mfgl2基因反义质粒对小鼠暴发型肝炎病程的干预作用

目的研究mfgl2反义质粒对暴发型肝炎小鼠体内mfgl2表达的改变,以及对小鼠暴发型肝炎病情发展的影响。方法为了建立一个有效的体内基因转移系统,分别于感染前1 d和当天尾静脉高压注射LacZ质粒,第1天收集小鼠肝脏标本,并做X-gal染色观察质粒在肝脏的转染效率;用同样的方法将mfgl2反义质粒转入MHV-3感染的Balb/cJ小鼠肝脏,对照组用空载体代替。观察两组动物生存率,并于感染后第2天收集肝脏标本,HE染色观察肝脏病理组织学改变,免疫组化方法和RT-PCR方法检测mfgl2 mRNA和蛋白的表达水平。结果尾静脉高压注射可以使得目的基因在小鼠肝脏达到20%的转染效率。基因治疗组小鼠于感染后第2天mfgl2 mRNA和蛋白表达均明显下降。MHV-3感染后,对照组小鼠3 d内全部死亡,而基因治疗组33%的小鼠存活10 d以上。显微镜观察基因治疗组小鼠肝组织坏死轻微,而对照组肝组织有大片坏死。结论mfgl2反义质粒可以明显抑制mfgl2基因的表达,并且显著提高暴发型肝炎小鼠的生存率。     

调节性T细胞在3型鼠肝炎病毒诱导的小鼠暴发型肝炎模型中作用的初步探讨

目的 检测调节性T细胞(Tr)在3型鼠肝炎病毒(MHV-3)诱导的小鼠暴发型肝炎模型中的比例变化及细胞因子表达,初步探讨Tr在该疾病模型中的作用.方法 通过腹腔注射MHV-3感染BALB/cJ小鼠诱导暴发型肝炎,观察小鼠的生存时间,检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)水平,利用苏木精-伊红(HE)染色法检测肝脏病理学改变,分离感染不同时间点外周血、脾脏以及肝脏中的淋巴细胞,利用流式细胞术来检测Tr的比例以及细胞因子IL-10表达水平.结果 BALB/cJ小鼠感染MHV-3后,全部在3～6d内死亡,血清ALT、AST水平随着感染时间延长逐渐升高,HE染色显示肝脏组织炎症及坏死程度逐渐加重,流式细胞术检测发现随着感染时间延长,小鼠肝脏中的Tr的比例明显升高.同时肝脏Tr分泌细胞因子1L-10的比例以及肝脏组织IL-10的mRNA水平逐渐升高.结论 MHV-3诱导的小鼠暴发型肝炎模型中Tr在肝脏中的比例和功能显著升高,这种代偿性升高提示Tr可能发挥调节机体过度免疫反应的功能.     

TNFSF14及其受体LTβR和HVEM在病毒肝炎中的作用

目的:探讨TNFSF14(即LIGHT)及其受体LTβR和HVEM在暴发性病毒肝炎中的作用。方法:MHV-3感染易感小鼠建立暴发性病毒肝炎模型,通过HE染色和血清ALT水平的测定比较了LIGHT KO和WT两组小鼠的肝损情况;定量PCR技术检测了MHV-3感染C57BL/6小鼠后各时相点(0 h、12 h、24 h、48 h、72 h)肝脏和脾脏组织HVEM和LTβR的mRNA水平。流式细胞术检测了临床病毒性重症肝炎病人PBMC中HVEM和LTβR的表达情况。结果:MHV-3感染72 h后,WT小鼠的肝脏出现明显坏死灶,而LIGHT KO小鼠的肝脏则未见异常;LIGHT KO小鼠血清中的ALT浓度也显著低于WT组(P0.01)。MHV-3感染48 h后,C57BL/6小鼠脾脏中HVEM及LTβR的mRNA水平均有显著升高(P0.05);肝脏中LTβR表达在12 h时就有显著上调(P0.01)。与之一致的是,临床重症肝炎病人PBMC中HVEM和LTβR的表达较正常人均有明显升高。结论:TNFSF14可能通过与其受体LTβR和HVEM的相互作用在病毒诱发的暴发性肝炎中扮演重要角色。     

