NT-3体外转导在小鼠耳蜗螺旋神经节细胞的表达

以新一代HSV－Amplicon做载体，探讨在体外培养的小鼠耳蜗螺旋神经节细胞（SGNC）中转导神经营养因子－3（NT－3）的表达和对听觉神经系统的神经元发育和存活的影响。实验构建了表达NT－3的HSV－Amplicon重组体，经过无辅助病毒的包装细胞系包装后，感染体外培养的小鼠SGNC。结果显示HSV－Amplicon转导的NT－3表达，能够刺激SGNC分泌较高水平的NT－3；SGNC的神经突起增长；耐受顺铂（DDP）的毒性作用。其作用的机制可能是抑制Caspase-3前体的活化，减少了SGNC进入调亡。     

顺铂对豚鼠耳蜗形态与Bcl-2表达的影响

目的 探讨顺铂对豚鼠耳蜗毛细胞耳毒性作用的形态学改变以及顺铂对螺旋神经节细胞Bcl-2蛋白表达的影响.方法 在实验动物被处死前,检测其畸变产物耳声发射（DPOAE）幅值变化.取左侧耳蜗,行耳蜗铺片,观察基底膜毛细胞.右侧耳蜗冰冻切片,用免疫组织化学方法检测Bcl-2的表达.结果 对照组耳蜗铺片显示内、外毛细胞结构完整,排列整齐.顺铂组毛细胞肿胀,纤毛缺失,DPOAE幅值和螺旋种经节的Bcl-2表达明显低于正常对照组（P＜0.01）.结论 顺铂引起耳蜗内、外毛细胞发生形态学改变,其耳毒性作用可能与抑制Bcl-2蛋白的表达有关.     

SOCS3通过抑制STAT3抑制受损伤大鼠初级感觉神经元生长

目的通过慢病毒转导SOCS3和作用相反的突变型SOCS3(mSOCS3),体外研究SOCS3在成年鼠的初级感觉神经元再生中的作用。方法将慢病毒载体质粒pRRL-SOCS3-IRES-GFP,pRRL-mSOCS3-IRES-GFP和pRRL-STAT3ER-IRES-GFP分别转染293T细胞,包装慢病毒载体并测定滴度。完全切断大鼠左侧坐骨神经,术后饲养8-128h,在不同时间点摘取双侧L5背根神经节。通过实时PCR和原位杂交检测DRGs中SOCS3 mRNA的存在;通过背根神经节分离神经元培养,分别将三种慢病毒载体感染神经元细胞,采用免疫荧光染色法观察神经元细胞核质反应和突起的长度。结果大鼠左侧坐骨神经损伤后,SOCS3 mRNA的表达在背根神经节神经元中明显增加,外源性SOCS3能阻止神经元中STAT3的磷酸化及核移位,mSOCS3增强了突起生长。结论 SOCS3可通过抑制STAT3抑制轴突生长。     

碱性成纤维细胞生长因子对豚鼠耳蜗螺旋神经节细胞谷氨酸钠毒性损伤的保护作用

目的　在体研究碱性成纤维细胞生长因子 (bFGF)对谷氨酸钠所致豚鼠耳蜗螺旋神经节细胞毒性损伤的拮抗作用。方法　谷氨酸钠组 :谷氨酸钠 2g·kg-1·d-1,ip× 18d ;bFGF组 :bFGF 80 0U·kg-1·d-1,ip ,谷氨酸钠 2g·kg-1·d-1,ip× 18d。停止用药后耳廓反射粗略测定动物听觉水平 ,后取耳蜗 ,固定包埋、切片染色 ,光镜观察。结果　谷氨酸钠组表现为基底转与顶转几乎均匀一致的耳蜗螺旋神经节细胞的消失 ,平均细胞密度为 (1976 3± 10 18 9)个 /mm2 ,占正常的 4 5 % ;bFGF组细胞密度为 (384 0 9± 373 1)个 /mm2 ,约为正常组的 89%。经方差分析 ,F 检验 ,谷氨酸钠组与bFGF组比较 ,耳蜗螺旋神经节存活细胞密度差异显著 ,P <0 0 5。结论　bFGF对豚鼠谷氨酸钠耳蜗旋神经节细胞在体毒性损伤具有保护作用     

