[The study of anti-tumor activities of DC vaccine loaded with multi-epotipes of survivin].

AIM: To observe the anti-tumor activity of dendritic cell (DC)vaccine loaded with multi-epitopes of survivin. METHODS: The recombinant plasmid pPIRESneo3.0-survivin (4)/Th which include four survivin HLA-A2-restricted CD8(+); CTL epitopes and a CD4(+);Th epitope, pPIRESneo3.0-survivin (4) which include four survivin CD8(+); CTL epitopes, were transfected into human dendritic cells respectively. There were five groups, which included survivin(4)/Th group, survivin(4)group, empty plasmid group, untransfected group and T lymphocytes group The expression of CD83 and CD86 on the surface of DCs, the expression of CD4 and CD8a on the surface of T lymphocytes, the apoptotic rates of MCF-7 cells after treated by DC vaccine were measured by flow cytometry; IFN-γ levels of all groups were detected by ELISA and the growth inhibition of MCF-7 cells after being treated with DC vaccine was tested by MTT colorimetry. RESULTS: The results of flow cytometry revealed that high levels CD83 and CD86 were expressed on the surface of DCs; high levels CD4 and CD8a were expressed on the surface of T lymphocytes; the IFN-γ levels in survivin(4)/Th group [(66.50±3.34)ng/L]were significantly higher than that in survivin(4)group[(46.10±1.35)ng/L], empty plasmid group[(25.17±0.32)ng/L], untransfected group [(25.47±0.95)ng/L] or T lymphocytes group[(23.73±0.50)ng/L](P<0.05). The inhibition rate of MCF-7 cells in survivin(4)/Th group was significantly higher than that in survivin(4)group, empty plasmid group, untransfected group or T lymphocytes group(P<0.05). The apoptotic rate of MCF-7 cells in survivin(4)/Th group was (10.63±0.29)% after treated by DC vaccine, which was significantly higher than that in in survivin(4)group, empty plasmid group, untransfected group or T lymphocytes group(P<0.05). CONCLUSION: The DCs vaccine loaded with multi- CD8(+); CTL epitopes of survivin has strong anti-tumor effects. CD4(+); Th cells can promote the anti-tumor activity of CD8(+);CTL.

[Construction of the eukaryotic expression vector encoding multi-epitope fusion protein of human survivin and its expression in dendritic cells].

AIM: To construct a eukaryotic expression vector encoding the multi-epitope fusion protein of human survivin, and express it in human dendritic cells. METHODS: Recombinant cDNA sequence encoding four HLA-A2-restricted CD8+ CTL epitopes and a CD4+ Th epitope was synthesized and cloned into pBluescript II SK (+) vector. After confirmed by sequencing, the cDNA fragment was inserted to eukaryotic expression vector pIRESneo3.0 to generate the recombinant plasmid pPIRESneo3.0-survivin (4)/Th. The pPIRESneo3.0-survivin (4)/Th was then transfected into human dendritic cells and the transfectants were selected for stable expression. RESULTS: The eukaryotic expression vector encoding the multi-epitope fusion protein of survivin was constructed, and successfully transfected into human dendritic cells. CONCLUSION: The eukaryotic expression vector encoding the multi-epitope fusion protein of survivin has been constructed successfully, and stably expressed in human dendritic cells, which provides clues for further research on multi-epitope cancer vaccine.

Comparative evaluation of two laparoscopic procedures for treating common bile duct stones.

Cholesystolithiasis is often associated with common bile duct stones (CBDS). In order to assess the choice of surgery in terms of effectiveness and complications in the treatment of CBDS, we have compared three surgical procedures, viz., laparoscopic choledocholithotomy T-tube drainage (LCH-TD), laparoscopic cholecystectomy with endoscopic sphincterotomy (LC-EST), and the traditional open choledocholithotomy with T-tube drainage (OCHTD). This study is a retrospective comparative analysis of LCH-TD (77 patients), LC-EST (43 patients), and OCHTD (60 patients) for CBDS. The success of the surgical procedures was assessed in terms of recovery duration, hospitalization, and post-operative complications. Both the micro-invasive procedures, LCH-TD and LC-EST, with a success rate of 92.5%, are found to be superior to the traditional OCHTD. Between the two micro-invasive procedures, patients in LCH-TD group had shorter operation time and hospital stay, and fewer post-operative complications. Although the size of the stones is comparable between these two groups, the CBD diameter was significantly larger in patients who underwent LCH-TD. In comparison to OCHTD, both LCH-TD and LC-EST are micro-invasive, safe, and suitable for routine use in patients with CBDS. Moreover, when the CBD diameter is wider than 1 cm, LCH-TD is strongly advocated.

siRNA mediated the type 1 insulin-like growth factor receptor and epidermal growth factor receptor silencing induces chemosensitization of liver cancer cells.

