背景:血管性血友病因子水解蛋白酶13(a disintegrin and metalloproteinase with a thrombospondin type 1motif,member 13,ADAMTS13)可结合并酶切血管性血友病因子,在防止血小板过多聚集和血栓的形成中起到关键作用。重组ADAMTS13通常...背景:血管性血友病因子水解蛋白酶13(a disintegrin and metalloproteinase with a thrombospondin type 1motif,member 13,ADAMTS13)可结合并酶切血管性血友病因子,在防止血小板过多聚集和血栓的形成中起到关键作用。重组ADAMTS13通常选择在真核细胞中表达分泌,然而对于其表达纯化目前仍缺乏一个明确高效的途径。目的:寻找合适的宿主细胞,得到稳定表达野生型ADAMTS13和功能增强型ADAMTS13的细胞株,建立高纯度ADAMTS13的纯化方法,进而研究其生物学功能。方法:以野生型ADAMTS13重组质粒为模板,利用位点突变试剂盒得到功能增强型ADAMTS13重组质粒。比较CHO-S细胞、CHO-K1细胞、293T细胞对ADAMTS13的表达效果,挑选出合适的宿主细胞。通过Ni柱亲和层析和分子筛纯化蛋白;采用7.5%SDS-PAGE、Western-blot鉴定野生型ADAMTS13和功能增强型ADAMTS13的纯度及特异性;利用原子力显微镜技术检测野生型及功能增强型ADAMTS13与血管性血友病因子A2的相互作用,鉴定两种蛋白的活性。结果与结论:相对于CHO-S和CHO-K1,293T细胞更适合作为野生型ADAMTS13和功能增强型ADAMTS13的表达宿主;经Ni柱亲和层析和分子筛纯化后,所得到的野生型ADAMTS13和功能增强型ADAMTS13质量浓度分别为103.7,149.7 mg/L,且两种蛋白纯度均约90%;原子力显微镜检测结果显示,野生型ADAMTS13、功能增强型ADAMTS13与血管性血友病因子A2的黏附频率分别为11.37%,14.70%,表明功能增强型ADAMTS13与血管性血友病因子A2的结合亲和力更高,这与近期ADAMTS13构象研究一致。展开更多
Near-infrared （NIR） photothermal therapy has developed very quickly in recent years. However, its clinical applications are hindered by many practical problems, such as low accumulation in tumors, high laser power d...Near-infrared （NIR） photothermal therapy has developed very quickly in recent years. However, its clinical applications are hindered by many practical problems, such as low accumulation in tumors, high laser power density and high biotoxicity in vivo. Herein, a versatile system combining chemotherapy with photothermal therapy for cancer therapy using ultrasmall Pd nanosheets （SPNS） has been developed. The SPNS can serve as pH-responsive drug carriers to efficiently deliver DOX into cancer cells and tumors. On the other hand, the coordinative loading of DOX on SPNS enhances its accumulation in tumor tissue. So we can efficiently ablate tumor using low-intensity laser radiation. Importantly, with ultrasmall size （-4.4 nm）, SPNS surface-functionalized with reduced glutathione （GSH） can be cleared from the body through the renal system into the urine. This cancer therapeutic nanosystem, which exhibits a significant synergistic effect and low systemic toxicity, has great potential for clinical applications.展开更多
文摘背景:血管性血友病因子水解蛋白酶13(a disintegrin and metalloproteinase with a thrombospondin type 1motif,member 13,ADAMTS13)可结合并酶切血管性血友病因子,在防止血小板过多聚集和血栓的形成中起到关键作用。重组ADAMTS13通常选择在真核细胞中表达分泌,然而对于其表达纯化目前仍缺乏一个明确高效的途径。目的:寻找合适的宿主细胞,得到稳定表达野生型ADAMTS13和功能增强型ADAMTS13的细胞株,建立高纯度ADAMTS13的纯化方法,进而研究其生物学功能。方法:以野生型ADAMTS13重组质粒为模板,利用位点突变试剂盒得到功能增强型ADAMTS13重组质粒。比较CHO-S细胞、CHO-K1细胞、293T细胞对ADAMTS13的表达效果,挑选出合适的宿主细胞。通过Ni柱亲和层析和分子筛纯化蛋白;采用7.5%SDS-PAGE、Western-blot鉴定野生型ADAMTS13和功能增强型ADAMTS13的纯度及特异性;利用原子力显微镜技术检测野生型及功能增强型ADAMTS13与血管性血友病因子A2的相互作用,鉴定两种蛋白的活性。结果与结论:相对于CHO-S和CHO-K1,293T细胞更适合作为野生型ADAMTS13和功能增强型ADAMTS13的表达宿主;经Ni柱亲和层析和分子筛纯化后,所得到的野生型ADAMTS13和功能增强型ADAMTS13质量浓度分别为103.7,149.7 mg/L,且两种蛋白纯度均约90%;原子力显微镜检测结果显示,野生型ADAMTS13、功能增强型ADAMTS13与血管性血友病因子A2的黏附频率分别为11.37%,14.70%,表明功能增强型ADAMTS13与血管性血友病因子A2的结合亲和力更高,这与近期ADAMTS13构象研究一致。
文摘Near-infrared （NIR） photothermal therapy has developed very quickly in recent years. However, its clinical applications are hindered by many practical problems, such as low accumulation in tumors, high laser power density and high biotoxicity in vivo. Herein, a versatile system combining chemotherapy with photothermal therapy for cancer therapy using ultrasmall Pd nanosheets （SPNS） has been developed. The SPNS can serve as pH-responsive drug carriers to efficiently deliver DOX into cancer cells and tumors. On the other hand, the coordinative loading of DOX on SPNS enhances its accumulation in tumor tissue. So we can efficiently ablate tumor using low-intensity laser radiation. Importantly, with ultrasmall size （-4.4 nm）, SPNS surface-functionalized with reduced glutathione （GSH） can be cleared from the body through the renal system into the urine. This cancer therapeutic nanosystem, which exhibits a significant synergistic effect and low systemic toxicity, has great potential for clinical applications.
