In this study, a recombinant pET28c-gBTB/POZ was constructed by cloning the sequence of the BTB/POZ domain of the zebrafish gcl （germ cell-less） into the expression vector pET28c, and pET28c-gBTB/POZ was transformed...In this study, a recombinant pET28c-gBTB/POZ was constructed by cloning the sequence of the BTB/POZ domain of the zebrafish gcl （germ cell-less） into the expression vector pET28c, and pET28c-gBTB/POZ was transformed into BL21（DE3） pLysS strain to express the fusion protein for the preparation of antibody. Polyclonal-antibody against the GCL-BTB/POZ domain was prepared by immunizing rabbit with the fusion protein, and the Western Blot and immuno-histochemical analysis were performed to detect the quantity of the polyclonal-antibody. The result indicates that the polyclonal-antibodies were of good quantity and specification. Further studies will be performed to demonstrate the function and expression pattern of the GCL protein during the development process of zebrafish with the polyclonal-antibody.展开更多
目的:观察Rho激酶抑制剂法舒地尔(Fasudil)对人高侵袭潜能HCC细胞株3(human high metastatic liver cancer cells 3,HCCLM3)侵袭转移的影响,并且探讨其作用的机制。方法:应用100 mol/L Fasudil作用于HCCLM3细胞,采用肌动蛋白微丝荧光染...目的:观察Rho激酶抑制剂法舒地尔(Fasudil)对人高侵袭潜能HCC细胞株3(human high metastatic liver cancer cells 3,HCCLM3)侵袭转移的影响,并且探讨其作用的机制。方法:应用100 mol/L Fasudil作用于HCCLM3细胞,采用肌动蛋白微丝荧光染色和侵袭小室实验观察HCCLM3细胞的运动侵袭能力。HCCLM3细胞经过处理后分为阴性对照组、Fasudil作用组、BTBD7干扰组,通过Western印迹检测BTB/POZ结构域蛋白7(BR-C,ttk and bab/pox virus domain containing 7,BTBD7)、Ras同系物家族成员C(ras homolog family member C,Rho C)、Rho关联卷曲螺旋蛋白激酶2(Rhoassociated,coiled-coil containing protein kinase 2,ROCK2)、MMP2和MMP9蛋白表达水平,酶谱分析法检测MMP2和MMP9活性水平。BTBD7干扰组作为阳性对照。结果:Fasudil处理后HCCLM3侵袭运动能力下降,BTBD7,Rho C,ROCK2蛋白表达下调,MMP2和MMP9活性降低,与阴性对照组比较差异有统计学意义(均P<0.01)。结论:Fasudil具有干预BTBD7-ROCK2信号通路、抑制HCC侵袭转移的重要作用。展开更多
目的:探讨BTB/POZ结构域蛋白7(BTBD7)假基因1(BTBD7P1)在肝细胞癌(HCC)中的表达及功能。方法:检测106例配对的HCC组织与癌旁组织标本中BTBD7P1 m RNA的表达,分析BTBD7P1m RNA表达与HCC患者临床病理特征及预后的关系。用BTBD7P1过表达慢...目的:探讨BTB/POZ结构域蛋白7(BTBD7)假基因1(BTBD7P1)在肝细胞癌(HCC)中的表达及功能。方法:检测106例配对的HCC组织与癌旁组织标本中BTBD7P1 m RNA的表达,分析BTBD7P1m RNA表达与HCC患者临床病理特征及预后的关系。用BTBD7P1过表达慢病毒载体转染HCC细胞系Bel7404后,检测细胞增殖率以及BTBD7 m RNA与蛋白的表达。结果:HCC组织中BTBD7P1相对表达量明显低于癌旁组织为(0.71 vs.2.14,P<0.05);BTBD7P1m RNA低表达与肿瘤大小、卫星灶、分化程度、静脉血管侵犯、出血坏死、HCC分期明显有关(均P<0.05);BTBD7P1 m RNA低表达患者的1、3、5年总体生存率及无瘤生存率均明显低于BTBD7P1m RNA高表达患者(均P<0.05)。与转染空载体质粒的对照组Bel7404细胞比较,转染BTBD7P1过表达慢病毒载体的Bel7404细胞,细胞增殖能力明显减低,BTBD7 m RNA表达明显下调(均P<0.05),但BTBD7蛋白表达无明显变化(P>0.05)。结论:BTBD7P1可能在m RNA水平对亲本基因BTBD7表达进行调控,从而参与了HCC发生与发展。展开更多
