BACKGROUND: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine ...BACKGROUND: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxification. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS: hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and amplified. Then hGS mRNA was measured by semi-quantitative RT-PCR; hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH(4)(+) were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS: The expression of hGS mRNA in HepG2/pLNChGS cells (8.306+/-0.336) and HepG2/pLNAFhGS cells (21.358+/-1.716) was much stronger than in control cells (P<0.05), and that in HepG2/pLNAFhGS cells was markedly stronger than in HepG2/pLNChGS cells (P<0.05). The hGS enzymatic activities of HepG2/pLNChGS cells (3.279+/-0.328 U/mg prot) and HepG2/pLNAFhGS cells (4.557+/-0.253 U/mg prot) were higher than those of control cells (P<0.05), and those of HepG2/ pLNAFhGS cells were also higher than the activities of HepG2/pLNChGS cells (P<0.05). In addition, the effect of hGS introduction on HepG2 cell proliferation was not significant. The amount of glutamine synthesis in HepG2/pLNChGS or HepG2/pLNAFhGS cells in three different concentrations of NH(4)(+) was higher than in the two control cells (P<0.05). The amount of glutamine synthesis and cell proliferation in the higher concentrations of NH(4)(+) (5 or 10 mmol/L) in HepG2/pLNAFhGS cells increased more than those in HepG2/pLNChGS cells (P<0.05). NH(4)(+) at a high concentration (10 mmol/L) was toxic to HepG2 and HepG2/pLNCX cells, but less toxic to HepG2/pLNChGS and HepG2/pLNAFhGS cells. CONCLUSION: The constructed hepatocytes (HepG2 cells) with specific high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.展开更多
Summary: The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic e...Summary: The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.展开更多
(2′-5′) oligoadenylate (2-5 A) synthetase is a key enzyme in the establishment of the antiviral and antieetlular states caused by interferon. 2-5 A synthesized by the enzyme is capable of activating a cellular laten...(2′-5′) oligoadenylate (2-5 A) synthetase is a key enzyme in the establishment of the antiviral and antieetlular states caused by interferon. 2-5 A synthesized by the enzyme is capable of activating a cellular latent endonuclease (RNase L ), leading to the degradation of viral mRNA and cellular rRNA, and inhibiting viral replicatio and cellular proliferation, The level of 2-5 A synthetase in human leukocytes could be enhanced during virus infection or展开更多
Using ion-pair reversed-phase HPLC (IPrHPLC), the PRPP synthetase activity inhuman erythrocytes was estimated. Methodological improvements have been taken on seeral aspects, including the removal of leukocytes and pla...Using ion-pair reversed-phase HPLC (IPrHPLC), the PRPP synthetase activity inhuman erythrocytes was estimated. Methodological improvements have been taken on seeral aspects, including the removal of leukocytes and platelets in preparation of hemolysates, lysis of erythrocyte membranes and raising of efficiency in separation of ADP from ATP peaks etc. Thismethod fulfills the requirements of simplicity, reproducibility and accuracy. The intrabatch coefficient of variation was ±3. 0 % on average, the mean relative deviation was 2. 3 %, with a recoveryrate of 94.3—98.6%. The mean value of PRPP sythetase activities in the erythrocytes of 28healthy Chinese was 2. 19 ± 0. 61 μmol·min－1·g－1.展开更多
