High-intensity focused ultrasound combined with herpes simplex virus thymidine kinase gene-loaded ultrasound-targeted microbubbles improved the survival of rabbits with VX2 liver tumor.

BACKGROUND: To explore the anti-tumor effect of high-intensity focused ultrasound (HIFU) combined with herpes simplex virus thymidine kinase (HSV-TK) gene-loaded ultrasound-targeted microbubbles on VX2 rabbit liver tumors. METHODS: Seventy-five New Zealand white rabbits were randomly divided into five groups after the models of VX2 rabbit liver tumors were established: (a) HIFU group; (b) HIFU and HSV-TK group (HIFU + HSV-TK); (c) HIFU, HSV-TK and ultrasound group (HIFU + HSV-TK + US); (d) HIFU, HSV-TK gene-loaded microbubbles and ultrasound group (HIFU + HSV-TK-MBs + US); and (e) HSV-TK gene-loaded microbubbles and ultrasound group (HSV-TK-MBs + US). After 2 weeks of VX2 liver tumor implantation, rabbits in groups (a), (b), (c) and (d) received HIFU to establish rabbit models of residual tumor by ablating 80% of the tumor volume. After HIFU ablation, rabbits in different groups received MBs wrapped around HSV-TK or HSV-TK solution via marginal ear veins and/or local ultrasonic irradiation to the tumor. Six rabbits in each group were sacrificed 48 h after the corresponding treatment, and tumors were extracted for in vitro experiments. Thymidine kinase mRNA was detected by the real-time polymerase chain reaction. The green fluorescent protein expression in liver tumor was detected by western blotting and immunohistochemistry. Tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling. The growth curves of VX2 liver tumors and survival curves of rabbits were compared. RESULTS: Forty-eight hours after treatment, TK mRNA and protein were the highest in the HIFU + HSV-TK + US + MBs group and the HSV-TK + US + MBs group (p < 0.05). At 48 h after treatment, the apoptotic index of tumor cells in HIFU + HSV-TK-MBs + US group was the highest (p < 0.05). Compared to other groups, HIFU combined with MBs wrapped HSV-TK suicide gene significantly inhibited tumor growth in vivo (p < 0.05) and prolonged the survival time of animals (p < 0.05). CONCLUSIONS: HIFU combined with HSV-TK gene-loaded ultrasound-targeted MBs significantly inhibited the growth of VX2 rabbit liver tumors in vivo and prolonged the survival time of the animals, providing a novel gene delivery method and a novel strategy for liver tumor treatment.

Effects of tumor necrosis factor, endothelin and nitric oxide on hyperdynamic circulation of rats with acute and chronic portal hypertension.

