Objective To investigate the expressions of platelet derived growth factor (PDGF) and survivin in hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT) and to evaluate the relationships among the expressions of PDGF, survivin and the proliferation of cancer cells. Methods The expression of PDGF mRNA in 16 cases in HCC with PVTT group was observed by in situ hybridization and the results were compared with that in HCC tissue group. The expressions of PDGF and survivin protein in 36 cases in HCC with PVTT group were detected with immunohistochemistry and it was found that there was a correlation between them. Flow cytometry (FCM) was used to measure the proliferation of cancer cells and it was also used to analyze the relations among PDGF, survivin and the proliferation of cancer cells. Results The expression level of PDGF mRNA in HCC with PVTT group was significantly higher than that in HCC tissue group (P＜0.01). There was a positive correlation between the expressions of PDGF and survivin protein in HCC with PVTT (P＜0.01). The degree of proliferation of cancer cells in PDGF and survivin protein positive group was significantly higher than that in negative group (P＜0.01). Conclusion PDGF and survivin gene over-expressed in HCC with PVTT group and there is a positive correlation between the expressions of PDGF and survivin protein. The proliferation of cancer cells increases as the expressions of PDGF and survivin increase.
Objective To construct the responsive plasmid PTRE-HIF-1αof Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the cDNA of HIF-1α, which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid PTRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing. The responsive plasmid PTRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid PTRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2Tet-on cells.
【 Abstract 】 Objective To observe the effect of disruption of hypoxia inducible factor-1 α (HIF-1 α ) pathway by small hairpin RNA (shRNA) on chemosensitivity of human hepatocellular carcinoma (HCC) cells and to reveal the correlative mechanisms. Methods Plasmid of pshRNA-HIF-1α was transfected into HepG2 cells by lipofectamine. HepG2/pshRNA-HIF-1α (HepG2/pshRNA) cell lines were obtained by selection of HepG2 cells in G418. Meanwhile, plasmid of empty vector (pHK) was transfected as a control (HepG2/pHK). The mRNA and protein expression levels of HIF-1α and mdr1 were investigated by RT-PCR and Western blot respectively. Using CoCl2 to simulate the hypoxia condition, growth inhibition and apoptosis rates of HepG2 cells under different dosages of chemotherapeutic agents (adriamycin) were measured by MTT assay and flow cytometry (FCM) ． ResultsCompared with HepG2/pHK cells, the mRNA and protein expression levels of HIF-1αand mdr1 were obviously down-regulated in HepG2/pshRNA cells. At the same time, the proliferation inhibition and apoptosis rates were evidently increased after transfection with pshRNA-HIF-1α(P＜0.05)，which decreased the expression of HIF-1αto 82.18% at mRNA level and 75.51% at protein level. There was no significant effect of transfection pHK (Pgt;0.05). Conclusion These data demonstrates that HIF-1α interference by shRNA increased the sensitivity of HCC chemotherapy and the reversal of multidrug resistance, which may be done by down-regulating the transcription of mdr1 and the translation of P-gp. Blocking HIF-1αin HCC cells may offer an new avenue for gene therapy．
Objective To establish a VX2 liver tumor model in rabbits under guidance of ultrasound mini-invasively, and to evaluate the imaging characteristics of VX2 liver tumor on ultrasound, CT scanning and angiography，respectively． Methods VX2 tumor tissues that were planted intramuscularly in the rabbit’s hind leg, were resected and cut into small pieces in 0.5 mm×0.5 mm×0.5 mm, and were inoculated into the left lobes of livers in 60 New Zealand rabbits under the guidance of ultrasound. The achievement ratio of the inoculated tumors growing in the rabbit livers were measured, and the imaging characteristics of the tumors on ultrasound, CT scanning and angiography were observed and then were evaluated 2 weeks later. Results The achievement ratio of establishing a liver tumor model under the guidance of ultrasound was 95% （57/60）. The average physio-life span of the model rabbits was (45±8) d. Ultrasound showed that the tumor in the rabbit liver was a single spherical or sphere-alike hypoechoic nodus, and there were higher blood flow signals in and around the nodus. VX2 tumor in the liver appeared as a solitary low density nodus on unenhanced CT scanning, and the hepatic arteriography showed that the tumor was rich of blood vessels, which built the disordered vasoganglion. Tumor was maily stained at the edge of the nodus. Conclusion It may be simple and effective to estabish a rabbit liver tumor model by inoculating VX2 tumor tissue under the guidance of ultrasound and the achievement ratio of such method was relatiely high. And the imaging characteristics of the tumor was similar to that of human primary liver carcinoma.