目的:将HIV-1的转录反式激活蛋白(trans-activator of transcription protein,TAT)中的片段短肽(RKKRRQRRR)偶联于聚乙烯亚胺-β-环糊精(polyethylenimine-β-cyclodextrin,PEI-β-CyD)聚合物,构建出低毒性、高转染率的新型基因载体。方...目的:将HIV-1的转录反式激活蛋白(trans-activator of transcription protein,TAT)中的片段短肽(RKKRRQRRR)偶联于聚乙烯亚胺-β-环糊精(polyethylenimine-β-cyclodextrin,PEI-β-CyD)聚合物,构建出低毒性、高转染率的新型基因载体。方法:β-环糊精(β-CyD)和低分子量树枝状聚乙烯亚胺(PEI600)通过羰基二咪唑(1,1’-carbonyldiim idazole,CDI)聚合形成骨架结构,通过琥珀酰亚胺-3-(2-嘧啶二硫)丙酸酯[N-succinimidy-3-(2-pyridyldithio)propionate,SPDP]将TAT短肽偶联于PEI-β-CyD,构成新的聚合物TAT-PEI-β-CyD。采用1H-NMR和FT-IR对聚合物进行化学结构表征;凝胶电泳阻滞实验、粒径测定和透射电镜观察TAT-PEI-β-CyD对DNA的浓缩能力,以及浓缩质粒DNA后颗粒形态和粒径大小;MTT法测定载体在A293和B16细胞上的毒性,并对A293和B16细胞进行体外细胞转染实验,以PEI25kDa作为对照。结果:1H-NMR和FT-IR结果显示,TAT短肽已成功偶联到PEI-β-CyD。凝胶电泳阻滞试验显示,TAT-PEI-β-CyD在N/P为4∶1时可以完全阻滞DNA的迁移。粒径测定结果和透射电镜图像表明,TAT-PEI-β-CyD/pDNA(N/P=30∶1)复合物粒径在100nm左右。细胞毒性实验表明,在B16和A293两种不同细胞中,聚合物毒性低于PEI25kDa。体外转染结果表明,在N/P为30∶1时,聚合物在A293、B16和B16BL6细胞中的基因转染效率最高;TAT短肽的偶联能提高PEI-β-CyD在B16、B16BL6细胞上的基因转染效率。结论:实验成功构建了TAT短肽修饰的PEI-β-CyD新型基因载体。该载体毒性低,基因转染效率高。展开更多
HIV整合至CD4＋T细胞、脾脏、淋巴结、胃肠相关淋巴组织中，成为HIV潜伏感染的储存库。学界提出“shock and kill”的治疗策略，即先激活释放再杀灭病毒。而实施该策略的前提是评估和测定HIV储存库的大小。本文介绍目前常用的测定HIV储...HIV整合至CD4＋T细胞、脾脏、淋巴结、胃肠相关淋巴组织中，成为HIV潜伏感染的储存库。学界提出“shock and kill”的治疗策略，即先激活释放再杀灭病毒。而实施该策略的前提是评估和测定HIV储存库的大小。本文介绍目前常用的测定HIV储存库方法如Alu-gagPCR、定量病毒产物分析及tat／rev诱导有限稀释法。Alu．gagPCR可区别整合与未整合病毒基因，但不能区分缺陷与有功能的前病毒，因此易高估储存库。定量病毒产物分析在纯化静止CD4＋T细胞中进行，被誉为HIV储存库检测的“金标准”，但实验成本高，技术要求高，所需标本量大，仅由一轮激活并不能完全诱导产物释放，因此会低估储存库。Tat／rev诱导有限稀释法主要测定经由刺激物诱导表达多拼接RNA等生物学标志的潜伏感染细胞频数，具有所需血标本少，不用抽提病毒RNA，实验时间短，对于不同大小储存库都适用等优点，但可能高估潜伏储存库。展开更多
Background Proteins or peptides can be directly transferred into cells when covalently linked to protein transduction domains （PTDs）. TAT is one of the most widely studied PTDs. The effect of fusion protein TAT and ...Background Proteins or peptides can be directly transferred into cells when covalently linked to protein transduction domains （PTDs）. TAT is one of the most widely studied PTDs. The effect of fusion protein TAT and heme oxygenase-1 （HO-1） on liver sinusoidal endothelial cells （SECs） apoptosis during cold storage is unknown. The present study aimed to determine whether fusion protein TAT-HO-1 would transduce efficiently into liver during cold storage, and, if so, to determine whether TAT-HO-1 would attenuate SECs apoptosis during preservation injury in rat. Methods Livers of Sprague-Dawley rats were harvested and randomly assigned to group 1 （HTK solution） and group 2 （HTK solution containing TAT-HO-1 fusion protein） according to the type of the preservation solution. The transduction efficiency of TAT-HO-1 was examined and the impairment of SECs was assessed during the period of cold storage followed by 1 hour of reperfusion. Results TAT-HO-1 can transduce efficiently into liver during cold storage. A significantly lower apoptotic index of SECs was observed in group 2, at 6, 12 and 18 hours of cold storage after 1 hour reperfusion, when compared with group 1. TAT-HO-1 reduced HA and ET levels in liver at each time point. Both Bcl-2 and Bax protein were expressed in hepatocytes and SECs at the periphery of the sinusoidal space. Moreover, higher Bcl-2 expression and lower Bax expression were observed in group 2. Conclusions TAT-HO-1 can transduce efficiently into rat livers and shows a protective effect on SECs by attenuating apoptosis during cold ischemia/reperfusion injury. Protein transduction will be a novel therapeutic strategy to reduce the risk of preservation injury in liver transplantation.展开更多
