Red cell membrane changes during storage

A great deal is known about the red cell membrane and its abnormalities in various pathologic states. During red cell storage there is a progressive development of spheroechniocytosis with eventual production of irreversibly nondeformable red cells. The loss of membrane function is most likely related to some abnormality in maintenance of the cytoskeleton of the red cell. These changes appear to occur independent of ATP levels. Despite the increasing knowledge of the structure and function of the red cell membrane very little as yet is known about the specific abnormality in the red cell membrane that occurs during storage in the blood bank. Recent evidence for abnormal spectrin-actin interaction and abnormal spectrin oxidation has been the most promising. Areas of interest for research include studies of the specific mechanisms by which the plasticizer DEHP interacts with the membrane, specific definition of the molecular defect in membrane proteins that occurs during storage, and means to prevent these. If such deterioration and membrane stiffening could be prevented then the quality of the red cells that are transfused would be improved both in their function and ability to survive in the microcirculation. A final need, while not of specific value to the red cell itself, is the development of media and additives that will allow for increased plasma and Factor VIII yields, one of the driving forces in the blood transfusion system.     

神经干细胞诱导分化相关因素的研究

目的:综合分析神经干细胞分化诱导机制的研究现状。资料来源:应用计算机检索万方数据库和中国期刊全文数据库2002-01/2006-10有关神经干细胞分化诱导机制的文章,检索词"神经干细胞,分化",限定语种为中文;同时应用计算机检索Pubmed数据库2002-01/2006-10期间相关文献,检索词为"neural stem cell,differentiation",限定文章语种为"English"。资料选择:对检索到的神经干细胞分化诱导机制研究方面的相关信息进行整理,选取针对性强的文章,排除综述类文献。资料提炼:共检索到80篇相关文献,其中31篇符合要求。资料综合:神经干细胞是一种终生具有自我更新能力的细胞,目前对于影响其分化的机制仍不清楚。现在得到的共识是,神经干细胞的定向分化是由细胞因子、特殊蛋白质和激素、化学物质等局部环境因素和自身基因信号共同调节的,二者互相作用,彼此补充,共同作用于神经干细胞,决定神经干细胞的分化方向。结论:诱导神经干细胞分化成需要的细胞表型,是神经干细胞移植应用于临床的必要条件。目前对于神经干细胞分化诱导机制的研究已取得了较大进展,但仍有一系列问题亟待解决。     

姜黄素对SW480细胞株诱导分化的研究

目的探讨姜黄素对SW480(人结肠癌细胞)细胞株诱导分化机制。方法应用MTT比色法和流式细胞仪测定细胞周期的变化。并通过电子显微镜观察诱导分化后的亚细胞结构。结果姜黄素对SW480细胞增殖有明显的抑制作用。且呈时间、剂量依赖性,流式细胞仪分析G1期细胞堆积、S期细胞减少、G2/M期细胞增多、亚细胞结构、细胞空化、核仁不规则、染色质趋边凝集。结论姜黄素能够抑制SW480细胞增殖,阻止G1期细胞向S期转化的进程,促进SW480细胞凋亡。     

Techniques for the induction of human pluripotent stem cell differentiation towards cardiomyocytes

The derivation of pluripotent stem cells from human embryos and the generation of induced pluripotent stem cells (iPSCs) from somatic cells opened a new chapter in studies on the regeneration of the post‐infarction heart and regenerative medicine as a whole. Thus, protocols for obtaining iPSCs were enthusiastically adopted and widely used for further experiments on cardiac differentiation. iPSC‐mediated cardiomyocytes (iPSC‐CMs) under in vitro culture conditions are generated by simulating natural cardiomyogenesis and involve the wingless‐type mouse mammary tumour virus integration site family (WNT), transforming growth factor beta (TGF‐β) and fibroblast growth factor (FGF) signalling pathways. New strategies have been proposed to take advantage of small chemical molecules, organic compounds and even electric or mechanical stimulation. There are three main approaches to support cardiac commitment in vitro: embryoid bodis (EBs), monolayer in vitro cultures and inductive co‐cultures with visceral endoderm‐like (END2) cells. In EB technique initial uniform size of pluripotent stem cell (PSC) colonies has a pivotal significance. Hence, some methods were designed to support cells aggregation. Another well‐suited procedure is based on culturing cells in monolayer conditions in order to improve accessibility of growth factors and nutrients. Other distinct tactics are using visceral endoderm‐like cells to culture them with PSCs due to secretion of procardiac cytokines. Finally, the appropriate purification of the obtained cardiomyocytes is required prior to their administration to a patient under the prospective cellular therapy strategy. This goal can be achieved using non‐genetic methods, such as the application of surface markers and fluorescent dyes. Copyright © 2016 John Wiley & Sons, Ltd.     

