Inhibitory effect of parvovirus H—1 on the formation of colonies of human hepatoma cell line in vitro and its tumors in nude mice

The inhibitory effect of parvovirus H-1 on the colonyforming ability.in vitro of QGY-7703,a cultured human hepatoma cell line,and on the formation and growth of its tumors in nude mice was studied.With higher multiplicity of infection(MOI) of H-1 given,survival of the QGY-7703 cells was found to be decreased.H-1 DNA amplification level at 30h postinfection(p.i.) was detected to be 7.4 times higher than that at 2h by dispersed cells assay,while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells(new-born human kidney cell line,NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay.The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2h postinfection was observed to by prevented in 2 proups with given MOI 25 and 50.The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20d latent period.It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.     

P-glycoprotein expression induced by glucose depletion enhanced the chemosensitivity in human hepatocellular carcinoma cell-lines

Chemoresistance in cancer cells is frequently associated with an over-expression of the P-glycoprotein (P-gp). The expression of P-gp can be regulated as the cells encounter a number of chemical, physical or environmental stimuli. In this study, P-gp was found gradually expressed in a human hepatocellular carcinoma (HCC) QGY-7703 cells after 48 h of culturing in glucose-free medium. This phenomenon disappeared after the removal of glucose deprivation culture conditions. Mdr1-cDNA isolated from the cell line cultured in glucose-free conditions (namely QGY-7703G), was transiently transformed into the parent QGY-7703 cells, and multi-drug resistance was eventually induced. Results from XTT cytotoxicity assays indicated that the mdr1 gene was functional and the P-gp could restore the QGY-7703 cell's ability to withstand high concentrations of a number of chemotherapeutic agents. A P-gp inhibitor, verapamil, could completely reverse the cellular drug resistance when applied to the QGY-7703G cells. Our results indicated that an alteration of a specific state in cells caused by an external stimulus in vitro may lead to an expression of stress proteins (e.g. P-gp), which may enhance the cells' survival in adverse conditions. The expressed P-gp induced by glucose deprivation has a functional role in affecting the chemosensitivity in HCC QGY-7703G cells. Inhibition of P-gp activity may enhance the effect of the cancer cells towards cancer chemotherapy.     

Some differentiating effects of selenium on the cultured human hepatoma cells and human pulmonary adenocarcinoma cells in vitro

Two human hepatoma cell lines, QGY-7703 and SMMC-7721, and two human pulmonary adenocarcinoma cell lines, LTEP-a-2 and SPC-A-1, were found to respond to 1 μg/mL Na₂SeO₃, 24 h, in-vitro treatment by decreasing its confluent saturation density. The same treatment was found to cause an increase in the adhesiveness of cells measured as resistance to detachment by trypsin/EDTA. The pathological features of tumors after heterotransplantation of treated and untreated cells were similar, but the size of tumor grown from treated cells was much smaller.     

MKi67-siRNA可抑制QGY-7703细胞的生长

为了探讨MKI67在肝癌细胞发生发展中的作用,采用实时定量 PCR 方法检测人肝细胞癌 QGY 7703 细胞中MKI67 基因表达水平, 以及 MKI67在肝细胞癌组织和癌旁正常组织中的表达情况,设计并合成针对MKI67 的siRNA,利用脂质体转染法将其转入QGY-7703 细胞内,通过MTT和细胞集落形成实验观察MKI67-siRNA 对QGY-7703细胞生长活性和增殖能力的影响.实时定量PCR结果表明,MKI67在肝细胞癌组织中的表达水平明显高于癌旁正常组织（P< 0.01）. MTT和细胞集落形成实验结果显示,转染MKI67-siRNA 的QGY-7703细胞生长活性和集落形成率明显低于对照组（P< 0.01）.由此得出结论：MKI67 在肝癌细胞系QGY-7703细胞中的表达水平较高,且它在肝癌组织中的表达水平明显上调. 同时,MKI67-siRNA 可以有效抑制QGY-7703细胞的生长活性和增殖能力,提示MKI67可能与肝细胞癌的发生、发展相关.     

AFP的亚细胞定位及对肿瘤细胞生长的影响

目的观察甲胎蛋白（AFP）在不同肿瘤细胞中的亚细胞定位及对肿瘤细胞生长的影响。方法运用免疫荧光的方法观察内源性AFP在HeI。a细胞、QGY-7703细胞、MCF-7细胞中的亚细胞定位。将构建的表达AFP的质粒pcDNA3-AFP及AFP腺病毒siRNA干涉载体Adv—AFPsiRNA作用于QGY-7703细胞,MCF-7细胞,运用M1Tr,集落形成实验检测细胞增殖状况。结果免疫荧光显示,内源性的AFP在HeLa细胞、QGY-7703细胞、MCF-7细胞均只在细胞质中表达。pcDNA3-AFP使QGY-7703的细胞活性增加了2l％（P〈0．05）及集落形成能力增加了32％（P〈0．01）,MCF-7实验组比对照组细胞活性降低了30％（P〈0．01）．克隆形成能力降低82％（P〈0．01）。Adv—AFPsiRNA使QGY-7703的细胞活性降低了22％（P〈0．05）,平均克隆形成能力降低52％（P〈0．01）,MCF-7细胞活性提高了24．5％（P〈0．05）,克隆形成能力提高了89％（P〈O．01）。结论内源性的AFP只在细胞质中表达。AFP能促进QGY-7703细胞的增殖及克隆形成能力,而在MCF-7细胞中发挥相反的作用。腺病毒介导的内源性的AFP表达的下调能降低QGY-7703的增殖,却增加了MCF-7的细胞活性及克隆形成能力。     

