Introduction: 68Ga-PSMA-11 is considered the gold standard in detection of micro and oligometastases in advanced prostate cancer, being used for therapeutic planning, as well as, potentially, for evaluating response t...Introduction: 68Ga-PSMA-11 is considered the gold standard in detection of micro and oligometastases in advanced prostate cancer, being used for therapeutic planning, as well as, potentially, for evaluating response to treatment. The development of ready-to-use lyophilized kit of PSMA-11 adds quality and safety to the routine use of this radiopharmaceutical and represents a pharmacotechnical challenge as it must preserve the integrity and specificity of the ligand. Methods: PSMA-11 kit formulation was proposed, considering radiolabeling parameters and the preservation of the peptide during the lyophilization process, using mannitol as an excipient. Critical temperature characterization studies were carried out using DSC equipment and the freeze-drying process was developed. The direct radiolabeling conditions were evaluated and standardized using 68Ge/68Ga generator eluate from two different manufacturers (ITG and Eckert & Ziegler). The radiochemical purity was evaluated by TLC and HPLC. Biological evaluation was carried out with lyophilized PSMA-11 to demonstrate the integrity of the peptide and preservation of biological activity after the lyophilization process. Results: Based on critical temperature characterization studies, the freeze-drying cycle was designed to reach a freezing temperature of around −40˚C and primary drying at 2˚C. Using 20 mg of mannitol, an intact and elegant lyophilized cake was obtained. PSMA-11 lyophilized kit was directly labeled with 68Ga eluate from 68Ge/68Ga GMP generators (ITG and Eckert & Ziegler) resulting in % RP > 95% at pH 4.0 to 4.5. The results obtained from in vitro and in vivo biological competition studies confirmed the preservation of PSMA-11 affinity for the receptor after lyophilization. Conclusion: A lyophilized formulation (Kit) of PSMA-11 was successfully obtained, which preserved the integrity and biological activity of the peptide and guaranteed radiolabeling efficiency.展开更多
目的:研究泛素特异性蛋白酶35(ubiquitin-specific protease 35,USP35)对类风湿关节炎成纤维样滑膜细胞(rheumatoid arthritis-fibroblast like synoviocytes,RA-FLS)铁死亡的作用及机制,加深对RA发病机制的理解,并为其治疗提供潜在的...目的:研究泛素特异性蛋白酶35(ubiquitin-specific protease 35,USP35)对类风湿关节炎成纤维样滑膜细胞(rheumatoid arthritis-fibroblast like synoviocytes,RA-FLS)铁死亡的作用及机制,加深对RA发病机制的理解,并为其治疗提供潜在的标靶。方法:(1)体外培养RA-FLS,用慢病毒载体感染以构建稳定敲低USP35的细胞系(short hairpin ribonucleic acid of USP35,shUSP35)和其对照细胞系(negtive control of short hairpin ribonucleic acid,shNC),以及稳定过表达USP35的细胞系(overexpression of USP35,USP35 OE)和其对照细胞系(Vector)。为了探究USP35在铁死亡调控中的作用,使用1μmol/L依拉司亭(Erastin)诱导RA-FLS细胞构建铁死亡模型。将细胞分为6组,第一组是shNC细胞,第二组是用Erastin处理shNC细胞(shNC+Erastin),第三组是用Erastin处理shUSP35细胞(shUSP35+Erastin),第四组是Vector细胞,第五组是用Erastin处理Vector细胞(Vector+Erastin),第六组是用Erastin处理USP35 OE细胞(USP35 OE+Erastin)。