一种新型肠上皮干细胞分离方法的建立

目的 探讨密度梯度离心法作为一种新型肠上皮干细胞分离方法的可行性.方法 通过腹腔注射大剂量5-氟尿嘧啶(5-FU)制备肠黏膜严重损伤小鼠模型,取出损伤的肠黏膜消化成单细胞悬液,利用Percoll密度梯度离心法分离细胞体积大小不同的细胞群.HE染色观察各群细胞形态特征,免疫细胞化学及RT-PCR检测各群细胞musashi-1(msi-1)的表达.结果 分离的大部分细胞位于50%和70%密度梯度的Percoll分离液体层,50%密度梯度分离液体层中的细胞符合干细胞的形态特征,免疫细胞化学检测其msi-1表达阳性率约为93%,RT-PCR显示该群细胞msi-1 mRNA表达较强.结论 建立了一种简便、有效的分离具有活性的肠上皮干细胞的方法.     

骨形态发生蛋白4对大鼠脑神经干细胞的胆碱能诱导分化效应

观察了骨形态发生蛋白4(BMP4)对原代分离培养的大鼠脑神经干细胞(NSC)的胆碱能诱导分化效应.将从2个月龄大鼠脑海马、纹状体等区域分离的细胞培养于含EGF和bFGF的DMEM/F12培养液中, 在光镜下观察细胞的形态特征, 并行神经巢蛋白(nestin)细胞化学染色.24h后, 改换成含有BMP4的培养液, 继续培养7或8天.在光镜下观察细胞的形态变化, 并行胆碱乙酰转移酶(choline acetyltransferase, ChAT)和nestin双标免疫细胞化学染色.结果表明, 加BMP4继续培养7或8天后,光镜下见有34%的培养细胞具有神经元的形态特征.用免疫细胞化学染色可见ChAT阳性的细胞与nestin阳性的细胞共存,其中ChAT阳性细胞占28%, nestin阳性细胞占38%.总之, 在培养液中加入BMP4, 可以诱导NSC分化成具有胆碱能特性的细胞.     

浆膜腔积液中转移癌原发部位的细胞学研究

目的探讨能检测浆膜腔转移性腺癌原发部位的有效方法。方法对89例浆膜腔转移性腺癌行常规HE染色,其中肺癌40例、乳腺癌6例、卵巢癌21例、胃肠及胰腺癌22例,从癌细胞群等18个形态学方面进行观察。其中75例采用免疫细胞化学SP法检测了CA125、CA199、SPB、甲状腺转录因子（TTF）-1的表达。结果通过形态学对比研究发现不同原发部位的转移癌的癌细胞总量、细胞群大小、细胞群与单个细胞的比例、细胞群形态及涂片背景等方面各有一些特点：肺癌、乳腺癌中小细胞群各占95％、100％;卵巢癌多以大群存在,占85．7％,并且在涂片中癌细胞数量较多,以群团为主;部分胃肠癌（45．5％）在涂片中数量较少,并以单个散在为主（40．9％）;肺癌及卵巢癌中都可见沙砾体。SPB、TTF-1可以用来支持在浆膜腔转移性肺腺癌的来源,CA125阳性支持卵巢癌来源。CA199在各组癌中都有表达,无明显统计学差异。但若用于腹水,对胃肠、胰腺来源的肿瘤还是有帮助的。结论浆膜腔转移性腺癌的形态学观察、辅以免疫细胞化学染色和基本临床资料三者结合对推测原发部位很有意义。     

尼克酰胺、β-细胞调节素、bFGF、HGF联合诱导人脐血CD34+细胞向胰岛素分泌细胞的分化

目的 探讨在体外培养条件下用尼克酰胺、β-细胞调节素(betacellulin)、bFGF、HGF诱导人脐血CD34 细胞向胰岛细胞的分化.方法 用磁性细胞分选试剂盒(MACS)分离出CD34 细胞后在含5?S,1×ITS,4.7mg/L亚油酸,10-4mol/L 2-磷酸抗坏血酸的低糖型DMEM中培养,扩增后于培养第5d加入尼克酰胺、betacellulin、bFGF、HGF进行诱导,并于诱导14d、24d留取细胞.应用RT-PCR、免疫细胞化学染色方法检测分化细胞中胰岛细胞标志物,并用ELISA方法检测培养液中胰岛素水平.结果 流式细胞仪检测结果显示,CD34 细胞的平均分离纯度＞90%,达到分离要求.RT-PCR检测到诱导后的CD34 细胞表达nestin、ngn3、IPF-1 mRNA.免疫细胞化学染色可见诱导的CD34 细胞中出现nestin和insulin阳性表达细胞.胰岛素分泌细胞的分化率平均为(9.8±2.7)%.ELISA检测发现诱导组和未诱导组培养液中的insulin含量有显著性差异(P＜0.01).结论 体外联合应用上述因子能诱发脐血CD34 造血干细胞向胰岛素分泌细胞的分化.     