PURPOSE: This study was to investigate if downregulation of IGF1R and EGFR by RNA interference (RNAi) would sensitize human liver cancer cells (HEPG2, Huh7 ) to adriamycin. METHODS: HEPG2, Huh7 cell lines were transfected IGF1R siRNAs and EGFR siRNAs and IGF1R or EGFR mRNA level was determined by RT-PCR and Western-blot analysis. We investigated the effects of the adriamycin-induced apoptosis of these cells by TUNEL assay. Also we analyze caspase3, 8 and the phosphorylation levels of Akt and Erk by Western-blot. The p53 effect of adriamycin-induced cell death by inhibitors of EGFR/IGF1R is investigated by cell growth curves. RESULTS: Transfection of an IGF1R and EGFR siRNAs resulted in substantial loss of IGF1R and EGFR mRNA of HEPG2, Huh7 cells relative to the control case. EGFR siRNA and IGF1R siRNA treatments increased the adriamycin-induced apoptosis of these cells. IGF1R siRNA and EGFR siRNA enhance a caspase-dependent cell death program. The phosphorylation levels of Akt and Erk were reduced by the combination of the two agents. The facilitation of adriamycin-induced cell death by inhibitors of EGFR/IGF1R is p53-independent. CONCLUSIONS: The results indicate that the siRNA for IGF1R has a great potential for cancer therapy when combined with either a chemotherapeutic agent or siRNAs that targets EGFR.

[Inhibitory effect of IGF1R siRNA on the growth of human liver cancer SMMC7721 cell xenograft in nude mice].

BACKGROUND & OBJECTIVE: Using small interfering RNA (siRNA) to inhibit mammal gene expression becomes an effective technique in studying gene function. This study was to investigate the effect of insulin-like growth factor 1 receptor (IGF1R) siRNA on the growth of human liver cancer SMMC7721 cell xenograft in nude mice. METHODS: siRNA targeting IGF1R was designed, and plasmid SMMC7721-IGF1R-siRNA was constructed and transfected into SMMC7721 cells (SMMC7721-IGF1R-siRNA cells); the cells transfected with SMMC7721-IGF1R-mutation (SMMC7721-IGF1R-mutation cells) were used as negative control, and untransfected cells as empty control. Stable cell clones were screened by G418, and transplanted into nude mice to establish cancer xenograft. Tumor growth was monitored. Tumor morphology was observed with HE staining. The expression of IGF1R protein in tumor tissues was detected by Western blot. Microvessel density (MVD) in tumor tissues was detected by SP immunohistochemistry. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. RESULTS: The tumor volume was significantly smaller in SMMC7721-IGF1R-siRNA group than in SMMC7721-IGF1R-mutation group and SMMC7721 group (P < 0.05). Necrosis and cell apoptosis were found in SMMC7721-IGF1R-siRNA group. The expression of IGF1R protein was significantly lower in SMMC7721-IGF1R-siRNA group than in SMMC7721-IGF1R-mutation group and SMMC7721 group (P < 0.05). MVD was significantly lower in SMMC7721-IGF1R-siRNA group than in SMMC7721-IGF1R-mutation group and SMMC7721 group (11.3+/-4.4 vs. 36.7+/-7.6 and 28.4+/-6.5, P < 0.05). The apoptosis rate of tumor cells was significantly higher in SMMC7721-IGF1R-siRNA group than in SMMC7721-IGF1R-mutation group and SMMC7721 group [(50.2+/-6.4)% vs. (5.4+/-1.0)% or (6.0+/-2.1)%, P < 0.05]. CONCLUSION: IGF1R siRNA can inhibit the growth of SMMC7721 cell xenograft in nude mice.

siRNA-mediated type 1 insulin-like growth factor receptor silencing induces chemosensitization of a human liver cancer cell line with mutant P53.

Overexpression of type 1 insulin-like growth factor receptor (IGF1R) contributes to the progression and metastasis of liver cancer, implying that IGF1R gene is a suitable target of RNA interference (RNAi) for liver cancer therapy. To investigate the possible regulation of IGF1R by P53, we examined the level of IGF1R expression in liver cancer cell lines in response to adriamycin. Levels of IGF1R mRNA and protein in cell lines with wild-type P53 decreased dramatically after P53 induction, but no such reduction of IGF1R was observed in cell lines with mutated P53. Inhibition of wild-type P53 in HEPG2 cells by small interfering RNA (siRNA) significantly upregulated the expression of IGF1R. IGF1R inhibition by siRNA in Huh7 cells with mutated P53 significantly depressed cell proliferation. To investigate the sensitivity of cancer cells to adriamycin after inhibition of IGF1R, we depressed IGF1R expression using siRNA, and then added adriamycin at an IC50 dose. After a further 48 h incubation with adriamycin, proliferation was significantly depressed in the cells treated with siRNA targeting IGF1R, in comparison with siRNA targeting scramble. Furthermore, both TUNEL and pro-caspase-3 expression assay showed a significant increase in apoptosis after combined treatment with adriamycin and siRNA targeting IGF1R. Our results demonstrate that IGF1R is downregulated by P53, and that siRNA targeting of IGF1R increases liver cancer cells sensitivity to adriamycin and promotes apoptosis. siRNA targeting of IGF1R could be potentially useful for increasing sensitivity to anti-cancer drugs, especially in drug-resistant cells with mutated P53.