文摘目的:将HIV-1的转录反式激活蛋白(trans-activator of transcription protein,TAT)中的片段短肽(RKKRRQRRR)偶联于聚乙烯亚胺-β-环糊精(polyethylenimine-β-cyclodextrin,PEI-β-CyD)聚合物,构建出低毒性、高转染率的新型基因载体。方法:β-环糊精(β-CyD)和低分子量树枝状聚乙烯亚胺(PEI600)通过羰基二咪唑(1,1’-carbonyldiim idazole,CDI)聚合形成骨架结构,通过琥珀酰亚胺-3-(2-嘧啶二硫)丙酸酯[N-succinimidy-3-(2-pyridyldithio)propionate,SPDP]将TAT短肽偶联于PEI-β-CyD,构成新的聚合物TAT-PEI-β-CyD。采用1H-NMR和FT-IR对聚合物进行化学结构表征;凝胶电泳阻滞实验、粒径测定和透射电镜观察TAT-PEI-β-CyD对DNA的浓缩能力,以及浓缩质粒DNA后颗粒形态和粒径大小;MTT法测定载体在A293和B16细胞上的毒性,并对A293和B16细胞进行体外细胞转染实验,以PEI25kDa作为对照。结果:1H-NMR和FT-IR结果显示,TAT短肽已成功偶联到PEI-β-CyD。凝胶电泳阻滞试验显示,TAT-PEI-β-CyD在N/P为4∶1时可以完全阻滞DNA的迁移。粒径测定结果和透射电镜图像表明,TAT-PEI-β-CyD/pDNA(N/P=30∶1)复合物粒径在100nm左右。细胞毒性实验表明,在B16和A293两种不同细胞中,聚合物毒性低于PEI25kDa。体外转染结果表明,在N/P为30∶1时,聚合物在A293、B16和B16BL6细胞中的基因转染效率最高;TAT短肽的偶联能提高PEI-β-CyD在B16、B16BL6细胞上的基因转染效率。结论:实验成功构建了TAT短肽修饰的PEI-β-CyD新型基因载体。该载体毒性低,基因转染效率高。
文摘HIV整合至CD4＋T细胞、脾脏、淋巴结、胃肠相关淋巴组织中，成为HIV潜伏感染的储存库。学界提出“shock and kill”的治疗策略，即先激活释放再杀灭病毒。而实施该策略的前提是评估和测定HIV储存库的大小。本文介绍目前常用的测定HIV储存库方法如Alu-gagPCR、定量病毒产物分析及tat／rev诱导有限稀释法。Alu．gagPCR可区别整合与未整合病毒基因，但不能区分缺陷与有功能的前病毒，因此易高估储存库。定量病毒产物分析在纯化静止CD4＋T细胞中进行，被誉为HIV储存库检测的“金标准”，但实验成本高，技术要求高，所需标本量大，仅由一轮激活并不能完全诱导产物释放，因此会低估储存库。Tat／rev诱导有限稀释法主要测定经由刺激物诱导表达多拼接RNA等生物学标志的潜伏感染细胞频数，具有所需血标本少，不用抽提病毒RNA，实验时间短，对于不同大小储存库都适用等优点，但可能高估潜伏储存库。
基金This study was supported by a grant from National Natural Science Foundation of China （No. 30672024）.
文摘Background Proteins or peptides can be directly transferred into cells when covalently linked to protein transduction domains （PTDs）. TAT is one of the most widely studied PTDs. The effect of fusion protein TAT and heme oxygenase-1 （HO-1） on liver sinusoidal endothelial cells （SECs） apoptosis during cold storage is unknown. The present study aimed to determine whether fusion protein TAT-HO-1 would transduce efficiently into liver during cold storage, and, if so, to determine whether TAT-HO-1 would attenuate SECs apoptosis during preservation injury in rat. Methods Livers of Sprague-Dawley rats were harvested and randomly assigned to group 1 （HTK solution） and group 2 （HTK solution containing TAT-HO-1 fusion protein） according to the type of the preservation solution. The transduction efficiency of TAT-HO-1 was examined and the impairment of SECs was assessed during the period of cold storage followed by 1 hour of reperfusion. Results TAT-HO-1 can transduce efficiently into liver during cold storage. A significantly lower apoptotic index of SECs was observed in group 2, at 6, 12 and 18 hours of cold storage after 1 hour reperfusion, when compared with group 1. TAT-HO-1 reduced HA and ET levels in liver at each time point. Both Bcl-2 and Bax protein were expressed in hepatocytes and SECs at the periphery of the sinusoidal space. Moreover, higher Bcl-2 expression and lower Bax expression were observed in group 2. Conclusions TAT-HO-1 can transduce efficiently into rat livers and shows a protective effect on SECs by attenuating apoptosis during cold ischemia/reperfusion injury. Protein transduction will be a novel therapeutic strategy to reduce the risk of preservation injury in liver transplantation.
