BACKGROUND: The mortality rate of heavy type hepatitisis high. No special treatment is available except generaltreatment. This multicenter clinical study was designed toobserve the safety and efficacy of promoting hep...BACKGROUND: The mortality rate of heavy type hepatitisis high. No special treatment is available except generaltreatment. This multicenter clinical study was designed toobserve the safety and efficacy of promoting hepatic growthfactor (PHGF) in the treatment of heavy type hepatitis andsevere chronic hepatitis.METHODS: 347 patients with heavy type hepatitis and 324with severe chronic hepatitis were subjected to administra-tion of 120 μg of PHGF per day for 4 weeks on the basis ofgeneral treatment. Those who were being effectively treat-ed would last additional 2 to 4 weeks. Blood routine, urineroutine, blood urea nitrogen (BUN), blood creatinine(Cr), blood ammonia, alpha fetoprotein (AFP), electro-lyte, alanine transaminase (ALT), aspartate transaminase(AST), serum total bilirubin (TBIL), serum direct biliru-bin (DBIL), prothrombin time activity (PTA), total pro-tein (TP) and albumin (ALB) were detected in the pa-tients before treatment, 2 weeks after treatment, and at theend of the treatment. Any side-effect would be recorded.RESULTS: In the patients with severe chronic hepatitis, thetotal effective rate of the treatment was 88. 9% The levelsof ALT, AST and TBIL decreased significantly (P<0.001),whereas those of PTA and ALB increased significantly (P <0.001), and the level of AFP increased slightly. In patientswith heavy type hepatitis, the total effective rate of thistreatment was 78.4%, and patients at different stage showeddifferent results. The total effective rates of patients withearly, medium and terminal stage heavy type hepatitis were89.9%, 84.8% and 27.5%, respectively. No severe side-effect was shown.CONCLUSION: PHGF is effective and safe in the treat-ment of patients with heavy type hepatitis and severe chro-nic hepatitis. But it should be administered early in patientswith heavy type hepatitis so as to get better curative effects.展开更多
AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cass...AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.展开更多
AIM: To transfer human HGF gene into the liver of rats by direct electroporation as a means to prevent radiationinduced liver damage.METHODS: Rat whole liver irradiation model was accomplished by intra-operative appro...AIM: To transfer human HGF gene into the liver of rats by direct electroporation as a means to prevent radiationinduced liver damage.METHODS: Rat whole liver irradiation model was accomplished by intra-operative approach. HGF plasmid was injected into liver and transferred by electroporation using a pulse generator. Control rats (n = 8) received electrogene therapy (EGT) vehicle plasmid and another 8rats received HGF-EGT 100 μg 48 h before WLIR.Expression of HGF in liver was examined by RT-PCR and ELISA methods. Apoptosis was determined by TUNEL assay. Histopathology was evaluated 10 wk after whole liver irradiation.RESULTS: Marked decrease of apoptotic cells and downregulation of transforming growth factor-beta 1 (TGF-β1)mRNA were observed in the HGF-EGT group 2 d after liver irradiation compared to control animals. Less evidence of radiation-induced liver damage was observed morphologically in liver specimen 10 wk after liver irradiation and longer median survival time was observed from HGF-EGT group (14 wk) compared to control rats (5 wk). (P = 0.031).CONCLUSION: For the first time it has been demonstrated that HGF-EGT would prevent liver from radiation-induced liver damage by preventing apoptosis and down-regulation of TGF-β1.展开更多
Objective： The aim of the study was to investigate the interference and anti-tumor effects of lentiviral vector of miRNA targeting IGF1R gene regulated by survivin promoter. Methods： The fragment of the survivin pro...Objective： The aim of the study was to investigate the interference and anti-tumor effects of lentiviral vector of miRNA targeting IGF1R gene regulated by survivin promoter. Methods： The fragment of the survivin promoter was acquired by PCR amplification and inserted into pPRIME to recombinant plasmid sur-pPRIME. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the sur-pPRIME vector, named sur-pPRIME-IGF1R-miR30-shRNA. Viruses were propagated on 293T cells. Viruses were purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. The effect of sur-pPRIME-IGF1R-miR30-shRNA on IGF1R expression of Hep3B cells was detected by RT-PCR and Western blot. The antitumor potential of sur-pPRIME-IGF1R-miR30-shRNA to Hep3B cells was evaluated by CCK-8 assay. Results： sur-pPRIME-IGF1R-miR30-shRNA was constructed successfully. Functional PFU titers of sur-pPRIME-IGF1R-miR30-shRNA were 4.58×10^9 PFU/rnL. Sur-pPRIME-IGF1R-miR30-shRNA was more effective to inhibit IGF1R expression in mRNA or protein levels and the proliferation of Hep3B cells. Conclusion： sur-pPRIME-IGF1R-miR30-shRNA expressing IGF1R-siRNA can inhibit IGF1R expression and may be used for further investigation of gene therapy of liver cancer.展开更多