(2)采用细胞计数试剂盒-8(cell counting kit-8,CCK8)检测细胞活力;(3)分别使用活性氧(reactive oxygen species,ROS)检测试剂盒、丙二醛(malondialdehyde,MDA)检测试剂盒、谷胱甘肽(glutathione,GSH)和氧化型谷胱甘肽(glutathione sulfide,GSSG)检测试剂盒、亚铁离子含量检测试剂盒检测细胞中ROS、MDA、Fe ^(2+)的含量以及GSH/GSSG比值;(4)采用蛋白免疫印迹实验(Western blotting)检测溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白的表达水平。结果:(1)与shNC+Erastin组相比,shUSP35+Erastin组的RA-FLS细胞活力显著降低(P<0.001),而与Vector+Erastin组相比,USP35 OE+Erastin组的细胞活力显著升高(P<0.001),表明USP35可显著缓解Erastin对RA-FLS细胞活力的抑制作用。(2)与shNC+Erastin组相比,shUSP35+Erastin组细胞的ROS(P<0.001)、MDA(P<0.05)和Fe ^(2+)水平(P<0.001)显著升高,GSH/GSSG比值显著增加(P<0.05);而与Vector+Erastin组相比,USP35OE+Erastin组细胞的ROS(P<0.001)、MDA(P<0.05)和Fe ^(2+)水平(P<0.05)显著降低,GSH/GSSG比值显著下降(P<0.05),表明USP35可显著抑制Erastin诱导的RA-FLS细胞氧化应激和脂质过氧化。(3)在Erastin诱导的RA-FLS中,USP35的表达与SLC7A11和GPX4蛋白水平呈正相关。结论:USP35抑制RA-FLS的铁死亡,可能与增加SLC7A11和GPX4表达相关。展开更多
目的:克隆粉尘螨过敏原Der f 11基因,表达、纯化该蛋白,并鉴定其免疫活性。方法:人工合成粉尘螨第11组过敏原Der f 11基因,将其连接至pET-32a表达载体,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后,通过Ni+亲和层析纯化重组过敏原Der f...目的:克隆粉尘螨过敏原Der f 11基因,表达、纯化该蛋白,并鉴定其免疫活性。方法:人工合成粉尘螨第11组过敏原Der f 11基因,将其连接至pET-32a表达载体,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后,通过Ni+亲和层析纯化重组过敏原Der f 11。以粉尘螨过敏患者血清作为一抗,经Western blot方法分析Der f 11的免疫学特性。结果:获得高纯度的重组Der f 11蛋白,SDS-PAGE结果显示表达产物pET-32a(+)-Der f 11分子质量约为118 ku。重组过敏原Der f 11检测15份尘螨过敏性患者血清中特异性IgE,阳性率为20%。结论:获得的Der f 11重组蛋白具有与天然蛋白相似的免疫学活性,为标准化抗原的临床特异性诊断和治疗及进一步的实验研究奠定基础。展开更多
目的对近年来脂多糖介导的炎症疾病含半胱氨酸的天冬氨酸蛋白水解酶11(cysteinyl aspartate specific proteinase-11,caspase-11)靶点药物进行综述。方法利用PubMed、中国知网等检索平台,检索脂多糖介导的不同炎症疾病中caspase-11靶向...目的对近年来脂多糖介导的炎症疾病含半胱氨酸的天冬氨酸蛋白水解酶11(cysteinyl aspartate specific proteinase-11,caspase-11)靶点药物进行综述。方法利用PubMed、中国知网等检索平台,检索脂多糖介导的不同炎症疾病中caspase-11靶向药物并进行了归纳汇总。结果根据文献查阅发现,若caspase-11激活,可诱发白介素1β、白介素18相关的炎症反应及Gasdermin D介导的细胞焦亡,从而导致大量炎症因子释放到胞外,引发强烈的炎症反应。caspase-11介导的炎症反应在帕金森病、阿尔兹海默症、肺纤维化、内毒素血症、脓毒症等疾病的发生发展中发挥了重要作用。结论对caspase-11靶点药物的探索,可能是未来脂多糖介导的相关炎症疾病药物研究的新方向。展开更多
This study investigated the expression and distribution of 2810408A11Rik in mouse testis and sperm, and explored its role in sperrnatogenesis and sperm function. The expression levels of 2810408A11Rik mRNA in multiple...This study investigated the expression and distribution of 2810408A11Rik in mouse testis and sperm, and explored its role in sperrnatogenesis and sperm function. The expression levels of 2810408A11Rik mRNA in multiple tissue samples were analyzed using bioinformatic resources and RT-PCR technique. A specific rabbit polyclonal antibody was prepared by prokaryotic expression of 2810408A11Rik recombinant protein and utilized for animal immunization. Western blotting, immunohistochemistry and immunofluorescence were used to detect the expression and distribution of 2810408A11Rik. The results of the bioinformatic analysi and RT-PCR showed that 2810408A11Rik mRNA was specifically expressed in mouse testis, and 2810408AllRik protein included a protein phosphatase inhibitor domain. Western blotting assays, immunohistochemistry and immunofluorescence confirmed the expression of 2810408A11Rik protein in mouse testis, especially in post-meiosis round and long spermatids, and that it is localized in the acrosome and the post-nucleus area of sperm. Our findings suggest that 2810408A11Rik may play an important role in spermatogenesis, sperm capacitation and fertilization.展开更多
