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Objective To explore the mechanism of melanoma differentiation associated gene-7(mda-7) in combination with adriamycin(ADM) killing the HCC HepG2 cells and reversing their multidrug resistance (MDR). Methods The experiment was conducted in three groups including the combined group, ADM group and mda-7 group. MTT assay and FCM were used to determine the differences among the 3 groups and clarify the reversing effect of combined treatment on multidrug resistance of the tumor cells. Expression levels of MDR-1, STAT-3, BCL-2, BAXmRNA were determined with real-time PCR. Western blotting was performed to observe the changes of proteins gp-l70, stat3,P-stat3, PKB, bcl-2,bax in all 3 groups. Result After transfection with 100VP/cell Ad. mda-7,the growth suppression rate of HepG2 treated by ADM (1.5 mg/L) rose from 17.46% to 79. 5%.According to the changes, killed HepG2 cells were increased by a factor of 4.55. times. MDR-1 mRNA was decreased from (16.49 ± 0. 11) to (5.48±0.05) and STAT-3 mRNA increased from (13.17±0. 08) to (21. 57±0. 11)(P＜0.05). Western blotting also showed that P-170 and PKB was decreased and the phosphorylation-stat-3 increased after the combined treatment. Conclusion Ad.mda-7 can reverse the multidrug resistance HepG2 cells. It inhibits the expression of MDR-1 mRNA,then arrests PKB protein and the signaling pathway of active stat-3 to induce apoptosis of HCC cells.
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<p><b>OBJECTIVE</b>To study the changes of immune function in liver cirrhosis patients after splenectomy combined with resection of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Sixteen patients with HCC associated with liver cirrhosis were divided into two groups: splenectomy combined with hepatectomy (n = 7) and hepatectomy (n = 9). T-lymphocyte subsets such as CD4, CD8, CD4/CD8 and Th-lymphocyte cytokines such as IFN-gamma, IL2, IL10 in 7 ml peripheral venous blood before operation and two months after operation were examined and compared between the two groups.</p><p><b>RESULTS</b>There was no significant difference in pre-operative CD4, CD4/CD8, IL2, IFN-gamma, IL10 levels in the two group. Two months after operation, the levels of CD4 (38.2% +/- 3.7%), CD4/CD8 (1.7 +/- 0.3), IFN-gamma [(104.4 +/- 14.9) pg/ml], IL2 [(98.6 +/- 18.6) pg/ml] were increased and those of CD8 (23.7 +/- 13.7) pg/ml and IL10 [(55.5 +/- 11.2) pn/ml] were decreased in the two groups, but the changes in the group of splenectomy combined with hepatectomy were more obvious than those in the hepatectomy group. The levels of CD4 (32.5% +/- 4.0%), CD4/CD8 (1.1 +/- 0.1), IFN-gamma [(70.5 +/- 12.6) pg/ml], IL2 [(80.9 +/- 13.5) pg/ml] in the group of splenectomy combined with hepatectomy, were much higher than those in the hepatectomy group; those of CD8 (29.4% +/- 4.0%), IL10 [(89.4 +/- 10.0) pg/ml] level were significantly lower than those in the hepatectomy (P < 0.05).</p><p><b>CONCLUSION</b>Splenectomy combined with hepatectomy for HCC associated with liver cirrhosis donor decrease but promote the recover T-lymphocyte subsets and Th1/Th2 cytokines from imbalance and improve the patient's antitumor immune function.</p>
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Humans , CD4 Lymphocyte Count , CD8-Positive T-Lymphocytes , Allergy and Immunology , Carcinoma, Hepatocellular , Allergy and Immunology , General Surgery , Interferon-gamma , Metabolism , Interleukin-10 , Metabolism , Interleukin-2 , Metabolism , Liver Cirrhosis , Allergy and Immunology , General Surgery , Liver Neoplasms , Allergy and Immunology , General Surgery , SplenectomyABSTRACT
To study the therapeutic effects of herpes simplex virus thymidine kinase (HSV-TK) gene transferred by the EBV-based expression vector (pDR2) on experimental hepatocellular carcinoma, pDR2-TK gene was delivered into human hepatocellular carcinoma cell line SMMC-7721 by using liposome-mediated transfection technique，and then gene expression was detected by RT-PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model，the antitumor effects of pDR2-TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2-TK /GCV had cytotoxic effect and about 70 % SMMC-7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2-TK gene was significantly smaller than that in control group (P<0.01). On the 10th day the tumor in 3 mice (60 %） disappeared completely after GCV treatment. It is concluded that the pDR2-TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.
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To study the therapeutic effects of herpes simplex virus thymidine kinase (HSV-TK) gene transferred by the EBV-based expression vector (pDR2) on experimental hepatocellular carcinoma, pDR2-TK gene was delivered into human hepatocellular carcinoma cell line SMMC-7721 by using liposome-mediated transfection technique，and then gene expression was detected by RT-PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model，the antitumor effects of pDR2-TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2-TK /GCV had cytotoxic effect and about 70 % SMMC-7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2-TK gene was significantly smaller than that in control group (P<0.01). On the 10th day the tumor in 3 mice (60 %） disappeared completely after GCV treatment. It is concluded that the pDR2-TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.