大鼠骨髓间充质干细胞向神经元样细胞定向诱导分化及免疫组化鉴定

目的建立体外分离、培养大鼠骨髓间充质干细胞(MSCs)的方法,体外诱导其向神经元样细胞方向分化。方法应用细胞培养技术从大鼠股骨、胫骨中分离、纯化骨髓间充质干细胞并在体外进行培养,以形态学方法鉴定间充质干细胞,在倒置显微镜下观察细胞的形态特征,利用含10 ng/ml bFGF的LG-DMEM及含200μmol/L BHA、2%DMSO的无血清DMEM诱导其向神经元样细胞分化,并通过免疫组化方法鉴定。结果经原代及传代培养的骨髓间充质干细胞呈梭形,类似于成纤维细胞;成神经元诱导24 h后,多数MSCs变为典型的神经元样细胞,胞体向胞核收缩并有较多的长突起,免疫组化显示神经元特异性标志NSE、神经巢蛋白Nestin染色阳性。结论体外成功的进行了MSCs原代及传代培养,细胞生长稳定、增殖迅速并可多次传代,经诱导后具有向神经元样细胞分化潜能。     

中药诱导干细胞分化为心肌细胞的效应

背景:经典干细胞诱导剂由于具有细胞毒性而仅仅应用在干细胞体外实验中,这对进一步提高干细胞移植疗效提出了挑战.越来越多的课题组证实,中药作为全新的干细胞诱导剂可以诱导胚胎干细胞和骨髓间充质干细胞分化为心肌细胞.课题组长期从事心肌干细胞研究,利用从中药川芎中提取的生物碱川芎嗪,已成功在体外诱导心肌干细胞分化为心肌细胞.目的:总结中药干细胞诱导剂的整体研究进展情况,为进一步提高心血管疾病干细胞移植治疗过程中干细胞诱导分化为心肌细胞的效率奠定基础.方法:以"traditional Chinese medicine,stem cells,cardiomyocytes"为英文关键词,以"中药,中草药,干细胞,心肌细胞"为中文关键词,检索2005/2009西文生物医学期刊文献数据库(www.kjmed.com.cn)、中国全文期刊数据库、中国博士学位论文全文数据库及中国优秀硕士学位论文全文数据库(www.cnki.net)有关中药诱导干细胞分化为心肌细胞的文献,排除重复性研究.检索文献类型包括研究论著、学位论文及综述.结果与结论:共保留30篇文献进一步分析,中文14篇,英文16篇.结果显示中药作为一种全新的干细胞诱导剂,不仅能够有效诱导干细胞分化为心肌细胞,而且具有无毒副作用的特点,因此很有希望应用于临床干细胞治疗中.中药在诱导干细胞分化为心肌细胞方面的研究较新,随着研究深入,将会有越来越多的中药诱导剂被发现.     

Lipolytic Enzymes as markers of induction and differentiation

Factors leading to microsomal enzyme induction are associated with hypertriglyceridemia in man. Phenobarbital (PB) increases hepatic synthesis of triglyceride but lowers its serum concentration in rats due to increased postheparin plasma activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL); these changes are accompanied by increased activity of these lipolytic enzymes in adipose tissue and liver. The present work explores the cellular mechanisms whereby PB increases the tissue content of these enzymes, using primary cultures of rat liver hepatocytes and a continuous cell line of mouse fibroblasts (preadipocytes) that undergo differentiation into mature fat cells.

Secretion and synthesis of HTGL in primary rat hepatocytes increased 50% with insulin; when PB was added with insulin, activity was enhanced an additional 50%. By contrast, insulin inhibited HTGL secretin from the well differentiated rat hepatoma cell line, FU-5-5, C8, and this inhibition was partly overcome by PB. These results suggest that different control mechanisms govern the synthesis and secretion of HTGL in normal rat liver cells and hepatoma.

In cultured pre-adipocytes (3T3-F442A) insulin promoted differentiation when added to confluent cultures. PB (0.5 mM) resulted in marked enhancement of conversion of adipocytes characterized by a two-to threefold increase in extracellular LPL and a 10-fold increase in intracellular enzyme. These results suggest that PB promotes conversion of uncommitted cells into pre-adipocytes at an early stage in the differentiation of adipose tissue.     

