应用二甲基亚砜体外诱导P19细胞分化为心肌细胞

目的观察P19细胞经二甲基亚砜（DMSO）诱导及不同培养方法,培养形成细胞聚集体并向心肌分化的过程.方法将P19细胞接种于Petri塑料培养皿或铺有0.5%软琼脂的培养皿中,DMSO诱导悬浮培养7 d形成细胞聚集体,将聚集体用生长培养基黏附培养至19 d.观察细胞跳动情况,用α-sarcomeric actin、cardiac Troponin T（cTnT）抗体进行免疫组织化学染色,鉴定细胞分化.结果经DMSO诱导7d,将形成的细胞聚集体用生长培养基黏附培养至15 d,在聚集体周围的生长晕中出现自发性节律跳动的细胞团片,α-sarcomeric actin及cTnT染色阳性,至19d,阳性率分别可达26%和15%.结论 DMSO诱导结合聚集体形成培养方法可诱导P19细胞向心肌细胞分化,并出现自发性有节律跳动.     

P19细胞体外向脂肪细胞诱导分化的实验

目的 探讨应用96孔板悬浮法诱导P19细胞体外向脂肪细胞分化的可行性和有效性。方法 P19细胞使用96孔板悬浮培养法制备拟胚体（EBs）,其中第3天至第5天使用诱导剂全反式维甲酸（RA）。第7天挑取EBs用胰岛素和三碘甲状腺原氨酸诱导,继续贴壁培养20d后,油红O染色鉴定分化细胞的特征。
结果 使用96孔板悬浮培养法可以形成大小均一的EBs,诱导结束后EBs生长晕周围部分细胞分化为脂肪细胞。细胞形态趋于类圆或圆形,胞质中出现小脂滴,可被油红O染色清晰显示。 结论 使用96孔板悬浮法获得的P19细胞EBs在体外可诱导分化为脂肪细胞。     

5-氮胞苷诱导体外培养的胚胎干细胞向心肌样细胞分化

目的研究5-氮胞苷(5-aza)体外诱导胚胎干细胞分化为心肌细胞的可行性。方法将P19细胞接种于培养板中或铺有琼脂的培养板中,5μmol/L5-aza培养7d后用不含5-aza的培养基继续培养。倒置显微镜观察细胞跳动情况,用免疫细胞化学、RT-PCR检测及电镜观察等方法鉴定细胞分化。结果5-aza暴露结合悬浮培养可诱导P19细胞分化为有节律跳动的心肌细胞。分化的细胞表达心肌特异的GATA-4、α-MHC mRNA及α-sarcomeric actin、cTnT蛋白,同时透射电镜观察到细胞质内有明显的肌丝。结论5-aza在体外可诱导胚胎干细胞向心肌样细胞分化,悬浮培养有助于细胞的心肌化过程。     

二甲基亚砜诱导胚胎干细胞分化伴随凋亡发生

目的：标准化二甲基亚砜（DMSO）诱导胚胎干细胞（ESC）向心肌细胞分化的方法,及DMSO是否同时诱导细胞凋亡。方法：MTT法确定DMSO的应用剂量,不同条件培养基对ESC进行诱导分化,并在形态学、蛋白质及基因水平鉴定ESC源心肌细胞。利用形态学、流式细胞仪等对细胞凋亡进行观测。结果;DMSO的最佳应用浓度为1％,拟胚体经诱导后跳动率为96．7％。该心肌细胞表达多种心肌蛋白,且肌小节结构发育成熟。DMSO的促分化效应可能与心肌转录因子GATA-4的表达有关,1％DMSO可诱导ESC部分产生凋亡,且具有时间和剂量依赖性。结论：1％DMSO不但能够高效诱导ES-D3分化为心肌细胞,且以时间依赖的方式诱导ES-D3细胞部分凋亡。     

