In order to construct plasmid of hypoxia-inducible factor-1alpha (HIF-1alpha), and transfect into human lung cancer cells A549, the change in sensitivity of lung cancer cells A549 to chemotherapy was observed. HIF-1alpha mRNA structure region was amplified by RT-PCR and inserted into plasmid pcDNA3. The expression plasmid pcDNA3/HIF-1alpha was transfected into A549 with Lipofec-tAMINE2000. The expression of HIF-1alpha protein was detected by Western blot. After A549 cells were transfected with HIF-1alpha prior to addition of 5-Fu, the growth activity was measured by growth curve, apoptosis was detected by flow cytometry at 48 h, and the levels of caspase3 and MDR-1 were determined by Western blot. The results showed that the constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two DNA lanes at 2.55 kb and 5.4 kb respectively were found, which were consistent with that expected. The growth rate in 5-Fu group was significantly inhibited, and the apoptosis index and caspase3 activity were increased significantly as compared with control group. After HIF-1alpha being transfected into A549, the activity of MDR-1 was increased and the effect of 5-Fu was weakened. In conclusion, HIF-1alpha can promote chemoresistance by increasing the activation of MDRI and suppressing apoptosis during lung cancer cells A549 induced with 5-Fu.

To study the role and mechanisms of hypoxia-inducible factor-1alpha (HIF-1alpha) on the growth and tumorigenicity of lung cancer cells A549, the antisense oligonucleotide of HIF-1alpha was transfected to A549 cells. The effect of the antisense oligonucleotide on tumor growth in vitro and in vivo was evaluated by the growth rate suppression of A549 cells and subcutaneous implanted tumor in nude mice, and the effect on tumorigenicity was evaluated by the expression inhibition of angiogenic factors, the microvessel density (MVD) and vascular endothelial growth factor (VEGF) protein expression which were detected by immohistochemistry and western blot respectively. This study revealed that in vitro the growth rate of antisense oligonucleotide group was significantly decreased as compared with that of control group, sense oligonucleotide group and false-sense oligonucleotide group; in vivo the weight of implanted tumors in nude mice of antisense oligonucleotide group was 1.51 +/- 0.40 g, which was significantly lower than that of control group (2.79 +/- 0.33 g), sense oligonucleotide group (2.81 +/- 0.45 g) and false-sense oligonucleotide group (2.89 +/- 0.39 g) and the inhibitory rate was 47%. Both MVD and VEGF protein expression were significantly inhibited in antisense oligonucleotide group compared with those in other groups. These results indicated that antisense oligonucleotide of HIF-1alpha could inhibit lung cancer cells A549 growth in vitro and in vivo, and the mechanism may be due to the inhibition of vascular growth and VEGF protein expression.

Objective To evaluate the usefulness of the "inserting" bilio-jejunostomy for iatrogenic bile duct injury. Methods A retrospective analysis was performed on 27 cases with iatrogenic bile duct injury treated by the "inserting" bilio-jejunostomy in the past 11 years. Results Among the 27 patients, bile duct injury was caused by open cholecystectomy in 21 and by laparoscopic cholecystectomy in 6. The diameter of injuried bile ducts in all patients was less than 1 centimeter (0.3～0.8 cm ). The end of remnant bile duct 2～3 cm in length was completely inserted into the jejunum loop in 10 cases, while the anterior wall of the remnant bile duct inserted into jejunum loop with the posterior wall anastomosed with the jejumum conventionally in 17 cases. There was no biliary fistula nor stenosis occurred after the operation. Conclusions The "inserting" bilio-jejunostomy is a simple,safe and useful method, and this new surgical technique should be applied to patients with iatrogenic bile duct injury when bilio-enteric reconstruction was needed.

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

Objective To study the effect of Fungi of Huai Er on human umbilicus vein endothelial cell((HUVEC)) and its inhibiting effect on liver cancer formation by liver cancer cells implanted under nude mice(subcutaneous) tissue, and to study the possible mechanism. Methods HUVEC was cultured with different concentrations of fungi of Huai Er and/or mitomycin(MMC) to observe the effect of Fungi of Huai Er on(HUVEC)′s hyperplasia , motility and adherence ability, and on the generation of blood vessels.In addition,the effect the Fungi of Huai Er on liver(cancer) formation by liver cancer cells implanted in nude mice(subcutaneous) tissue was also observed. Results Fungi ≥2mg/ml, can apparently reduce the hyperplasia(ability),inhibit motility and adherence ability of HUVEC,and the formation of blood vessels. The strongest effect on inhibiting the subcutaneous tumor formation was Fungi(3g/kg)plus MMC(500?g/kg),followed by MMC,and Fungi(3g/kg). Conclusions Possible mechanism of the inhibiting effect of Fungi on liver(cancer) is that the Fungi inhibits the(hyperplatic) ability, motility and adherence ability of HUVEC, and this(inhibits) the growth of blood vessels. Therefore, the growth of blood vessels is reduced and the development of liver cancer is suppressed.