目的探讨薏苡仁油对人前列腺癌PC-3细胞的抑制作用。方法建立人前列腺癌PC-3细胞裸鼠移植瘤模型,以2、6 m L/kg剂量的薏苡仁油连续ig给药26 d,绘制肿瘤生长曲线,实验结束后摘除肿瘤组织,称质量,计算抑瘤率,并采用紫外-可见分光光度法测...目的探讨薏苡仁油对人前列腺癌PC-3细胞的抑制作用。方法建立人前列腺癌PC-3细胞裸鼠移植瘤模型,以2、6 m L/kg剂量的薏苡仁油连续ig给药26 d,绘制肿瘤生长曲线,实验结束后摘除肿瘤组织,称质量,计算抑瘤率,并采用紫外-可见分光光度法测定肿瘤组织中脂肪酸合成酶(FAS)活性。体外培养人前列腺癌PC-3细胞,应用SYBR Green I实时荧光定量RT-PCR法测定各组FAS m RNA的相对表达量。结果薏苡仁油6 m L/kg组抑瘤率为43.9%,肿瘤组织中FAS活性与模型组比较下降44.7%(P<0.05)。薏苡仁油20μL/m L水平上的FAS m RNA表达明显下降(P<0.05)。结论薏苡仁油对人前列腺癌PC-3细胞的荷瘤裸鼠模型有明显的抑瘤作用,该作用可能与下调FAS m RNA的表达和降低FAS的活性有关。展开更多
文摘BACKGROUND: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxification. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS: hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and amplified. Then hGS mRNA was measured by semi-quantitative RT-PCR; hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH(4)(+) were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS: The expression of hGS mRNA in HepG2/pLNChGS cells (8.306+/-0.336) and HepG2/pLNAFhGS cells (21.358+/-1.716) was much stronger than in control cells (P<0.05), and that in HepG2/pLNAFhGS cells was markedly stronger than in HepG2/pLNChGS cells (P<0.05). The hGS enzymatic activities of HepG2/pLNChGS cells (3.279+/-0.328 U/mg prot) and HepG2/pLNAFhGS cells (4.557+/-0.253 U/mg prot) were higher than those of control cells (P<0.05), and those of HepG2/ pLNAFhGS cells were also higher than the activities of HepG2/pLNChGS cells (P<0.05). In addition, the effect of hGS introduction on HepG2 cell proliferation was not significant. The amount of glutamine synthesis in HepG2/pLNChGS or HepG2/pLNAFhGS cells in three different concentrations of NH(4)(+) was higher than in the two control cells (P<0.05). The amount of glutamine synthesis and cell proliferation in the higher concentrations of NH(4)(+) (5 or 10 mmol/L) in HepG2/pLNAFhGS cells increased more than those in HepG2/pLNChGS cells (P<0.05). NH(4)(+) at a high concentration (10 mmol/L) was toxic to HepG2 and HepG2/pLNCX cells, but less toxic to HepG2/pLNChGS and HepG2/pLNAFhGS cells. CONCLUSION: The constructed hepatocytes (HepG2 cells) with specific high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.
文摘Summary: The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.
基金Project supported by the National Natural Science Foundation of China
文摘(2′-5′) oligoadenylate (2-5 A) synthetase is a key enzyme in the establishment of the antiviral and antieetlular states caused by interferon. 2-5 A synthesized by the enzyme is capable of activating a cellular latent endonuclease (RNase L ), leading to the degradation of viral mRNA and cellular rRNA, and inhibiting viral replicatio and cellular proliferation, The level of 2-5 A synthetase in human leukocytes could be enhanced during virus infection or
文摘Using ion-pair reversed-phase HPLC (IPrHPLC), the PRPP synthetase activity inhuman erythrocytes was estimated. Methodological improvements have been taken on seeral aspects, including the removal of leukocytes and platelets in preparation of hemolysates, lysis of erythrocyte membranes and raising of efficiency in separation of ADP from ATP peaks etc. Thismethod fulfills the requirements of simplicity, reproducibility and accuracy. The intrabatch coefficient of variation was ±3. 0 % on average, the mean relative deviation was 2. 3 %, with a recoveryrate of 94.3—98.6%. The mean value of PRPP sythetase activities in the erythrocytes of 28healthy Chinese was 2. 19 ± 0. 61 μmol·min－1·g－1.
文摘目的探讨薏苡仁油对人前列腺癌PC-3细胞的抑制作用。方法建立人前列腺癌PC-3细胞裸鼠移植瘤模型,以2、6 m L/kg剂量的薏苡仁油连续ig给药26 d,绘制肿瘤生长曲线,实验结束后摘除肿瘤组织,称质量,计算抑瘤率,并采用紫外-可见分光光度法测定肿瘤组织中脂肪酸合成酶(FAS)活性。体外培养人前列腺癌PC-3细胞,应用SYBR Green I实时荧光定量RT-PCR法测定各组FAS m RNA的相对表达量。结果薏苡仁油6 m L/kg组抑瘤率为43.9%,肿瘤组织中FAS活性与模型组比较下降44.7%(P<0.05)。薏苡仁油20μL/m L水平上的FAS m RNA表达明显下降(P<0.05)。结论薏苡仁油对人前列腺癌PC-3细胞的荷瘤裸鼠模型有明显的抑瘤作用,该作用可能与下调FAS m RNA的表达和降低FAS的活性有关。