IRF-7在急性HBV感染小鼠模型肝脏中的表达变化分析

目的本文拟通过构建急性乙型肝炎病毒(HBV)感染小鼠模型,观察干扰素调节因子-7(IRF-7)的mRNA及蛋白表达水平在急性HBV感染小鼠模型中的变化,以期探讨IRF-7在HBV急性感染清除机制中的作用。方法用高压水动力法经小鼠尾静脉注射HBV基因组1.3倍体真核表达质粒(pcDNA3.1-HBV1.3)构建急性乙型肝炎病毒感染小鼠模型,用RT-PCR和Western blot检测正常组(NC组)、模型组(pHBV组)及空载体组(pcDNA组)小鼠肝脏中IRF-7的mRNA和蛋白表达水平。结果 pHBV组在6 h即可检测到肝脏内HBsAg及HBcAg阳性,抗原表达在12 h达峰值,至第5天逐渐消失。pHBV组肝脏组织中IRF-7 mRNA和蛋白表达在6 h均显著上调(P0.05),12 h达最高,随后下降,48 h恢复至正常水平。而pcDNA组与正常组的IRF-7表达在各时间点均无显著变化。结论成功构建了急性HBV感染小鼠模型,该模型肝脏组织IRF-7 mRNA及蛋白水平变化与肝脏内HBV抗原表达变化趋势一致,提示肝脏内IRF-7可能参与了HBV的急性感染清除过程。     

干扰Itk表达抑制CVB3感染小鼠的炎症反应

目的 研究在病毒性心肌炎小鼠模型上敲减Itk蛋白的表达对小鼠炎症反应的影响.方法 BALB/c小鼠尾静脉注射表达Itk-shRNA的质粒后感染CVB3,第4天后观察Itk基因的抑制情况、小鼠存活率,PBMC增殖率及部分炎症性细胞因子的分泌水平.结果 Itk-shRNA转染至小鼠体内后脾细胞中Itk基因的mRNA水平与空白对照组及转染shRNAnon的对照组相比,敲减率约40％(P＜0.05).与转染shRNAnon组相比,Itk-shRNA组中Itk基因表达的抑制导致小鼠PBMC增殖活性降低约41％,小鼠的存活率提高(P＜0.05).Itk-shRNA组血清中细胞因子水平降低.结论 抑制Itk表达能有效降低小鼠PBMC的增殖,同时减少炎症相关细胞因子的分泌,提高小鼠的存活率.     

重型肝炎小鼠模型肝组织凋亡相关蛋白的表达及其促肝细胞凋亡的研究

目的研究肝凋亡相关蛋白在重型肝炎小鼠模型肝组织中的表达及其促肝细胞凋亡的作用。方法100PFU鼠3型肝炎病毒（MHV-3）分别感染敏感鼠BALB／cJ系和耐受鼠A／J系，感染后24、48、72h取小鼠肝组织，固定后石蜡包埋并切片，常规HE染色病理诊断计算肝组织炎症活动度积分（HAI）；采用原位缺口末端标记（TUNEL）法检测肝细胞凋亡；免疫组化SP法检测mfg12、Fas、TNFR1蛋白的表达及纤维素的沉积；Western blot法检测小鼠肝组织Bcl-2类蛋白的表达，并计算Bax／Bcl-2的值。结果MHV-3感染后BALB／cJ鼠肝组织出现大量炎性细胞浸润和肝细胞凋亡和坏死，而A／J鼠肝组织炎性反应轻微，且仅有少量肝细胞凋亡和坏死，两者差异有统计学意义（P〈0．01）；感染后24h BALB／cJ鼠肝组织出现大量mfg12、Fas、TNFR1阳性细胞，并有大量纤维素沉积出现，而A／J鼠无阳性细胞和纤维素的沉积；感染后24h BALB／cJ鼠肝组织Bax蛋白的相对表达水平要远高于A／J鼠（P〈0．01），Bcl-2蛋白的相对表达水平差异无统计学意义，BALB／cJ鼠肝组织Bax／Bcl-2值要远高于A／J鼠（P〈0．01）。结论肝组织凋亡相关蛋白的表达及其促肝细胞凋亡在重型肝炎的发病机理中具有重要作用。     

重组9型腺相关病毒转染小鼠骨髓间充质干细胞

背景:重组9型腺相关病毒对心肌细胞具有良好的亲和力,是目前研究基因治疗心肌梗死的理想载体。目的:观察携带增强型绿色荧光蛋白基因的重组9型腺相关病毒(r AAV9-e GFP)对小鼠骨髓间充质干细胞的转染效率。方法:将携带增强型绿色荧光蛋白基因的重组9型腺相关病毒以不同感染复数(1×105,1×106,1×107)转染体外培养的第4代小鼠骨髓间充质干细胞,并在转染后1-7 d连续用荧光倒置显微镜观察骨髓间充质干细胞中增强型绿色荧光蛋白阳性表达情况,寻找最佳感染条件;采用流式细胞仪检测最佳感染复数下携带增强型绿色荧光蛋白基因的重组9型腺相关病毒对小鼠骨髓间充质干细胞的转染效率。结果与结论:转染后第1天,感染复数为1×107组可见增强型绿色荧光蛋白开始表达,转染后第2天,感染复数为1×105、1×106组开始表达;各组增强型绿色荧光蛋白表达强度随感染复数值的增高而增强,同时增强型绿色荧光蛋白的表达强度随着时间延长而逐渐增强;转染后第5天达到高峰,此时感染复数为1×107组转染效率约8%,结果表明携带增强型绿色荧光蛋白基因的重组9型腺相关病毒对小鼠骨髓间充质干细胞转染效率较低。     

