慢病毒介导酪氨酸激酶受体 A过表达载体促进神经干细胞增殖和分化的实验研究

目的探究酪氨酸激酶受体 A(Trk A)过表达载体对神经干细胞增殖和分化能力的影响。方法取 SD大鼠海马组织进行提取神经干细胞,采用免疫荧光的方法对神经球进行鉴定,利用 siRNA技术构建 Trk A过表达载体,将培养的神经干细胞分成三组,空白对照组为未做任何处理的细胞组,阴性对照组是经空白载体的慢病毒转染的细胞组,实验组是由 Trk A的过表达载体构建的慢病毒转染细胞组。逆转录 PCR(RT?PCR)检测 Trk A和神经干细胞分化标志基因 Tuj1,GFAP和 CNPase的表达, CCK?8试剂盒检测各组细胞的增殖率。结果免疫荧光染色显示神经干细胞细胞球 Nestin(红色荧光), Brud染色(绿色荧光)阳性,且占细胞量的 90%以上;荧光双标染色(棕色荧光)显示双染细胞量占总细胞量的 90%以上,明培养的细胞是神经干细胞。慢病毒滴度的检测结果: Trk A过表达慢病毒载体转染滴度为 3×108 TU/mL。RT?PCR结果说表明,空白对照组(1.09±0.09)和阴性对照组(1.11±0.05)的 Trk A的 mRNA相对表达量比较差异无统计学意义(P＞0.05),实验组的 Trk A的 mRNA相对表达量(7.12±1.32)要明显高于空白对照组和阴性对照组(P＜0.05);神经干细胞分化标志基因 Tuj1,GFAP和 CNPase的表达与 Trk A相同。 CCK?8试剂盒检测结果表明,空白对照组、阴性对照组及实验组的细胞增殖率分别为(87.92±9.21)%、(88.22±9.37)%、(176.01±11.33)%(F＝18.082,P＜0.05)其中空白对照组和阴性对照组的细胞增殖率比较差异无统计学意义(P＞0.05)实验组的细胞增殖率要明显高于空白对,照组和阴性对照组(P＜0.05)。结论 Trk A过表达载体可以促进神经元干细胞向,神经元细胞,星型胶质细胞和少突胶质细胞的分化,并且可以促进神经元干细     

胚胎大鼠神经干细胞体外分化的激光共聚焦显微镜观察

目的:采用激光扫描共聚焦显微镜观察体外培养胎鼠大脑皮质神经干细胞(neural stem cells,NSCs)分化情况。方法:利用无血清培养方法,分离培养胚胎大鼠大脑NSCs,进行体外扩增培养、传代;采用溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)掺入、双重免疫荧光细胞化学标记方法和激光扫描共聚焦显微镜,用神经细胞的特异性抗体(神经元β-微管蛋白、胶质纤维酸性蛋白),鉴定NSCs向神经元与星形胶质细胞分化的情况。结果:从胎鼠大脑皮质及皮质下分离的组织,经原代和传代培养均可形成细胞克隆,并表达神经上皮干细胞蛋白(Nestin)抗原。在血清诱导下,分化后的细胞表达神经元、星形胶质细胞2种神经细胞的特异性抗原,NSCs分化为星形胶质细胞神经元的比例分别为(43.70±8.55)%和(23.00±3.69)%。结论:从胎鼠大脑皮质分离出的细胞可获得呈集落样生长的神经干细胞团,并能表达NSCs的特异性抗原;激光扫描共聚焦显微镜可清晰地观察到培养细胞具有分化为神经元和星形胶质细胞的潜能。     

胎鼠多巴胺能神经元前体细胞体外扩增和诱导分化的初步研究

目的　尝试体外扩增胎鼠多巴胺能神经元前体细胞并用L 抗坏血酸 2 磷酸酯倍半镁盐 (AA 2P)促其分化成多巴胺能神经元。方法　体外培养来自胎鼠中脑腹侧组织的前体细胞 ,用bFGF促其增殖 7天 ,而后用AA 2P促其分化 ,并进行免疫荧光染色分析。结果　细胞总数平均增长了 6 8倍 ,其中星形胶质细胞只占 ( 4 15± 1 37) % ,而神经元占 ( 2 0 38± 11 6 0 ) % ,多巴胺能神经元占所有神经元的 ( 2 9 4 7± 13 79) %。结论　该方法能体外扩增胎鼠多巴胺能神经元前体细胞并促其分化为多巴胺能神经元     