基金Supported by the National Natural Science Foundation of China (30570968, 30370744)
文摘In this study, a recombinant pET28c-gBTB/POZ was constructed by cloning the sequence of the BTB/POZ domain of the zebrafish gcl （germ cell-less） into the expression vector pET28c, and pET28c-gBTB/POZ was transformed into BL21（DE3） pLysS strain to express the fusion protein for the preparation of antibody. Polyclonal-antibody against the GCL-BTB/POZ domain was prepared by immunizing rabbit with the fusion protein, and the Western Blot and immuno-histochemical analysis were performed to detect the quantity of the polyclonal-antibody. The result indicates that the polyclonal-antibodies were of good quantity and specification. Further studies will be performed to demonstrate the function and expression pattern of the GCL protein during the development process of zebrafish with the polyclonal-antibody.
文摘目的:观察Rho激酶抑制剂法舒地尔(Fasudil)对人高侵袭潜能HCC细胞株3(human high metastatic liver cancer cells 3,HCCLM3)侵袭转移的影响,并且探讨其作用的机制。方法:应用100 mol/L Fasudil作用于HCCLM3细胞,采用肌动蛋白微丝荧光染色和侵袭小室实验观察HCCLM3细胞的运动侵袭能力。HCCLM3细胞经过处理后分为阴性对照组、Fasudil作用组、BTBD7干扰组,通过Western印迹检测BTB/POZ结构域蛋白7(BR-C,ttk and bab/pox virus domain containing 7,BTBD7)、Ras同系物家族成员C(ras homolog family member C,Rho C)、Rho关联卷曲螺旋蛋白激酶2(Rhoassociated,coiled-coil containing protein kinase 2,ROCK2)、MMP2和MMP9蛋白表达水平,酶谱分析法检测MMP2和MMP9活性水平。BTBD7干扰组作为阳性对照。结果:Fasudil处理后HCCLM3侵袭运动能力下降,BTBD7,Rho C,ROCK2蛋白表达下调,MMP2和MMP9活性降低,与阴性对照组比较差异有统计学意义(均P<0.01)。结论:Fasudil具有干预BTBD7-ROCK2信号通路、抑制HCC侵袭转移的重要作用。
文摘目的:探讨BTB/POZ结构域蛋白7(BTBD7)假基因1(BTBD7P1)在肝细胞癌(HCC)中的表达及功能。方法:检测106例配对的HCC组织与癌旁组织标本中BTBD7P1 m RNA的表达,分析BTBD7P1m RNA表达与HCC患者临床病理特征及预后的关系。用BTBD7P1过表达慢病毒载体转染HCC细胞系Bel7404后,检测细胞增殖率以及BTBD7 m RNA与蛋白的表达。结果:HCC组织中BTBD7P1相对表达量明显低于癌旁组织为(0.71 vs.2.14,P<0.05);BTBD7P1m RNA低表达与肿瘤大小、卫星灶、分化程度、静脉血管侵犯、出血坏死、HCC分期明显有关(均P<0.05);BTBD7P1 m RNA低表达患者的1、3、5年总体生存率及无瘤生存率均明显低于BTBD7P1m RNA高表达患者(均P<0.05)。与转染空载体质粒的对照组Bel7404细胞比较,转染BTBD7P1过表达慢病毒载体的Bel7404细胞,细胞增殖能力明显减低,BTBD7 m RNA表达明显下调(均P<0.05),但BTBD7蛋白表达无明显变化(P>0.05)。结论:BTBD7P1可能在m RNA水平对亲本基因BTBD7表达进行调控,从而参与了HCC发生与发展。
