小鼠脑微血管内皮细胞的原代培养与鉴定

目的探索和建立一种纯度高、操作简单且重复性强的小鼠脑微血管内皮细胞原代培养方法。方法通过剪碎、过细胞筛网等物理方法,结合白蛋白密度梯度离心及化学酶消化法,并以严格控制1 g/LⅡ型胶原蛋白酶消化作用时间15～20 min为首要,从2～3周龄ICR小鼠脑组织中分离培养出小鼠脑微血管内皮细胞。通过细胞形态学观察和免疫细胞化学染色检测Ⅷ因子相关抗原鉴定所培养的细胞。结果接种24 h,贴壁的血管段周围爬出多角形或短梭状细胞,并逐渐扩散成团簇状; 7 d后细胞融合,呈典型的单层、"铺路石样"镶嵌式排列。Ⅷ因子相关抗原细胞免疫化学染色鉴定为阳性,胞质呈棕红色,内皮细胞纯度达95%以上。结论成功建立了一种操作简单、重复性强、目的细胞纯度高的小鼠脑微血管内皮细胞原代培养方法。     

大鼠脑皮质微血管内皮细胞的分离和培养

目的： 本研究旨在探索1种有效分离、培养和获取较高纯度大鼠脑微血管内皮细胞（BMECs）的方法。方法: 自12 d SD大鼠脑分离出皮质，采用二次酶消化、BSA和Percoll非连续梯度离心获得较纯的脑微血管段后，接种于涂布有明胶的培养皿进行原代培养；相差显微镜观察细胞的形态学特性，进行血管内皮细胞特异性标志物Ⅷ因子相关抗原免疫组化检测。结果: 培养24 h即可见细胞从贴壁的脑微血管段周围爬出，细胞呈短梭形，集落呈典型的“鹅卵石样”，区域性单层生长，6-7 d内皮细胞开始融合，血管内皮细胞特异性标志物Ⅷ因子相关抗原表达阳性，纯度达92.6%。结论: 成功地自大鼠脑皮质分离并培养出纯度较高的BMECs，为进一步开展脑微血管内皮细胞的生物学特性的相关研究提供有用的方法。     

大鼠脑微血管内皮细胞的培养

目的:探索一种简便易行、可培养出高纯度大鼠脑微血管内皮细胞的方法。方法:1月龄SD大鼠,解剖得到大脑皮质,密度离心法获得较纯的脑微血管段后进行原代培养,传代采用差速消化和贴壁方法进行纯化。通过形态学观察及第Ⅷ因子相关抗原免疫荧光检测对培养细胞进行鉴定。结果:密度离心法分离出的大量微血管段呈"串珠样"结构,培养24h可见短梭形、多角形细胞,8～10天基本融合。第2代细胞经免疫荧光染色,第Ⅷ因子相关抗原呈阳性,阳性率达90%。结论:原代细胞采用低分子量葡聚糖加Percoll密度离心法、传代细胞采用差速消化和差速贴壁法纯化可成功培养高纯度的脑微血管内皮细胞。     

原代大鼠脑微血管内皮细胞的培养及鉴定

建立一种纯度较高的原代大鼠脑微血管内皮细胞的分离培养方法。取7～10日龄SD大鼠脑组织,经匀浆,牛血清白蛋白密度梯度离心,Ⅱ型胶原酶消化获得脑微血管段后,置于CO_2培养箱中进行原代培养。通过细胞形态学观察、第Ⅷ因子相关抗原免疫细胞化学染色鉴定所培养的细胞。在倒置显微镜下,体外培养24～48 h后,细胞从贴壁的脑微血管段周围爬出,单个细胞为短梭形或多角形,呈团簇状单层贴壁生长,待细胞密集融合后,则成典型的"铺路石样"涡旋状排列;Ⅷ因子免疫细胞化学染色检测,相关抗原表达为阳性,细胞胞质显棕黄色,阳性细胞率达99%以上。该方法能够成功分离培养出原代大鼠脑微血管内皮细胞。     

大鼠脑微血管内皮细胞的原代培养

目的分离、培养原代脑微血管内皮细胞,建立体外血脑屏障模型。同时探索高纯度、活性好的脑微血管内皮细胞的分离培养方法。方法取3周大鼠大脑,分离皮质,经过匀浆、葡聚糖离心以及消化后,取纯度较高的脑微血管段种植于胶原蛋白包被过的塑料培养瓶中进行培养。显微镜观察及检测Ⅷ因子相关抗原。结果镜下细胞呈长梭形。7d左右细胞可融合,Ⅷ因子相关抗原免疫组化检测为阳性．且阳性细胞占绝大部分。结论本实验成功分离大鼠脑微血管内皮细胞,并进行原代培养,为脑微血管内皮细胞的进一步研究提供了模型．也为构建更高级的大鼠体外血脑屏障奠定基础。     

