腺苷促进体外培养神经干细胞增殖作用研究

目的研究腺苷促进体外培养神经干细胞（NSCs）增殖作用。方法取新生小鼠大脑进行神经干细胞原代培养,观察神经球生成情况,特异性蛋白质免疫细胞化学染色和BrdU标记鉴定并判断其增殖能力;将腺苷按0.4,2.0,10.0 μmol•L 13个浓度加入神经干细胞培养基,同时设立溶媒对照组和阳性对照组,通过观察神经球形态、神经球生成数目及 MTT法定量比较,分析不同浓度腺苷对神经干细胞分裂增殖的影响。结果培养物中有大量神经球生成,神经干细胞特异性蛋白质免疫细胞化学染色呈阳性,BrdU标记亦呈阳性反应,所培养的细胞为神经干细胞,具有增殖能力;腺苷中、高浓度组神经球数目、MTT比色结果明显高于溶媒对照组。结论腺苷具有促进NSCs增殖作用。     

骨骼肌提取液促进体外培养神经干细胞增殖作用研究

目的寻找促神经干细胞分裂增殖物质,为神经系统发育研究和神经系统疾病的治疗研究提供新的依据。方法用硫酸铵盐析及超滤离心法分离骨骼肌蛋白质,体外培养新生小鼠神经干细胞,通过神经球生成的观察、特异性蛋白质免疫细胞化学染色和MTT比色检测法鉴定神经干细胞并判断其增殖能力。结果在培养物中有大量神经球生成,且与对照组差异有显著性(P<0.01),神经干细胞的特异性蛋白质(Nestin)免疫细胞化学染色呈阳性。结论骨骼肌提取液可促进体外培养神经干细胞增殖分裂,且呈一定量效关系,其有效成分为分子量≤100 ku的蛋白质分子。     

托吡酯促进体外培养神经干细胞增殖的实验研究

目的:研究托吡酯对体外培养神经干细胞的促增殖作用。方法:取新生小鼠大脑进行神经干细胞原代培养,托吡酯按0.4、2、10μmol.L-13个量级(或3种浓度)加入神经干细胞培养基中,同时设立溶媒对照组和阳性对照组,通过神经球生成数目及MTT法定量比较,分析不同浓度托吡酯对神经干细胞分裂增殖的影响。结果:2、10μmol.L-1托吡酯组神经球数目分别为20.67±4.04和49.68±3.79;MTT比色结果分别为0.3546±0.0705和0.5018±0.0369,明显高于溶媒对照组(7.34±2.31、0.2880±0.0093,P<0.01)。结论:托吡酯具有促进神经干细胞增殖的作用。     

黄芪甲苷对体外神经干细胞增殖作用影响的研究

目的探讨黄芪甲苷(astragaloside,AS)对神经干细胞增殖的影响。方法体外培养大鼠胎鼠神经干细胞,Nestin免疫化学染色鉴定NSCs,BrdU标记鉴定NSCs增殖。BrdU标记免疫细胞化学染色检测神经干细胞增殖能力。实时定量PCR技术探讨黄芪甲苷促进NSCs增殖的作用机制。结果Nestin鉴定为阳性,Brdu标记亦呈阳性;BrdU标记结果表明,黄芪甲苷高、中、低剂量组NSCs的增殖率明显提高(P<0.05,P<0.01)。实时定量PCR结果表明在诱导NSCs增殖过程中,黄芪甲苷高剂量组Hes5基因表达增加(P<0.05)。结论黄芪甲苷对NSCs增殖具有明显的诱导作用,其可能通过上调与细胞增殖相关基因Hes1、Hes5、cyclinD1表达而起作用,但也可能调节与其它增殖通路有关。     

双嘧达莫可能通过Trk受体促进神经干细胞增殖

目的:探讨双嘧达莫促进体外培养神经干细胞增殖的机制。方法:取新生小鼠大脑进行神经干细胞原代培养,以2.5μmol·L~(-1)双嘧达莫组作为阳性对照组,分别加入腺苷受体拮抗剂CGS15943和Trk受体拮抗剂K252a,通过观察神经球形态、神经球生成数目、MTT法定量,初步探讨双嘧达莫促神经干细胞增殖的机制。结果:K252a 双嘧达莫组神经球数量和细胞活性均明显低于双嘧达莫组(P<0,01),而CGS15943 双嘧达莫组与双嘧达莫组相比差异无统计学意义。结论:双嘧达莫的促神经干细胞增殖作用可能是通过Trk受体介导的。     

胎鼠神经干细胞培养方法的建立及药物对干细胞增殖的影响

目的建立神经干细胞培养模型，观察药物对其增殖能力的影响。方法建立大鼠胎脑神经干细胞的培养方法，并用MTT法和3H-胸腺嘧啶核苷参入法观察黄皮酰胺、丹酚酸A及人参皂苷Rg1等对第2代神经球细胞状态的影响。结果免疫细胞化学结果显示，培养的神经细胞具备干细胞的基本特性；MTT和液闪结果则表明，上述3种药物可不同程度地影响神经干细胞的存活率和/或增殖活性。结论本实验所建立的胚胎大鼠神经干细胞培养模型可以观察到一些有脑保护作用的药物对干细胞存活和增殖有一定影响。     

