AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning t...AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αBcrystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli(E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.RESULTS: Compared with the gene bank(GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin issuccessfully constructed, and the recombinant human αBcrystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.展开更多
目的探讨下调αB-晶状体蛋白(αB-crystallin)对人皮肤角质形成细胞(Ha Ca T)在应激条件下的存活和凋亡的影响。方法设计并筛选特异性靶向下调αB-crystallin表达的siRNA;噻唑蓝法检测siRNA下调αB-crystallin表达对过氧化氢(H2O2)处理...目的探讨下调αB-晶状体蛋白(αB-crystallin)对人皮肤角质形成细胞(Ha Ca T)在应激条件下的存活和凋亡的影响。方法设计并筛选特异性靶向下调αB-crystallin表达的siRNA;噻唑蓝法检测siRNA下调αB-crystallin表达对过氧化氢(H2O2)处理的细胞相对存活率的影响;流式细胞术检测siRNA下调αB-晶状体蛋白表达对H2O2诱导的细胞凋亡率的影响。结果筛选获得可有效下调αB-crystallin表达的siRNA(si-CRYAB-1);si-CRYAB-1下调αB-crystallin表达可降低H2O2处理后的Ha Ca T细胞存活率(P<0.01),并明显增加H2O2诱导的Ha Ca T细胞凋亡率(P<0.05)。结论下调αB-crystallin的表达后,人角质形成细胞在氧化应激条件下的存活能力明显减弱。αB-crystallin可能具有抗人角质形成细胞凋亡的作用。展开更多
目的探讨晶状体蛋白αb(Cryab)在结直肠癌组织中的表达及其临床意义。方法收集兰州大学第一医院早复发和晚复发的结直肠癌标本10对,用RT-PCR法检测Cryab m RNA在早复发和晚复发患者中的表达差异;另收集兰州大学第一医院本部及东岗院区2...目的探讨晶状体蛋白αb(Cryab)在结直肠癌组织中的表达及其临床意义。方法收集兰州大学第一医院早复发和晚复发的结直肠癌标本10对,用RT-PCR法检测Cryab m RNA在早复发和晚复发患者中的表达差异;另收集兰州大学第一医院本部及东岗院区2005年1月至2008年12月期间100例行根治性手术切除患者的结直肠癌组织及其相应的癌旁组织,用免疫组织化学SP法检测100例结直肠癌组织和癌旁组织中Cryab蛋白的表达,并分析Cryab蛋白表达与结直肠癌患者的年龄、性别、肿瘤数目、肿瘤大小、TNM分期、分化程度、淋巴结转移及浸润深度的关系。使用Cox回归方法分析Cryab蛋白表达对结直肠癌患者预后的影响。结果 1 Cryab m RNA在早复发患者中的表达明显高于晚复发患者(P<0.05)。2 Cryab蛋白在结直肠癌组织中的表达明显高于其在癌旁组织中的表达(P<0.001)。3 Cryab蛋白表达与结直肠癌患者的肿瘤大小、分化程度、浸润深度、淋巴结转移及TNM分期有关(P<0.05),而与患者的性别、年龄及肿瘤数目无关(P>0.05)。4单因素分析结果显示,肿瘤大小、淋巴结转移及Cryab蛋白表达是结直肠癌患者总生存时间和无瘤生存时间的影响因素(P<0.05);进一步的Cox回归分析结果显示,淋巴结转移及Cryab蛋白表达是影响结直肠癌患者预后的独立危险因素。5 Cryab蛋白高表达患者的总生存率及无瘤生存率均明显低于Cryab蛋白低表达患者,差异有统计学意义(P=0.003,P=0.005)。结论 Cryab蛋白的高表达与结直肠癌的侵袭和发展有关,其可能参与了结直肠癌的演进,Cryab可能作为判断结直肠癌恶性程度和预后的指标。展开更多