IL-1β诱导中脑神经干细胞向多巴胺能神经元分化的研究

在胚鼠中脑曲分离神经干细胞 ,研究 IL-1β对中脑神经干细胞诱导分化为多巴胺神经元的效能 ,运用体外分离、培养小鼠胚胎中的中脑神经经干细胞 ,加入 IL -1β诱导其分化 ,通过巢蛋白 ( Nestin)、神经元特异性烯醇化酶 ( NSE)、胶质纤维酸性蛋白( GFAP)、酪氨酸羟化酶 ( TH)免疫组化鉴定。结果显示 ,小鼠胚胎的腹侧中脑曲部位分离获得了大量未分化、呈球状悬浮生长的细胞 ,且能在有血清的培养基中分化为神经元和神经胶质细胞。经 IL-1β诱导后 ,多巴胺能神经元分化比例达 16%左右。     

大黄素对去卵巢大鼠和经β淀粉样蛋白作用的培养神经元的保护作用

目的研究大黄素对去卵巢大鼠和经β淀粉样蛋白(β amyloid protein，β AP)作用的培养神经元的保护作用。方法 ① 将29只Wistar大鼠随机分为3组：模型组10 只，手术切除双侧卵巢后饲喂普通食物；给药组10只，手术切除双侧卵巢后饲喂含大黄素的食物，大鼠摄入大黄素按80 μg·kg 1·d 1计；对照组9只，给予假手术后饲喂普通食物。70 d后通过水迷宫实验测定3组大鼠行为改变情况。②取经过上述实验的3组大鼠的脑额叶皮质细胞，以流式细胞仪检测早期凋亡细胞百分数。③取乳鼠大脑皮质细胞，培养12 d后分为4组：实验组1加入β AP1 40；实验组2加入β AP1 40和17 β雌激素，实验组3加入β AP1 40和大黄素；正常组不加入任何药物。观察4组培养细胞的早期和晚期存活数，测定标记Annexin V FITC的细胞的荧光强度。结果① 给药组和对照组大鼠比模型组大鼠更容易找到实验平台的位置。给药组和对照组到达平台的平均时间分别为(1519±224) 、(1448±199) s，均比模型组大鼠到达平台所用的平均时间［(2245±242) s］短 (P＜0.01)；给药组和对照组到达平台的游泳距离分别为(43291±299)、(42658±266) cm，均比模型组到达平台的游泳距离［(76666±357) cm］明显缩短(P＜001)。② 给药组、对照组、模型组大鼠大脑皮质神经细胞凋亡比例分别为(54 ±07)%，(53±07)%，(128±25)%，前两者与后者比较，均 P＜001。③ 实验组1神经元和突起被抑制，早期凋亡细胞百分数比正常组、实验组2、实验组3高；实验组1标记Annexin V FITC的荧光强度(619±027)比正常组(41±011)、实验组2(43±029)、实验组3(48±09)明显增高。结论大黄素能保护去卵巢大鼠，并能拮抗β AP对培养神经元的毒性作用。     

丹参注射液诱导大鼠骨髓间充质干细胞分化为神经元样细胞的研究

目的:探讨丹参注射液在体外诱导大鼠骨髓间充质干细胞(MSCs)向神经元样细胞分化的作用。方法:采用差速贴壁法体外分离培养大鼠MSCs,流式细胞仪检测其表面标志,经碱性成纤维细胞生长因子(bFGF)预诱导24h,采用含丹参注射液的无血清L-DMEM培养液诱导MSCs,倒置显微镜下观察细胞形态的变化,免疫荧光细胞化学检测巢蛋白、神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-M)、胶质纤维酸性蛋白(GFAP)的表达。结果:体外培养的大鼠MSCs表达CD29、CD44、CD105、CD166。丹参注射液诱导后MSCs胞体收缩,突起伸出,较密的部分神经元拉成网状,免疫细胞化学显示巢蛋白、NSE、NF-M表达阳性,阳性率分别为(58.68±4.50)%、(61.23±5.72)%、(63.47±6.38)%,GFAP阳性细胞率(1.47±0.23)%。结论:丹参注射液在体外可以将大鼠MSCs诱导分化为神经元样细胞。     

星形胶质细胞对神经干细胞分化的影响实验研究

目的研究星形胶质细胞对神经干细胞分化的影响。方法随机在我院动物实验中心选取3只大鼠为研究对象,将本次实验均分为普通星形胶质细胞的培养的对照组以及星形胶质细胞与神经干细胞互不接触状态下共同培养的实验组。对比两组星形胶质细胞对神经干细胞分化的影响效果。结果实验组的纯化星形胶质细胞胶质纤维酸性蛋白抗体标记为阳性,其细胞纯度高到96%,且在神经元特异性烯醇化酶阳性细胞与酪氨酸羟化酶阳性细胞均比对照组多。结论星形胶质细胞不仅可以加快神经干细胞向神经元细胞的分化以及向多巴胺神经元细胞的分化,而且可以延长神经元存活时间,降低神经元凋亡率。     

