封闭蛋白与炎症性肠病相关性研究进展

肠黏膜上皮细胞与细胞间的紧密连接(tight junction, TJ)是构成肠黏膜机械屏障的结构基础,封闭蛋白(occludin)是TJ相关蛋白,对TJ有重要的调节作用,但具体机制尚不清楚。有研究表明,封闭蛋白可以通过调节TJ改变肠黏膜屏障功能,而肠黏膜屏障功能的改变参与了炎症性肠病(inflammatory bowel disease, IBD)的发生、发展。文章就封闭蛋白与IBD的相关性作一综述,以期为IBD的治疗提供新思路。     

细胞焦亡在肠黏膜屏障损伤机制中的作用

肠上皮细胞、免疫系统及微生物的相互联系维持肠黏膜屏障完整性和自我更新。肠黏膜屏障损伤易导致难以预测的严重疾病发生。细胞焦亡是一种由活化的半胱天冬酶切割Gasdermin家族蛋白介导的炎症细胞死亡形式,在急慢性疾病控制中发挥重要作用。病理生理状态下,细胞焦亡参与肠黏膜屏障损伤及异常免疫调节,最终导致多种肠道疾病发生。本文综述细胞焦亡在肠道感染、肠缺血再灌注、坏死性小肠结肠炎及炎症性肠病中的最新调控机制,针对此类肠道疾病提供新的有效治疗策略。     

缺血再灌注条件下BMP2/4参与肠黏膜屏障损伤的研究

目的探讨缺血再灌注条件下BMP2/4参与肠黏膜屏障损伤的机制。方法制作肠上皮细胞缺氧模型、大鼠肠缺血再灌注损伤模型。取空肠切片,Western blot、免疫荧光检测肠上皮细胞内BMP的变化; Western blot、免疫荧光、RT-PCR检测外源性BMP蛋白对肠上皮细胞内NF-κB信号及TNF-a、IL-6的表达变化的影响; Western blot检测外源性BMP蛋白对紧密连接蛋白occludin表达变化的影响。结果缺氧及缺血再灌注条件下肠上皮细胞内的BMP2/4表达明显增加,BMP2/4通过激活肠上皮细胞内的NF-κB信号导致炎症介质TNF-a、IL-6表达增加,增加肠黏膜屏障的通透性及减少紧密连接蛋白occludin的表达,损害肠黏膜物理屏障的完整性,从而造成肠黏膜屏障的损伤。结论缺血再灌注条件下,BMP信号通过激活NF-κB参与肠黏膜屏障损伤。     

SIRT1通过调节FOXO1/RAB7信号通路促进低氧诱导的胰腺癌细胞自噬

目的:探讨SIRT1对低氧诱导的胰腺癌细胞自噬的影响及FOXO1/RAB7信号通路在其中的作用。方法:利用Western blot和免疫荧光法检测SIRT1在低氧诱导的自噬胰腺癌细胞核内的表达情况。将下调SIRT1基因的小干扰RNA和过表达SIRT1的质粒转染到胰腺癌Panc-1细胞中,敲减和过表达SIRT1基因;激光共聚焦显微镜追踪双色LC3荧光蛋白表达,Western blot实验检测自噬相关蛋白LC3、p62及FOXO1/RAB7信号通路的蛋白表达情况。通过生物信息学GEPIA网站关联性分析预测SIRT1与FOXO1的相互作用,并进一步利用免疫共沉淀技术检测SIRT1与FOXO1蛋白的相互作用。结果:SIRT1在低氧诱导的胰腺癌细胞核内表达增加。敲减SIRT1的表达可以引起胰腺癌细胞自噬受抑制。过表达SIRT1能够增强Panc-1细胞中FOXO1和RAB7的蛋白表达水平(P0.05),而当SIRT1表达下调时,FOXO1和RAB7表达受到抑制(P0.05)。SIRT1与FOXO1蛋白存在相互作用。结论:SIRT1可能参与胰腺癌细胞自噬的调控,其机制可能与FOXO1/RAB7信号通路有关。     

ROCK参与乙醇诱导肠上皮细胞屏障通透性增加

目的探讨Rho相关的卷曲蛋白激酶(ROCK)参与乙醇诱导的肠上皮细胞屏障通透性增加的作用。方法应用ROCK特异性抑制剂Y-27632预处理Caco-2细胞后,测定跨上皮细胞阻抗值(TEER)及荧光黄透过率,分析肠上皮细胞屏障的通透性的变化。应用Western blot的方法检测肌球蛋白轻链(MLC)、p-MLC及紧密连接相关蛋白(occludin)的表达。结果 Y-27632显著抑制乙醇诱导的TEER值降低、荧光黄透过率升高、pMLC/MLC及occludin(S/IS)比值的增加。结论 ROCK参与乙醇诱导肠上皮细胞屏障通透性增加。     

