原发性肝癌组织中KruPPel样因子6(KLF6)的表达及其对肝癌细胞增殖的抑制作用

目的了解Kruppel样因子6（KLF6）基因在原发性肝癌组织中的表达水平及其对肝癌细胞增殖的抑制作用。方法分别以逆转录聚合酶链反应（RT-PCR）和Wstern blot检测原发性肝癌及其癌旁组织KLF6 mRNA及蛋白质的表达水平。构建KLF6真核表达质粒并转染肝癌细胞SMMC-7721,流式细胞仪及MTT试验观察KLF6基因对细胞周期及细胞增殖活性的影响。以Western blot技术检测外源KLF6基因对p21WAF1表达的影响。结果与对应的癌周组织比较,23例原发性肝癌组织中8例（34.8%）KLF6 mRNA,9例（39.1%）KLF6蛋白质水平明显下降。与对照组比较,转染KLF6基因的SMMC-7721细胞G0~G1期所占细胞的比例明显增高,转染KLF6基因SMMC-7721的增殖率明显下降。外源KLF6基因能上调肝癌细胞p21WAF1表达。结论KLF6基因表达水平的下降可能在原发性肝癌发病机制中起到一定作用。上调p21WAF1表达是KLF6基因抑制肝癌细胞增殖的重要途径.     

p16、p21基因表达缺失在原发性肝癌中的临床意义

背景与目的:P16,P21蛋白是调控细胞周期的抑制蛋白,在调节细胞增殖中起非常重要的作用,其基因缺失,失活与多种肿瘤的发生,发展有关,本研究观察原发性肝癌中P16、P21蛋白表达及其基因缺失情况,探讨p16、p21基因表达缺失的临床意义.方法:采用回顾性分析方法,收集原发性肝癌42例,正常肝组织(距离癌组织≥2 cm)36例,应用Western印迹试验、逆转录聚合酶链式反应(RT-PCR)检测肝癌和正常肝组织中P16、P21蛋白的表达和相应mRNA扩增情况.结果:在癌组织中,P16蛋白缺失率为24例(24/42,57.1%),正常肝组织中P16蛋白缺失率为13.9%(5/36);而相应组织中P21蛋白缺失率分别为45.2%(19/42)和19.4%(7/36).两者在癌和正常肝组织中缺失差异均有显著性(P＜0.05).14例癌组织(38.9%.14/36)仅p16 mRNA转录而未表达蛋白.P16、P21蛋白的低表达与肝癌大小、分化程度关系密切(P＜0.05),而与是否有包膜、是否肝硬化等无关.结论:P16、P21蛋白的表达缺失与肝癌发生发展密切相关,两者可作为判断肝癌恶性程度的参考指标.     

In vitro and in vivo studies of adenovirus-mediated human norepinephrine transporter gene transduction to hepatocellular carcinoma

The clinical value of (131)I-MIBG for targeted imaging and targeted radiotherapy is limited to neural crest-derived tumors expressing human norepinephrine transporters (hNET) protein. To extend (131)I-MIBG-targeted therapy to other nonexpressed hNET tumors, this study investigated the hNET expression in vitro and in vivo in HepG2 hepatoma mediated by recombinant adenovirus encoding the hNET gene (Ad-hNET). For this purpose, the HepG2 cells showed a 4.87-fold increase in (125)I-MIBG uptake after infection with Ad-hNET, and the uptake of (125)I-MIBG could be specifically inhibited by maprotiline. Immunohistological analysis, in vivo biological study and (131)I-MIBG scintigraphic imaging also revealed the high expression of hNET protein in hepatoma. This in vitro and in vivo studies demonstrate the feasibility of hNET gene transfer, meditated by adenovirus vector, could extend to tumors other than those derived from the neural crest, which provides a sound foundation for further investigation of hepatocellular carcinoma-targeted radiotherapy mediated by adenovirus transfection with hNET gene.     

Aberrations of p53 gene in human hepatocellular carcinoma from China

Allele losses and mutations have been examined in 38 cases ofprimary hepatocellular carcinomas (HCC) from different geographicareas of China by Southern, single-strand conformational polymorphism(SSCP) and direct DNA sequencing analyses. Two of 12 samplesfrom Qi-Dong and six of 18 HCCs from Shanghai showed loss ofheterozygosity (LOH) at the loci on chromosome 17p13.3. Allof the nine mutations in the p53 gene detected in HCC from Qi-Dongwere clustered at the third base of codon 249, i.e. G:C to T:A,leading to an arginine to serine change. In contrast, 18 HCCsamples from Shanghai contained three mutations at codons 249,255 and 279. These results suggested a relationship betweenthe spectrum of p53 aberration and environmental risk factorsin these two geographic areas. Since no correlation betweenthe state of HBV DNA and p53 abberration was observed, otherfactors such as dietary exposure to aflatoxin B1 (AFB1) mightbe responsible for the mutational hotspot at codon 249 in HCCsfrom Qi-Dong area.     

