Runx3敲减抑制BMP9诱导小鼠胚胎成纤维细胞iMEFs向成骨分化

目的研究Runx3敲减对BMP9诱导的小鼠胚胎成纤维细胞iMEFs成骨分化的作用。方法 BMP9腺病毒感染iMEFs,用Western blot检测内源性Runx3蛋白的表达。Runx3干扰腺病毒ad-siRunx3和BMP9条件培养基共同处理iMEFs,用碱性磷酸(ALP)染色和活性定量测定检测成骨早期指标ALP;用茜素红S染色检测成骨晚期指标钙盐沉积;用RT-PCR检测成骨相关基因Id1、Id2、Id3和Runx2,用Western blot检测成骨相关蛋白Dlx5;用SBE荧光素酶报告质粒检测smad1/5/8的转录活性。结果 BMP9可以使Runx3表达降低(P0.05);干扰Runx3可以抑制早期成骨指标ALP活性(P0.05)和晚期成骨指标钙盐沉积;ad-siRunx3抑制BMP9诱导的成骨相关转录因子Id1、Id2、Id3和Runx2基因的表达(P0.05)及DLX5蛋白的表达(P0.05)。结论敲减Runx3可以抑制BMP9诱导小鼠胚胎成纤维细胞向成骨分化。     

动态轴向压应变促进丝素蛋白支架内MC3T3-E1细胞成骨分化

目的 观察动态轴向压应变对三维丝素蛋白支架内成骨细胞成骨相关基因表达的影响。方法 应用动态力学加载仪对实验组小鼠胚胎成骨细胞MC3T3-E1加载动态轴向压应变（5%应变幅度,1 Hz,30 min/d,共21 d）,对照组细胞常规静置培养,不施加力学刺激。应用定量PCR检测细胞成骨基因碱性磷酸酶（ALP）、I型胶原（COLⅠ）、骨特异性转录因子（Runx2）、成骨相关转录因子（Osx）、骨钙蛋白（OCN） mRNA表达量。结果 成骨细胞在周期性轴向压应力刺激下,Runx2、Osx及COLⅠ表达分别增加280%、68.9%和79.6%,ALP及OCN表达也分别增加10.7%和26.9%。实验组成骨相关基因mRNA表达与对照组比较,差异具有统计学意义（P<0.05）。结论 成骨细胞复合丝素蛋白生物支架材料在周期性轴向压应力刺激下,成骨基因COLⅠ、Runx2、Osx及OCN表达明显上调,可能是生理状态下压应力刺激促进骨折愈合的重要机制之一。研究结果对于以力学信号为基础的细胞疗法修复骨缺损等疾病具有重要临床价值。     

不同氧浓度对骨髓基质细胞向成骨细胞分化的影响

目的:观察不同低氧环境对在向成骨细胞分化培养系中的骨髓基质细胞核心结合因子α1(Cbfα1/Runx2)、骨形态发生蛋白2(BMP2)和过氧化物酶体增殖物激活受体γ2(PPAR-γ2)表达的影响,探讨低氧环境对成骨细胞分化的影响。方法:取4月龄雌性SD大鼠骨髓基质细胞在生长培养基中培养传代后,随机分成4组,每组样本数为8,加入分化培养基,分别将细胞放入4个不同氧浓度中继续培养,3d后进行下面的实验:用Trizol提取细胞总RNA,用半定量逆转录PCR(RT-PCR)检测(Cbfα1/Runx2)、BMP2和PPARγ2mRNA的表达;用Western blotting检测(Cbfα1/Runx2)、BMP2蛋白的表达。结果:1.与常氧组(20%)相比,各低氧组Runx2mRNA和Runx2蛋白的表达明显增加。2.与常氧组(20%)相比,各低氧组BMP2mRNA的表达明显增加,低氧可促进BMP2蛋白的表达。3.与常氧组(20%)相比,各低氧组PPARγ2mRNA的表达明显降低。结论:低氧能明显促进Runx2mRNA、BMP2mRNA和Runx2蛋白的表达,且氧浓度越低,Runx2mRNA、BMP2mRNA和Runx2、BMP2蛋白表达越多,相反,低氧能明显抑制骨髓基质细胞PPARγ2mRNA的表达,且氧浓度越低,PPARγ2mRNA的表达越低,表明低氧明显抑制骨髓基质细胞向脂肪细胞分化而促进其向成骨细胞分化。     

活性维生素D3联合力学拉伸对成骨细胞MC3T3-E1增殖、分化及OPG和RANKL表达的影响

目的体外模拟成骨细胞在体内的生存环境,考察活性维生素D3(VD3)、力学拉伸以及两者联合对成骨细胞MC3T3-E1增殖、分化及破骨细胞抑制因子(OPG)和破骨细胞分化因子(RANKL)表达的影响。方法将10 nmol/L VD3、间断性力学拉伸以及两者联合作用于成骨细胞。流式细胞术检测细胞增殖。荧光探针试剂盒检测碱性磷酸酶(ALP)活性。实时定量PCR检测核心转录因子Runx2、OPG、RANKL mRNA水平,Western blotting检测其蛋白表达。结果 VD3抑制成骨细胞增殖,力学拉伸不能改变这种抑制效应。力学拉伸和VD3单独作用成骨细胞均能增加ALP活性及提高ALP、Runx2 mRNA水平,但当联合刺激后这些指标均降低,且成强度依赖性。力学拉伸增加OPG/RANKL比值,增强成骨作用,联合VD3后,OPG/RANKL比值下降。结论力学拉伸能有效诱导成骨分化,增加骨形成。VD3与力学拉伸联合抑制成骨细胞增殖和分化,并通过增加RANKL表达而影响骨重建。研究结果为骨质疏松及相关骨疾病的理论和治疗提供有意义的探索。     