EB病毒潜伏膜蛋白1对鼻咽癌细胞P53蛋白表达的影响

目的:研究EB病毒潜伏膜蛋白1(LMP1)对鼻咽癌细胞P53蛋白表达的影响。方法:将LMP1基因真核表达质粒转染至鼻咽癌CNE1细胞,脂质体介导端粒酶反义核酸处理转染细胞,MTT法检测细胞增殖能力,免疫组化法检测LMP1和P53蛋白表达,原位杂交技术检测端粒酶逆转录酶(hTERT)mRNA表达。 结果:对照组,转染并表达LMP1基因的细胞的增殖能力、P53蛋白和hTERT mRNA表达水平均显著高于未转染细胞和转染空载质粒的细胞。端粒酶反义核酸作用组,LMP1基因转染细胞的LMP1蛋白表达水平显著低于对照组(P＜0.01);LMP1基因转染细胞与未转染细胞和转染空载质粒的细胞的增殖能力、P53蛋白和hTERT mRNA表达水平均显著低于对照组(P＜0.01),但LMP1基因转染细胞的增殖能力和P53蛋白表达水平仍显著高于未转染细胞和转染空载质粒的细胞(P＜0.01)。 结论:EB病毒LMP1可促进鼻咽癌细胞P53蛋白的表达。     

乙型肝炎病毒感染小鼠肝组织视黄醇结合蛋白4表达降低

目的观察乙型肝炎病毒(HBV)感染对小鼠体内视黄醇结合蛋白4(RBP4)表达的影响。方法将C57BL/6小鼠随机分为正常组,HBV感染组(尾静脉高压注射rAAV8-1.3HBV病毒载体1×10~(11) vg/只),HBV感染+拉米夫定喂养组(拉米夫定150 mg/kg),分别于6周、12周和18周麻醉后处死6只小鼠。荧光定量PCR(RT-qPCR)检测血清HBV DNA含量;全自动生化仪以及ELISA检测血清相关生化指标以及RBP4含量;ELISA检测肝脏组织中肿瘤坏死因子α(TNF-α)以及白细胞介素6(IL-6)炎性因子水平;RT-qPCR、Western blot分别检测肝脏组织中RBP4 mRNA以及蛋白的表达。结果 HBV感染组小鼠血清中HbsAg均保持较高水平且阳性率稳定在80%以上,小鼠血清HBV DNA含量显著高于对照组(P0.05),HBV感染小鼠模型构建成功;感染组小鼠肝脏组织中TNF-α以及IL-6水平均随时间的增加而升高,其中第12周、18周时要显著高于正常组(P0.05);此外,感染组小鼠肝脏组织中RBP4蛋白以及RBP4 mRNA的表达水平均随感染时间的增加而下降,其水平在第6周、12周、18周均要低于正常组以及拉米夫定喂养组(P0.05)。结论 HBV感染小鼠后能够显著降低小鼠肝脏组织中视黄醇结合蛋白4的表达水平。     

人反义VEGF基因对肾癌细胞VEGF表达的影响

目的：构建反义VEGF基因真核表达载体，研究其对肾癌细胞VEGF表达的影响。 方法： 克隆人VEGF基因，将反义VEGF基因定向克隆于质粒pcDNA3.1(-)真核表达载体，酶切鉴定；转染人肾癌细胞，G418筛选阳性克隆。RT-PCR方法检测VEGFmRNA的表达，免疫组化法检测VEGF基因的蛋白表达，MTT法测细胞生长，流式细胞术检测细胞周期。 结果： 成功构建反义VEGF基因真核表达载体；反义 VEGF组VEGFmRNA的表达受到抑制，明显低于空载体组和对照组VEGFmRNA的表达,空载体组VEGFmRNA的表达没有受到影响；反义 VEGF组的VEGF蛋白表达明显低于空载体组和对照组，空载体组和对照组VEGF蛋白的表达无显著差异；反义 VEGF组细胞生长减慢，G1期细胞比例增加，S期细胞比例减少。 结论： 人反义VEGF基因可明显降低肾癌细胞VEGF基因在转录和翻译水平的表达，抑制肾癌细胞生长，为肾癌基因治疗提供一定的实验依据。     

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.