Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liv...Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.展开更多
Objective:To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7.Methods:Hepatocellular carcinoma Huh7 was cultured in vitro and lipido...Objective:To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7.Methods:Hepatocellular carcinoma Huh7 was cultured in vitro and lipidosome was used to transfect miR-520c-3p and miR-132 respectively or together.The effects of transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of Huh7 were detected by CCK8 and Annexin V staining and flow cytometry,and the expression level of the targeted gene of over-expressed miR-520c-3p and miR-132 was determined by Western blot and realtime PCR.Results:Compared with the control group,the proliferation ability of Huh7 of the single transfected and co-transfected miR-520c-3p and miR-132 decreased significantly,and the apoptosis ratio increased distinctly(P<0.05).Besides,the affect of the co-transfection group was better than that of the single transfection group.The protein levels of GPC3(Glypican-3) and YAP(Yes-associated protein),the target genes transfected only by miR-520c-3p and miR-132,respectively,reduced obviously(P<0.05),which was similar with the co-infected cells,but cells transfected by miR-132 only showed a decrease of YAP.Conclusions:The co-transfection of miR-520c-3p and miR-132 can target-regulate the expression of GPC3 and YAP,enhance the exhibition effect on proliferation of hepatocellular carcinoma Huh7 and induce cell apoptosis synergistically.展开更多
Objective: To study the effect of lipoteichoic acid(LTA) and 5-FU on the expression of caspase-3, EGFR, TGF-α proteins of tumor tissue of H22 cancer bearing mice and its antitumor mechanism. Methods: A total of 40 SP...Objective: To study the effect of lipoteichoic acid(LTA) and 5-FU on the expression of caspase-3, EGFR, TGF-α proteins of tumor tissue of H22 cancer bearing mice and its antitumor mechanism. Methods: A total of 40 SPF grade Kunming mice were selected to establish H22 liver cancer model, and then the mice were divided into 4 groups at random with ten mice in each group. Group A was given saline lavage treatment, Group B was treated with 5-FU by intraperitomeal injection, Group C was treated with LTA by lump body injection; Group D was treated with LTA by lump body injection and 5-FU by intraperitomeal injection. Two weeks after the treatment, the mice in each group were executed and the tumor tissue was stripping and weighted, and the tumor growth inhibition ratio was calculated. Then the tumor tissue was processed for conventional embedding, sectioned to observe the expression of caspase-3, EGFR, TGF-α by immunohistochemical staining method. Results: The tumor inhibitory rate o f Group D was significantly higher than Groups B and C(P<0.05); B, the tumor inhibitory rate o f Group B had no statistical difference compared with Group C(P>0.05). The IDO values of TGF-α, EGFR proteins in Groups B, C, D mice tumor tissue were significantly lower than that in group A(P<0.05); while IDO value of caspase-3 in Groups B, C, D group mice tumor tissue was significantly higher than that in Group A(P<0.05). The IDO value of TGF-α, EGFR in Group D mice tumor tissue were significantly lower than that in Groups B and C; While IDO value of aspase-3 in Group D was significantly higher than that in Groups B and C(P<0.05). Conclusions: LTA combined with 5-FU can effectively inhibit the tumorigenesis of H22 tumor bearing mice, increase the caspase-3 protein expression, inhibit TGF –α and EGFR protein expression, further promote tumor cell apoptosis and play a synergistic antitumor effect.展开更多
Objective:To study the change of the peripheral blood immune pattern and its correlation with prognosis in patients with liver cancer after treated by sorafenib.Methods:Patients with advanced liver cancer admitted in ...Objective:To study the change of the peripheral blood immune pattern and its correlation with prognosis in patients with liver cancer after treated by sorafenib.Methods:Patients with advanced liver cancer admitted in our hospital were enrolled and treated with sorafenib.After two months of the treatment,their peripheral blood was collected.The immune cell subset and cytokines level were determined by flow cytometry and luminex technology.According to the reaction expressed by patients towards sorafenib,patients were divided into the response group and the no response group.The changes of the peripheral blood immune pattern and its correlation with prognosis of patients in the two groups were compared.Results:Before and after treatment of sorafenib,there was no significant difference in the ratios of T cells,NK cells and their subtypes in peripheral blood of patients between the two groups;while after treatment the ratio of B cells and regulatory B cells(Breg) of patients in the response group was significant higher than that of the no response group(P<0.05),and the prognosis conditions of patients with decreased ratio of Breg cells were better than other patients after undergoing chemotherapy.The levels of plasma cytokines IL-6,IL-10,IL-12,IL17,FIL-3L,IFN- γ,TNF-α,MCP-1 and VEGF showed no significant differences.Conclusions:After treatment of sorafenib,the prognosis conditions of patients of advanced liver cancer with a reduced Breg ratio are better than patients with an unaltered or increased Breg ratio.The ratio of Breg in peripheral blood may be considered as early biological indicator for the prediction of the curative effects of sorafenib.展开更多
基金supported by Wuhan Municipal Science and Technology Bureau of applied basic research project(No.2013062301010823)Wuhan City health planning medieal research project(No.WX14A11)
文摘Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.