骨形态发生蛋白2诱导P19细胞体外向心肌样细胞分化

目的 探讨骨形态发生蛋白2(BMP-2)诱导P19细胞形成细胞聚集体并向心肌细胞分化的作用.方法 将P19细胞接种于铺有0.5%软琼脂的培养皿中,BMP-2诱导悬浮培养4d形成细胞聚集体,将细胞聚集体用生长培养基黏附培养至19d.相差显微镜观察细胞形态学变化,应用免疫细胞化学、激光扫描共焦显微镜技术检测α-横纹肌肌动蛋白(α-sarcomeric actin)和心肌特异性肌钙蛋白T(C-TnT)的表达,透射电镜观察分化细胞的超微结构.结果 经BMP-2诱导悬浮培养24h后即可见多个悬浮的细胞团形成,至第4天形成细胞聚集体/类胚体(Ebs).将Ebs再用生长培养基黏附培养后,可见位于Ebs中央的细胞密集、颜色较深,为Ebs的内核.Ebs贴壁生长后 8h,细胞由Ebs边缘逐渐爬出,在周边形成单层的生长晕,中央细胞多层排列,增殖速度较快,细胞体积较小,具有高核质比,为未分化细胞.培养至11d后,细胞生长晕中一些细胞变形,胞体伸长,体积增大,呈放射状排列.培养至第19天,Ebs边缘生长晕中变形的细胞表达α-横放肌肌动蛋白和c-TnT蛋白.免疫荧光双标染色显示α-横纹肌肌动蛋白和C-TnT蛋白均定位于细胞质并且呈共表达.透射电镜下观察到,分化细胞呈杆状,细胞核呈卵圆形,位于细胞中央,细胞器丰富,并可见到平行排列的肌丝.结论 BMP-2暴露结合悬浮培养可以诱导P19细胞向心肌样细胞分化.     

抗坏血酸体外诱导小鼠胚胎干细胞向心肌细胞分化

目的:探讨抗坏血酸作为诱导剂对小鼠胚胎干细胞(mESC)分化为心肌细胞的影响,建立一种体外诱导mESC分化为心肌细胞的实验方法。方法:采用直接悬浮培养法使mESC形成拟胚体(EBs),用含不同浓度抗坏血酸的分化培养基对其进行诱导分化,摸索最佳的诱导分化条件。结果:抗坏血酸诱导mESC分化为心肌细胞的最佳浓度为0.1mg/ml,约为70%的EBs出现跳动的心肌合胞体,显著高于不添加任何诱导剂的对照组。抗坏血酸诱导产生的心肌细胞表达多种心肌蛋白,具有心肌细胞的结构特征。结论:抗坏血酸能够促进mESC向心肌细胞分化,应用抗坏血酸体外诱导mESC向心肌细胞分化是一种较为理想的体外诱导方法。     

催产素诱导P19细胞分化为心肌细胞的作用

目的研究催产素(OT)在体外诱导P19细胞分化为心肌细胞中的作用。方法将P19细胞用含OT的诱导培养基悬浮培养4d,取其形成的拟胚体(EBs)用不含OT的生长培养基贴壁培养10~12d。用免疫细胞化学、透射电镜等方法对分化的细胞进行鉴定。结果EBs周边生长晕中部分细胞变形、伸长,呈放射状排列。免疫细胞化学染色显示,变形分化的细胞表达α-横纹肌肌动蛋白(α-SCA)和心肌肌钙蛋白T(cTnT),并以OT终浓度1×10-7mol/L的实验组阳性率最高(P<0.01)。透射电镜观察分化细胞具有心肌细胞发育早期的超微结构特征。结论在体外OT能够诱导P19细胞分化为早期的心肌细胞。     

PMA体外诱导小鼠多能干细胞向心肌细胞的分化

目的研究丙二醇甲醚醋酸酯(PMA)对小鼠诱导多能干细胞体外分化为心肌细胞的影响,以建立一种高效安全的体外诱导i PSC分化为心肌细胞的实验方法。方法用悬滴法形成拟胚体(EBs),PMA诱导其向心肌细胞定向分化。免疫细胞学标记检测心肌肌钙蛋白T(c Tn T)和α横纹肌辅肌动蛋白(α-actinin)的表达;RT-PCR和q-PCR检测Brachyury,c Tn T,MLC2a,NKX2.5和GATA4等mRNA的表达。以添加相应DMSO作为对照组,观察各组出现搏动拟胚体的数量,计算分化比率。结果 PMA诱导小鼠诱导多能干细胞分化为心肌细胞的最佳浓度为100 nmol/L,此时拟胚体搏动率可达53%,显著高于对照组(12%),且PMA诱导产生的心肌细胞表达多种心肌蛋白及基因,具有心肌细胞的结构特征。结论 PMA能够促进mi PSC在体外定向分化为心肌样细胞。     