脂肪干细胞的体外诱导与分化

背景:应用脂肪干细胞来作为组织工程的种子细胞是近年来组织工程研究中的一个活跃领域,大量的研究表明在不同的条件下脂肪干细胞可以向机体的不同组织分化.目的:对国内外脂肪干细胞体外诱导分化的现状及新进展作一综述.方法:应用计算机检索CNKI和PubMed数据库中2000-01/2010-10关于脂肪干细胞的文章,在标题和摘要中以"脂肪干细胞,体外分化潜能,种子细胞,转基因"或"Adipose-derived stem cells,cell differentiation in vitro,seeded cell,gene transfaction"为检索词进行检索.选择文章内容与脂肪干细胞体外分化有关者,同一领域文献则选择近期发表或发表在权威杂志文章.初检得到112篇文献,最终入选28篇文献进行综述.结果与结论:脂肪干细胞是存在于脂肪组织中的具有高度自我更新能力和多向分化潜能的干细胞群体.它具有多向系分化潜能,除可以分化为间充质来源的脂肪、骨、软骨以及骨骼肌、心肌等细胞,也可诱导分化为来源于外胚层的神经细胞以及具有功能性的血管内皮细胞.此外脂肪干细胞还具有造血支持作用以及可被腺病毒等高度转染等优点,可以作为基因转移良好的靶细胞.     

脂肪干细胞的体外诱导与分化

背景：应用脂肪干细胞来作为组织工程的种子细胞是近年来组织工程研究中的一个活跃领域,大量的研究表明在不同的条件下脂肪干细胞可以向机体的不同组织分化。目的：对国内外脂肪干细胞体外诱导分化的现状及新进展作一综述。方法：应用计算机检索CNKI和PubMed数据库中2000-01/2010-10关于脂肪干细胞的文章,在标题和摘要中以＂脂肪干细胞,体外分化潜能,种子细胞,转基因＂或＂Adipose-derived stem cells,celldifferentiation in vitro,seeded cell,genetrans faction＂为检索词进行检索。选择文章内容与脂肪干细胞体外分化有关者,同一领域文献则选择近期发表或发表在权威杂志文章。初检得到112篇文献,最终入选28篇文献进行综述。结果与结论：脂肪干细胞是存在于脂肪组织中的具有高度自我更新能力和多向分化潜能的干细胞群体。它具有多向系分化潜能,除可以分化为间充质来源的脂肪、骨、软骨以及骨骼肌、心肌等细胞,也可诱导分化为来源于外胚层的神经细胞以及具有功能性的血管内皮细胞。此外脂肪干细胞还具有造血支持作用以及可被腺病毒等高度转染等优点,可以作为基因转移良好的靶细胞。     

联合培养法优化K562细胞红系诱导分化及红系相关基因和膜蛋白表达分析

目的 优化K562细胞体外红系诱导分化条件,分析红系相关基因和膜蛋白的表达,探讨K562细胞的分化潜能.方法 采用红系诱导联合培养法,优化诱导试剂丁酸钠、红细胞生成素、氯化高铁血红素等成分组合和剂量组合.诱导效率鉴定指标选用红系ALAS mRNA和粒系bcr/abl mRNA表达丰度分析、细胞形态学观察、联苯胺染色阳性率计数.红系分化进一步鉴定指标选用Realtime-PCR检测10种红系分化相关基因的表达;流式细胞术测定红系细胞膜标志物.结果 优化红系诱导组合联合培养K562细胞120 h后,联苯胺染色计数阳性细胞可达100%.Realtime-PCR检测Spectrina、Spectrinβ、band3、eALAS、CD47、RhD、EPB4.2的mRNA水平分别为对照组的8.05、14.58、22.87、14.52、26.53、3.48、1.94倍,而ber/abl mRNA水平为对照组的0.39倍.流式细胞术测定红系膜蛋白CD47,band3、RhD水平明显增高(P<0.01),GPA无明显差异.结论优化组合体外红系诱导分化法可使K562细胞稳定向红系分化,良好细胞状态利于转外源基因操作,进行红系统疾病研究.RhD比GPA更早在红系细胞表达,可作为早期红系特异标记.     