胶质瘤细胞诱导神经干细胞迁移作用的实验研究

目的观察胶质瘤细胞在体外培养条件下对神经干细胞是否有诱导迁移的作用。方法①从新生1～2dSD大鼠脑皮层分离培养神经干细胞,进行神经干细胞及其增殖能力鉴定。②胶质瘤细胞与神经干细胞限定区域联合培养,观察神经干细胞的生长及其形态学变化。结果①所获得神经细胞球Nestin染色阳性,传代神经细胞球抗BrdU染色阳性。②可观察到神经干细胞球周围长出细胞突起,在靠近胶质瘤细胞的一侧,细胞突起的密度及长度均大于其他方向上的突起并且可见部分干细胞向胶质瘤细胞方向的移动。结论胶质瘤细胞在体外对神经干细胞有诱导迁移作用。     

体外神经干细胞三维药物筛选模型的构建

研究目的：建立中药对神经干细胞诱导转变为神经细胞的体外模型，不仅对研究脑的发育和分化以及神经系统疾病的治疗具有重要价值，而且也有助于了解中药的作用机制，为中药的临床应用提供坚实的科学依据。本研究评价体外海马神经干细胞在胶原凝胶内的生长情况，探讨胶原凝胶作为神经系统组织支架材料的可行性，以建立适合于干细胞药物筛选及作用机理研究的三维培养模型。研究方法：①细胞培养：体外培养来源于孕13天胎鼠的海马部位神经干细胞，传至第三代；根据胶原的特性，把神经干细胞均匀混合于胶原的预凝胶溶液中，便可形成“细胞．胶原”的凝胶三维结构。②检测指标：倒置相差显微镜和激光共聚焦显微镜观察胶原凝胶中神经干细胞的生长、伸展情况；通用免疫细胞化学方法及荧光显微技术鉴定神经干细胞，BrdU染色标记增殖的细胞；采用CCK-8法及Live／DeadViability／CytotoxicityKit试剂盒检测神经干细胞不同培养条件下的生存和增殖能力，筛选出最佳的胶原工作浓度。结果：①倒置相差显微镜下观察发现胶原凝胶中的神经干细胞生长状态良好；     

GM-1对新生大鼠神经干细胞增殖和分化的影响

目的:观察不同剂量神经节苷脂(GM—1)对神经干细胞增殖和分化的影响。方法:从新生大鼠海马组织分离出神经干细胞,分别培养于基础培养液(阴性对照组) ,加入三种不同剂量GM—1(实验组)及含有b FGF(阳性对照组)的神经干细胞培养液。采用神经干细胞球直接计数法测定细胞的增殖能力,并应用免疫细胞化学技术对神经干细胞分化情况进行鉴定。结果:3个实验组神经干细胞的增殖能力较阳性对照组无明显差异,较阴性对照组差异显著,贴壁培养细胞分化发现3个实验组分化能力明显低于阳性对照组。结论:GM—1对新生鼠神经干细胞的增殖起促进作用,对神经干细胞无明显的诱导分化作用。     

成年大鼠嗅球神经干细胞的培养和鉴定

目的建立成年大鼠嗅球神经干细胞分离培养和鉴定方法,探索新的成年神经干细胞种子来源。方法用无血清方法分离培养成年大鼠嗅球来源的神经干细胞;用克隆培养、BrdU整合的方法检验培养细胞的干细胞特性;用免疫荧光细胞化学的方法检测BrdU、神经干细胞标记物Nestin和SOX2,分化的细胞标记物Tuj1、GFAP、NG2的表达。结果从成年大鼠嗅球能够分离、培养出具有自我更新、增殖的神经球,构成神经球的细胞呈Nestin和SOX2阳性,它们分化后产生Tuj1阳性的神经元、GFAP阳性的星形胶质细胞、NG2阳性的少突胶质细胞。结论成年大鼠嗅球存在神经干细胞,能够在体外进行培养、增殖、分化,是神经干细胞的新的种子来源。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

---

Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Pharmacological treatment of COVID-19: an opinion paper

The precocity and efficacy of the vaccines developed so far against COVID-19 has been the most significant and saving advance against the pandemic. The development of vaccines has not prevented, during the whole period of the pandemic, the constant search for therapeutic medicines, both among existing drugs with different indications and in the development of new drugs. The Scientific Committee of the COVID-19 of the Illustrious College of Physicians of Madrid wanted to offer an early, simplified and critical approach to these new drugs, to new developments in immunotherapy and to what has been learned from the immune response modulators already known and which have proven effective against the virus, in order to help understand the current situation.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.