Determination of phospholipidosis potential based on gene expression analysis in HepG2 cells.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.     

超声引导下细针穿刺检测甲状腺乳头状癌 ret/PTC基因重组突变

目的了解ret／PTC基因突变与超声、病理诊断在甲状腺乳头状癌诊断中的关系。方法术前超声检查，并在超声引导下细针穿刺，活体取材进行病理诊断和逆转录聚合酶链（RT-PCR）方法检测ret／PTC基因突变。结果在34例超声与病理诊断的甲状腺乳头状癌（PTC）患者中，16例分子生物学检测发现ret／PTC基因突变（47％），其中8例为PTC-1基因突变（50％），2例PTC-2基因突变（12．5％），2例PCT-3基因突变（12．5％），3例PTC-1和PTC-2突变同时存在（18．75％），1例PTC-1和PTC-3突变同时存在（6．25％）。结论ret／PTC基因重组突变可存在于散发的甲状腺乳头状癌中，主要表现PTC-1型。应用超声引导下细针穿刺与甲状腺乳头状癌基因突变的检测相结合是早期诊断甲状腺乳头状癌的有效方法。     

Up-regulation of urinary UPIII mRNA levels in vesicoureteral reflux patients: Potential application as a screening test for vesicoureteral reflux

OBJECTIVES: Vesicoureteral reflux (VUR) is the most common congenital urinary tract anomaly. This disease can pose a major threat to the kidneys as twenty percent of patients with endstage renal disease are reported to have VUR. Although genetic studies for uroplakin III (UPIII) have been reported recently, no study has focused on UPIII gene expression in VUR patients. We describe here the up-regulation of UPIII mRNA in exfoliated urinary cells from primary VUR patients. METHODS: A real-time RT-PCR for UPIII mRNA was performed on exfoliated urothelial cells from 18 primary VUR and 38 control samples. UPIII mRNA copies were calculated for each sample. The statistical differences were assessed by the Mann-Whitney U test. Receiver operator characteristic curves were constructed for analysis of the diagnostic values. RESULTS: UPIII mRNA was found to be up-regulated to a greater extent in VUR than in control exfoliated urinary cells (mean +/- SE: 497.0 +/- 178.5 copies vs. 69.0 +/- 10.0 copies, respectively, P < 0.001). In evaluating the measurement of urinary UPIII mRNA as a screening test for VUR, the sensitivity was 77.8% and the specificity was 76.3% by the best diagnostic cutoff point. CONCLUSIONS: This is the first report demonstrating up-regulation of UPIII in mRNA levels in VUR patients. We submit that the quantitative measurement of urinary UPIII mRNA has a potential of developing into the first non-invasive screening test for VUR.     

Oncolytic herpes viral therapy is effective in the treatment of hepatocellular carcinoma cell lines

The rising incidence of hepatocellular carcinoma (HCC) in western countries, along with the poor prognosis offered by present-day treatment modalities, makes novel therapies for this disease necessary. Oncolytic herpes simplex viruses (HSV) are replication-competent viruses that are highly effective in the treatment of a wide variety of experimental models of human malignancies. This study seeks to investigate the effectiveness of oncolytic herpes viruses in the treatment of primary HCC cell lines. Sixteen commercially available human HCC cell lines were studied. G207 is an attenuated, replication-competent, oncolytic HSV engineered to selectively replicate within cancer cells. Cell lines were tested for viral sensitivity to G207 and their ability to support viral replication using standard cytotoxicity and viral replication assays. Eleven of 16 cell lines were moderately to highly sensitive to G207 viral oncolysis. HCC cell lines additionally demonstrated the ability to support viral replication in vitro with as high as 800-fold amplification of the administered viral dose observed. G207 is cytotoxic to, and efficiently replicates within, HCC cell lines in vitro. From these data, we suggest that oncolytic HSV therapy may have a role in the treatment of HCC, and in vivo studies are warranted. Presented in part at the 2005 American Hepato-Pancreato-Biliary Association Congress, Hollywood, Florida, April 14–17, 2005. Supported by grants R01CA75461 and R01CA72632 from the National Institutes of Health, and by grant MBC-99366 from the American Cancer Society (Yuman Fong).     

