AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell pr...AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P【0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.展开更多
AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human...AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 micromol/L, 300 micromol/L, 400 micromol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%+/-1.62% vs 2.24%+/-0.26% and 21.23+/-1.78 vs 2.01+/-0.23 (P【0.05). CONCLUSION:The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 micromol and 400 micromol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.展开更多
AIM To determine the activities ofpolysaccharide extracts from Flammulina velutipes (Curt. ex Fr. ) Sing (FV), Lentinusedodes (LE) and Agaricus bisporus Sing (AB)on the proliferation of human hepatoma SMMC-7721 cells ...AIM To determine the activities ofpolysaccharide extracts from Flammulina velutipes (Curt. ex Fr. ) Sing (FV), Lentinusedodes (LE) and Agaricus bisporus Sing (AB)on the proliferation of human hepatoma SMMC-7721 cells in vitro and on mouse implanted S-180tumors in vivo.METHODS The polysaccharide extracts were isolated from the fruit bodies of FV, LE and AB by the methods of hot-water extraction, Sevag’sremoval of proteins, ethanol precipitation,trypsin digestion and ethanol fractionalprecipitation. Human hepatoma SMMC-7721 cells were treated with 50 mg/L Polysaccharide extracts, and the mitosis index, mitochondria activity and cell proliferation were detected at different times in both control and experimental groups. The mice with S-180 implanted tumors were injected with the polysaccharide extracts at 24 mg/ kg body weight for 9 d and the tumorweight was measured on the 15th day.RESULTS The mitosis index of hepatoma cells in vitro could be significantly decreased by treatment with the polysaccharide extracts fromthe three kinds of edible fungi (P < 0 .005 ). Thecell numbers and mitochondria activity of SMMC7721 cells treated with polysaccharide extracts were lower than those in control groups (P <0.005). The inhibition rates of polysaccharide extracts against implanted S-180 tumors in mice were 52.8%, 56.6% and 51 .9% respectivelycompared with that in c0ntrol gr0ups.CONCLUSI0N The POIysaccharide extractsfrom the three kinds of edible fungi could inhibitnot only the Cultured malignant cells in vitfO butalso impIanted Sl80 tum0r i0 vivo.展开更多
AIM To determine the relationship between serum deprivation or serum levels and cell proliferation of human hepatoma SMMC 7721 cells. METHODS Human hepatoma SMMC 7721 cells were grown in RPMI 1640 supplemented wi...AIM To determine the relationship between serum deprivation or serum levels and cell proliferation of human hepatoma SMMC 7721 cells. METHODS Human hepatoma SMMC 7721 cells were grown in RPMI 1640 supplemented with 10% fetal calf (FCS) in 5% CO 2 incubator at 37℃ for 24h , and culture media were replaced to serum free or different serum FCS levels (2 5%, 5%, 10%, 20% and 25%). Six h, 12h , 18h and 24h after the culture, the cells were incorporated TdR for 4h . At last TdR incorporation was detected with liquid scintillation counting. RESULTS DNA synthesis of SMMC 7721 cells could be sharply stimulated by short time (6h) serum deprivation (the cpm value of 3H TdR incorporation of cells in serum free was 39 32 fold higher than cells in 25% serum), and the incorporation of 3H TdR was negatively related to the serum levels. Longer time serum starvation ( 12h , 18h and 24h ) also greatly stimulated DNA synthesis, although the cpm value of 3H TdR incroporation was less than that in 6h serum deprivation. Morphology of cells cultured in different serum levels also showed significant difference. CONCLUSIONS Compared with other cell lines such as BEL7404 and Swiss 3T3, human hepatoma SMMC 7721 cells had different response to the serum deprivation. Short time serum deprivation could greatly stimulate DNA synthesis of human hepatoma SMMC 7721 cells. Precautions must be given to the changes of serum levels for the detection of growth factors and drugs using SMMC 7721 cells as a model.展开更多
AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patie...AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.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
文摘AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P【0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.
文摘AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 micromol/L, 300 micromol/L, 400 micromol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%+/-1.62% vs 2.24%+/-0.26% and 21.23+/-1.78 vs 2.01+/-0.23 (P【0.05). CONCLUSION:The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 micromol and 400 micromol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.
文摘AIM To determine the activities ofpolysaccharide extracts from Flammulina velutipes (Curt. ex Fr. ) Sing (FV), Lentinusedodes (LE) and Agaricus bisporus Sing (AB)on the proliferation of human hepatoma SMMC-7721 cells in vitro and on mouse implanted S-180tumors in vivo.METHODS The polysaccharide extracts were isolated from the fruit bodies of FV, LE and AB by the methods of hot-water extraction, Sevag’sremoval of proteins, ethanol precipitation,trypsin digestion and ethanol fractionalprecipitation. Human hepatoma SMMC-7721 cells were treated with 50 mg/L Polysaccharide extracts, and the mitosis index, mitochondria activity and cell proliferation were detected at different times in both control and experimental groups. The mice with S-180 implanted tumors were injected with the polysaccharide extracts at 24 mg/ kg body weight for 9 d and the tumorweight was measured on the 15th day.RESULTS The mitosis index of hepatoma cells in vitro could be significantly decreased by treatment with the polysaccharide extracts fromthe three kinds of edible fungi (P < 0 .005 ). Thecell numbers and mitochondria activity of SMMC7721 cells treated with polysaccharide extracts were lower than those in control groups (P <0.005). The inhibition rates of polysaccharide extracts against implanted S-180 tumors in mice were 52.8%, 56.6% and 51 .9% respectivelycompared with that in c0ntrol gr0ups.CONCLUSI0N The POIysaccharide extractsfrom the three kinds of edible fungi could inhibitnot only the Cultured malignant cells in vitfO butalso impIanted Sl80 tum0r i0 vivo.
文摘AIM To determine the relationship between serum deprivation or serum levels and cell proliferation of human hepatoma SMMC 7721 cells. METHODS Human hepatoma SMMC 7721 cells were grown in RPMI 1640 supplemented with 10% fetal calf (FCS) in 5% CO 2 incubator at 37℃ for 24h , and culture media were replaced to serum free or different serum FCS levels (2 5%, 5%, 10%, 20% and 25%). Six h, 12h , 18h and 24h after the culture, the cells were incorporated TdR for 4h . At last TdR incorporation was detected with liquid scintillation counting. RESULTS DNA synthesis of SMMC 7721 cells could be sharply stimulated by short time (6h) serum deprivation (the cpm value of 3H TdR incorporation of cells in serum free was 39 32 fold higher than cells in 25% serum), and the incorporation of 3H TdR was negatively related to the serum levels. Longer time serum starvation ( 12h , 18h and 24h ) also greatly stimulated DNA synthesis, although the cpm value of 3H TdR incroporation was less than that in 6h serum deprivation. Morphology of cells cultured in different serum levels also showed significant difference. CONCLUSIONS Compared with other cell lines such as BEL7404 and Swiss 3T3, human hepatoma SMMC 7721 cells had different response to the serum deprivation. Short time serum deprivation could greatly stimulate DNA synthesis of human hepatoma SMMC 7721 cells. Precautions must be given to the changes of serum levels for the detection of growth factors and drugs using SMMC 7721 cells as a model.
基金Suppprted by the Mational Natural Science Foundation of China,No.39670672.
文摘AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.
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.