雷公藤单体T10对Aβ1-42所致PC12细胞凋亡的抑制作用

阿尔茨海默病(Alzheimer's disease,AD)是发病率最高的中枢神经系统退变性疾病.目前AD的病因不清,亦无有效的防治手段,其重要的原因是尚无适宜的AD模型.因此,本实验首先建立了PC12细胞系β淀粉样蛋白(p-amyloid,Aβ)细胞损伤模型,在此基础上,探讨了中药免疫抑制剂雷公藤单体T10对细胞的保护作用及其机制.首先用不同浓度的Aβ(5×10、5×10-3、5×10-2、5×10、5、50 μmol/L)与PC12细胞共孵育48 h,用MTT法检测细胞存活率.选取Aβ致使细胞存活率降低的浓度(0.5、5、50 μmol/L)与PC12细胞共孵育48 h,通过流式细胞仪检测凋亡细胞百分比.用1×10-11mol/L的T10预孵育PC12细胞48 h后,加入50μmol/L Ap共孵育48 h,亦用流式细胞仪检测凋亡细胞百分比,激光共聚焦显微镜检测细胞内钙离子浓度变化.结果显示,Aβ的浓度存50μmol/L时可使细胞存活率降低至55.1%,凋亡细胞比例显著增加,而1×10-11mol/L的T10可明显降低50 μmol/L Aβ诱导的PC12细胞死亡.50 μmol/L Aβ可促进PC12细胞胞外钙离子内流,1×10-11mol/L的T10对Ap诱导的胞外钙离子内流有抑制作用.这些观察结果表明T10对Ap导致的PC12细胞损伤具有明显的保护作用,其机制可能与抑制Aβ诱导的胞内钙离子浓度升高和细胞凋亡有关.     

p53 gene expression of human hepatoma cell lines and their sensitivities to parvovirus H-1]

DNA structure and expression of p53 gene in human hepatoma cell lines SMMC-7721, YY-8103 and a spontaneously transformed liver cell line L-02 were analysed using the following method: analysis of allelic losses on chromosome 17p, PCR/SSCP, Northern blot and immunoprecipitation. There was no point mutation found in the exons 4-9 of the p53 gene, and a low level of expression of p53 gene was detected in the three cell lines. These observations were in agreement to the reported results of the relevant experiment using the human hepatoma cell line QGY-7703. Sensitivities of these cell lines and other eight human hepatoma cell lines (QGY-7703, PLC/PRF/5, Tong/HCC, Huh-7, FOCUS, Hep3B, SK-Hep-1, HepG2) with known p53 backgrounds to parvovirus H-1 was assayed using MTT method. Abnormality in the structure and/or function was observed in all of the cell lines examined except HepG2. The cell line HepG2 with normal structure and function of the p53 gene was found to be the least sensitive to H-1 in comparison to all the cell lines which have defeated structure and/or function of the p53 gene. The present study serves as a preliminary evidence that enhancement of the sensitivity of human hepatoma cell lines to H-1 is correlated to the abnormality of the structure and/or function of the p53 gene.     

人肝癌细胞p53基因的表达及其对细小病毒H-1的敏感性

本文利用单链构象多态性分析,17号染色体短臂等位基因杂合性分析,Northern印迹,免疫沉淀,p53基因第7外显子酶切等技术检测了两个中国人肝癌细胞系SMMC-7721,YY-8103和一个自发转化的人肝细胞系L-02的p53基因结构与表达。实验表明,这三个细胞系中没有出现17号染色体短臂等位基因杂合性缺失,第4—9外显子也没发生突变,但其mRNA和蛋白表达水平很低。利用MTT比色分析法研究了这三个细胞系和其他已知p53基因背景的八个人肝癌细胞系(QGY-7703、PLC/PRF/5、Huh-7、Hep3B、FOCUS、Tong/ HCC、SK-Hep-1、HepG2)对自主性细小病毒H-1的敏感性。除HepG2细胞外,其他十个细胞系p53基因的结构和/或表达都不正常。经H-1感染(moi=20)后,其敏感性均高于HepG2细胞。本研究初步表明了p53基因结构或表达的不正常可能导致人肝癌或转化细胞对H-1的敏感性的提高。     

尼氟灭酸对肝癌细胞增殖的影响

为了观察氯通道阻断剂尼氟灭酸(NFA)对人肝癌细胞(kuman hepatoma cell,HHCC)增殖的影响,我们将NFA作用于HHCC,应用细胞计数法及噻唑兰(MTT)比色分析法观察细胞增殖情况;用流式细胞仪检测细胞周期时相;并用激光扫描共聚焦显微镜检测[Ca^2 ]i的变化。结果发现,NFA使HHCC细胞数及MTT光吸收值(OD)较对照组都显著降低,去除NFA后,OD值逐渐恢复。经100μmol/L NFA处理48h的HHCC细胞G1期细胞比例比对照组明显增高,S期及G2期细胞比例明显低于对照组。细胞外应用NFA(100μmol/L)使[Ca^2 ]i快速降低,去除NFA后,[Ca^2 ]i可恢复。这些结果表明,尼氟灭酸能抑制细胞增殖,其机制可能与细胞内信号转导Ca^2 /CaM途径被抑制有关。