α和VEGF的mRNA和蛋白表达水平升高,细胞上清液中VEGF含量明显高于对照组[(654.5±43.7) pg/ml与(365.9±26.8)pg/ml,t=653.1％,P＜ 0.01)];Huh7.5.1细胞共转染flag2B-core和HIF-1 α siRNA后,HIF-1 α和VEGF的mRNA和蛋白表达水平降低,细胞上清液VEGF含量明显低于对照组[(389.2±29.6) pg/ml与(768.8±47.3) pg/ml,t=1330.22,P＜0.01).结论 HCV核心蛋白能够上调HIF-1 α和VEGF的表达;HCV可能通过核心蛋白来调节HIF-1 α和VEGF的表达.     

常氧下高低浓度葡萄糖培养对不同密度肝癌HepG2细胞HIF-1α、HIF-2α表达的影响

目的观察常氧下高低浓度葡萄糖对不同浓度肝癌HepG2细胞缺氧诱导因子1α（HIF-1α）、HIF-2α表达的影响。方法将HepG2细胞分成高中低三个细胞密度组,常氧下分别在高糖和低糖DMEM中培养16h,采用免疫细胞化学法检测各组HepG2细胞中的HIF-1α和HIF-2α。结果常氧下高糖培养,高、中、低密度组HepG2细胞中HIF-1α的光密度值分别为0．270±0．014、0．318±0．015、0．052±0．012,常氧下低糖培养分别为0．300±0．012、0．242±0．015、0．205±0．018。三组间比较,P均〈0．05。常氧下高糖培养,高、中、低密度组HepG2细胞中HIF-2α的光密度值分别为0．076±0．013、0．175±0．011、0．310±0．014,常氧下低糖培养分别为0．120±0．017、0．1874-0．014、0．347±0．015。三组间比较,P均〈0．05。结论常氧下低糖培养能诱导HepG2细胞中的HIF-1α、HIF-2α高表达;HIF-1α在适中的细胞密度下表达最强,HIF-2α的表达随着细胞密度的增加而降低。     

Antiangiogenic effects of ganetespib in colorectal cancer mediated through inhibition of HIF-1α and STAT-3

Hypoxia-inducible factors (HIFs) and STAT-3 play essential roles in angiogenesis. HIF-1α and STAT-3 are clients of the heat shock protein 90 (HSP90). We hypothesized that ganetespib, a potent HSP90 inhibitor, would disrupt angiogenesis in colorectal cancer (CRC) through inhibition of HIF-1α and STAT-3. CRC cell lines (HCT116 and HT29) were used in all the experiments. Egg CAM and HUVEC assays revealed decreased angiogenesis in ganetespib treated cell lines. Ganetespib inhibited matrigel plug vascularization and tumor growth of xenografts. Significant inhibition of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, HIF-1α and STAT-3 expression was observed in both cell lines treated ganetespib. HIF-1α overexpression resulted in the increase VEGF and STAT-3 expression and this was inhibited by ganetespib. HIF-1α knockdown inhibited VEGF and STAT-3 expression. STAT-3 knockdown inhibited VEGF but not HIF-1α expression. HSP90, STAT-3 and VEGF expression was significantly higher in CRC compared to adjacent normal tissue. Significant downregulation of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, STAT-3 and HIF-1α mRNA was observed in the post ganetespib treatment tumor samples from patients with rectal cancer. These results collectively suggest that inhibition of HSP90 is a promising antiangiogenic strategy in CRC. HSP90 angiogenic effects are mediated through HIF-1α and STAT-3.     