[Changes in intrahepatic portal systemic shunt flow in a rat model of acute intrahepatic presinusoidal portal hypertension].

OBJECTIVES: To investigate the changes in intrahepatic portal systemic shunt flow (IHSF) and their relationship with microspheres induced acute portal hypertension. METHODS: Following acute intrahepatic presinusoidal obstruction by intraportal injection of 15 microm diameter microspheres in male Wistar rats, functional hepatic blood flow (FHBF) and IHSF were determined by hepatic sorbitol uptake methods. The percentage of large shunts of diameter > 15 microm were estimated by intraportal injection of 51Cr labeled 15 mum diameter microspheres. RESULTS: In normal control rats, hepatic sorbitol uptake was 97.9%+/-0.5% and IHSF was negligible, with FHBF equaling total hepatic blood flow [(2.52 +/- 0.23) ml/min x 100 g body weight-1]. Microsphere injection decreased sorbitol uptake to 12.8% +/- 4.3% and further to 4.1% +/- 0.7% when followed by hepatic arterial ligation. In the latter two groups, IHSF (1.46 +/- 0.15 and 1.16 +/- 0.19 ml/min x 100 g body weight-1, respectively) was not significantly different from portal venous flow [(1.36 +/- 0.20) and (1.20 +/- 0.20) ml/min x 100 g body weight-1, respectively; t = 2.013 and t = 2.116]. Portal venous flow remained at 70% of basal values and portal venous pressure only increased by 50% from baseline. 51Cr labeled microsphere shunt fraction through large shunts (> 15 microm) was less than 1.0%. CONCLUSION: Intrahepatic portasystemic shunts in the normal rat liver predominantly have diameters less than 15 microm and, when activated by intraportal injection of microspheres, they divert up to 70% of portal venous blood flow away from hepatic sinusoids and thereby they reduce acute increases in portal venous pressure.

Hepato-cardiovascular response and its regulation.

AIM: To determine the possible existence of a hepato-cardiovascular response and its regulatory mechanism in normal rats. METHODS: Systemic hemodynamic changes following intraportal injection of latex microspheres were studied in two modified rat models of hepatic circulation, in which the extrahepatic splanchnic circulation was excluded by evisceration and the liver was perfused by systemic blood via either the portal vein (model 1) or hepatic artery (model 2) in vivo. RESULTS: In model 1, intraportal injection of two sized microspheres (15-mum and 80-mum) induced a similar decrease in mean arterial pressure, while extrahepatic portal venous occlusion induced an immediate increase in mean arterial pressure. In model 2, microsphere injection again induced a significant reduction in mean arterial pressure, aortic blood flow and aortic resistance. There were no significant differences in these parameters between liver-innervated rats and liver-denervated rats. The degrees of microsphere-induced reduction in mean arterial pressure (-38.1+/-1.9% in liver-innervated rats and -35.4+/-2.1% in liver-denervated rats, respectively) were similar to those obtained by withdrawal of 2.0 mL of blood via the jugular vein (-33.3+/-2.1%) (P>0.05). Injection of 2.0 mL Haemaccel in microsphere-treated rats, to compensate for the reduced effective circulating blood volume, led to a hyperdynamic state which, as compared with basal values and unlike control rats, was characterised by increased aortic blood flow (+21.6+/-3.3%), decreased aortic resistance (-38.1+/-3.5%) and reduced mean arterial pressure (-9.7+/-2.8%). CONCLUSION: A hepato-cardiovascular response exists in normal rats. It acts through a humoral mechanism leading to systemic vasodilatation, and may be involved in the hemodynamic disturbances associated with acute and chronic liver diseases.

A new rat model of portal hypertension induced by intraportal injection of microspheres.

AIM:To produce a new rat model of portal hypertension by intraportal injection of microspheres.METHODS: Measured aliquots of single or different-sized microspheres (15,40,80&mgr;m) were injected into the portal vein to block intrahepatic portal radicals. The resultant changes in arterial,portal,hepatic venous and splenic pulp pressures were monitored.The liver and lungs were excised for histological examination.RESULTS: Portal venous pressure was elevated from basal value of 0.89-1.02 kPa to a steady-state of 1.98-3.19 kPa following the sequential injections of single or different-sized microspheres, with a markedly lowered mean arterial pressure. However, a small-dose injection of 80&mgr;m microspheres (1.8X10(5)) produced a steady-state portal venous pressure of 2.53 plus minus 0.17 kPa,and all rats showed normal arterial pressures. In addition, numerous microspheres were found in the lungs in all experimental groups.CONCLUSION: Portal hypertension can be reproduced in rats by intraportal injection of microspheres at a small dose of 80&mgr;m (1.8X105).Intrahepatic portal-systemic shunts probably exist in the normal rat liver.