亚纳秒脉冲电场联合温度杀伤人宫颈癌细胞的实验研究

为研究亚纳秒脉冲联合温度对人宫颈癌(HeLa)细胞的杀伤效应, 将脉冲电场参数组合(电场强度25、50、100 kV/cm, 半峰值脉宽1 ns, 脉冲个数分别为4 000、1 000、250, 频率5 Hz), 作用于处于不同温度(25℃、43℃、48℃)的HeLa细胞, 利用MTT检测亚纳秒脉冲对细胞的生长抑制情况, 通过AO/EB染色和透射电镜观察细胞形态学变化, 采用免疫细胞化学S-P法检测caspase-3蛋白表达活性。实验发现:当细胞悬液温度到达48℃后, 随着时间推移细胞死亡率不断增加;采用亚纳秒脉冲电场(电场强度50 kV/cm, 半峰值脉宽1 ns, 脉冲个数1 000, 频率5 Hz)处理不同温度下HeLa细胞, 细胞悬液温度达到43℃后, 细胞死亡率达到18.07%±1.98%, 细胞悬液温度达到48℃, 细胞死亡率增至25.11%±6.05%;采用相同能量脉冲电场处理处于相同温度(48℃)的细胞悬液时, 细胞死亡率与脉冲场强正相关, 100 kV/cm电场作用下得到最高细胞死亡率为31.09%±5.03%, 进一步的分子生物学检测结果显示HeLa细胞发生凋亡效应。适当场强的亚纳秒脉冲联合温度通过诱导细胞凋亡, 进而杀死HeLa细胞。     

纳秒脉冲用于细胞内电处理的研究进展

近年来，国内外学者对纳秒脉冲电场应用于细胞内电处理效应进行了一些较为深入的实验和机制研究，实验结果表明纳秒脉冲电场不会导致细胞膜电穿孔，但能够诱导凋亡、钙离子释放、基因表达增强、DNA和染色体破碎等细胞内电处理效应。为了解释细胞内电处理，研究者们认为当脉冲电场的脉宽大于细胞膜充电时间常数时，脉冲电场主要作用在细胞外膜上产生电穿孔。随脉冲宽度变小，电场对细胞核及核膜影响增强，当脉宽小于细胞膜充电时间常数时，脉冲电场主要作用于膜内细胞器，进行细胞内电处理。     

电脉冲致离体S-180细胞电穿孔的观察与研究

扫描电镜下我们观察到了一定强度电脉冲致 S- 180细胞电穿孔的现象。同时分别改变电压、电容、脉冲个数 ,对 S- 180离体细胞进行电脉冲作用 ,通过苔盼蓝追踪 ,发现在一定的条件下 ,随着对 S- 180离体细胞电穿孔的电压越大、电容越小、脉冲个数越多 ,其穿孔百分率越大。     

利用双向电泳比较3种提取户尘螨蛋白的方法

为改进户尘螨过敏原提取方法,在采用Coca^ s液、裂解液及Trizol法提取纯种户尘螨螨体蛋白后,用二喹啉甲酸(BCA)检测法测定蛋白质含量,双向电泳比较上述蛋白提取方法的效果。结果显示,在蛋白质回收率方面,裂解液优于Trizol法优于Coca^ s液。经过双向电泳,Coca^ s液提取的蛋白仅仅有少量低分子量的蛋白点;用裂解液提取的蛋白可见明显增多低分子量的蛋白点,但无中分子量的蛋白点;Trizol法提取的蛋白可见中分子量的蛋白点如174～178ku和133．0ku的蛋白质。另外,纯种户尘螨螨体经Trizol液提取后进行双向电泳,在考染和银染时均出现5个高丰度的特殊蛋白点,一个为酸性中分子量蛋白,位置孤立的;另有4个位置彼此非常接近的中性蛋白点。比较后结论为使用Coca^ s液提取的尘螨蛋白质浓度低,蛋白点少。使用裂解液提取的尘螨蛋白质浓度虽然较高,但是没有中分子量的蛋白点,不能全面反映尘螨的过敏原。使用Trizol法提取的蛋白质浓度中等,有中分子量蛋白点,反映的蛋白点更加全面。纯种户尘螨螨体经Trizol液提取后进行双向电泳,在考染和银染时出现的特征性蛋白点,作为一种双向电泳的指纹图谱,在鉴定此种纯种户尘螨螨体时具有一定的价值。     

Fascin与上皮组织肿瘤

Fasc in基因属于fasc ins家族,其蛋白质编码产物是一种结构独特、进化保守的肌动蛋白(actin)交联蛋白,位于细胞膜皱褶、微棘及应力纤维,在各种转化细胞中促使细胞膜突起并增加细胞运动性。近年来的研究发现,fasc in在许多上皮来源的肿瘤组织细胞中表达上调,在肿瘤的进展中起重要作用。本文综述了fasc in的研究现状及其在上皮组织肿瘤中的研究进展。     

