小鼠骨髓间充质干细胞分离培养方法的建立

目的建立培养小鼠骨髓间充质干细胞(none mesenchymal stem cells,BMSCs)的分离培养方法。方法采用全骨髓体外分离并采用差速贴壁法纯化扩增C57BL/6小鼠BMSCs,形态学观察细胞生长情况,流式细胞仪检测其表面抗原情况并观察其多项分化潜能。结果使用该方法纯化扩增的BMSCs形态均一,生长良好,表达CD29、CD44和Sca-1,不表达CD11b、CD45和CD34,并具有成骨成脂潜能。结论通过全骨髓贴壁法可以成功的分离培养出小鼠BMSCs。     

兔骨髓间充质干细胞体外诱导血管内皮细胞

目的:探讨兔骨髓间充质干细胞(BMSCs)体外诱导为血管内皮细胞(VECs)的方法,为心血管疾病的血管重建提供治疗策略。方法:抽取兔骨髓,采用全骨髓贴壁法加内皮细胞培养液诱导培养细胞,记录并观察诱导后细胞的形态及生长过程,并进行荧光标记;采用免疫细胞化学显色法鉴定诱导后细胞表面标志物CD31、CD34,利用支架材料Matrigel基质胶观察VECs三维血管化形态。结果:兔BMSCs经诱导后48 h,呈小圆形,可见贴壁生长;3～4d可见少量细胞贴壁,多为短梭形;6～8d完全贴壁,多为长梭形、锥形、不规则形;10～12d后细胞呈集落样生长;第15天成片生长的细胞集落相互连接,呈现典型的"鹅卵石样"或"铺路石样"外观;CM-DiI细胞荧光标记显示VECs的胞质、胞膜呈现红色荧光,DAPI细胞荧光标记显示VECs胞核呈蓝色荧光,细胞形态为长梭形,且细胞生长状况良好;免疫细胞化学显色显示CD31、CD34均呈阳性;在Matrigel基质胶上VECs呈现典型的环状或管状。结论:以全骨髓贴壁法和经内皮细胞培养液诱导法可获得VECs。     

先天性心脏病患儿骨髓单核细胞分离培养内皮祖细胞的实验研究

目的探索自先天性心脏病患儿骨髓单核细胞中分离培养内皮祖细胞的方法，期望为儿童组织工程血管、补片或带瓣管道的制备找到新的种子细胞来源。方法采集先天性心脏病患儿骨髓，梯度密度离心法分离单核细胞，内皮细胞培养液EGM-2培养，种植于提前包埋了纤维连接蛋白的培养皿进行体外扩增，取48h后贴壁细胞，应用免疫组织化学和免疫荧光技术鉴定内皮细胞系列标志：CD34、CD31、FLK-1、ve-Cadherin和Ⅷ因子。结果经过梯度密度离心和贴壁法选择的细胞表达内皮细胞特异性抗原：CD34、CD31、FLK-1、ve—Cadherin和Ⅷ因子。培养至第5代细胞形态基本相似，细胞总数可以达到10^8以上。结论自先天性心脏病患儿骨髓单核细胞中可以分离培养出内皮祖细胞并能体外扩增，可以作为先天性心脏病患儿构建组织工程血管的种子细胞来源。     

不同条件下骨髓间充质干细胞分离培养及其生物学特性研究

目的:探讨并优化大鼠骨髓间充质干细胞(BMSCs)体外分离培养的方法,筛选出最佳培养时间,并鉴定其生物学特性,为BMSCs的应用奠定基础。方法:在不同季节,采用贴壁筛选法分离纯化BMSCs,并通过传代培养对其进行扩增。倒置光学显微镜下观察细胞形态及生长特征,并按时间段绘制细胞生长曲线。流式细胞术检测BMSCs抗原分子CD90及造血干细胞抗原分子CD45的表达;用β-巯基乙醇诱导BMSCs分化。结果:生长曲线分析,冬季培养的BMSCsF3代潜伏期为4d,对数期为5～9d,第10天后进入平台期,细胞出现接触抑制现象;春、夏、秋三季培养的BMSCsF3代潜伏期为2d,对数增殖期为3～6d,第7天后进入平台期,细胞出现接触抑制现象。流式细胞术检测F0代细胞中,CD90+CD45-细胞为3.5%,F3代细胞中CD90+CD45-细胞达到97%;F3代BMSCs镜下呈梭形、多角形成纤维细胞样形态,大小均一,漩涡状生长。β-巯基乙醇诱导BMSCs5h后表现出明显的神经元样细胞形态。结论:不同培养季节对BMSCs生长曲线影响明显,春夏秋三季细胞生长较快,而冬季细胞生长缓慢,体现了"春生、夏长、秋收、冬藏"的生物规律;贴壁筛选法是理想的体外分离扩增BMSCs的培养体系,所培养的BMSCs纯度高、生物学性状稳定。     