乙型肝炎病毒X蛋白对HepG2细胞羧末端结合蛋白反应蛋白表达的影响

目的 研究乙型肝炎病毒X蛋白(HBx)对博莱霉素诱导HepG2细胞DNA双链断裂(DSB)损伤修复关键因子羧末端结合蛋白反应蛋白(CtIp)的影响.方法 建立稳定表达HBx的HepG2肝癌细胞株(HepG2-HBx)及空质粒对照细胞株(HepG2-vec).用博莱霉素诱导细胞DSB损伤后,用流式细胞仪检测细胞周期和细胞凋亡情况,用实时定量PCR及Western blot检测CtIP的mRNA与蛋白表达水平,并用激光共聚焦显微镜观察CtIp蛋白在细胞内的定位情况.多组数据采用单因素方差分析和SNK-q检验;两组数据间比较用t检验.结果 经检测转染HBx基因的HepG2细胞(HepG2-HBx)能稳定表达HBx.博莱霉素处理后,HepG2-HBx细胞与HepG2-vec细胞的凋亡比例分别为16.90%±0.89%和15.30%±0.86%,差异无统计学意义(q=2.074,P＞0.05),但死亡细胞比例分别为8.71%±0.74%和4.90%±0.46%,差异有统计学意义(q=7.126,P＜0.01) ;同时两种细胞株都出现了细胞周期G2/M期阻滞,差异有统计学意义(F=11.401,P＜0.05).HBx使CtIP蛋白表达水平和mRNA表达水平均下调,HepG2-HBx细胞与HepG2-vec细胞CtIp蛋白的相对表达量分别为0.66±0.04、0.73±0.05,差异有统计学意义(t=2.314,P＜0.05); CtIP mRNA相对表达量分别为1.00±0.06、1.23±0.08,差异有统计学意义(t=2.732,P＜0.05).同时观察到CtIP蛋白主要在细胞胞核内表达.结论 HBx能干扰肿瘤抑制蛋白CtIp的表达,可能影响细胞DSB损伤的修复.     

过氧化物酶体增殖物辅助激活因子1 α基因多态性对糖异生基因转录的影响

目的 研究过氧化物酶体增殖物辅助激活因子1α（PGC-1α）基因482号密码子甘氨酸-丝氨酸突变（Gly482Ser）多态性对糖异生关键基因磷酸烯醇式丙酮酸羧基酶（PEPCK）转录的影响.方法 （1）应用寡核苷酸诱导的定点突变和PCR技术构建PGC-1α 1444位点基因多态性表达质粒,并用普通PCR和酶切方法构建连接荧光素酶报告基因的人PEPCK启动子报告质粒PGL3-hPCK-luc.分别将PGC-1α基因482号密码子为甘氨酸的野生型表达质粒pcDNA3.1-PGC-1α（G）或482号密码子为丝氨酸的突变型表达质粒pcDNA3.1-PGC-1α（S）,与转录因子肝细胞核因子4α（HNF4α）表达质粒pcDNA3.0-HNF4α共转染进培养的人肝癌细胞及人正常肝细胞株.（2）转染48 h后检测细胞株内PGC-1α及PEPCK的mRNA水平及蛋白水平.进一步按不同组合联合转染PGL3-hPCK-luc及内参质粒PRL-SV40,培养48 h后用双荧光素酶检测试剂盒检测细胞荧光素酶的相对活性.多组间比较用单因素方差分析,2组组间比较用独立样本t检验.结果 成功构建PGC-1α 1444位点突变质粒及PGL3-hPCK-luc荧光素酶报告质粒.瞬时转染进HepG2细胞中,PGC-1α（G）与PGC-1α（S）质粒转染组PGC-1α mRNA及蛋白表达水平均明显增加,但2组间无明显差异（P＞0.05）;联合转染PGC-1α和HNF4α组的PEPCK的mRNA及蛋白表达水平较单转染PGC-1α明显增高（分别为10.40±0.70、4.50±0.50和0.86±0.18、0.99±0.09,均P＜0.05）;转染PGC-1α（G）＋HNF4α组较PGC-1α（S）＋HNF4α组PEPCK的mRNA及蛋白表达水平分别增高1.83倍和1.4倍（分别为10.40±0.70、5.35±0.23和4.50±0.50、3.00±0.40,均P＜0.05）.转染PGC-1α＋HNF4α组可显著促进PEPCK启动子活性（与单转染PGL3-hPCK-luc组比较）（分别为28.0±5.0和2.4±0.4,F=23.41,P＜0.05）;在HepG2中联合转染HNF4α时,PGC-1α（G）组较PGC-1α（S）可使PEPCK启动子相对活性增高2倍（分别为83±10和41±5,F=23.41,P＜0.001）;在L02细胞中联合转染HNF4α时,PGC-1α（G）组较PGC-1α（S）可使PEPCK启动子相对活性增高2.25倍（分别为28.0±5.0和12.6±1.5,F=60.75,P＜0.001）.结论PGC-1α可辅助激活HNF4α对PEPCK转录的促进作用,而PGC-1α基因Gly482 较Ser482对HNF4α的蛋白辅助激活作用更强,这可能为基因多态性引起蛋白表达后不同构型影响蛋白-蛋白相互作用有关.     