叶酸聚乙二醇接枝支化聚乙烯亚胺纳米载体转染螺旋神经节细胞

背景:新型阳离子纳米材料叶酸聚乙二醇接枝支化聚乙烯亚胺易于合成和改性,稳定性好,结构及性能容易调控,已被作为基因治疗转染载体应用于多种疾病的研究。目的:观察叶酸聚乙二醇接枝支化聚乙烯亚胺转染体外培养小鼠内耳螺旋神经节细胞的可行性及特点。方法:分别以叶酸聚乙二醇接枝支化聚乙烯亚胺(实验组)、阳离子脂质体Lipofectamine?2000(对照组)作为载体,制备携带增强型绿色荧光蛋白的转染载体系统。将昆明小鼠螺旋神经节细胞分别培养于含实验组和对照组转染载体系统的DMEM/F-12培养基中,培养24 h后,MTT法观察载体系统的细胞毒性,倒置荧光显微镜观察细胞形态及绿色荧光蛋白表达强度,流式细胞仪检测细胞转染率。结果与结论:两种载体分别转染后,螺旋神经节细胞形态均未发生明显改变,但两组载体对螺旋神经节细胞均有一定的毒性,实验组载体毒性较小且转染效率较高(P0.05)。结果说明叶酸聚乙二醇接枝支化聚乙烯亚胺基因转染体外螺旋神经节细胞的转染效率及细胞毒性均优于传统的阳离子脂质体。     

神经再生素促大鼠背根神经节生长作用的研究

目的研究神经再生素（NRF）对体外培养新生大鼠背根神经节生长及NF—H表达的影响。方法体外大鼠背根神经节（DRG）培养;免疫荧光细胞化学、实时荧光定量PCR和Western blot等方法。结果免疫荧光细胞化学结果提示,NRF能促进背根神经节神经突起的生长,浓度为2．0mg／L时生长状况最佳;实时荧光定量PCR和Western blot结果提示,NRF能增加体外培养的背根神经节NF-H mRNA和蛋白的表达,在浓度为2．0mg／L时表达最高。结论NRF能促进体外培养背根神经节神经突起的生长和NF—H的表达,表明NRF对发育期感觉神经元具有神经营养作用。     

HPV16E6E7转导的卵巢癌细胞系顺铂耐药性的变化

目的　人乳头瘤病毒 16型 E6 E7区基因编码的蛋白具有转导人上皮细胞 ,使之获得永生化特性的作用。为建立卵巢癌永生细胞系 ,我们对 HPV16 E6 E7转导卵巢癌细胞的方法及转导细胞对顺铂的耐药性进行了研究。　方法　采用脂质体方法 ,将含有 HPV16 E6 E7的重组逆转录病毒载体 p L XSN16 E6 E7,转染逆转录病毒包装细胞 PT6 7,经 G418筛选 ,病毒滴度测定 ,筛选出 1株高滴度 (1.9× 10 7/m l)的产病毒克隆 ,将之转导卵巢癌细胞系SKOV3、3AO,各筛选出 1株稳定表达 HPV16 E6 E7的克隆 ,SKOV3/E6 E7、3AO/E6 E7。经 RT- PCR、免疫细胞化学检测证实 HPV16 E6 E7的表达 ,之后采用 MTT法检测了 HPV16 E6 E7转导的卵巢癌细胞系对顺铂耐药性的变化。结果　 HPV16 E6 E7转导的卵巢癌细胞系 SKOV3/E6 E7、3AO/E6 E7对顺铂的耐药性无改变。　结论　HPV16 E6 E7转导的卵巢癌细胞没有发生顺铂耐药性的改变。本研究为采用此法建立卵巢癌永生细胞系 ,特别是建立耐药卵巢癌细胞系奠定了基础。     

单纯疱疹病毒扩增子载体转导的ATM基因在活体的异位表达(英文)