与心肌细胞共培养的小鼠胚胎干细胞向心肌样细胞分化

目的 探讨心肌细胞促进胚胎干细胞（ESCs）分化为心肌样细胞的诱导作用。方法 收集小鼠3.5d胚龄的囊胚,将其培养在小鼠胚胎成纤维细胞饲养层上,4～5d后取内细胞团接种在饲养层上分离培养出ESCs。取3～5代ESCs,先将ESCs悬浮培养形成2～3d的拟胚体(EBs),再与新生大鼠心肌细胞共培养诱导向心肌细胞分化,相差显微镜下观察分化细胞的形态学变化,免疫细胞荧光技术检测心肌细胞特异性肌钙蛋白T(TnT)、α－肌动蛋白（α-Actin）的表达。结果 诱导第3天起可见自发性、有节律跳动的拟胚体出现,12d时第2组约有93%的拟胚体出现节律性收缩,显著高于其他各组,均表达心肌细胞特异性蛋白cTnT、а-actin,心肌细胞直接接触诱导组其分化比率达56.5%,高于其他各组,并且分化的细胞形态较单一。结论 心肌细胞和心肌细胞裂解液均可诱导ESCs向心肌细胞定向分化,且心肌细胞的诱导作用强于心肌细胞裂解液。     

淫羊藿苷体外诱导小鼠胚胎干细胞分化为心肌细胞

目的研究淫羊藿苷(icariin,ICA)体外诱导小鼠胚胎干细胞(embryonic stem cells,ESCs)ES-E14细胞分化为心肌细胞的作用。方法复苏的ESCs经直接悬浮法形成拟胚体(EBs),应用ICA定向诱导,相差显微镜下观察分化细胞的形态学变化,免疫细胞荧光技术及Western blot(WB)分别检测心肌细胞特异性肌钙蛋白I(TnI)、心室肌球蛋白轻链(Mlc-1v)的蛋白表达及表达量的变化;透射电镜观察分化细胞的超微结构。结果经ICA诱导后第5天的EBs出现了细胞跳动点,ICA诱导后第3天的细胞心肌细胞特异性肌钙蛋白I(TnI)、心室肌球蛋白轻链(Mlc-1v)的蛋白表达均阳性,于诱导后的第3天(早期)、第12天(中期)及第20天(晚期)心肌细胞特异性肌钙蛋白I(TnI)、心室肌球蛋白轻链(Mlc-1v)的表达逐渐增多,晚期明显高于早期及中期;透射电镜下可见大量平行排列的肌丝。结论用直接悬浮法,ICA体外能诱导鼠ESCs分化为心肌细胞,分化的心肌细胞两种心肌特异性结构蛋白量的表达随分化进程的进展逐渐增多。     

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.     

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.     

The universal muscular chain reaction,muscle spasm,Torsions, ruptures and extravasations. Chameleons of pathology and some manifestations of simple muscular disorders

Most bodily functions require the coordinated actions of complementary and supplementary paired muscle groups. Where this essential muscular cooperation is lacking, hollow organs may burst and others become literally screwed up, giving rise to many similar spastic diseases such as Torticollis, Twisted ovarian cyst, Torsion of the Testis, Volvulus of the intestines, Varicose Veins, Megacolon, Aortamegaly, Scoliosis, Erb's Palsy, Peyronie's Disease, Main-en-Griffe, Undescended Foot (Pes Cavus), Talipes, Strabismus. Spasm is “panenepidemic” and unclassified examples of Torsion Dystonia and Dyskinesia really are as common as debt and taxes.     

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.     