文摘Introduction: 68Ga-PSMA-11 is considered the gold standard in detection of micro and oligometastases in advanced prostate cancer, being used for therapeutic planning, as well as, potentially, for evaluating response to treatment. The development of ready-to-use lyophilized kit of PSMA-11 adds quality and safety to the routine use of this radiopharmaceutical and represents a pharmacotechnical challenge as it must preserve the integrity and specificity of the ligand. Methods: PSMA-11 kit formulation was proposed, considering radiolabeling parameters and the preservation of the peptide during the lyophilization process, using mannitol as an excipient. Critical temperature characterization studies were carried out using DSC equipment and the freeze-drying process was developed. The direct radiolabeling conditions were evaluated and standardized using 68Ge/68Ga generator eluate from two different manufacturers (ITG and Eckert & Ziegler). The radiochemical purity was evaluated by TLC and HPLC. Biological evaluation was carried out with lyophilized PSMA-11 to demonstrate the integrity of the peptide and preservation of biological activity after the lyophilization process. Results: Based on critical temperature characterization studies, the freeze-drying cycle was designed to reach a freezing temperature of around −40˚C and primary drying at 2˚C. Using 20 mg of mannitol, an intact and elegant lyophilized cake was obtained. PSMA-11 lyophilized kit was directly labeled with 68Ga eluate from 68Ge/68Ga GMP generators (ITG and Eckert & Ziegler) resulting in % RP > 95% at pH 4.0 to 4.5. The results obtained from in vitro and in vivo biological competition studies confirmed the preservation of PSMA-11 affinity for the receptor after lyophilization. Conclusion: A lyophilized formulation (Kit) of PSMA-11 was successfully obtained, which preserved the integrity and biological activity of the peptide and guaranteed radiolabeling efficiency.
文摘目的:研究泛素特异性蛋白酶35(ubiquitin-specific protease 35,USP35)对类风湿关节炎成纤维样滑膜细胞(rheumatoid arthritis-fibroblast like synoviocytes,RA-FLS)铁死亡的作用及机制,加深对RA发病机制的理解,并为其治疗提供潜在的标靶。方法:(1)体外培养RA-FLS,用慢病毒载体感染以构建稳定敲低USP35的细胞系(short hairpin ribonucleic acid of USP35,shUSP35)和其对照细胞系(negtive control of short hairpin ribonucleic acid,shNC),以及稳定过表达USP35的细胞系(overexpression of USP35,USP35 OE)和其对照细胞系(Vector)。为了探究USP35在铁死亡调控中的作用,使用1μmol/L依拉司亭(Erastin)诱导RA-FLS细胞构建铁死亡模型。将细胞分为6组,第一组是shNC细胞,第二组是用Erastin处理shNC细胞(shNC+Erastin),第三组是用Erastin处理shUSP35细胞(shUSP35+Erastin),第四组是Vector细胞,第五组是用Erastin处理Vector细胞(Vector+Erastin),第六组是用Erastin处理USP35 OE细胞(USP35 OE+Erastin)。