大鼠骨髓内皮祖细胞的分离培养与鉴定

背景：内皮祖细胞参与血管新生,但其分离、培养、鉴定方法目前并不统一。 目的：探索大鼠骨髓内皮祖细胞的分离培养及鉴定方法。 方法：使用密度梯度离心法及差速贴壁法联合的方法培养内皮祖细胞,在倒置显微镜下观察细胞生长情况及形态变化,使用Dil标记的乙酰化低密度脂蛋白和FITC标记的荆豆凝集素1双荧光染色、流式细胞仪检测细胞表面抗原CD34、CD133表达情况。 结果与结论：培养前4 d细胞增殖不明显,第5~10天迅速增殖,并可见细胞集落及线状结构形成。培养第7天的内皮祖细胞具有吞噬Dil标记的乙酰化低密度脂蛋白和FITC标记的荆豆凝集素1的功能。流式细胞仪检测体外培养第10天的细胞,CD133+ 细胞占19.2%,CD34+ 细胞占28.7%,CD34+/CD133+ 细胞占19.1%。说明密度梯度离心法联合差速贴壁法可在体外有效分离培养大鼠骨髓内皮祖细胞。 关键词：内皮祖细胞;细胞分离;细胞培养;骨髓;细胞鉴定 doi:10.3969/j.issn.1673-8225.2012.10.006     

全骨髓贴壁法分离培养大鼠骨髓间充质干细胞及其生物学特性

背景：培养出生长状态良好、数量足够多的大鼠骨髓间充质干细胞是其作为种子细胞在组织工程等领域广泛应用的重要前提。 目的：寻求快捷有效的大鼠骨髓间充质干细胞体外培养条件,观察培养的间充质干细胞生物学特性。 方法：全骨髓法体外分离培养大鼠骨髓间充质干细胞,贴壁筛选法进行纯化,倒置相差显微镜下观察细胞形态及生长特征,免疫荧光分析细胞骨架,MTT法绘制细胞生长曲线,流式细胞仪检测其表面标记。 结果与结论：分离培养的细胞呈长梭形或多边形;细胞生长曲线呈S形;微丝免疫荧光染色结果显示,培养的骨髓间充质干细胞具有良好的细胞骨架系统。第3代骨髓间充质干细胞CD29、CD44、CD71、CD90均呈阳性表达,而CD13、CD34、CD45、CD133呈阴性。提示,经全骨髓贴壁法体外分离培养的细胞在形态学和细胞表面标志物表达方面具有干细胞生物学特性,经鉴定为骨髓间充质干细胞,其第3代活性最佳,可用于后续实验。     

大鼠骨髓间充质干细胞对免疫耐受的作用

目的:体外分离培养大鼠骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMSCs),观察和检测其生长特性及表面标记,探讨它对免疫耐受的作用.方法:通过密度梯度离心、贴壁筛选法分离培养大鼠 BMSCs,观察细胞形态学变化,应用流式细胞术测定其表面分子 CD34、CD44 的表达.、将培养第 3 代的 BMSCs 经尾静脉输注大鼠,应用流式细胞技术检测大鼠外周血中CD4+CD25+Treg 比率,通过3H-TdR 掺入实验检测 BMSCs 对大鼠胸腺细胞自发增殖反应的影响.结果:体外培养的大鼠 BMSCs 呈贴壁生长,似成纤维细胞样,表面分子 CD44 表达阳性率为98%,但 CD34 表达阴性.输注 BMSCs 的大鼠外周血中CD4+CD25+Treg 占2.5%±O.69%,明显高于对照组(0.8%±0.14%)和PBS输注组(1.0%±0.23%),P<0.05;胸腺细胞白发增殖活性(cpm值3 275.41±1 736.05)明显低于正常对照组(7 368.25±2 532.13)和PBS输注组(6 143.71±2 380.47),P相似文献   

大鼠骨髓间充质干细胞的培养与鉴定

背景：骨髓间充质干细胞在骨髓中含量极低,体外培养难度较大。体外分离培养纯度高、活力强、生物特性均一的间充质干细胞,对组织工程及细胞的体内、体外实验显得至关重要。 目的：建立大鼠骨髓间充质干细胞的分离、培养、纯化方法,并进行细胞形态学观察、表面标志物鉴定及多向分化能力检测。 方法：通过全骨髓贴壁法体外分离、培养、纯化大鼠骨髓间充质干细胞,进行形态学观察,绘制生长曲线,细胞周期分析,流式细胞仪检测细胞表面标记物,分别向成骨、成脂方向诱导分化。 结果与结论：大鼠骨髓间充质干细胞生长以梭形细胞为主,呈放射状排列的细胞集落,细胞生长旺盛,可连续稳定传代10代以上。生长曲线及细胞周期显示骨髓间充质干细胞符合正常细胞生长特征且生长活跃。第3代骨髓间充质干细胞CD44,CD90,CD105均呈阳性表达,而CD34,CD45呈阴性表达。成脂、成骨诱导后,油红O染色、碱性磷酸酶染色、von Kossa法染色和茜素红染色均呈阳性。全骨髓贴壁培养法操作简单,可大量分离、纯化、扩增骨髓间充质干细胞,所获细胞具有间充质干细胞的一般生物学特性,经诱导培养后具有多向分化潜能。实验所用的全骨髓贴壁法法为组织工程提供充足的种子细胞来源具有重要的现实意义。     