肝细胞生长因子预处理对过氧化氢诱导大鼠神经干细胞凋亡的保护作用

目的研究肝细胞生长因子(HGF)对神经干细胞(NSC)凋亡的保护作用及其作用机制,为HGF用于NSC移植提供实验基础。方法分离培养大鼠NSC。细胞分为正常对照、模型(H2O2100μmo.lL-1)、HGF+H2O2(HGF15,30及60μg.L-1预处理24h后,再加入H2O2100μmol.L-1处理4h),LY294002(PI3K/Akt通路抑制剂)+HGF+H2O2(先加入LY29400220μmol.L-1处理30min,再加入HGF60μg.L-1处理24h,最后再加入100μmo.lL-1H2O2培养4h)组。MTT法检测细胞存活率;TUNEL法检测细胞凋亡率;比色法检测半胱氨酸天冬氨酸蛋白酶(caspase)-3活性;Western印迹分析Bcl-2,Bax蛋白表达。结果MTT检测发现,随着HGF浓度的增加,NSC细胞的存活率也增加。与模型组〔(63.5±2.4)%〕比较,HGF15,30及60μg.L-1预处理组细胞存活率明显升高〔(79.1±7.5)%,(83.8±6.1)%和(86.6±8.2)%;n=3,P<0.05〕。TUNEL法检测发现,HGF预处理组凋亡细胞明显减少,模型组的凋亡率为(43.5±6.2)%,HGF预处理组则分别为(34.2±8.6)%,(21.7±3.8)%及(19.4±4.0)%。Caspase-3活性检测表明,与模型组相比,HGF预处理组细胞caspase-3活性降低。Western印迹分析结果显示,与模型组比较,HGF预处理使细胞的Bcl-2蛋白表达升高,但Bax蛋白表达不受影响;HGF的抗凋亡效应可被PI3K/Akt通道阻滞剂LY294002阻断。结论HGF可减轻H2O2所诱导的大鼠NSC凋亡,且呈一定的浓度依赖关系,其作用机制可能与NSC的PI3K/Akt通路激活和Bcl-2表达增强有关。     

不同细胞因子组合对神经干细胞分化的影响

目的:探讨不同细胞因子组合对神经干细胞(NSCs)分化的影响。方法:采用不同组合的细胞因子(bFGF EGF; bFGF PDGF;bFGF BDNF;bFGF)分离培养大鼠海马神经干细胞,并用免疲细胞化学技术研究神经干细胞的分化情况。结果:在bFGF、bFGF EGF、bFGF BDNF及bFGF PDGF不同因子或因子组合中,均能诱导神经干细胞分化,分化后的神经元比例分别为(14.5±1.2)%、(14.7±1.8)%、(23.3±2.1)%、(35.5±2.4)%,神经干细胞的分化比例趋势为bFGF PDGF >bFGF BDNF>bFGF EGF>bFGF(P<0.05)。姑论:PDGF对bFGF促进神经干细胞向神经元定向分化有较好的协同作用。     

生长激素及其受体在结直肠癌中的表达及临床意义

目的 探讨生长激素(GH)和生长激素受体(GHR)在结直肠癌患者血清及其癌组织中的表达水平及临床意义。方法 采用酶免疫吸附(ELISA)和免疫组织化学(IHC)法分别检测实验组与对照组血清GH和组织 GHR的表达水平。结果 实验组血清GH水平(5.98±3.48) μg·L-1明显高于对照组(2.39±1.63) μg·L-1,并且GH水平与肿瘤大小、分化程度及TNM分期密切相关(P=0.017,P＜0.001,P=0.005);其中血清实验组0+Ⅰ+Ⅱ/TNM分期GH水平(5.07±3.44)μg·L-1明显高于血清对照组(2.39±1.63)μg·L-1;实验组组织GHR表达(79.2%)明显高于对照组(61.1%),并且GHR表达与肿瘤大小、分化程度及TNM分期密切相关(P=0.019,P=0.015,P=0.035)。结论 GH-GHR轴与结直肠癌的发生发展密切相关,检测其表达水平,有望为结直肠癌的早期诊断及个体化治疗提供理论依据。     

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.

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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.     

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.     

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.