干细胞共培养的方法及其分化诱导

目的:体细胞与干细胞共培养可以诱导干细胞向该种体细胞表型分化,文章就干细胞共培养的不同方法及其诱导分化的机制进行综述。资料来源:应用计算机检索PUBMED1990-01/2006-11期间的相关文章,检索词为"stem cells,coculture",并限定文章语言种类为English。同时计算机检索中国期刊全文数据库1990-01/2006-11期间的相关文章,检索词为"干细胞,共培养",并限定文章语言种类为中文。资料选择:对资料进行初审,并查看每篇文献后的引文。纳入标准:文章所述内容应与干细胞共培养分化诱导研究相关。排除标准:重复研究或Meta分析类文章。资料提炼:共收集到40篇相关文献,30篇文献符合纳入标准,排除的10篇文献为内容陈旧或重复。符合纳入标准的30篇文献中,分别涉及干细胞与体细胞共培养的不同方法、体细胞的旁分泌作用、细胞与细胞接触介导诱导以及干细胞与体细胞之间的缝隙连结诱导干细胞分化的可能机制。资料综合:干细胞可以通过特定的培养条件被诱导分化成具有不同表型的细胞,这为组织工程技术的应用提供了又一有效的种子细胞来源。用体细胞与干细胞共培养同样可以诱导干细胞向该种体细胞表型分化。干细胞与体细胞共培养的方法主要有六种,分别为单层共培养(monolayer co-culture)、分层渗透培养(transwells)、悬浮培养(suspension culture)、细胞团块培养(cell pellet)、细胞层培养(cell sheet)以及三维支架培养。干细胞分化的可能机制是体细胞通过分泌一些细胞因子,从而激活蛋白激酶,如PI3-激酶、促分裂原活化蛋白激酶、蛋白激酶C等,作用于相关的受体或抑制体,为干细胞的分化增殖提供了合适的环境。同时,有研究显示细胞与细胞接触反应是干细胞分化的独立机制。缝隙连接介导的细胞与细胞间反应也是干细胞分化中必需的。结论:干细胞与体细胞共培养诱导干细胞定向分化已经成为一种确实可行且非常有研究前景的方法,但共培养诱导分化的机制尚未完全明确。     

心肌细胞裂解液诱导人脂肪基质干细胞分化为心肌样细胞的研究

目的分离培养人脂肪基质干细胞（ASCs），观察心肌细胞裂解液诱导ASCs分化为类心肌细胞的情况。方法人脂肪组织用胶原蛋白酶I消化，贴壁法培养获得ASCs，流式细胞术鉴定其表型，向ASCs培养体系中加入乳鼠心肌细胞裂解液，培养2周后，利用免疫荧光技术和RT—PCR法分析分化后细胞的心肌特异性蛋白和基因的表达情况。结果培养获得ASCs，表型为CD44^＋、CD105^＋、CD31^-、CD45^-。心肌细胞裂解液诱导培养2周后，细胞表达心脏特异性肌钙蛋白T（cTnT）、结蛋白，同时也表达心脏特异性基因cTnT、ANP、aMHC。结论心肌细胞裂解液可在体外诱导ASCs分化为类心肌细胞。     

Red cell membrane protein changes caused by freezing and the mechanism of cryoprotection by glycerol

Membranes isolated from frozen-thawed erythrocytes and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate have significantly decreased band six, which is the glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase. The total membrane protein and sialic acid contents of these membranes are also significantly decreased. The red blood cell membrane protein abnormality is reproduced by suspending membranes in NaCl solutions of increasing molarity. Glycerol prevents the elution of band six in NaCl solutions less than 0.2 M and ameliorates it in solutions of higher ionic strength. When intact cells are suspended in hypertonic salt solution, there is no elution of band six, indicating that exposure of the inner surface of the membrane to toxic concentrations of solutes results in this elution. The data indicate that freezing with its associated hypertonicity induces a specific membrane change which is ameliorated by the addition of glycerol.     

Establishment,maintenance and differentiation induction of embryonic stem cells

Embryonic stem(ES) cells are the cell lines derived from the inner cell mass of early preimplantation embryos at blastocyst stage. Mouse ES cells are pluripotent since the cells can be differentiated into all lineages of the cells once re-introduced into blastocysts. Human ES cells have been established from the inner cell mass of blastocysts which are developed from surplus embryos obtained by in vitro fertilization. ES cells are considered valuable resource of regeneration medicine because of their vigorous proliferative activity and broad differentiation capacity. Establishment, maintenance and in vitro differentiation of the cells are key points for the utilization of ES cells in regeneration medicine.     

新型氨基甾体类对人粒系白血病HL-60细胞系的增殖抑制和分化诱导的作用

研究发现一种新型氨基甾体化合物,2β-(4’-甲基-1’-哌嗪基)-3α,17β二羟基-5α-雄甾烷(HY)可抑制HL-60细胞的增殖,并诱导该类细胞向巨噬样细胞分化。其主要证据如下:①细胞计数,集落计数及MTT检测显示抑制HL-60细胞的增殖;②液体培养6天后形态学显示诱导HL-60细胞向巨噬样细胞分化;③可诱导NBT反应阳性;④可诱导α-萘酚醋酸酯酶反应阳性;⑤流式细胞术显示可诱导CD11b和CD14的表达。结论提示该化合物具有治疗白血病的潜在价值。