小脑性共济失调与血管扩张症 (A-T)是一种常染色体隐性遗传性疾病 ,具多效表型。此病的特征是小脑变性、免疫缺陷、生长迟缓、早熟性衰老、染色体不稳定、肿瘤易患体质和对辐射的过度敏感。目前尚未发现有效的方法阻断此病的进行性发展。基因治疗是此病的潜在希望。ATM是此病的致病基因 ,编码 ATM蛋白激酶。本研究将 ATM c DNA完整的读码框架克隆进 1型单纯疱疹病毒 (HSV-1)扩增子载体 (p TO-ATM)。培养的 A-T细胞经 p TO-ATM病毒载体转导后 ,用免疫组织化学方法证明了 ATM在这些细胞的异位表达。为了进一步研究在活体应用此载体系统的可能性 ,选择大鼠脑作为实验靶区。先用免疫组织化学方法观察了内源性 ATM在成年大鼠前脑和小脑的正常分布 ,发现在尾壳核有低水平的 ATM出现 ,而在其余脑区 ,ATM水平则显著地高于尾壳核区。将 p TO-ATM病毒载体注射到成年大鼠的一侧尾壳核 ,3 d后在注射区 (包括针道周围的区域 )观察到密集的 ATM免疫反应阳性神经元。本研究结果证明 ,HSV扩增子病毒载体可以稳定地运载相当大的 c DNA片段 ,转基因在离体和活体均能有效地表达。本研究为使用基因治疗的方法治疗 A-T病人的神经变性迈出了第一步     

玻璃体注射腺相关病毒2和慢病毒转染大鼠视网膜的比较

目的通过玻璃体注射重组腺相关病毒2载体(r AAV2)和慢病毒载体(LV),比较两者转染成年大鼠视网膜的异同。方法实验组大鼠60只,每组12只玻璃体内分别注射重组腺相关病毒2载体-增强绿色荧光蛋白(r AAV2-EGFP)、重组腺相关病毒2载体-神经突起素-增强绿色荧光蛋白(r AAV2-neuritin-EGFP)、慢病毒载体-红色荧光蛋白(LV-RFP)和慢病毒载体-神经突起素-红色荧光蛋白(LV-neuritin-RFP),对照组注射等量的生理盐水,4周后取材,采用免疫荧光和CTB-FITC检测2种病毒载体在视网膜中转染的细胞及转染率,然后分别采用Real-time PCR和Western blotting检测神经突起素(neuritin)在视网膜中的表达变化。结果 r AAV2能够转染约70%视网膜节细胞(RGCs),LV主要转染色素上皮细胞,RGCs转染率仅为30%;r AAV2-neuritin-EGFP组神经突起素mRNA表达约是r AAV2-EGFP组和对照组的16倍,蛋白表达约为3倍;LV-neuritin-RFP组神经突起素mRNA表达约是LVRFP组和对照组的5.5倍,蛋白表达约为1.7倍。结论 r AAV2玻璃体注射后,转染大部分RGCs且神经突起素表达量高于LV,LV主要转染色素上皮细胞,提示基因治疗眼科疾病和损伤时,涉及到转染RGCs时应采用r AAV2载体,转染色素上皮时宜采用LV载体。     

神经生长颗粒含药血清对大鼠背根神经节生长的影响

目的: 研究神经生长颗粒(NGG)含药血清对体外培养新生大鼠背根神经节生长及高分子量神经丝蛋白(NF-H)和生长相关蛋白43(GAP43)表达的影响.方法: 采用体外大鼠背根神经节(DRG)植块培养,通过免疫荧光细胞化学法,观察不同剂量的含药血清对DRG神经突起生长的影响;采用DRG单细胞分离培养,通过实时荧光定量PCR和免疫印迹法分别观察不同剂量的含药血清对DRG细胞NF-H和GAP43基因及蛋白表达的影响.结果: 免疫荧光细胞化学法提示NGG含药血清能促进DRG神经突起的生长;实时荧光定量PCR和免疫印迹法结果提示NGG含药血清能增加体外培养的DRG细胞NF-H、 GAP43 mRNA和蛋白的表达.结论: NGG含药血清能促进体外培养DRG神经突起的生长并促进NF-H和GAP43的表达,表明NGG对发育期感觉神经元具有一定的神经营养作用.     

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.