New developments in lentiviral vector design,production and purification

Introduction: Lentiviruses are a very potent class of viral vectors for which there is presently a rapidly growing interest for a number of gene therapy. However, their construction, production and purification need to be performed according to state-of-the-art techniques in order to obtain sufficient quantities of high purity material of any usefulness and safety.

Areas covered: The recent advances in the field of recombinant lentivirus vector design, production and purification will be reviewed with an eye toward its utilization for gene therapy. Such a review should be helpful for the potential user of this technology.

Expert opinion: The principal hurdles toward the use of recombinant lentivirus as a gene therapy vector are the low titer at which it is produced as well as the difficulty to purify it at an acceptable level without degrading it. The recent advances in the bioproduction of this vector suggest these issues are about to be resolved, making the retrovirus gene therapy a mature technology.     

Revolutionary Impact of Nanodrug Delivery on Neuroscience

Brain research is the most expanding interdisciplinary research that is using the state of the art techniques to overcome limitations in order to conduct more accurate and effective experiments. Drug delivery to the target site in the central nervous system (CNS) is one of the most difficult steps in neuroscience researches and therapies. Taking advantage of the nanoscale structure of neural cells (both neurons and glia); nanodrug delivery (second generation of biotechnological products) has a potential revolutionary impact into the basic understanding, visualization and therapeutic applications of neuroscience. Current review article firstly provides an overview of preparation and characterization, purification and separation, loading and delivering of nanodrugs. Different types of nanoparticle bioproducts and a number of methods for their fabrication and delivery systems including (carbon) nanotubes are explained. In the second part, neuroscience and nervous system drugs are deeply investigated. Different mechanisms in which nanoparticles enhance the uptake and clearance of molecules form cerebrospinal fluid (CSF) are discussed. The focus is on nanodrugs that are being used or have potential to improve neural researches, diagnosis and therapy of neurodegenerative disorders.     

连续性血液净化中阿加曲班与枸橼酸钠的抗凝效果及安全性比较

 目的 比较阿加曲班和枸橼酸钠在连续性肾脏替代治疗(continuous renal replacement therapy,CRRT)中的有效性和安全性。方法 选取2018-09至2019-05医院行CRRT治疗的患者132例,随机分为阿加曲班组60例和枸橼酸钠组72例,分别给予阿加曲班和枸橼酸钠抗凝治疗,阿加曲班组监测体内活化凝血时间(activated clotting time,ACT),枸橼酸钠监测ACT及血钙,同时记录两组患者透析前后血常规、血生化指标,尿素下降率、滤器及管路凝血情况,以及治疗后24 h内组织器官出血情况。结果 两组患者透析前ACT均在正常范围内,阿加曲班组ACT 5 min、2 h和4 h均显著升高,差异有统计学意义(P＜0.05),透析1 h后ACT显著下降至正常范围;枸橼酸钠组透析4 h和透析后ACT均无明显变化。枸橼酸钠组透析前、透析中、透析后体内游离钙均无明显变化;透析中体外游离钙均低于同时间体内游离钙水平。两组患者治疗前后血红蛋白及血小板较治疗前均无明显变化,血肌酐水平均显著下降,差异有统计学意义(P＜0.05)。两组患者治疗时间、URR值、管路滤器凝血情况及出血事件对比差异无统计学意义。结论 阿加曲班和枸橼酸钠在CRRT治疗中均为有效和安全的抗凝剂。对有高出血风险的患者选择枸橼酸钠更佳。     

金黄色葡萄球菌锰超氧化物歧化酶的表达纯化及活性分析

目的克隆表达、纯化金黄色葡萄球菌(Staphylococcus aureus)锰超氧化物歧化酶(Mn-SOD)并测定其活性。方法使用Clustal Omega比对不同来源Mn-SOD的序列,使用Swiss model网站预测金黄色葡萄球菌Mn-SOD的三级结构。提取细菌DNA,以此为模板PCR扩增获得Mn-SOD基因并将其与pET28a连接,构建pET28a-Mn-SOD重组质粒。将重组质粒转化大肠埃希菌BL21(DE3)获得重组菌BL/pET28a-Mn-SOD,使用IPTG诱导表达重组Mn-SOD,通过镍柱亲和层析纯化Mn-SOD蛋白并测定不同pH条件下的酶活性。结果金黄色葡萄球菌Mn-SOD具有Mn-SOD的特征序列和锰离子结合位点。成功得到重组质粒pET28a-Mn-SOD,转化大肠埃希菌BL21后表达相对分子质量为24.8×10^3的Mn-SOD,使用Ni^2+柱纯化后得到高纯度的Mn-SOD,且在pH值为7.5时活性较高,约为3 200 U/mL。结论成功构建了pET28a-Mn-SOD重组质粒能,表达的Mn-SOD重组蛋白纯化后仍具有SOD活性。     