(2)采用细胞计数试剂盒-8(cell counting kit-8,CCK8)检测细胞活力;(3)分别使用活性氧(reactive oxygen species,ROS)检测试剂盒、丙二醛(malondialdehyde,MDA)检测试剂盒、谷胱甘肽(glutathione,GSH)和氧化型谷胱甘肽(glutathione sulfide,GSSG)检测试剂盒、亚铁离子含量检测试剂盒检测细胞中ROS、MDA、Fe ^(2+)的含量以及GSH/GSSG比值;(4)采用蛋白免疫印迹实验(Western blotting)检测溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白的表达水平。结果:(1)与shNC+Erastin组相比,shUSP35+Erastin组的RA-FLS细胞活力显著降低(P<0.001),而与Vector+Erastin组相比,USP35 OE+Erastin组的细胞活力显著升高(P<0.001),表明USP35可显著缓解Erastin对RA-FLS细胞活力的抑制作用。(2)与shNC+Erastin组相比,shUSP35+Erastin组细胞的ROS(P<0.001)、MDA(P<0.05)和Fe ^(2+)水平(P<0.001)显著升高,GSH/GSSG比值显著增加(P<0.05);而与Vector+Erastin组相比,USP35OE+Erastin组细胞的ROS(P<0.001)、MDA(P<0.05)和Fe ^(2+)水平(P<0.05)显著降低,GSH/GSSG比值显著下降(P<0.05),表明USP35可显著抑制Erastin诱导的RA-FLS细胞氧化应激和脂质过氧化。(3)在Erastin诱导的RA-FLS中,USP35的表达与SLC7A11和GPX4蛋白水平呈正相关。结论:USP35抑制RA-FLS的铁死亡,可能与增加SLC7A11和GPX4表达相关。
文摘目的:克隆粉尘螨过敏原Der f 11基因,表达、纯化该蛋白,并鉴定其免疫活性。方法:人工合成粉尘螨第11组过敏原Der f 11基因,将其连接至pET-32a表达载体,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后,通过Ni+亲和层析纯化重组过敏原Der f 11。以粉尘螨过敏患者血清作为一抗,经Western blot方法分析Der f 11的免疫学特性。结果:获得高纯度的重组Der f 11蛋白,SDS-PAGE结果显示表达产物pET-32a(+)-Der f 11分子质量约为118 ku。重组过敏原Der f 11检测15份尘螨过敏性患者血清中特异性IgE,阳性率为20%。结论:获得的Der f 11重组蛋白具有与天然蛋白相似的免疫学活性,为标准化抗原的临床特异性诊断和治疗及进一步的实验研究奠定基础。
文摘目的对近年来脂多糖介导的炎症疾病含半胱氨酸的天冬氨酸蛋白水解酶11(cysteinyl aspartate specific proteinase-11,caspase-11)靶点药物进行综述。方法利用PubMed、中国知网等检索平台,检索脂多糖介导的不同炎症疾病中caspase-11靶向药物并进行了归纳汇总。结果根据文献查阅发现,若caspase-11激活,可诱发白介素1β、白介素18相关的炎症反应及Gasdermin D介导的细胞焦亡,从而导致大量炎症因子释放到胞外,引发强烈的炎症反应。caspase-11介导的炎症反应在帕金森病、阿尔兹海默症、肺纤维化、内毒素血症、脓毒症等疾病的发生发展中发挥了重要作用。结论对caspase-11靶点药物的探索,可能是未来脂多糖介导的相关炎症疾病药物研究的新方向。
基金supported by the National 973 Project of China (No.2011CB944304)
文摘This study investigated the expression and distribution of 2810408A11Rik in mouse testis and sperm, and explored its role in sperrnatogenesis and sperm function. The expression levels of 2810408A11Rik mRNA in multiple tissue samples were analyzed using bioinformatic resources and RT-PCR technique. A specific rabbit polyclonal antibody was prepared by prokaryotic expression of 2810408A11Rik recombinant protein and utilized for animal immunization. Western blotting, immunohistochemistry and immunofluorescence were used to detect the expression and distribution of 2810408A11Rik. The results of the bioinformatic analysi and RT-PCR showed that 2810408A11Rik mRNA was specifically expressed in mouse testis, and 2810408AllRik protein included a protein phosphatase inhibitor domain. Western blotting assays, immunohistochemistry and immunofluorescence confirmed the expression of 2810408A11Rik protein in mouse testis, especially in post-meiosis round and long spermatids, and that it is localized in the acrosome and the post-nucleus area of sperm. Our findings suggest that 2810408A11Rik may play an important role in spermatogenesis, sperm capacitation and fertilization.