miR-146a调控骨髓间充质干细胞成骨分化的机制研究

目的探讨miR-146a调控骨髓间充质干细胞(BMSC)成骨分化的作用及其分子机制。方法贴壁法分离培养小鼠BMSC,检测成骨分化早期标志物Runx 2的变化,观察BMSC体外成骨分化,利用miRNA特异性的聚合酶链式反应(miRNAspecific qPCR)观察miR-146a的变化情况,并干预miR-146a表达,明确miR-146a对BMSC成骨分化的调控作用。结果成功建立了稳定的BMSC体外培养体系,该细胞能够成功分化为脂肪细胞和成骨细胞;在成骨诱导培养条件下,随着成骨分化,miR-146a水平降低,过表达miR-146a,成骨分化早期标志分子Runx 2表达降低;转染miR-146a拮抗体antago-miR-146a可以补救Runx 2表达的降低。结论 miR-146a负向调控BMSC成骨分化,拮抗miR-146a可以补救BMSC成骨分化的降低。     

活性维生素D3联合力学拉伸对成骨细胞MC3T3-E1增殖、分化及OPG和RANKL表达的影响（英文）

目的体外模拟成骨细胞在体内的生存环境,考察活性维生素D3(VD3)、力学拉伸以及两者联合对成骨细胞MC3T3-E1增殖、分化及破骨细胞抑制因子(OPG)和破骨细胞分化因子(RANKL)表达的影响。方法将10 nmol/L VD3、间断性力学拉伸以及两者联合作用于成骨细胞。流式细胞术检测细胞增殖。荧光探针试剂盒检测碱性磷酸酶(ALP)活性。实时定量PCR检测核心转录因子Runx2、OPG、RANKL mRNA水平,Western blotting检测其蛋白表达。结果 VD3抑制成骨细胞增殖,力学拉伸不能改变这种抑制效应。力学拉伸和VD3单独作用成骨细胞均能增加ALP活性及提高ALP、Runx2 mRNA水平,但当联合刺激后这些指标均降低,且成强度依赖性。力学拉伸增加OPG/RANKL比值,增强成骨作用,联合VD3后,OPG/RANKL比值下降。结论力学拉伸能有效诱导成骨分化,增加骨形成。VD3与力学拉伸联合抑制成骨细胞增殖和分化,并通过增加RANKL表达而影响骨重建。研究结果为骨质疏松及相关骨疾病的理论和治疗提供有意义的探索。     

M2型巨噬细胞外泌体诱导骨髓间充质干细胞的成骨分化北大核心

背景:目前研究表明,M2型巨噬细胞能够促进成骨分化并呈网络状调控,外泌体可携带大量信息参与细胞间的信号传导,M2型巨噬细胞来源外泌体是否能促进骨髓间充质干细胞的成骨分化,有待研究。目的:探究M2型巨噬细胞来源外泌体对骨髓间充质干细胞成骨分化的影响。方法:培养鼠源性巨噬细胞系RAW264.7与鼠骨髓间充质细胞系CP-M131,培养至第3代,应用白细胞介素4诱导巨噬细胞极化为M2型巨噬细胞,从M2型巨噬细胞培养上清中提取外泌体,然后用终质量浓度为0,30,60,90 mg/L的外泌体与骨髓间充质干细胞共培养72 h后收集样本,以及60 mg/L M2型巨噬细胞来源外泌体与骨髓间充质干细胞共培养24,48,72 h后收集样本,Western blot检测骨髓间充质干细胞中成骨相关因子RUNX2和碱性磷酸酶蛋白表达,茜素红染色检测矿物质沉积情况。结果与结论:①成骨相关因子RUNX2及碱性磷酸酶的表达水平与巨噬细胞外泌体质量浓度密切相关,与空白对照组比较,60 mg/L外泌体组RUNX2、碱性磷酸酶表达明显升高(P<0.05),干预72 h RUNX2、碱性磷酸酶表达明显升高(P<0.05);②茜素红实验结果表明,与空白对照组比较,60 mg/L外泌体组钙结节含量较高(P<0.05),干预72 h钙结节含量较高(P<0.05);③结果表明,M2型巨噬细胞分泌的外泌体能够诱导骨髓间充质干细胞向成骨细胞分化。     

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 case for allele‐specific recognition by the TCR

There is a sharp difference in how one views TCR structure–function–behaviour dependent on whether its recognition of major histocompatibility complex‐encoded restriction elements (R) is germline selected or somatically generated. The generally accepted or Standard model is built on the assumption that recognition of R is by the V regions of the αβ TCR, which is not driven by allele specificity, whereas the competing model posits that recognition of R is allele‐specific. The establishing of allele‐specific recognition of R by the TCR would rule out the Standard model and clear the road to a consideration of a competing construct, the Tritope model. Here, the case for allele‐specific recognition (germline selected) is detailed making it obvious that the Standard model is untenable.