SIRT2沉默对胃癌SGC-7901细胞增殖、迁移和侵袭的影响

目的探究沉默信息调节蛋白2(SIRT2)对人胃癌SGC-7901细胞增殖、迁移和侵袭的影响及机制。方法采用实时荧光定量PCR(q RT-PCR)检测SIRT2在胃癌SGC-7901细胞和正常胃上皮GES-1细胞中的表达。采用RNA干扰技术处理SGC-7901细胞,SIRT2沉默组转染靶向SIRT2的干扰小RNA(SIRT2-siRNA1组、SIRT2-siRNA2组),阴性对照组转染SIRT2-si RNA阴性对照序列,空白对照组不转染。CKK-8法和平板细胞克隆形成实验检测SGC-7901细胞增殖, Transwell法检测SGC-7901细胞侵袭迁移能力,蛋白印迹检测SGC-7901细胞中SIRT2、丝氨酸/苏氨酸蛋白激酶(Akt)和磷脂酰肌醇-3激酶(PI3K)蛋白表达。结果人胃癌细胞SNU-1、KATO III、SGC-7901中SIRT2m RNA和蛋白水平均高于正常胃上皮GES-1细胞(P0.05)。转染SIRT2-si RNA的SGC-7901细胞中SIRT2m RNA和蛋白水平均低于阴性及空白对照组(P0.05);转染SIRT2-si RNA后,SGC-7901细胞增殖能力、克隆形成能力、迁移和侵袭能力均降低(P0.05);转染SIRT2-siRNA后SGC-7901细胞中Akt、PI3K蛋白磷酸化程度显著下降(P 0.05)。结论 SIRT2在胃癌细胞中表达上调,沉默SIRT2可能通过调节PI3K/Akt通路抑制胃癌细胞增殖、迁移和侵袭,推测SIRT2可能作为潜在靶标应用于胃癌的基因治疗。     

SIRT2在浆液性卵巢癌细胞系中的表达及其对增殖、迁移和侵袭的影响

目的:研究SIRT2在卵巢表层上皮(ovarian surface epithelium,OSE)及浆液性卵巢癌(serous ovarian carcinoma,SOC)细胞系中的表达情况并从细胞增殖、迁移和侵袭这3个方面探讨SIRT2对SOC恶性生物学行为的影响。方法:运用Western blot技术检测OSE和SOC细胞系中SIRT2蛋白的表达水平;设计靶向SIRT2的siRNA,构建SIRT2过表达载体,分别瞬时转染OSE细胞系HOSEpi C和SOC细胞系HO8910,平板克隆形成实验和CCK-8实验研究SIRT2对细胞生长的影响;细胞划痕实验考察SIRT2在SOC细胞迁移中的作用;Transwell实验研究SIRT2对SOC细胞侵袭能力的影响。结果:5株SOC细胞系中SIRT2的表达水平显著低于OSE细胞系。在HOSEpi C细胞中沉默SIRT2,细胞克隆形成数增多,细胞活力增强。相反,在HO8910细胞中过表达SIRT2,细胞克隆形成数减少,细胞活力降低。沉默SIRT2促进HOSEpi C细胞的迁移,而过表达SIRT2则抑制HO8910细胞的迁移。沉默SIRT2的HOSEpi C细胞侵袭能力明显增加,而过表达SIRT2的HO8910细胞侵袭能力则显著降低。结论:SIRT2在SOC细胞中表达显著下调。SIRT2在OSE细胞中是肿瘤抑制蛋白,抑制细胞增殖、迁移和侵袭。     

鸡肠神经胶质细胞超微结构特征及钙结合蛋白S100β在小肠中的分布

目的观察10只健康黄羽肉鸡肠神经胶质细胞(EGCs)结构特征及钙结合蛋白S100β(S100β)蛋白在鸡小肠的分布特点,为探讨鸡肠神经胶质细胞的形态学特征提供实验依据。方法采用透射电子显微镜技术观察神经胶质细胞的超微结构特点,免疫组织化学SABC-AP法研究S100β蛋白的分布特征。结果电子显微镜下观察表明,鸡肠神经胶质细胞在小肠各段均呈星形,形态上属于星形胶质细胞,细胞核不规则,胞质中分布有大小不一的圆形或椭圆形无髓神经纤维。免疫组织化学显示,S100β在鸡小肠各段黏膜上皮细胞、肠腺表达较强,其中上皮细胞基膜和肠腺上皮细胞顶端为强阳性,固有膜为阴性,在黏膜下神经丛和肌间神经丛均呈强阳性表达。结论鸡肠神经胶质细胞属于星形胶质细胞,其在小肠的分布较为广泛,除具有营养、保护神经节细胞的功能外,可能还参与调节肠腺细胞分泌及黏膜免疫屏障功能。     