缺氧诱导因子-1α对非小细胞肺癌血管生成及其对预后的影响

目的分析缺氧诱导因子-1α(HIF-1α)对非小细胞肺癌病情、血管生成和预后的影响。方法采用回顾性研究,纳入笔者医院2014年8月-2016年2月期间收治的100例非小细胞肺癌患者。运用免疫组化分析HIF-1α和Ang-2在不同性别、年龄、TNM分析、组织学类型、淋巴结转移、分化程度中的阳性率;对比HIF-1α阳性组和阴性组患者VEGF、VEGFR2、Ang-2阳性率;对比HIF-1α阳性组和HIF-1α阴性组患者治疗1年转移+复发率、死亡率,分析HIF-1α、VEGF、VEGFR2、Ang-2阳性表达与非小细胞肺癌患者不良预后(转移=死亡+复发)的关系,统计HIF-1α阳性组和HIF-1α阴性组患者无病生存时间。结果①Ang-2以及TNMⅢ期高阳性率均出现在TNMⅠ+Ⅱ期、有淋巴结转移的患者、低分化程度的患者中,P<0.05。②HIF-1α阳性组VEGF阳性率为97.45%、VEGFR2阳性率为78.08%,均较HIF-1α阴性组VEGF阳性率(51.85%)、VEGFR2阴性率(48.15%)高,P<0.05。HIF-1α阳性组Ang-2阳性率为72.60%,HIF-1α阴性组Ang-2阳性率为33.33%,P<0.05。③HIF-1α阳性组转移+复发率为17.34%,HIF-1α阴性组转移+复发率为5%;死亡患者中HIF-1α阳性组患者占比为66.78%(4/6),HIF-α阴性组患者占比为33%,P<0.05。④HIF-1α阳性、VEGF阳性、VEGFR2阳性、Ang-2阳性是非小细胞肺癌不良预后的独立因素,P<0.05。⑤HIF-1α阳性表达组平均无病生存时间为11.23±2.54个月,HIF-1α阴性表达组平均无病生存时间为31.23±5.98个月,P<0.05。结论HIF-1α主要在TNMⅢ、淋巴结转移、低分化的非小细胞肺癌患者癌灶组织中成阳性。其阳性表达与VEGF、VEGFR2、Ang有关,即HIF-1α可能参与了肿瘤新生血管,此四个指标均提示不良预后几率增高,HIF-1α阳性患者无病生存率相对HIF-α阴性者短,临床可通过联合HIF-1和肿瘤血管生成因子评估预后。     

HIF-1α对乳腺癌MCF-7细胞增殖的影响及其机制探讨

目的 观察缺氧诱导因子1α（HIF-1α）基因对乳腺癌MCF-7细胞增殖的影响,并探讨其可能的机制.方法 构建并筛选HIF-1 α基因的RNAi表达质粒,以脂质体LipofectamineTM 2000介导转染MCF-7细胞.转染后48h,采用实时定量RT-PCR技术检测转染细胞中HIF-1α mRNA的转录水平,筛选有效的HIF-1 αRNAi质粒;CCK-8法比较转染前后乳腺癌细胞增殖变化,实时定量RT-PCR检测顺滑蛋白（SMO）mRNA表达.结果 成功构建含HIF-1α短发夹状RNA（shRNA）1～4的RNAi表达质粒,其中HIF-1 αshRNA-4干扰抑制效率为74％,干扰效果最强（P均＜0.05）.HIF-1 αshRNA-4干扰48、72、96 h后,MCF-7细胞的生长抑制率明显升高（P均＜0.05）.HIF-1αshRNA-4转染后MCF-7细胞SMO mRNA的相对表达量为0.56 ±0.06,低于转染前的1.07 ±0.16（P ＜0.05）.结论 HIF-1α表达可促进乳腺癌细胞增殖,可能与其参与调控SMO的表达有关.     