The Experiment of Gene Therapy for Gallbladder Carcinoma Cell in Vitro with Herpes Simplex Virus Thymidine Kinase/Ganciclovir

Objective To evaluate preliminarily the effect of HSV - tk/GCV system on gallhladder carcinoma cells in vitro.Methods Recombinant retroviral vector PLtkSN containing tk suicide gene was transfacted into gallbladder carcinoma cells, mediated by LipofectAMINETM 2000 liposome, and the growth - inhibiting rotes of GBC, SD/tk cells in the different GCV concentrations were measured by MTr methods; GBC - SD cells were mixed in the different proportions, and death rotes of the mixed cells treated with GCV were detected to verify by stander effect. Resuts GCV could lead GBC - SD/tk cells to significant death, and there was striking difference compared with the control cells （p〈0.01）. When the concentration of GCV was 1,10, 50,100,500ug/ml, the killing rate of GBC - SD/tk cells was respecfively 6.8%, 2.5.2%, 54.5%, 66.3%, 89.3%, indicating dose - dependent phenomenon; With GBC - SD/tk cells in the proportion of 0, 10%, 20% ,50%, 70%, 100%, the killing rate of the mixed cells treated with GCV was 0, 19.7%, 40.3%, 77.7%, 88.0%, 93.5%, respectively. It was apparent that bystander effect was observed in the experiment. Condusion HSV - tk/GCV system could be a potential tool for the treatment of carcinoma.     

自发性高血压大鼠颈动脉血管平滑肌中抑癌基因P53和原癌基因c-jun、c-fos、c-myc mRNA表达的研究

目的探讨自发性高血压大鼠颈动脉中抑癌基因P53和原癌基因c-jun、c-fos、c-myc mRNA的表达.方法用逆转录聚合酶链式反应检测两种基因的表达水平.正常雄性大鼠作为对照组.结果 SHR颈动脉中,抑癌基因P53和原癌基因c-jun、c-fos、c-myc均有高表达,较WKY差异有显著性(P<0.05).结论自发性高血压大鼠颈动脉组织中抑癌基因P53和原癌基因c-jun、c-fos、c-myc均有高表达,癌基因的活化可能与自发性高血压大鼠颈动脉血管重构有关.     

Human Tumor–infiltrating Lymphocytes Transfected with Tumor Necrosis Factor Gene Could Augment Cytotoxicity to Autologous Tumor Cells

Human tumor–infiltrating lymphocytes (TILs) derived from pleural or ascitic fluid were incubated with recombinant interleukin 2 and transfected with human tumor necrosis factor (TNF) a gene by the lipofection procedure. The resulting TILs secreted significant amounts of TNF in the culture supernatant and exhibited cytotoxicity against established cell lines, such as K562 and Daudi, and autologous tumor cells. The TNF gene–transfected TILs exhibited an augmented killing of autologous tumor cells.     

Rb1基因第16内含子内21个碱基缺失1例

目地研究双眼视网膜母细胞瘤患者Rb1基因杂合性突变的分子生物学特性。方法应用PCR—SSCP直接测序技术检测双眼视网膜母细胞瘤患者白细胞DNA中Rb1基因杂合性突变。结果50例证实有Rb1基因杂合性突变的病例中有1例发生于第16内含子中可以用3种定位方法解释、具有相同序列的21个碱基缺失。结论这种极为少见的Rb1基因突变方式可能是由于破坏了正常拼接位点的结构而激活了“隐蔽拼接位点”，导致异常的Rb1基因mRNA产生或由此影响整个拼接过程。     

Cationic Lipid-Based Gene Delivery Systems: Pharmaceutical Perspectives

Gene delivery systems are designed to control the location of administered therapeutic genes within a patient's body. Successful in vivo gene transfer may require (i) the condensation of plasmid and its protection from nuclease degradation, (ii) cellular interaction and internalization of condensed plasmid, (iii) escape of plasmid from endosomes (if endocytosis is involved), and (iv) plasmid entry into cell nuclei. Expression plasmids encoding a therapeutic protein can be, for instance, complexed with cationic liposomes or micelles in order to achieve effective in vivo gene transfer. A thorough knowledge of pharmaceutics and drug delivery, bio-engineering, as well as cell and molecular biology is required to design optimal systems for gene therapy. This mini-review provides a critical discussion on cationic lipid-based gene delivery systems and their possible uses as pharmaceuticals.     

肿瘤转移相关蛋白在甲状腺乳头状腺癌伴淋巴结转移组织中的原位表达研究

利用免疫组化ＡＢＣ法，研究甲状腺乳头状腺癌，甲状腺腺瘤和正常甲状腺组织中的肿瘤转移相关基因蛋白ＣＤ４４ｖ６，ＥＧＦＲ，转移抑制基因ｎｍ２３－Ｈ１和抑癌基因ｐ５３蛋白的原位表达。结果发现ＣＤ４４ｖ６和ＥＧＦＲ表达上调与肿瘤转移密切相关（Ｐ＜０．０５，Ｐ＜０．０１），而ｎｍ２３－Ｈ１的表达与肿瘤转移抑制密切相关（Ｐ＜０．０１）。这提示肿瘤转移相关基因和转移抑制基因之间的表达失衡是甲状腺乳头状腺癌易发生转移的重要原因。