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ObjectiveTo study the changes of immune function after hepatectomy for hepatocellular carcinoma(HCC) in patients with liver cirrhosis.MethodsPatients with liver cirrhosis were used as control group,CD4?CD8?CD4/CD8? and protein expression level of IFN ??IL2?IL10 in peripheral blood from 18 advanced HCC cases with liver cirrhosis before and after heaptectomy were detected by flow cytometry(FCM) and ELISA.Results Postoperative CD4(33?3)%?CD4/CD8(1 1?0 1)?IL2(71?11)?pg/ml?IFN ?(90?15)?pg/ml level elevated compared to that of pre operation 〔CD4(29?4)%?CD4/CD8(0 9?0 3)?IL2(57?15)?pg/ml?IFN ?(78?13)?pg/ml〕(all P
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0.05). Second and third generation of the implanted tumor were obtained in 8 nude mice respectively,and all transplanted tumors survived. Liver,bone and lung metastasis were found in 100% of nude mice between 10 w～14 w. [WT5”HZ] Conclusions This nude mouse HCC model holds the biologic features of human HCC and serves the purpose of in vivo study investigation .
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Objective To investigate the reversal of MDR by using anti-MDR1 ribozyme N2A + tRNAimet-iMDR1- sRz (sRz) in nude mice bearing human hepatocellular carcinoma. Methods The nude mice model with implanted human hepatocellular carcinoma was randomly devided into group A ( saline 40?l + Lipofect AMINE?000 10?l), B( N2A + tRNAimet10?g/40?l + Lipofect AMINE?000 10?l) and C(sRz 10?g/40?l + Lipofect AMINE?000 10?l). After one week, mice was peritoneally injected E-ADM 15 mg?kg-1 once a week for 4 weeks. The size of tumors was measured with B-ultrasound and the tumor inhibition rate was calculated. One week after chemotherapy mice was sacrificed and MDR1 mRNA and P-gp were investigated with RT-PCR and Western blot. Results In group C tumor shrank upon each chemotherapy (F = 659. 99, P
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Objective To investigate the selective killing effect of MDA/IL-24 on human hepatocellular carcinoma line HepG2 in vitro and provide a theoretical basis for gene therapy of hepatocellular carcinoma.Methods The MDA-7/IL-24 gene was transfected into human hepatocullular carcinoma cell line HepG2 and normal liver cell line L02 with a replication-incompetent adenovirus vector.The mRNA and protein expression of MDA7/IL-24 in HepG2 and L02 cells was examined by RT-PCR and ELISA assay respectively.MTT assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro.Hoechst and Annexin-V and PI staining were studied to indicate the apoptosis.Results RT-PCR confirmed that the exogenous MDA-7/IL-24 gene was expressed in HepG2 and L02 cells.The protein product was confirmed by assay of the supernatant with ELISA.MTT and apoptosis test indicated MDA-7/IL-24 induced growth suppression and cell apoptosis of the HepG2 cell in vitro but not in cell line L02,and cell cycle test revealed MDA-7/IL-24 could block HepG2 cell in G2/M but not in L02.Conclusions MDA-7/IL-24 selectively induces growth suppression and apoptosis in lines HepG2 in vitro but not in L02 cell,which indicates that adenovirus mediated MDA-7/IL-24 can be an excellent tool for gene therapy in hepatocellular carcinoma.
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Objective To investigate the effects of radix salviae miltiorrhiaze(RSM) on the expression of Fas, perforin and bcl 2 mRNA in liver ischemia reperfusion(I R). Methods 45 patients were divided into three groups: Control group(group 1, n=5); I R group(group 2, n=20), and RSM pretreatment group(group 3, n=20). The patients in group 2 and group 3 subjected to hepatic resection with “Pringle procedure” for 15?min. Expression of Fas, perforin and bcl 2 mRNA were examined with reverse transcription polymerase chain reaction(RT PCR) after liver reperfusion for 40?min. Results Expression of Fas, perforin were very weak in control group(0.144?0.037; 0.106?0.032), but increased in group 2(0.928?0.135; 1.099?0.364); and in group 3 was significantly lower(0.347?0.081; 0.454?0.116)than that in group 2(P
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Objective To study T lymphocyte subsets immunological condition of spleen in primary hepatocellular carcinoma (PHC) with liver cirrhosis. Methods T lymphocyte subsets such as CD4,CD8,CD4/CD8 in peripheral and spleen venous blood in 31 cases of PHC with liver cirrhosis were detected by FCM.and 13 patients with liver cirrhosis were observed as the control group. Results In 6 patients with stage Ⅰ liver cancer, CD8 of peripheral venous blood significantly decreased (P
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ObjectiveTo study the clinical significance of splenectomy in cirrhotic patients undergoing hepatic resection for PLC.Methods26 PLC patients with portal hypertension and hypersplenism were randomly divided into two groups: incidental splenectomy group ( n =11), and hepatectomy only group ( n =15). Postoperative blood picture and liver function was compared.ResultsThe WBC and PLT count significantly elevated after splenctomy, (8 9?1 6)?10 9 /L, (310?32)?10 9 /L; vs. (3 7?1 4)?10 9 /L and (104?41)?10 9 /L respectively (all P