心肌转染缺氧诱导因子1α对大鼠心肌梗死的保护作用

目的 目的 评价心肌内注射缺氧诱导因子1α进行基因转染对大鼠心肌梗死的保护作用.方法 选择雄性大鼠72只进行实验,将实验分为转染腺病毒缺氧诱导因子1α组,转染空质粒对照组和假手术组.每组以术后的不同时间处死,取材分3、7、14天三个亚组,每个亚组8只.建立心肌梗死模型.当左前降支被结扎后,在室游离壁的三个不同部位分别注射50μg腺病毒缺氧诱导因子1α质粒和50μg的空质粒.假手术组不结扎左前降支.观察缺氧诱导因子1α的基因表达、心肌毛细血管密度和大鼠心肌梗死面积.结果 缺氧诱导因子1α组与对照组相比,心肌组织中缺氧诱导因子1α基因表达明显增强,梗死区毛细血管密度明显增加(P<0.01),心肌梗死面积分别为23.85%±2.25%和38.74%±3.12%,心肌梗死面积明显减少(P<0.01).结论 心肌内注射缺氧诱导因子1α进行基因转染可以增加缺氧诱导因子1α基因表达,增加心肌毛细血管生成,明显减少心肌梗死面积,对大鼠心肌梗死起保护作用.     

肝癌组织缺氧诱导因子-1α的动态表达特征及其临床价值

目的 探讨缺氧诱导因子-1α(HIF-1α)及转录异常对肝癌的临床价值.方法 以肝癌模型观察肝病理学、HIF-1α转录及表达水平的动态变化;以自身配对法收集手术切除的肝癌和癌周组织,抽提总RNA并扩增分析HIF-1α mRNA表达;以免疫组织化学和Western blot分析HIF-1α表达、胞内分布及临床病理学特征.根据不同资料采用方差分析、确切概率法、Pearson或Spearman等级相关分析进行统计学分析. 结果癌变过程中肝HIF-1α仅和HIF-1αmRNA均呈梯度表达.在对照组、变性组、癌前组和癌变组中,肝HIF-1α阳性率为0、77.8%、88.90/o和100%;HIF-1α/β-肌动蛋白比值为0.16±0.02、0.29±0.04、0.52±0.03和0.84±0.02,均显著高于对照组(P=0.000).人肝癌组织HIF-1α阳性率为80.0%(28/35),癌旁为100%(35/35),且与瘤体大小呈正相关,与分化程度呈负相关,与肿瘤数目及HBsAg阳性无关.结论 HIF-1表达与肝癌发生、发展相关,是肝癌治疗的分子靶目标.     

缺氧诱导因子1α对肝癌细胞HepG2干细胞特性及表阿霉素敏感性的影响

目的探讨缺氧诱导因子(HIF)1α对肝癌细胞HepG2干细胞特性及表阿霉素敏感性的影响。方法以肝癌细胞为研究对象,肝癌细胞HepG2脂质体转染过表达HIF-1α的质粒作为实验组,转染pcDNA3.1空质粒作为对照组,单独HepG2细胞为HepG2组。实时荧光定量PCR检测HIF-1αmRNA表达,Western Blot检测HIF-1α蛋白表达;流式细胞术检测细胞表面CD133表达。不同浓度表阿霉素(0、6.25、12.5、25、50μmol/L)作用3组细胞24 h,MTT法检测细胞活性,流式细胞术检测表阿霉素(50μmol/L)处理后细胞凋亡情况。计量资料多组间比较采用单因素方差分析,进一步两两比较采用t检验。结果相较于HepG2组及对照组,实验组HIF-1αmRNA的表达水平明显升高,差异具有统计学意义(P值均<0.001);Western Blot结果显示实验组HIF-1α蛋白高表达。HepG2组、对照组和实验组细胞的CD133比例分别为0.040%±0.003%、0.030%±0.010%、20.110%±0.600%,实验组的CD133阳性率显著高于HepG2组和对照组(P值均<0.001)。表阿霉素浓度为25、50μmol/L时,HepG2组和对照组的细胞活性明显受到抑制,显著低于实验组(P值均<0.05)。50μmol/L表阿霉素作用48 h后,实验组的细胞凋亡率(67.9%±2.5%)较HepG2组(93.6%±1.5%)和对照组(93.0%±1.2%)明显降低(P值均<0.001)。结论过表达HIF-1α的质粒成功转染至HepG2细胞,HIF-1α可提高肝癌细胞干细胞比例使其对表阿霉素耐药。