γ-H2AX分析用于检测电离辐射致HepG2细胞DNA双链断裂的研究

目的 应用γ-H2AX分析检测电离辐射导致肝癌细胞株HepG2基因组DNA双链断裂的情况，并检测在不同细胞周期时相中的表达差异，从而了解不同时相HepG2细胞株的辐射敏感性。方法利用胸腺嘧啶核苷（TdR）阻断HepG2细胞于G1期末，于不同时间点分别得到同步化的S期和G2/M期细胞，用60Coγ射线照射细胞，建立DNA双链断裂模型，采用免疫荧光法和Westemblotting检测γ-H2AX的表达。结果TdR阻断后继续培养至34和40h，分别得到了较高同步化程度的S期和G2／M期HepG2细胞；受照后的HepG2细胞中Y-H2AX表达较照射前显著增高，处于S期的细胞γ-H2AX表达增加更为突出。结论不同周期时相的HepG2细胞受照后均检测出不同程度的DNA双链断裂，其中S期细胞尤为敏感。γ-H2AX对DNA双链断裂快速敏感的反应使γ-H2AX分析在检测早期DNA双链损伤中具有广泛的廊用前景。     

Screening of Efficient siRNA Target Sites Directed against Gatekeeper Genes for DNA Repair

DNA double-strand breaks (DSBs) are common le-sions that occur in cells. They are caused by exogenous sources such as ionizing radiation, and by endogenous sources such as radicals generated during metabolic processes[1]. In mammalian cells, DSBs are repaired ei-ther by the homologous recombination (HR) pathways or by the non-homologous end joining (NHEJ)[2] pathways to maintain the fidelity of human genome. The basic mechanism and factor requirements of the two pathways are different. V…     

白花丹醌对肝癌细胞 HepG2 增殖及血管内皮生长因子表达的影响

目的 研究白花丹醌对人肝癌细胞 HepG2 的增殖、克隆形成及血管内皮生长因子 (VEGF) 表达的影响。方法 MTT 法检测白花丹醌对人肝癌细胞 HepG2 的增殖抑制率;平板克隆形成实验检测白花丹醌对 HepG2 细胞克隆形成率的影响;RT-PCR 及免疫荧光细胞化学检测白花丹酯对 HepG2 细胞 VEGF mRNA 和蛋白表达的影响。结果 MTT 法结果显示白花丹醌对人肝癌细胞 HepG2 的增殖有抑制作用,且随着药物浓度增加 (2、8、32、128 μmol/L) 和作用时间 (24、48、72 h) 延长,其抑制作用逐渐增强,与细胞对照组相比有显著差异 (〖WTBX〗P ＜0.05);平板克隆形成实验显示白花丹醌对 HepG2 细胞克隆形成有显著抑制作用 (〖WTBX〗P ＜0.05);RT-PCR 及免疫荧光细胞化学均显示随白花丹醌药物浓度增高,HepG2 细胞 VEGFmRNA 及蛋白表达水平下调。结论 白花丹醌对人肝癌细胞 HepG2 的增殖、克隆形成及 VEGF 表达均有显著抑制作用,其可能成为一种新的抗肿瘤药物。     

针对细胞周期蛋白E的有效小干扰RNA靶向位点的筛选

乳腺癌是威胁女性健康的常见恶性肿瘤,寻找乳腺癌分子靶向治疗的新靶点,对于乳腺癌的基因治疗尤为重要[1,2].细胞周期蛋白E(Cyclin E)是细胞周期中G1/s期的关键调节蛋白[3].     

The association between RASSF1 gene polymorphisms and lung cancer susceptibility among people in Hubei Province of China

The relationship between Ala/Ser polymorphism in 133 codon of exon 3 region of the RASSF1 gene and genetic susceptibility of lung cancer in Hubei province Han population was investigated by a case-control study. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） technique was adopted to analyze the polymorphism of codon 133 of exon 3 in the RASSF1 gene of 100 pathologically diagnosed lung cancer patients, and 100 healthy controls. The relationship between different genotypes and the susceptibility of lung cancer was analyzed. Among 200 blood samples from Han people in Hubei Province, including 100 from lung cancer patients and 100 from healthy controls, the frequencies of Ala/Ala, Ala/Ser, Ser/Ser genotype of the RASSF1 in lung cancer patients were 83%, 16%, 1%, and those in healthy controls was 93%, 7%, 0% respectively, with the difference being statistically significant between two groups （P〈0.05）. The individuals with Ala/Ser genotype had higher risk of suffering from lung cancer, with an OR of 2.341, and 95% CI of 1.009-6.393 respectively. It was concluded that RASSF1Ala133Ser was a susceptible genetic factor of lung cancer. Ala/Ser genotype increased the risk of lung cancer.     

