目的研究Gli1在髓母细胞瘤(MB)中的表达和作用。方法用不同浓度(正常对照组、10μmol/L组、20μmol/L组、40μmol/L组)的Gli1特异性抑制剂GANT61作用Daoy细胞,采用细胞增殖法检测Daoy细胞在GANT61作用下的增殖活性的变化;Real-time PCR...目的研究Gli1在髓母细胞瘤(MB)中的表达和作用。方法用不同浓度(正常对照组、10μmol/L组、20μmol/L组、40μmol/L组)的Gli1特异性抑制剂GANT61作用Daoy细胞,采用细胞增殖法检测Daoy细胞在GANT61作用下的增殖活性的变化;Real-time PCR法和Western blot法分别检测不同组Daoy细胞中Gli1和Bcl-2 m RNA表达量和蛋白表达量的差异。结果 GANT61作用细胞后,细胞间隙增大,异常突起增多,细胞增殖活性明显下降,与对照组相比,GANT61作用组的Daoy细胞增殖数显著降低(P<0.05),并且随着浓度的增加,增殖数显著降低(P<0.05),提示具有浓度依赖性;不同浓度GANT61组的Gli1和Bcl-2 m RNA表达量均较对照组明显下降(P<0.05),下降程度随着GNAT61浓度的增加而增加(P<0.05);不同浓度GANT61组的Gli1和Bcl-2蛋白表达水平较对照组明显下降(P<0.05),下降程度随着GANT61浓度的增加而增加(P<0.05)。结论 GANT61能通过抑制Gli1的表达,进而下调下游靶基因Bcl-2的表达,从而明显抑制MB细胞的增殖活性,达到促进MB细胞凋亡的作用。展开更多
Based on the protein comparison between zebrafish Gli2 and fruit fly C_i and the analysis of hydrophilicity/hydrophobicity of Gli2 protein by computer programmer, the recombination N-terminal partial protein of Gli2 p...Based on the protein comparison between zebrafish Gli2 and fruit fly C_i and the analysis of hydrophilicity/hydrophobicity of Gli2 protein by computer programmer, the recombination N-terminal partial protein of Gli2 protein was designed.After the DNA fragment encoding the N-terminal part was cloned out by PCR, it was ligated with an expression vector containing His-tag and transformed into BL-21, a bacterial expression strain, to express this protein.After the expression condition was optimized, the protein can be expressed at high level after having been induced for 3 hours when the inducer—IPTG was at 0.6 mmol/L.The protein is soluble in solution, a bacterial protein extraction solution.After the protein solution was diluted in B-per solution, the recombination protein can bind to the Ni-NTA column and be purified highly.展开更多
文摘目的研究Gli1在髓母细胞瘤(MB)中的表达和作用。方法用不同浓度(正常对照组、10μmol/L组、20μmol/L组、40μmol/L组)的Gli1特异性抑制剂GANT61作用Daoy细胞,采用细胞增殖法检测Daoy细胞在GANT61作用下的增殖活性的变化;Real-time PCR法和Western blot法分别检测不同组Daoy细胞中Gli1和Bcl-2 m RNA表达量和蛋白表达量的差异。结果 GANT61作用细胞后,细胞间隙增大,异常突起增多,细胞增殖活性明显下降,与对照组相比,GANT61作用组的Daoy细胞增殖数显著降低(P<0.05),并且随着浓度的增加,增殖数显著降低(P<0.05),提示具有浓度依赖性;不同浓度GANT61组的Gli1和Bcl-2 m RNA表达量均较对照组明显下降(P<0.05),下降程度随着GNAT61浓度的增加而增加(P<0.05);不同浓度GANT61组的Gli1和Bcl-2蛋白表达水平较对照组明显下降(P<0.05),下降程度随着GANT61浓度的增加而增加(P<0.05)。结论 GANT61能通过抑制Gli1的表达,进而下调下游靶基因Bcl-2的表达,从而明显抑制MB细胞的增殖活性,达到促进MB细胞凋亡的作用。
文摘Based on the protein comparison between zebrafish Gli2 and fruit fly C_i and the analysis of hydrophilicity/hydrophobicity of Gli2 protein by computer programmer, the recombination N-terminal partial protein of Gli2 protein was designed.After the DNA fragment encoding the N-terminal part was cloned out by PCR, it was ligated with an expression vector containing His-tag and transformed into BL-21, a bacterial expression strain, to express this protein.After the expression condition was optimized, the protein can be expressed at high level after having been induced for 3 hours when the inducer—IPTG was at 0.6 mmol/L.The protein is soluble in solution, a bacterial protein extraction solution.After the protein solution was diluted in B-per solution, the recombination protein can bind to the Ni-NTA column and be purified highly.