Objective To study the relationship between Th1/Th2 cytokine profiles and the survival time of cardiac allografts in mice.Methods The ventral heterotopic cardiac transplantation models were divided into three groups: rejection group, treated group, isograft group, each group with 20 recipients. Mean survival time(MST), pathologic histological changes, the mRNA expression of IFN-?,IL-2,IL-4 and IL-10 were measured.Results MST of heart allografts in rejection and treated group was (7.8?0.77)d, and (14.80?1.01)d respectively. The survival time of grafts in isograft group were all more than 28d. The difference among the three groups was significant. In rejection group, the number of infiltrating cells was much more than that in the other groups, and also, the extent of pathologic histological changes was more severe. The mRNA expression of IFN-? and IL-10 in graft and spleen in rejection group was much stronger than that in the others. There was no obvious mRNA expression of IL-2 and IL-4 in grafts of all three groups. The mRNA expression of IL-2 in the spleens of rejection group was the strongest, while the mRNA expression of IL-4 in the spleens of treated group was the strongest.Conclusions The dynamic equilibrium of Th1/Th2 cytokines plays an important role in the survival time prolongation of cardiac allografts in mice. IL-10 can also participate in the process of graft rejection.

Objective To study the role of PPARa gene in human hepatocellular carcinoma. Methods Transmission elctron microscopy was used to study the morphologic differences between the MDR cell line HepG2/ADM and its mother cell line HepG2;Real-time RT-PCR and Western blotting were used to check the expression of PPARa in mRNA level and protein level in the two cell lines respectively. Results Transmission elctron microscopy showed that the HepG2/ADM cells had more viliformed maculas on their nuclear memberanes, more vacuoles in their cytoplasmas, more rough endoplastic reticulums; the PPARa gene was down regulated in the HepG2/ADM cells. Conclusion PPARa is related with the multidrug resistance phenomenon in the HepG2/ADM cells. The down regulation of PPARa may be one of the mechanisms conferring the forming of MDR in tumor cells.

Objective To study the effects of HIF-1α expression on the growth of subcutaneously implanted human hepatocellular carcinoma (HCC) cell lines in nude mice. Methods A nude mouse model of subcutaneously implanted HCC cell line HepG2Tet-on-HIF-1α was established. This cell lines characterizes the inducible expression of HIF-1α by doxycycline (Dos). The impact of HIF-1α expression induced by Dox on the growth of subcutaneously implanted HCC cell lines in the nude mouse model was observed. Results The mRNA and protein expression of HIF-1α were significantly up-regulated in the nude mouse model by oral administration of Dox. Compared with that model in which Dox was not administrated ie Dos( - ) group, the tumor volume(513.545 ± 276. 229) mm3 vs. ( 166. 506 ± 110. 142) mm3 ( P < 0. 05 ), tumor weight ( 1.251 ± 0. 438 ) g vs. (0. 640 ± 0. 296) g ( P < 0. 05 ), and tumor growth velocity were significantly enhanced in Dox ( + ) group, while tumor necrosis was inhibited ( 31. 360% ±2. 728% vs. 36. 640% ± 3. 804% ) (P<0. 05). The weight loss of nude mice was larger in Dox( + )group( P < 0. 01 ). There was no liver or lung metastasis in either group. Conclusion The expression of HIF-1αin subcutaneously implanted HCC in a nude mouse model is up-regulated by oral Dox. High grade expression of HIF-1α promotes the growth of implanted HCC.

To investigate the expression of KLF6mRNA in primary hepatocellular carcinoma (HCC), nomal liver tissues and the tissues adjacent to the cancers, reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of the KLF6 gene in HCC, the corresponding adjacent non-cancerous tissues and normal liver tissue. Our results showed that an amplified fragment of 427 bp DNA was detected in 18 of 19 (94.7%) adjacent non-cancerous tissues and normal liver tissue, and in 12 (85.7%) of 14 HCC. There were no significant differences in the levels of KLF6 mRNA between normal liver and liver tumors (P>0.05). It is concluded that KLF6 mRNA is generally expressed in HCC.
Carcinoma, Hepatocellular/*metabolism ; Kruppel-Like Transcription Factors/*biosynthesis ; Kruppel-Like Transcription Factors/genetics ; Liver/metabolism ; Liver Neoplasms/*metabolism ; Proto-Oncogene Proteins/*biosynthesis ; Proto-Oncogene Proteins/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction

Objective To investigate the effects of HBx on proliferation and apoptosis of human hepatocellular carcinoma cell line HepGz in vitro and in vivo. Methods The expression plasmids of pHA-HBx encoding full length of HBx gene was transfected into HepG2 cells with Lipofectamine 2000. The proliferation activity of transformed cells was measured by calculating the growth curve,population doubling time and by the 3H-TdR incorporation rate assay. The nude mouse model of HepG2 expressing HBX gene was established and the apoptosis rate of tumor cells was detected with TUNEL assay. Results RT-PCR indicated that the HBx gene was integrated into the genome DNA of HepG2 cells and transcripted. The growth curve and population doubling time manifested that the proliferation activity of transformed cells was much higher than that of control group cells. The apoptosis rate of tumor cells expressing HBx gene was much lower than that of control group cells. ConclusionHBx facilitates the proliferation activity of hepatoma cells in vitro and in vivo and inhibits the apoptosis of hepatoma cells induced by adriamycin in nude mice.