温度开环和闭环反馈在高压脉冲电场消融中的对照研究

目的:对比温度开环和闭环反馈在高压脉冲电场消融中的区别。方法:温度开环组应用高压方波脉冲发生器以场强1.5 kV/cm、频率1 Hz、脉宽100 μs、脉冲数20个的条件对细胞悬液、3D细胞模型和离体猪肝组织3种不同负载施加高压脉冲电场。闭环组在相同脉冲条件下增加温度闭环算法对微控制器及温度传感器编程控制脉冲发放。分别从电场消融后的电极温度、细胞悬液电导率和细胞死亡率,3D细胞模型消融截面积以及猪肝组织消融区病理特点进行定量比较和定性分析。结果:消融后开环组电极温度、细胞悬液电导率、细胞死亡率和3D细胞模型消融截面积分别为（53.2±5.5）℃、（1.43±0.03） S/m、（86.8±5.5）%和（0.46±0.15） cm2,均高于闭环组对应的（38.5±1.6） ℃、（1.21±0.02） S/m、（73.6±10.2）%和（0.37±0.08） cm2,差异均有统计学意义（P<0.05）;开环组消融区猪肝组织H-E染色可见热损伤导致的凝固性坏死区,闭环组表现为肝血窦增宽、细胞质脱水丢失和核聚集。结论:该实验条件下,温度开环和闭环反馈高压脉冲电场消融存在差异。传统温度开环高压脉冲电场消融效果略强于温度闭环,这可能与电导率增加和热效应介导的电场杀伤效应增强有关。温度闭环反馈能避免热损伤效应,实现真正意义上的非热性消融。     

脉冲电场联合野生型p53基因诱导人宫颈癌Hela细胞凋亡

背景：在前期研究基础上,设想将基因电转染作为脉冲电场治疗恶性肿瘤的辅助方法。 目的：探讨脉冲电场联合抑癌基因野生型p53基因诱导人宫颈癌hela细胞发生凋亡及相互作用。 方法：采用空质粒及含有野生型p53的质粒转染Hela细胞得到Hela-vector、Hela-p53细胞,将Hela细胞、Hela-vector和Hela-p53细胞分别施加固定脉宽100 μs、频率1 Hz、脉冲数8个、电场强度为1 500 V/cm的脉冲电场处理。 结果与结论：相同参数脉冲电场作用于各组细胞12 h后,MTT结果显示Hela-p53组细胞吸光度明显低于Hela组(P < 0.05);流式细胞仪及RT-PCR检测结果显示Hela-p53组的早期凋亡率和p53 mRNA表达较Hela组明显增加;Western bolt及激光共聚焦显微镜结果显示与Hela组比较,Hela-p53组细胞Bax蛋白表达明显上升,而Bcl-2蛋白则明显下降,Casepase-3相对荧光强度明显增强。表明野生型p53基因对宫颈癌Hela细胞生长有明显的抑制作用,野生型p53基因可以提高脉冲电场诱导宫颈癌Hela细胞凋亡的作用。     

直流脉冲场作用下穿孔悬液细胞跨膜电压计算模型

细胞膜穿孔期间,膜电导率大幅度提高,正常生理条件下的悬液细胞跨膜电压(Transmembranevoltage,TMV)计算模型不再适用。我们建立了高强度直流脉冲电场作用下,均匀悬液中穿孔细胞跨膜电压的计算模型。首先将膜电导率不均匀分布的穿孔悬液细胞等效为一小球,然后根据有效介质理论求出细胞悬液的平均场,用其代替难于精确求解的实际场,最后利用穿孔单细胞膜电压计算公式,建立悬液中穿孔细胞膜电压的计算模型。模型表明在穿孔期间,细胞膜跨膜电压和外加电场大小,细胞膜穿孔临界角,细胞半径,细胞质、细胞膜和膜外溶液的电导率、细胞排列以及细胞浓度等参量有关。     

基于腔内压力评价的植入式结肠电刺激系统设计与动物实验

目的测试自行研制的植入式结肠起搏点电刺激系统对增加盲肠运动的效果。方法植入式结肠电刺激系统由植入式体内刺激器、便携式体外控制器和计算机参数设置分析软件组成。刺激参数及指令经体外控制器,以无线通信模式发送至体内刺激器;表征结肠收缩活动的压力信息由结肠腔内压力感受器采集后通过无线通信模式发送至体外控制器存储卡储存。选择46kg健康白猪1只,麻醉后开腹,将体内刺激器植入右侧腹股沟皮下,刺激电极穿过腹壁植入盲肠壁内固定,刺激参数选择电压为10、15、20 V,双脉冲频率为10、40、120 Hz,脉冲时间为0.5、1.0、3.0、5.0 ms,每次刺激前后连续观察记录5 min。检测完成后植入器固定,关腹后饲养1个月再次手术取出植入器。结果植入式结肠起搏点电刺激系统工作正常,在上述的电压、脉冲频率和双脉冲宽度的任意组合参数刺激下,发现电压和频率相同时脉宽越长,肠壁收缩活动的幅度越大;脉宽和电压相同时脉冲频率越高,肠壁收缩活动的潜伏期越短;频率和脉宽相同时电压越大,肠壁收缩活动的潜伏期越短,收缩幅度越大。在脉宽5.0ms、频率120Hz、电压15V的脉冲电刺激下,盲肠处于强直收缩状态。体内刺激器植入1个月,无感染及异物反应,取出时见植入物周围有少许包裹性积液。结论自行研制的电刺激系统初步适合进行结肠电刺激动物实验,选择合适刺激参数的结肠起搏点电刺激可明显增加结肠收缩运动,为进一步探索结肠电刺激治疗慢传输性便秘提供了研究基础。     

穿孔素

穿孔素是由具杀伤功能的淋巴细胞产生的一种杀伤蛋白,无种属特异性。穿孔素合成后贮存在细胞的胞浆颗粒中,当杀伤细胞与靶细胞接触时被细胞排出。人、鼠穿孔素分子量基本一致。在非还原条件下为60kd,在还原条件下为70kd。穿孔素是一种钙依赖蛋白,当它被释出细胞后,在钙存在时吸附在靶细胞膜上,集聚成聚穿孔素,并在靶细胞膜上形成跨膜通道,最终导致靶细胞裂解。穿孔素与补体C9成分在结构和功能上有许多相似之处,具有一定的同源性。     

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.     

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.     

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.