p53p21双基因转染对肝癌细胞生长的抑制作用研究

 目的　研究 p5 3和 p2 1双基因对肝癌细胞的联合基因治疗效果。 方法　通过磷酸钙 -DNA共沉淀法将p5 3和 p2 1双基因真核共表达载体及单基因表达载体引入肝癌细胞SMMC 772 1,用直接镜检 ,DNAladder检测法和四甲基偶氮唑 (MTT)法等研究细胞生长情况。结果　转染外源 p5 3p2 1双基因的肝癌细胞SMMC 772 1与未转染及单基因转染的肝癌细胞SMMC 772 1相比 ,其增殖速度显著下降。结论　p5 3和p2 1双基因对肝癌细胞的联合基因治疗效果比较明显 ,对肝癌基因治疗的进一步研究具有指导意义。     

p16基因治疗对肝癌细胞生物学行为的影响

背景与目的：p16基因是原发性肝癌（hepatocellular carcinoma,HCC）中失活频率很高的抑癌基因之一,应是治疗HCC的理想靶基因。本研究的目的是探讨转入野生型p16cDNA对肝细胞性肝癌细胞生物学行为的影响。方法：用我们构建的p16基因的逆转录病毒表达载体pcLXSN-p16,分别感染p16蛋白表达阴性与表达阳性的肝癌细胞株SNU-449与HepG2.2.15,筛选出稳定的表达株,对转基因后肿瘤细胞的生物学行为进行观察。结果：p16蛋白表达阴性的SNU-449细胞转入野生型p16基因后,细胞生长速度明显减慢,G0-G1期细胞明显多于转基因前,其裸鼠首次接种成瘤率（可在一定程度上反映瘤细胞对组织的侵袭能力）、在裸鼠体内的生长速度以及对裸鼠的致死性均低于未转基因者。而p16蛋白表达阳性的HepG2.2.15细胞转入外源p16cDNA后,其生长状况及细胞周期则未发现有明显改变。就SNU-449与HepG2.2.15细胞株而言,转入p16cDNA均未能诱导细胞凋亡。结论：转入外源野生型p16基因可抑制p16蛋白表达阴性的肝癌细胞的生长并降低其侵袭能力,但对p16蛋白表达阳性的肝癌细胞的生长则无影响。     

In vitro and in vivo suppression of hepatocellular carcinoma growth by chitosan nanoparticles

Chitosan nanoparticles (CNP), a kind of widely used drug carrier, have shown potent cytotoxic effects on various tumour cell lines in vitro and in vivo. This study sought to evaluate the antitumour effect of CNP on growth of human hepatocellular carcinoma (BEL7402) and the possible mechanisms involved. Cells were grown in the absence and presence of various concentrations of CNP with mean particle size of about 40nm. Cell viability, ultrastructural changes, surface charge, mitochondrial membrane potential, reactive oxygen species (ROS) generation, lipid peroxidation, DNA fragmentation and fatty acid composition were analysed by MTT assay, electron microscopy, zetasizer analysis, flow cytometry, spectrophotometric thiobarbituric (TBA) assays, DNA agarose gel electrophoresis and GC/MS respectively. For in vivo experiments, male BABL/c nude mice were implanted with BEL7402 cells subcutaneously to establish human hepatoma model. Chitosan, saline, and CNP with different mean particle size (40, 70 and 100nm) were administrated by oral administration (1mg/kg body weight). Tumour and body weight were measured, morphologic changes of tumour and liver tissues were studied under electron microscope. In vitro, CNP exhibited high antitumour activities with an IC(50) value of 15.01microg/ml, 6.19microg/ml and 0.94microg/ml after treatment for 24h, 48h and 72h respectively. CNP could induce cell necrosis observed by electron microscope and DNA fragmentation. The antitumour mechanism was mediated by neutralisation of cell surface charge, decrease of mitochondrial membrane potential and induction of lipid peroxidation. The tumour growth inhibitory rates on BEL7402 cells in nude mice treated with chitosan and CNP with different mean particle size (40, 70 and 100nm) were 24.07%, 61.69%, 58.98% and 34.91% respectively. Typical necrotic morphological changes of tumour tissues and no liver abnormalities were found under electron microscope. In this paper, results show a strong antitumour effect of CNP on human hepatoma cell line BEL7402 in vitro and in vivo. These findings suggest that CNP could be a kind of promising agent for further evaluations in the treatment of hepatocellular carcinoma.     

