Objective To explore the effect of cyclopamine （Cyc） which is the inhibitor of the Hedgehog signaling pathway on portal venous pressure of normal and liver cirrhosis rats, and it’s possible mechanisms. Moreover, to provide the experimental basis of drug efficacy and clinical treatment. Methods Thirty two healthy male SD rats were randomly average divided into four groups：normal control group, normal treatment group, liver cirrhosis control group, and liver cirrhosis treatment group. The liver cirrhosis models of rat were established by using the thioacetamide （TAA） method, which made 0.03% of TAA as the initial water concentration, and then the concentration of TAA in drinking water was adjusted according to the changes of the weekly body weight of rats lasting for twelve weeks. In thirteenth week, intraperitoneal injection of corn oil （0.1 ml/100 g body weight, 1 time/d） were performed lasting for a week in rats of the normal control group and liver cirrhosis control group; intraperitoneal injection of Cyc 〔1 mg （0.1 ml）/100 g body weight, 1 time/d〕were performed lasting for a week in rats of the normal treatment group and liver cirrhosis treatment group. In fourteenth week, the liver function, portal venous pressure （PVP）, and the ration of liver or spleen weight to body weight were detected, the expressions of α-smooth muscle actin （α-SMA） and typeⅠcollagen α1 （Col1α1） of hepatic stellate cell were detected by using immunohistochemistry. Results PVP were （10.7±0.9） and （12.3±1.3） cm H2O （1 cm H2O=0.098 kPa） in normal control group and normal treatment group, respectivly, the latter was higher than the former （t=-2.918，P=0.011）. PVP were （21.8±0.7） and （14.3±1.4） cm H2O in liver cirrhosis control group and liver cirrhosis treatment group, respectivly, the latter was lower than the former（t=13.602，P=0.000）. The expressions of α-SMA and Col1α1 in liver cirrhosis treatment group was lower than the liver cirrhosis control group. There were no significant difference of the liver function and ration of liver or spleen weight to body weight between the treatment group and the control group （P＞0.05）. Conclusion Cyclopamine could signally reduce the PVP of liver cirrhosis rats through reducing the expressions of α-SMA and Col1α1.
Objective To establish hepatocellular carcinoma (HCC) cell lines which olig-expressed IGF1R gene stably． Methods An eukaryotic expressing vector pSUPER-IGF1R-siRNA that could block IGF1R expressing was transferred into hepatocellular carcinoma cell lines SMMC7721 and Hep3B with Lipofectamine 2000 reagents． After transferred, cells were selected with G418 to obtain positive clones. The expressions of IGF1R, cyclin D1 and cyclin B1 were detected by RT-PCR and Western-blot． Cell growth curve were painted. Results Two cell lines clones were screened olig-expressing IGF1R gene stably． The experimental cell lines grew more slowly than control cell lines and the expression of cyclin D1 decreased (P＜0.05). Conclusion The HCC cell lines for olig-expressing IGF1R gene stably are established successfully．The plasmid pSUPER-IGF1R-siRNA can inhibit the growth of SMMC7721 and Hep3B cell lines, and the expression of cyclin D1.
Objective To study the interferencing and anti-tumor effects of lentiviral vector of siRNA targeting IGF1R and EGFR gene of the liver cancer cell. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and connected to the pLVTHM vector, named pLVTHM-IGF1R, into whom the EGFR-siRNA expression frame containing H1 promotor synthesized by RT-PCR was cloned to generate pLVTHM-IGF1R-EGFR-siRNA. The 293T cells were cotransfected by 3 plasmids of pLVTHM-IGF1R-EGFR-siRNA, psPAX2 and pMD2G to enclose LVTHM-IGF1R-EGFR-siRNA, which was amplified in large amount and purified by caesium chloride density gradient centrifugation for measurement of virus titer. SMMC7721 cells infected by LVTHM-IGF1R-EGFR-siRNA were infection group, the untreated SMMC7721 cells and blank vector plasmid LVTHM were two control groups (SMMC7721 cell group and blank vector group). The effect of LVTHM-IGF1R-EGFR-siRNA on IGF1R and EGFR expressions of SMMC7721 cells were detected by RT-PCR and Western blot. The antitumor potential of LVTHM-IGF1R-EGFR-siRNA to SMMC7721 cells was evaluated by Cell Counting Kit-8 assay for cell growth and TUNEL for apoptosis respectively. Results LVTHM-IGF1R-EGFR-siRNA was constructed successfully. Functional pfu titers of LVTHM-IGF1R-EGFR-siRNA was 4.58×109 pfu/ml. Protein and mRNA expression of IGF1R and EGFR of infection group were less than those of blank vector group and SMMC7721 cell group （P＜0.05）, LVTHM-IGF1R-EGFR-siRNA was more effective to inhibit the proliferation and promote apoptosis of SMMC7721 cells （P＜0.05）. Conclusion LVTHM-IGF1R-EGFR-siRNA expressing IGF1R-EGFR-siRNA can inhibit the expression of IGF1R and EGFR, and may be used for further investigation of gene therapy of liver cancer.