AIM: To evaluate the effect of tumor necrosis factor (TNF), endothelin (ET) and nitric oxide (NO) on hyperdynamic circulation (HC) of rats with acute and chronic portal hypertension (PHT). METHODS: Chronic portal hypertension was induced in Wistar rats by injection of carbon tetrachloride. After two weeks of cirrhosis formation, L-NMMA (25 mg/kg) was injected into one group of cirrhotic rats via femoral vein and the experiment was begun immediately. Another group of cirrhotic rats was injected with anti-rat TNFalpha (300 mg/kg) via abdominal cavity twice within 48 h and the experiment was performed 24 h after the second injection. The blood concentrations of TNFalpha, ET-1 and NO in portal vein and the nitric oxide synthase (NOS) activity in hepatic tissue were determined pre-and post-injection of anti-rat TNFalpha or L-NMMA. Stroke volume (SV), cardiac output (CO), portal pressure (PP), superior mesenteric artery blood flow (SMA flow) and iliac artery blood flow (IAflow) were measured simultaneously. Acute portal hypertension was established in Wistar rats by partial portal-vein ligation (PVL). The parameters mentioned above were determined at 0.5 h, 24 h, 48 h, 72 h and 120 h after PVL. After the formation of stable PHT, the PVL rats were injected with anti-rat TNFalpha or L-NMMA according to different groups, the parameters mentioned above were also determined. RESULTS: In cirrhotic rats, the blood levels of TNFalpha, NO in portal vein and the liver NOS activity were significantly increased (P<0.05) while the blood level of ET-1 was not statistically different (P>0.05) from the control animals (477.67+/-83.81 pg/mL vs 48.87+/-32.79 pg/mL, 278.41+/-20.11 micromol/L vs 113.28+/-14.51 micromol/L, 1.81+/-0.06 u/mg.prot vs 0.87+/-0.03 u/mg.prot and 14.33+/-4.42 pg/mL vs 8.72+/-0.79 pg/mL, respectively). After injection of anti-rat TNFalpha, the blood level of TNFalpha was lower than that in controls (15.17+/-18.79 pg/mL vs 48.87+/-32.79 pg/mL). The blood level of NO and the liver NOS activity were significantly decreased, but still higher than those of the controls. The blood level of ET-1 was not significantly changed. PP, SV, CO, SMAflow and IAflow were ameliorated. After injection of L-NMMA, the blood level of NO and the liver NOS activity were recovered to those of the controls. PP and CO were also recovered to those of the controls. SV, SMAflow and IAflow were ameliorated. In PVL rats, the blood levels of TNFalpha, NO in portal vein and the liver NOS activity were gradually increased and reached the highest levels at 48 h after PVL. The blood level of ET-1 among different staged animals was not significantly different from the control animals. PP among different staged animals (2.4+/-0.18 kPa at 0.5 h, 1.56+/-0.08 kPa at 24 h, 1.74+/-0.1 kPa at 48 h, 2.38+/-0.05 kPa at 72 h, 2.39+/-0.16 kPa at 120 h) was significantly higher than that in controls (0.9+/-0.16 kPa). After injection of anti-rat TNFalpha in 72 h PVL rats, the blood level of TNFalpha was lower than that in controls (14+/-14 pg/mL vs 48.87+/-32.79 pg/mL). The blood level of NO and the liver NOS activity were significantly decreased, but still higher than those of the controls. The blood level of ET-1 was not significantly changed. PP was decreased from 2.38+/-0.05 kPa to 1.68+/-0.12 kPa, but significantly higher than that in controls. SV, CO, SMAflow and IAflow were ameliorated. After injection of L-NMMA in 72 h PVL rats, the blood level of NO and the liver NOS activity were recovered to those of the controls. PP, SV, CO, SMAflow and IAflow were also recovered to those of the controls. CONCLUSION: NO plays a critical role in the development and maintenance of HC in acute PHT and is a key factor for maintenance of HC in chronic PHT. TNFalpha may not participate in the hemodynamic changes of HC directly, while play an indirect role by inducing the production of NO through activating NOS. No evidence that circulating ET-1 plays a role in both models of portal hypertension has been found.

[Genetic characterization analysis on epidemic rubella virus strains isolated in Liaoning from 2007 to 2012].

To analyze the genetic characterization of epidemic rubella virus strains isolated in Liaoning from 2007-2012, a total of 145 rubella virus strains were isolated using Vero/Slam cell line from the patients' throat swabs during rubella outbreaks and sporadics cases in Liaoning Province from 2007 to 2012. Fragments of 945 nucleotides containing 1E gene from 145 rubella virus isolates were amplified by RT-PCR, the PCR products were sequenced and analyzed. Based on the 739 nucleotides of 1E gene, the phylogenetic trees were constructed with 32 WHO rubella reference strains of 13 genotypes downloaded from GenBank and 145 rubella virus strains. The results showed that the 145 rubella virus strains in 2007 -2012 belonged to genotype 1E, nucleotide acids and amino acids similarities were 97.2%-100.0% and 97.6%-100.0%, respectively. Compared to the 1E reference strains(Rvi/ Dezhou.CHN/02, RVi/MYS/01), the nucleotide acids and amino acids similarities were 96.6%-99.2% and 98.2%-100.0%, respectively except for one amino acid change (Val246-Ala246) of RVi/Shenyang. Liaoning. CHN/13.11/13, and Asp262-Asn262 of RVi/Shenyang. Liaoning. CHN/13.11/4 and RVi/Liaoyang. Liaoning. CHN/26. 11/2. there had no change found in the important antigenic epitope sites, the hemagglutination inhibition and neutralization epitopes of the other rubella viruses. All the 145 strains isolated had the same amino acid change (Leu338--Phe338) in E1 protein. These findings suggested that genotype 1E of rubella virus was the predominant genotype in Liaoning province. the rubella prevailed in recent six years was mainly caused by rubella viruses genotype 1E with multi-transmission routes.