miR-34a 通过靶向SIRT1 对MPP+ 诱导的帕金森病模型细胞凋亡和氧化应激损伤的影响

目的:探讨miR-34a通过靶向SIRT1对MPP~+诱导的帕金森病模型细胞凋亡和氧化应激损伤的影响。方法:以MPP~+处理SH-SY5Y细胞建立帕金森病细胞模型,MTT法检测细胞存活率,RT-PCR检测细胞中miR-34a和SIRT1 mRNA的表达,Western blot检测SIRT1蛋白的表达。脂质体介导转染miR-34a模拟物和miR-34a抑制剂,RT-PCR和Western blot检测miR-34a对SIRT1 mRNA和蛋白表达的影响。采用荧光素酶实验检测miR-34a与SIRT1的靶向关系。将miR-34a抑制剂和SIRT1干扰序列转染至SH-SY5Y细胞后,采用流式细胞仪和LDH、SOD、MDA试剂盒分别检测miR-34a低表达靶向SIRT1对MPP~+诱导的SH-SY5Y细胞凋亡和氧化应激损伤的影响。结果:与对照组相比,500μmol/L、1 000μmol/L和2 000μmol/L MPP~+能够降低SH-SY5Y细胞的存活率、上调miR-34a和下调SIRT1表达(P0.05),且呈浓度依赖性。miR-34a模拟物可引起SIRT1 mRNA和蛋白表达降低,miR-34a抑制剂可引起SIRT1 mRNA和蛋白表达升高;荧光素酶实验证实SIRT1是miR-34a的靶基因。2 000μmol/L MPP~+可诱导SH-SY5Y细胞凋亡,细胞上清液中MDA含量和LDH活性显著升高,而SOD活性降低;转染miR-34a抑制剂成功下调miR-34a表达后可逆转MPP~+引起的上述变化,而转染SIRT1干扰序列成功下调SIRT1表达后可减弱miR-34a低表达引起的上述变化。结论:miR-34a低表达可通过靶向调控SIRT1抑制MPP~+诱导的帕金森病模型细胞凋亡和氧化应激损伤。     

党参多糖联合沉默调节蛋白4对体外人脐静脉糖尿病血管内皮细胞凋亡的影响

目的:研究党参多糖联合SIRT4对体外人脐静脉糖尿病血管内皮细胞凋亡的影响。方法:血管内皮细胞分成空白对照组(常规细胞培养液培养)、高糖模型组(30mmol/L葡萄糖处理)、转染阴性对照组(转染阴性对照载体并经30mmol/L葡萄糖处理)、转染SIRT4组(转染SIRT4过表达载体并经30mmol/L葡萄糖处理)、转染阴性+党参多糖组(转染阴性对照载体并经30mmol/L葡萄糖处理和100μg/ml党参多糖处理)、转染SIRT4+党参多糖组(转染SIRT4过表达载体并经30mmol/L葡萄糖处理和100μg/ml党参多糖处理)。MTT检测细胞增殖,流式细胞术检测细胞凋亡,ELISA检测ROS、MDA、SOD、CAT水平,JC-1染色法检测线粒体膜电位,Western blot检测C-Caspase-3蛋白和cyt-c蛋白表达。结果:与空白对照组比较,高糖模型组细胞增殖能力降低,细胞凋亡和C-Caspase-3蛋白表达增多,细胞中ROS和MDA水平升高,SOD、CAT水平下降,胞浆cyt-c蛋白增多,线粒体cyt-c蛋白减少,线粒体膜电位降低。与转染阴性对照组比较,转染SIRT4组和转染阴性+党参多糖组细胞增殖能力升高,细胞凋亡和C-Caspase-3蛋白表达减少,细胞中ROS和MDA水平降低,SOD、CAT水平升高,线粒体膜电位升高,胞浆cyt-c蛋白减少,线粒体cyt-c蛋白增多。与转染SIRT4组和转染阴性+党参多糖组比较,转染SIRT4+党参多糖组细胞增殖能力升高,细胞凋亡和C-Caspase-3蛋白表达减少,细胞中ROS和MDA水平降低,SOD、CAT水平升高,线粒体膜电位升高,胞浆cyt-c蛋白减少,线粒体cyt-c蛋白增多。结论:党参多糖联合SIRT4可抑制体外糖尿病血管内皮细胞凋亡,作用机制可能与其抑制线粒体凋亡途径有关。     

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.     

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.