基金supported by Supported by Education Department of Hubei Province science and technology research project(NO.B2015230)Applied Fundamental Research Project of Wuhan Municipal Science and Technology Bureau(Grant No.2015061701011630)+1 种基金Medical Scientific Research Project of Health and Familly Planning Commission of Wuhan Municipality(Grant No.WX16E12)the fourth batch of "Hanyang Talent Associate Program"
文摘Objective:To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7.Methods:Hepatocellular carcinoma Huh7 was cultured in vitro and lipidosome was used to transfect miR-520c-3p and miR-132 respectively or together.The effects of transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of Huh7 were detected by CCK8 and Annexin V staining and flow cytometry,and the expression level of the targeted gene of over-expressed miR-520c-3p and miR-132 was determined by Western blot and realtime PCR.Results:Compared with the control group,the proliferation ability of Huh7 of the single transfected and co-transfected miR-520c-3p and miR-132 decreased significantly,and the apoptosis ratio increased distinctly(P<0.05).Besides,the affect of the co-transfection group was better than that of the single transfection group.The protein levels of GPC3(Glypican-3) and YAP(Yes-associated protein),the target genes transfected only by miR-520c-3p and miR-132,respectively,reduced obviously(P<0.05),which was similar with the co-infected cells,but cells transfected by miR-132 only showed a decrease of YAP.Conclusions:The co-transfection of miR-520c-3p and miR-132 can target-regulate the expression of GPC3 and YAP,enhance the exhibition effect on proliferation of hepatocellular carcinoma Huh7 and induce cell apoptosis synergistically.
基金supported by Applied Basic Research Program,Science and Technology Bureau of Wuhan City,(No.2013062301010823)Medical Care and Science Research Project of Health and Family Planning Commission of Wuhan(No.WX14A11,WX15D26)The third group of"Hanyang Talents’Plan"
文摘Objective: To study the effect of lipoteichoic acid(LTA) and 5-FU on the expression of caspase-3, EGFR, TGF-α proteins of tumor tissue of H22 cancer bearing mice and its antitumor mechanism. Methods: A total of 40 SPF grade Kunming mice were selected to establish H22 liver cancer model, and then the mice were divided into 4 groups at random with ten mice in each group. Group A was given saline lavage treatment, Group B was treated with 5-FU by intraperitomeal injection, Group C was treated with LTA by lump body injection; Group D was treated with LTA by lump body injection and 5-FU by intraperitomeal injection. Two weeks after the treatment, the mice in each group were executed and the tumor tissue was stripping and weighted, and the tumor growth inhibition ratio was calculated. Then the tumor tissue was processed for conventional embedding, sectioned to observe the expression of caspase-3, EGFR, TGF-α by immunohistochemical staining method. Results: The tumor inhibitory rate o f Group D was significantly higher than Groups B and C(P<0.05); B, the tumor inhibitory rate o f Group B had no statistical difference compared with Group C(P>0.05). The IDO values of TGF-α, EGFR proteins in Groups B, C, D mice tumor tissue were significantly lower than that in group A(P<0.05); while IDO value of caspase-3 in Groups B, C, D group mice tumor tissue was significantly higher than that in Group A(P<0.05). The IDO value of TGF-α, EGFR in Group D mice tumor tissue were significantly lower than that in Groups B and C; While IDO value of aspase-3 in Group D was significantly higher than that in Groups B and C(P<0.05). Conclusions: LTA combined with 5-FU can effectively inhibit the tumorigenesis of H22 tumor bearing mice, increase the caspase-3 protein expression, inhibit TGF –α and EGFR protein expression, further promote tumor cell apoptosis and play a synergistic antitumor effect.
基金supported by the Applied Basic Research Project of Wuhan Municipal Science and Technology Bureau(Grant No.2015061701011630,NO.2013062301010823)Medical Scientific Research project of the Education Department of Hubei Province(Grant No.B2015230)the fourth batch of Hanyang Concert Talent Plan
文摘Objective:To study the change of the peripheral blood immune pattern and its correlation with prognosis in patients with liver cancer after treated by sorafenib.Methods:Patients with advanced liver cancer admitted in our hospital were enrolled and treated with sorafenib.After two months of the treatment,their peripheral blood was collected.The immune cell subset and cytokines level were determined by flow cytometry and luminex technology.According to the reaction expressed by patients towards sorafenib,patients were divided into the response group and the no response group.The changes of the peripheral blood immune pattern and its correlation with prognosis of patients in the two groups were compared.Results:Before and after treatment of sorafenib,there was no significant difference in the ratios of T cells,NK cells and their subtypes in peripheral blood of patients between the two groups;while after treatment the ratio of B cells and regulatory B cells(Breg) of patients in the response group was significant higher than that of the no response group(P<0.05),and the prognosis conditions of patients with decreased ratio of Breg cells were better than other patients after undergoing chemotherapy.The levels of plasma cytokines IL-6,IL-10,IL-12,IL17,FIL-3L,IFN- γ,TNF-α,MCP-1 and VEGF showed no significant differences.Conclusions:After treatment of sorafenib,the prognosis conditions of patients of advanced liver cancer with a reduced Breg ratio are better than patients with an unaltered or increased Breg ratio.The ratio of Breg in peripheral blood may be considered as early biological indicator for the prediction of the curative effects of sorafenib.