改良原代大鼠脑微血管内皮细胞的培养方法及鉴定

目的 改良原代大鼠脑微血管内皮细胞分离培养方法。方法 选取4～6周龄SD大鼠6只,经开颅取脑、漂洗剪碎、过筛、牛血清白蛋白密度梯度离心、Ⅱ型胶原酶及胶原酶-分散酶两次连续酶消化后进行原代培养。通过细胞形态学观察和第Ⅷ因子免疫细胞化学染色鉴定所培养的目的细胞。结果 体外培养12～24 h后,细胞以贴壁的脑微血管段为中心,放射状向外周移行,并逐渐扩大成团簇状;细胞融合后则呈典型的单层、扁平、“铺路石样”镶嵌式排列。第Ⅷ因子免疫细胞化学染色检测,胞质呈棕红色,表达为阳性,阳性细胞率达99％以上。结论 改良方法能够成功高效分离培养出原代大鼠脑微血管内皮细胞。     

人胎盘微血管内皮细胞的分离培养及鉴定

背景:建立高纯度的人胎盘微血管内皮细胞体外培养体系对研究胎盘功能是十分必要的。目前,国内外研究多采用三步酶消化法结合免疫磁珠法分离胎盘微血管内皮细胞,然而步骤过于繁琐且对细胞损伤较大。因而,如何简化人胎盘微血管内皮细胞分离步骤,同时又能提高目标细胞纯度的体外培养方法成为研究热点。目的:探索一种简便高效的从早期绒毛组织中分离人胎盘微血管内皮细胞的体外培养方法,观察细胞生长状态并进行鉴定。方法:利用健康孕6-8周孕妇行人工流产后的绒毛组织,经两步酶消化法和非连续Percoll密度梯度离心得到人胎盘微血管内皮细胞,传代时利用胰酶消化法和反复贴壁法对细胞进行纯化。结果与结论:实验成功获取人胎盘微血管内皮细胞,原代培养的人胎盘微血管内皮细胞在培养24 h后基本完全贴壁,第10天进入对数生长期,第12至13天细胞浓度达80%,传代细胞较原代细胞生长活跃,培养5-7 d可长满培养瓶底,呈"铺路石样"排布。免疫荧光化学结果显示,培养的细胞中FⅧ因子与CD31相关抗原双荧光染色呈阳性,细胞阳性率达100%。MTT法测得培养第5代人胎盘微血管内皮细胞的生长曲线呈倒"S"形。结果证实,应用两步酶消化法、非连续Percoll密度梯度离心法分离细胞,并利用胰酶消化法和反复贴壁法纯化细胞,可获得大量高纯度的人胎盘微血管内皮细胞。     

大鼠脑皮质微血管内皮细胞培养和形态学观察

为获取较纯净脑微血管内皮细胞进行血脑屏障的病理生理研究，我们采用脑组织匀浆、过滤和酶消化技术分离大鼠脑微血管，对分离的脑微血管内皮细胞进行了体外培养和形态学观察。倒置显微镜下，细胞具有单层“卵石样”排列的典型特征、电镜观察可见细胞间连接，免疫酶技术显示，95％以上的细胞为第Ⅷ因子相关抗原反应阳性，进一步证实为血管内皮细胞。     

乳鼠心肌膜微血管内皮细胞的体外培养

目的 改进心肌膜微血管内皮细胞的体外培养方法,为进一步研究其结构和功能提供实验数据.方法 选取10～15d龄的SD大鼠,采用植块法培养原代心肌膜微血管内皮细胞,在继代培养中通过差速消化和差速贴壁方法、结合倒置显微镜下形态学的观察进行继代培养和纯化,利用免疫细胞化学方法检测第Ⅷ因子相关抗原对培养细胞进行了鉴定.结果 成功培养了大鼠心肌膜微血管内皮细胞,细胞呈多边形或鹅卵石状;免疫细胞化学染色第Ⅷ因子相关抗原显阳性.结论 改良了心肌膜微血管内皮细胞的体外培养方法,获得了较高纯度的心肌膜微血管内皮细胞,可用于进一步的科学研究.     

大鼠原代脊髓微血管内皮细胞的体外分离培养及鉴定

目的:建立一种简单高效的大鼠原代脊髓微血管内皮细胞培养方法。方法:分离大鼠脊髓组织,经剪碎、细胞筛网过滤、Ⅱ型胶原酶消化后接种培养。通过细胞形态学观察、FⅧRag免疫细胞化学染色鉴定所培养的目的细胞。结果:接种的脊髓微血管段呈“短棍样”,培养24 h后有少量短梭形细胞从血管段周围迁移爬出,48 h后逐渐形成“岛屿状”的细胞集落,72 h后细胞铺满皿底,呈典型的单层、“鹅卵石样”、镶嵌式排列生长;细胞FⅧRag免疫细胞化学染色显示细胞质呈棕红色,表达为阳性。结论:该方法可成功分离培养出活性好、纯度高的大鼠原代脊髓微血管内皮细胞。     

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.     

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.     

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.     

What will studies of Fulani individuals naturally exposed to malaria teach us about protective immunity to malaria?

There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with Plasmodium falciparum infection in sub-Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide-treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.     

'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.     

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.     

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.     

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.