毛囊培养上清液诱导大鼠骨髓间充质干细胞分化的研究

目的探讨毛囊培养上清液诱导体外培养的大鼠骨髓间充质干细胞(MSCs)向毛囊干细胞横向分化的潜能。方法采用完全贴壁法分离培养大鼠MSCs,取第3代MSCs,用免疫细胞化学染色技术鉴定CD44和CD29。用毛囊培养上清液为条件培养液诱导MSCs,倒置相差显微镜观察细胞形态学变化,对诱导后的细胞进行角蛋白15的免疫细胞化学染色和免疫荧光细胞化学染色,逆转录-聚合酶链反应(RT-PCR)进一步鉴定诱导后细胞角蛋白15的表达。结果体外培养的MSCs,CD44、CD29表达阳性。经毛囊上清液诱导后,免疫细胞化学、免疫荧光细胞化学染色鉴定,部分细胞角蛋白15表达阳性;同时RT-PCR也检测到keratin 15 mRNA的表达。结论体外培养的骨髓间充质干细胞经毛囊培养上清液诱导可以分化为毛囊干细胞样的细胞。     

乳鼠肌源性干细胞向许旺样细胞的诱导分化

背景：有证据显示,肌源性干细胞能够促进肌纤维的再生,增强再生肌组织的功能,并具有分化为神经外膜组织细胞和具有许旺细胞表型细胞的潜能。 目的：分析许旺细胞条件培液诱导小鼠乳鼠源肌源性干细胞向许旺细胞的分化,探讨其作为神经组织工程的种子细胞的可行性。 方法：取C57BL/6乳鼠四肢骨骼肌,采用改良的Preplate技术纯化培养肌源性干细胞,利用抗体Desmin和Sca-1对纯化后的细胞进行免疫细胞化学鉴定。采用许旺细胞条件培液诱导其向许旺细胞分化,并行许旺细胞特异性抗体S100β、P75NTR免疫细胞化学鉴定。 结果与结论：从小鼠骨骼肌中分离得到肌源性干细胞,能在体外稳定增殖传代,Desmin和Sca-1免疫细胞化学染色阳性。肌源性干细胞经体外诱导分化后部分细胞S100β、P75NTR染色阳性。说明应用改良的Preplate方法,可从新生小鼠骨骼肌中分离得到肌源性干细胞;采用许旺细胞条件培液能将其诱导分化为许旺样细胞,有可能成为神经组织工程神经较理想的许旺细胞替代物。     

C-kit+细胞体外增殖、分化的研究

分离纯化卵圆细胞并对其体外增殖、分化进行研究。SD大鼠喂饲2-乙酰氨基芴(2-acetainofluorene,2-AAF)10mg/kg,以促进肝脏干细胞增殖,通过免疫磁珠法(Magnetic activated cell sorting,MACS)标记C-kit阳性细胞以纯化卵圆细胞,进行体外培养,采用免疫细胞化学、逆转录聚合酶链反应(RT-PCR)等方法观察其体外增殖、分化特性。以MACS纯化的C-kit阳性卵圆细胞,90%以上为卵圆细胞抗原(OV6)阳性,大部分甲胎蛋白(AFP)阳性。C-kit阳性细胞在体外增殖能力较强,能形成集落样生长,并向胆管细胞及肝细胞分化,表达角蛋白19(CK19)和/或白蛋白(ALB)标记。细胞集落RT-PCR结果显示AFP、CK19及ALB基因均有表达。以上结果表明利用C-kit标记通过MACS纯化的C-kit 细胞为具有双向分化潜能的肝脏干细胞。     

LCT与ICC结合在胸腹水细胞学鉴别诊断中的应用

目的:探讨液基薄层细胞检测(LCT)技术与免疫细胞化学(ICC)技术在胸腹水细胞学鉴别诊断中的意义.方法:在87例胸腹水液基细胞涂片中应用免疫细胞化学技术检测癌胚抗原(CEA)、上皮细胞膜抗原(EMA)、间皮细胞(MC)抗体及波形蛋白(Vimentin)的表达并与HE染色比较.结果:腺癌中CEA、EMA、MC、Vimentin阳性表达率分别为88.2%、90.2%、5.9%和3.9%.增生性间皮细胞中CEA、EMA、MC、Vimentin阳性表达率分别为5.6%、2.8%、97.2%和88.9%.结论:免疫细胞化学技术可应用于胸腹水LCT涂片.选择一组特异的抗体(CEA、EMA、MC、Vimentin)并结合脱落细胞HE染色可以在转移性腺癌与增生性间皮细胞的鉴别诊断中起重要作用.     

子宫内膜异位症在位内膜腺上皮细胞及基质细胞的分离培养和鉴定

目的分离培养子宫内膜异位症在位内膜组织中子宫内膜腺上皮细胞及基质细胞,建立研究子宫内膜异位症的细胞模型。方法对分泌期子宫内膜异位症在位内膜组织用混合酶消化,滤网过滤和差速梯度离心的方法分离,体外培养后通过光镜观察及免疫细胞化学、免疫荧光化学方法对分离细胞鉴定。结果分离的细胞角质蛋白阳性子宫内膜腺细胞百分率约90～95%;分离的骨架蛋白形成蛋白阳性的基质细胞百分率达90%以上。结论本研究获得较高纯度的子宫内膜腺细胞和基质细胞,成功建立了研究子宫内膜异位症的细胞模型。     

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.     

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.     

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.