目的:探讨α-晶状体蛋白B链(Alpha-crystallin B chain,CRYAB)在乳腺癌中表达的临床病理学意义。方法:收集乳腺癌病例及相应的临床资料包括随访资料,应用IHC染色方法检测CRYAB在乳腺良性病变(BBD)、无淋巴结转移乳腺癌(NMBC)、有淋巴结...目的:探讨α-晶状体蛋白B链(Alpha-crystallin B chain,CRYAB)在乳腺癌中表达的临床病理学意义。方法:收集乳腺癌病例及相应的临床资料包括随访资料,应用IHC染色方法检测CRYAB在乳腺良性病变(BBD)、无淋巴结转移乳腺癌(NMBC)、有淋巴结转移乳腺癌(MBC)及配对淋巴结转移灶(PMLN)中的表达,分析CRYAB表达与乳腺癌临床病理指标(患者年龄、肿块大小、淋巴结转移情况、临床分期、组织学分型和分级、雌孕激素受体和c-cerb B2表达情况、绝经情况)间及生存状态的关系。结果:CRYAB在对照组BBD组、NMBC组、MBC组、PMLN组的阳性表达率分别为97.9%(46/47)、44.6%(37/83)、13.1%(14/107)、10.8%(11/107),其中BBD组和NMBC组,BBD组、NMBC组分别与MBC组、PMLN组均存在显著性差异。CRYAB表达与淋巴结转移(P<0.001)、临床分期(P=0.001)、组织学分级(P=0.037)和雌孕激素受体表达情况(P<0.001)有显著相关,无淋巴结转移组的阳性表达率显著高于有淋巴结转移组,临床晚期的阳性表达率低于临床早期,雌孕激素受体阳性病例的阳性表达率显著低于雌孕激素受体阴性病例。生存分析结果显示CRYAB阳性表达的患者生存期比CRYAB阴性表达的患者生存期更长(p=0.037)。结论:CRYAB与乳腺癌的转移、临床分期、生存状态、雌孕激素受体表达有关。展开更多
基金Supported by National Natural Science Foundation of China Grant (No.81270996)Science and Technology Project Foundation of Hainan Province (No.ZDYF201631)Health Science and Technology Innovation Project Foundation of Sanya (No.2016YW22)
文摘AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αBcrystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli(E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.RESULTS: Compared with the gene bank(GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin issuccessfully constructed, and the recombinant human αBcrystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.
文摘目的探讨下调αB-晶状体蛋白(αB-crystallin)对人皮肤角质形成细胞(Ha Ca T)在应激条件下的存活和凋亡的影响。方法设计并筛选特异性靶向下调αB-crystallin表达的siRNA;噻唑蓝法检测siRNA下调αB-crystallin表达对过氧化氢(H2O2)处理的细胞相对存活率的影响;流式细胞术检测siRNA下调αB-晶状体蛋白表达对H2O2诱导的细胞凋亡率的影响。结果筛选获得可有效下调αB-crystallin表达的siRNA(si-CRYAB-1);si-CRYAB-1下调αB-crystallin表达可降低H2O2处理后的Ha Ca T细胞存活率(P<0.01),并明显增加H2O2诱导的Ha Ca T细胞凋亡率(P<0.05)。结论下调αB-crystallin的表达后,人角质形成细胞在氧化应激条件下的存活能力明显减弱。αB-crystallin可能具有抗人角质形成细胞凋亡的作用。
文摘目的:探讨α-晶状体蛋白B链(Alpha-crystallin B chain,CRYAB)在乳腺癌中表达的临床病理学意义。方法:收集乳腺癌病例及相应的临床资料包括随访资料,应用IHC染色方法检测CRYAB在乳腺良性病变(BBD)、无淋巴结转移乳腺癌(NMBC)、有淋巴结转移乳腺癌(MBC)及配对淋巴结转移灶(PMLN)中的表达,分析CRYAB表达与乳腺癌临床病理指标(患者年龄、肿块大小、淋巴结转移情况、临床分期、组织学分型和分级、雌孕激素受体和c-cerb B2表达情况、绝经情况)间及生存状态的关系。结果:CRYAB在对照组BBD组、NMBC组、MBC组、PMLN组的阳性表达率分别为97.9%(46/47)、44.6%(37/83)、13.1%(14/107)、10.8%(11/107),其中BBD组和NMBC组,BBD组、NMBC组分别与MBC组、PMLN组均存在显著性差异。CRYAB表达与淋巴结转移(P<0.001)、临床分期(P=0.001)、组织学分级(P=0.037)和雌孕激素受体表达情况(P<0.001)有显著相关,无淋巴结转移组的阳性表达率显著高于有淋巴结转移组,临床晚期的阳性表达率低于临床早期,雌孕激素受体阳性病例的阳性表达率显著低于雌孕激素受体阴性病例。生存分析结果显示CRYAB阳性表达的患者生存期比CRYAB阴性表达的患者生存期更长(p=0.037)。结论:CRYAB与乳腺癌的转移、临床分期、生存状态、雌孕激素受体表达有关。