重组人肝再生增强因子的克隆、表达及纯化

目的通过构建原核表达系统，在大肠埃希菌中表达重组人肝再生增强因子（hALR）蛋白，为各种急慢性肝损伤及肝衰竭的治疗提供新的治疗方法奠定基础。方法利用正常人肝组织提取总RNA，经一步法逆转录聚合酶链反应（RT—PCR）获取hALR cDNA全序列，构建原核表达载体hALR—pET28a（＋）转化大肠埃希菌（BL21），诱导表达重组hALR蛋白，以组氨酸为标签进行蛋白纯化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western Blot鉴定重组蛋白。结果经双酶切和DNA测序证实hALR cDNA正确插入表达载体pET28a（＋），成功表达并纯化得相对分子质量为23000的重组蛋白，低温诱导表达使可溶蛋白明显增加，与预期结果相符。结论成功表达和纯化获重组hALR蛋白。     

粉尘螨Der f4变应原的提纯及其淀粉酶活性分析

采用上海医科大学医学螨类研究室培养的粉尘螨提纯粉尘螨４类变应原，并对其作淀粉活性分析。     

日本血吸虫成虫组分抗原的分离及小鼠免疫试验

目的 分离日本血吸虫可溶性成虫抗原(AWA)中的组分抗原，分析其诱导的抗血吸虫的免疫保护性。方法 电泳层析法分离日本血吸虫AWA中的组分抗原，计算各组分抗原的分子量，并分析其免疫学活性。将各组分抗原分别免疫小鼠，攻击感染日本血吸虫尾蚴，感染后45d剖杀，观察各组分抗原的免疫保护效果。结果 采用电泳层析法分离获得多个组分抗原，选择收集其中4个较纯的组分抗原，分子量分别为52kD、63kD、67kD和74kD；其中63kD和67kD抗原与感染血清有强的反应，而52kD和74kD抗原则反应弱；4种组分抗原均与AWA的免疫血清反应。63kD抗原诱导的免疫保护力水平较52kD、67kD和74kD抗原好。结论 电泳层析法分离的4个血吸虫组分抗原有良好的免疫原性，且63kD抗原有一定的免疫保护性。     

血液净化室常见的护理风险分析与干预策略

目的探讨血液净化室常见的护理风险分析并分析干预策略。方法选取固定在我科进行血液透析患者共220例,将患者随机分为两个小组,每组各110例。对照组护理上依照常规血液净化程序进行,不采用护理风险分析与干预。观察组则在对照组的基础上加入护理风险分析与干预。观察两组患者护理风险发生率的变化。结果观察组护理风险发生率为3．64％,低于对照组的16．36％,两组差异有统计学意义（P〈0．05）。结论通过对血液净化室常见的护理风险进行分析,提出合理的干预策略,可以有效减少临床护理风险的发生,值得各级医院进一步研究与推广。     

连续性血液净化(CBP)治疗危重症患者对机体氧化抗氧化指标的影响研究

目的:分析危重症采用连续性血液净化对治疗机体氧化抗氧化指标的影响。方法:选我院22例重症监护室行CBP治疗的患者,不同时间段取血,并检测血清过氧脂质(LPO)与谷胱甘肽氧化物酶(GSH-Px)的水平,行APACHEIII的评分。结果:相较于健康对照组,LPO升高,GSH-Px降低;在CBP的不同时间段治疗后,APACHEIII评分有着明显改善;LPO、GSH-Px与APACHEIII的评分分别呈正、负相关。结论:CBP可改善患者氧化抗氧化紊乱,从而减轻氧化损伤,促进危重症患者病情得到较好的改善,而LPO及GSH-Px是检测危重症患者病情轻重的关键指标,可作为判断预后与CBP停止的时机起到了重要的依据。