The p53 tumor-suppressor gene and ras oncogene mutations in oral squamous-cell carcinoma.

The frequencies of mutations in the p53 tumor-suppressor gene and ras proto-oncogenes were investigated systematically in surgically resected oral squamous-cell carcinomas (SCCs) using single-strand conformation polymorphism (SSCP) and/or dot-blot hybridization analysis of DNA fragments which had been amplified by the polymerase chain reaction (PCR). p53 gene mutations, within the region of exons 5 to 8, were detected in 17 out of 27 (63%) tumor specimens. The role of p53 mutations in cell-line establishment was investigated. p53 gene mutations were detected in 5 out of 6 tissue samples from which cell lines were established and in 4 out of 5 specimens from which cell lines could not be established, suggesting that the presence of p53 gene mutations is not by itself sufficient for cell-line establishment. Tumor samples were also analyzed for point mutational activation of the ras proto-oncogenes. One out of 30 (3%) tumors showed an activating point mutation in codon 12 of H-ras, this being consistent with reports from Europe and USA but not with any from India. Compared to frequencies of the other genetic changes so far reported for oral SCC, the p53 mutations have been observed most often to undergo genetic change. p53 gene mutation is thus intimately involved in the genesis of oral SCC and consequently should be useful as a marker for the diagnosis of this neoplasm.     

层黏连蛋白与其α6整合素受体对人肝癌细胞表型的调控作用

目的 利用自制的抗整合素α6亚基胞外区的单克隆抗体(IA6ED McAb)研究人肝癌细胞表面α6整合素胞外区对人肝癌细胞系BEL-7402黏附、铺展的影响以及在控制细胞增殖与分化中的作用。方法 以层黏连蛋白(LN)、纤黏连蛋白(FN)、Matrigel分别作为细胞外基质,检测IA6ED McAb对人肝癌细胞系BEL-7402细胞在不同基质上的黏附、铺展的影响;以MTT法检测McAh对BEL-7402细胞存活或增殖的影响;以明胶酶谱法检测McAb对BEL-7402细胞分泌基质金属蛋白酶(MMPs)的影响;以微粒子免疫吸附法检测对AFP分泌的影响。结果 LN可以明显促进BEL-7402细胞AFP及MMPs的分泌;IA6ED McAb的封阻不但可以特异性抑制BEL-7402细胞在LN上的黏附及铺展,而且对BEL-7402细胞以LN为基质的存活或增殖、去分化及异常分化表型有非常显著的抑制作用。结论 人肝癌BEL-7402细胞的增殖、分化表型受LN及整合素α6的调节。人肝癌组织细胞外基质成分中LN及细胞表面整合素α6的过表达,可以增强肝癌细胞的去分化及异常分化表型;而用IA6ED McAb阻断IN与肝癌细胞表面α6β1整合素受体的相互作用,可以逆转其去分化及异常分化表型,从而可能降低癌细胞的转移潜能。     

Donor interleukin 6 gene polymorphisms predict the recurrence of hepatocellular carcinoma after liver transplantation

Background

Application of the Milan criteria is an effective strategy to select patients with hepatocellular carcinoma (HCC) for liver transplantation, but HCC recurrence is still a major concern. The aim of this study was to determine whether interleukin 6 (IL6) polymorphisms and clinical variables are potential predictors for HCC recurrence and prognosis after transplantation.

Methods

A total of 110 consecutive patients with HCC undergoing liver transplantation were enrolled in the study. Six tag single nucleotide polymorphisms in IL6 were genotyped in both the donors and recipients. Demographic characteristics, HCC features, and IL6 polymorphisms were assessed against HCC recurrence.

Results

Pretransplant hepatitis B virus DNA (P = 0.014), pretransplant serum alpha-fetoprotein (P = 0.035), number of nodules (P = 0.011), diameter of main nodule (P = 0.001), macrovascular invasion (P = 0.001), microvascular invasion (P = 0.001), HCC exceeding the Milan criteria (P < 0.001), and donor rs2069852 AA genotype (P = 0.010) were associated with HCC recurrence. Recurrence-free survival rate and overall survival rate were significantly lower (P = 0.011 and P = 0.026, respectively) in patients whose donor had the rs2069852 AA genotype than in those whose donor had the AG and GG genotypes. Independent risk factors for recurrence-free survival and overall survival were microvascular invasion (P = 0.003; P = 0.002), HCC exceeding the Milan criteria (P < 0.001; P = 0.001), and donor rs2069852 AA genotype (P = 0.002; P = 0.010).

Conclusions

Our data suggest that donor IL6 rs2069852 polymorphisms may be a potential genetic marker for HCC recurrence after liver transplantation in the Han Chinese population.