Hypo- und Hypermagnesämie aus kardiologischer Sicht

Zusammenfassung Eine Reihe pathologischer Zustände bedingen Magnesiummangel. Zustände mit Hypermagnesämie sind ebenfalls bekannt, doch wesentlich seltener. Für den Kardiologen beachtenswert ist, daß unter Therapie mit bestimmten Diuretica bei Herzinsuffizienz, bei Herzinfarkt, Kardiomyopathie, Digitalisintoxikation und bestimmten Herzrhythmusstörungen Hypomagnesämie beobachtet wurde. Leider kann in der klinischen Routine nur ein extracelluläres Magnesiumdefizit durch Serumbestimmungen gemessen werden; über Magnesiummangel einzelner Organe kann nichts ausgesagt werden. Hinweise für Magnesiummangel geben aber neben der Messung des Serumspiegels Anamnese, klinischer Befund, bestimmte EKG-Veränderungen wie auch evtl. Hypokalämie, ein Zustand, bei dem sich oft — besonders bei Aldosteronismus — parallele Veränderungen zeigten.Tierexperimente deuten darauf hin, daß infarktähnliche Läsionen unter Magnesiummangel entstehen, doch ob Herzinfarkt beim Menschen durch Magnesiummangel ausgelöst werden kann, ist noch ungeklärt. In Leichenherzen zeigte sich im Infarktgebiet neben Calciumakkumulation signifikanter Magnesiumverlust, wobei unklar blieb, ob sich Ursache oder Folge des Infarktes widerspiegelten. Falls ein ursächlicher Zusammenhang besteht, ist er im Myokardstoffwechsel selbst zu suchen, wie bei der Alkoholkardiomyopathie, wo myokardialer Magnesiummangel zumindest als pathogenetischer Teilfaktor anerkannt wird. Andererseits versucht man aber auch Beziehungen zwischen Atherosklerose, Blutgerinnung und Hypomagnesämie herzustellen, in der Meinung, daß Magnesiummangel auch über den coronaren Pathomechanismus des Herzinfarktes wirken könnte. Sicher scheint, daß gewisse EKG-Veränderungen und Herzrhythmusstörungen durch einen irritierten Magnesiumhaushalt bedingt sein können, da sie bei Gabe bzw. Entzug von Magnesium verschwinden. Daß Magnesiummangel die Glykosidtoleranz verringert, wird tierexperimentell bestätigt. Unter Hypomagnesämie bewirkt Acetylstrophanthidin eher und länger Rhythmusstörungen als ohne, außerdem lassen diese sich durch Magnesiumgaben eliminieren. Da in gewissen Fällen spontane und digitalisinduzierte Herzrythmusstörungen durch Magnesiuminjektionen beseitigt wurden, scheint Magnesium als Therapeuticum angebracht. Einsatz verschiedener Magnesiumsalze bei Angina pectoris, degenerativen Herzerkrankungen und Herzinsuffizienz ohne geprüften und offensichtlich gestörten Magnesiumhaushalt ist fragwürdig, weil keine eindeutigen klinischen Erfolgsbeweise vorliegen. Immerhin mag es aber larvierte, durch Serumbestimmungen nicht erfaßbare Mangelzustände geben. Allgemein erscheint es aus kardiologischer Sicht ratsam, den Magnesiumhaushalt zu überwachen und in entsprechenden Fällen auszugleichen, um möglichen Myokardläsionen oder fatalen Herzrhythmusstörungen entgegenzuwirken.     

Environmental risk factors and their role in the management of atopic dermatitis

Introduction: The etiology of atopic dermatitis (AD) is multifactorial with interaction between genetics, immune and environmental factors.

Areas covered: We review the role of prenatal exposures, irritants and pruritogens, pathogens, climate factors, including temperature, humidity, ultraviolet radiation, outdoor and indoor air pollutants, tobacco smoke exposure, water hardness, urban vs. rural living, diet, breastfeeding, probiotics and prebiotics on AD.

Expert commentary: The increased global prevalence of AD cannot be attributed to genetics alone, suggesting that evolving environmental exposures may trigger and/or flare disease in predisposed individuals. There is a complex interplay between different environmental factors, including individual use of personal care products and exposure to climate, pollution, food and other exogenous factors. Understanding these complex risk factors is crucial to developing targeted interventions to prevent the disease in millions. Moreover, patients require counseling on optimal regimens for minimization of exposure to irritants and pruritogens and other harmful exposures.     

Class II HLA in Georgia Caucasus Tbilisi Georgians and their Mediterranean ancestry: The Usko Mediterranean languages

Georgia (or Sakartvelo in its own language) is a South Caucasus Mts. country with its easternmost part is enigmatically named Iberia, like the Iberian Peninsula, which may refer to rivers “Kura” and “Ebro” or their valleys respectively. Most of their inhabitants speak Georgian which is included within Dene-Caucasian group and Usko-Mediterranean subgroup of languages. The latter includes Basque, Berber, ancient Iberian-Tartessian, Etruscan, Hittite, Minoan Lineal A and others. In the present paper, HLA class II -DRB1 and -DQB1 alleles has been studied and extended haplotypes calculated. Most frequent haplotypes are also of Mediterranean origin (i. e.: (A*02-B*51)-DRB1*11:01-DQB1*03:01, (A*02-B*51)-DRB1*13:01-DQB1*06:03, or (A*24-B*35)-DRB1*01:01-DQB1*05:01) and DA genetic distances show that closest world populations to Georgians are Mediterraneans. Georgians also show common extended haplotypes ((A*02-B*51)-DRB1*11:01-DQB1*03:01, (A*02-B*13)-DRB1*07:01-DQB1*02:01 and (A*03-B*35)-DRB1*11:01-DQB1*03:01) with Svan people, a secluded population in North Georgia mountains. We can conclude that Georgians belong to a very old Mediterranean substratum according to both linguistics (Usko Mediterranean languages) and HLA genetics.     

'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.