Association between CHRNA3 rs1051730 genotype and lung cancer risk in Chinese Han population: A case-control study

Recent population-based genome wide association studies have revealed potential suscepti- bility loci of lung cancer at the region of chromosome 15q25.1 containing nicotinic acetylcholine re- ceptor genes. The loci increasing lung cancer risk has been widely identified in Caucasians, but whether this association also exists in Asians and whether this association is a direct role or mediated via tobacco smoking indirectly has not been fully established. We conducted a case-control study comprising of 210 histologically confirmed lung cancer cases and 200 healthy controls to examine rsl051730 genotyping, a single nucleotide polymorphism receiving much attention recently, and its influence on lung cancer risk as well as nicotine dependence in a Chinese Han population. Our results showed that the heterozy- gous C/T genotype and minor allele T conferred a significant higher risk of lung cancer than the CC homozygotes and allele C （adjusted OR=2.25, 95% CI=1.0~4.89, P=-0.040 and OR=2.18, 95% CI=1.02~4.67, P=0.045 respectively）. However, no association between the smoking habit and the CHRNA3 rs1051730 polymorphism was observed in this study. The results suggested that the rs1051730 polymorphism may modify susceptibility to lung cancer via a smoking-independent manner among Chinese Han population. Additional studies in vitro and in vivo are warranted to further elucidate the impact ofrsl051730 on lung cancer susceptibility.     

β1-509C/T基因多态性与中国人群NSCLC遗传易感相关性的研究

目的:探讨转化生长因子β1基因(TGF-β1)-509C/T位点多态性与中国人群非小细胞肺癌(non-small cell lungcancer,NSCLC)遗传易感性的关系。方法:采用聚合酶链反应-限制性片段长度多态性PCR-RFLP方法检测210例NSCLC患者和208例健康对照者的TGF-β1-509C/T基因型分布,并分析两组之间的差异。结果:TGF-β1-509CT+TT基因型相对于CC基因型是NSCLC发生的独立危险因素(P=0.007,OR=2.297,95%CI:1.250～4.219);携带T等位基因者患NSCLC的风险是携带C等位基因者的1.617倍(P=0.001,95%CI:1.210～2.161);重度吸烟者相对于不吸烟和轻度吸烟者是NSCLC发生的独立危险因素(P=0.021,OR=1.783,95%CI:1.089～2.918)。结论:TGF-β1-509C/T位点多态性在中国人群中与NSCLC遗传易感性相关,可作为NSCLC发病风险评估的筛选指标。     

DNA double-strand breaks,potential targets for HBV integration

Hepatitis B virus(HBV)-induced hepatocellular carcinoma(HCC) is one of the most fre-quently occurring cancers.Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis.More and more researches were designed to find the relationship of the two.In this study,we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks(DSBs),one of the most detrimental DNA damage.An 18-bp I-SceI homing endonuclease recognition site...     

缺氧条件下HMGB1对HepG2细胞线粒体功能的影响

目的:探讨缺氧条件下高迁移率族蛋白B1(HMGB1)对人肝癌细胞系HepG2线粒体功能的影响及其机制。方法:将HMGB1小干扰RNA (HMGB1-siRNA)和阴性对照si RNA (si RNA-NC)分别转染入HepG2细胞,实验分为:缺氧(hypoxia)组、hypoxia+HMGB1-siRNA组和hypoxia+si RNA-NC组。流式细胞术检测各组细胞线粒体活性氧簇(mt ROS)含量和线粒体膜电位水平,RT-qPCR检测各组细胞线粒体DNA(mt DNA)拷贝数,ATP检测试剂盒检测各组细胞内ATP产生量,Western blot检测各组细胞线粒体生物合成相关分子表达的变化。结果:与hypoxia组和hypoxia+si RNA-NC组相比,当HMGB1表达被抑制后,细胞线粒体活性氧簇含量明显升高,线粒体膜电位水平、mt DNA拷贝数和ATP产生量明显下降(P 0. 05); Western blot结果显示,hypoxia+HMGB1-siRNA组的线粒体生物合成相关分子表达量下降(P 0. 05)。结论:缺氧环境下,HMGB1通过调控线粒体生物合成,诱导新生线粒体形成并维持细胞线粒体的正常功能。     

细胞周期蛋白E siRNA真核表达载体的构建与鉴定

目的构建细胞周期蛋白E(Cyclin E)小干扰RNA(siRNA)的真核表达载体,并检测其抑制乳腺癌细胞株MCF-7中Cyclin E表达的效果。方法根据基因文库中Cyclin E的cDNA序列,设计并合成两段siRNA,分别转入到pGENESIL-1质粒中,并对重组质粒(命名为pGENESIL/Cyclin E-1和pGENESIL/Cyclin E-2)进行测序鉴定。脂质体介导下转染MCF-7细胞株,RT-PCR检测转染后Cyclin E的表达。结果Cyclin E siRNA真核表达载体构建成功,测序结果显示插入片段的方向和序列与预想一致。RT-PCR检测显示Cyclin E的表达显著降低。结论Cyclin E siRNA真核表达载体的成功构建为进一步研究以Cyclin E为靶位点对乳腺癌进行基因治疗奠定了重要的实验基础。