大鼠骨髓间充质干细胞体外分离培养与CM-DiI荧光标记

背景：目前,对骨髓间充质干细胞的分离、纯化和扩增还没有统一的、标准化的方法。CM-DiI作为荧光标记物稳定、可靠、标记率高、标记简便。 目的：建立SD大鼠骨髓间充质干细胞体外分离培养及标记的方法。 方法：取2只体质量50-100 g雄性SD大鼠,无菌条件下采集双侧股骨、胫骨骨髓,用全骨髓贴壁分离法和密度梯度离心法培养出原代骨髓间充质干细胞,通过及时、反复传代对细胞进行扩增纯化,在体外用荧光活性染料CM-DiI标记第3代骨髓间充质干细胞后作为供体细胞来源。 结果与结论：用全骨髓贴壁分离法和密度梯度离心法两种方法均能成功体外分离培养骨髓间充质干细胞,经流式细胞仪分析,培养出的细胞CD34阳性率为17.5%,CD44阳性率为97.9%、CD90阳性率为91%,与骨髓间充质干细胞表面抗原一致。但培养出的细胞数量全骨髓贴壁分离法明显多于密度梯度离心法,两种方法培养骨髓间充质干细胞的细胞活力和增殖能力无明显差异。CM-DiI能够成功荧光标记骨髓间充质干细胞,CM-DiI作为荧光标记物稳定、可靠、标记率高、标记简便。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

密度梯度离心法结合贴壁法体外分离培养大鼠骨髓间充质干细胞

目的 研究密度梯度离心法结合贴壁法分离大鼠骨髓间充质干细胞(BMSCs)及不同首次换液时间对细胞增殖的影响,探索一种简单易行、扩增效率高的方法。方法 3月龄雄性SD大鼠提取原代BMSCs,密度梯度离心法结合贴壁法分离培养,并应用3组不同首次半量换液时间(24h,48h,72h)进行培养传代,测定生长曲线和细胞的存活率;免疫细胞化学染色分析细胞表面抗原CD44和CD166的表达。结果 分离的BMSCs48h后细胞完全贴壁生长,呈形态均一的纺锤形单核细胞状,第3代细胞培养1w后呈长梭型,呈平行排列生长。48h半量换液组细胞增殖活性及生存率最高,其次为24h和72h组;表面标志物CD44和CD166均呈阳性。结论 联合应用密度梯度离心法及贴壁筛选法可成功分离BMSCs,且效率高、纯度高,48h首次半量换液更适合BMSCs的生长繁殖。     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.     

Der Einfluß von Nahrungsmitteln und Rauchen auf die klinisch-chemische Diagnostik von Phäochromozytom,Neuroblastom und Karzinoid-Syndrom

Zusammenfassung Der Einfluß von verschiedenen Nahrungsmitteln auf Methoden zur Bestimmung von Adrenalin (AD), Noradrenalin (NA), Vanillinmandelsäure (VMS), Metanephrinen (MN), Homovanillinsäure (HVS) und 5-Hydroxyindolessigsäure (5-HIE) im 24 h-Harn zur Diagnose des Phäochromozytoms bzw. Karzinoid-Syndroms wurde untersucht. Die in die Untersuchung einbezogenen Nahrungsmittel waren: Tee, Kaffee, Mandeln, Ananas, Käse, Walnüsse, Vanillepudding, Bananen, Tomaten und Milchschokolade. Außerdem wurde der Einfluß des Zigarettenrauchens auf die Bestimmung von AD, NA, VMS und MN untersucht.Walnüsse führten zu einer starken Erhöhung der 5-HIE-Ausscheidung. Bananen erhöhten die Ausscheidung von AD, NA, VMS, MN und 5-HIE. Kaffee und Ananas bewirkten eine geringe Zunahme der MN-Werte. Rauchen von 20–30 Zigaretten/Tag beeinflußte keine der vier Variablen.Wenn die beschriebenen Methoden benutzt werden, sollte lediglich auf den Verzehr von Bananen und Walnüssen vor und während der Harnsammelperioden verzichtet werden, da die oberen Normgrenzen im Harn überschritten werden könnten. Ein Verzicht auf Kaffee und Ananas in normalen Mengen ist nicht erforderlich. Es besteht kein Anlaß, weiterhin die bisherigen umfangreichen Restriktionen der übrigen Nahrungsmittel beizubehalten.