     

p53和p21WAF1蛋白在肝细胞肝癌中表达的意义

 【摘要】　目的　探讨p53和p21WAF1蛋白在肝细胞肝癌（HCC）中的表达及其意义。方法　应用免疫组织化学SP法检测41例HCC及相应30例癌旁肝组织中p53和p21WAF1蛋白的表达。结果　p53和p21WAF1蛋白在HCC中阳性表达率分别为43.9 ％和75.6 ％，明显高于癌旁组织，差异有统计学意义（P＜0.01）。HCC中p53蛋白表达与p21WAF1蛋白表达无明显相关性（P＞0.05）。p53蛋白表达在不同组织学分级、有无肝内或门静脉转移之间差异有统计学意义，而p21WAF1蛋白表达在不同临床病理特征间差异无统计学意义。结论　p53和p21WAF1蛋白高表达均在HCC发生、发展中起作用，但两者间并无相关性。p21WAF1的诱导存在着不依赖p53的途径。     

野生型p53基因对肝癌细胞多药耐药的逆转作用及其相关机制

背景与目的：细胞凋亡的发生机制在肿瘤多药耐药中起重要作用,野生型p53基因是细胞凋亡的激活剂,与肿瘤多药耐药密切相关。本研究旨在探讨野生型p53基因能否实现对肝癌细胞多药耐药的逆转以及逆转的相关机制。方法：构建野生型P53蛋白表达序列的真核表达载体pCR3．1-p53,用脂质体转染技术建立人肝癌细胞Bel-7402的p53转染细胞株,对转染细胞株进行MTT实验,观察p53基因对肿瘤细胞生长的抑制作用和对肝癌细胞对长春新碱（vincristine,VCR）药物敏感性的影响,姬姆萨染色法观察细胞形态,免疫组织化学SP法检测细胞P糖蛋白（P—glycoprotein,P—gp）的表达,逆转录PCR法检测细胞内mdr1、MRP、GSTπ、TopoⅡ mRNA的表达。结果：转染p53的Bel—p53细胞生长明显慢于未转染的Bel-7402细胞。转染野生型p53的Bel-p53细胞在VCR浓度为0．01μg／ml,0．1μg／ml,1．0μg／ml,10μg／ml,25μg／ml时,其药物敏感性增加;VCR作用最佳浓度为1．0μg／ml。形态学检查转染细胞,在VCR作用下细胞数明显减少,细胞散在不成片、细胞高度水肿、胞体不规则突起,可见核固缩、核裂解、核溶解。p53基因对Bel-7402细胞的mdr1／P—gP的表达有明显的抑制作用．对TopoⅡα基因的表达有上调作用,对GSTπ、MRP基因表达没有影响。结论：p53基因对肝癌细胞多药耐药有逆转作用．其逆转机制可能与降低mdr1／P—gp的表达、上调TopoⅡα的表达．从而增加细胞内VCR药物浓度和VCR药效有关。     

p21对肝癌细胞中survivin转录的影响及调控机制的探讨

目的 研究细胞周期依赖性激酶抑制蛋白p21对人肝癌细胞survivin转录抑制的调控作用,并探讨其相应机制.方法 使用多柔比星处理人肝癌细胞株HepG2,采用脂质体法将质粒pEGFP-C2-p21导入HepG2细胞,用G418筛选转染后的HepG2-p21和HepG2-pEGFP细胞;采用实时定量聚合酶链反应(RQ-PCR)检测p21、p53和survivin的mRNA表达;采用流式细胞仪分析转染后细胞周期的变化;采用逆转录聚合酶链反应(RT-PCR)检测转染后E2F-1和p300的mRNA表达.结果 经多柔比星处理后,HepG2细胞中p21和p53 mRNA的表达先增高,然后逐渐降低;survivin mRNA的表达则相反.转染后,HepG2-p21细胞中p21 mRNA的表达明显增高,分别为HepG2和HepG2-pEGFP细胞的2100.1倍和980.9倍;survivin mRNA的表达却明显降低,分别为HepG2和HepG2-pEGFP细胞的0.5%和0.6%;p53 mRNA的表达差异无统计学意义.流式细胞仪检测显示,HepG2-p21细胞的细胞周期明显阻滞在G0/G1期.HepG2-p21细胞中E2F-1和p300 mRNA的表达水平分别为0.75±0.04和0.18 ±0.06,与HepG2和HepG2-pEGFP细胞比较,含量均明显减低(P＜0.05).结论 p21可能通过抑制survivin的转录调控因子E2F-1和p300 mRNA的表达,从而抑制细胞中survivin的表达,并导致细胞出现G0/G1期阻滞.