文摘BACKGROUND: The mortality rate of heavy type hepatitisis high. No special treatment is available except generaltreatment. This multicenter clinical study was designed toobserve the safety and efficacy of promoting hepatic growthfactor (PHGF) in the treatment of heavy type hepatitis andsevere chronic hepatitis.METHODS: 347 patients with heavy type hepatitis and 324with severe chronic hepatitis were subjected to administra-tion of 120 μg of PHGF per day for 4 weeks on the basis ofgeneral treatment. Those who were being effectively treat-ed would last additional 2 to 4 weeks. Blood routine, urineroutine, blood urea nitrogen (BUN), blood creatinine(Cr), blood ammonia, alpha fetoprotein (AFP), electro-lyte, alanine transaminase (ALT), aspartate transaminase(AST), serum total bilirubin (TBIL), serum direct biliru-bin (DBIL), prothrombin time activity (PTA), total pro-tein (TP) and albumin (ALB) were detected in the pa-tients before treatment, 2 weeks after treatment, and at theend of the treatment. Any side-effect would be recorded.RESULTS: In the patients with severe chronic hepatitis, thetotal effective rate of the treatment was 88. 9% The levelsof ALT, AST and TBIL decreased significantly (P<0.001),whereas those of PTA and ALB increased significantly (P <0.001), and the level of AFP increased slightly. In patientswith heavy type hepatitis, the total effective rate of thistreatment was 78.4%, and patients at different stage showeddifferent results. The total effective rates of patients withearly, medium and terminal stage heavy type hepatitis were89.9%, 84.8% and 27.5%, respectively. No severe side-effect was shown.CONCLUSION: PHGF is effective and safe in the treat-ment of patients with heavy type hepatitis and severe chro-nic hepatitis. But it should be administered early in patientswith heavy type hepatitis so as to get better curative effects.
基金Project supported by National Natural Science Foundation of China,No.863 Z2001-04
文摘AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.
基金Supported by National Science Council grant NSC-91-275-9075-001 for the development of Boron Neutron Capture Therapy for Hepatoma Treatment
文摘AIM: To transfer human HGF gene into the liver of rats by direct electroporation as a means to prevent radiationinduced liver damage.METHODS: Rat whole liver irradiation model was accomplished by intra-operative approach. HGF plasmid was injected into liver and transferred by electroporation using a pulse generator. Control rats (n = 8) received electrogene therapy (EGT) vehicle plasmid and another 8rats received HGF-EGT 100 μg 48 h before WLIR.Expression of HGF in liver was examined by RT-PCR and ELISA methods. Apoptosis was determined by TUNEL assay. Histopathology was evaluated 10 wk after whole liver irradiation.RESULTS: Marked decrease of apoptotic cells and downregulation of transforming growth factor-beta 1 (TGF-β1)mRNA were observed in the HGF-EGT group 2 d after liver irradiation compared to control animals. Less evidence of radiation-induced liver damage was observed morphologically in liver specimen 10 wk after liver irradiation and longer median survival time was observed from HGF-EGT group (14 wk) compared to control rats (5 wk). (P = 0.031).CONCLUSION: For the first time it has been demonstrated that HGF-EGT would prevent liver from radiation-induced liver damage by preventing apoptosis and down-regulation of TGF-β1.
基金Supported by the grants from the Education Departmental Natural Science Research Funds of Jiangsu Provincial Higher School of China(No.09KJB320018,No.09KJB310016)
文摘Objective： The aim of the study was to investigate the interference and anti-tumor effects of lentiviral vector of miRNA targeting IGF1R gene regulated by survivin promoter. Methods： The fragment of the survivin promoter was acquired by PCR amplification and inserted into pPRIME to recombinant plasmid sur-pPRIME. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the sur-pPRIME vector, named sur-pPRIME-IGF1R-miR30-shRNA. Viruses were propagated on 293T cells. Viruses were purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. The effect of sur-pPRIME-IGF1R-miR30-shRNA on IGF1R expression of Hep3B cells was detected by RT-PCR and Western blot. The antitumor potential of sur-pPRIME-IGF1R-miR30-shRNA to Hep3B cells was evaluated by CCK-8 assay. Results： sur-pPRIME-IGF1R-miR30-shRNA was constructed successfully. Functional PFU titers of sur-pPRIME-IGF1R-miR30-shRNA were 4.58×10^9 PFU/rnL. Sur-pPRIME-IGF1R-miR30-shRNA was more effective to inhibit IGF1R expression in mRNA or protein levels and the proliferation of Hep3B cells. Conclusion： sur-pPRIME-IGF1R-miR30-shRNA expressing IGF1R-siRNA can inhibit IGF1R expression and may be used for further investigation of gene therapy of liver cancer.
