肿瘤抑素30肽的重组表达及抗肿瘤活性研究

目的：重组表达肿瘤抑素30肽并研究其抗肿瘤活性。方法设计并合成30肽基因序列,将此序列与融合蛋白表达载体 PTYB21重组,转化到大肠埃希菌 BL-21(DE3)。 IPTG 诱导表达,几丁质柱纯化得到30肽,经 SDS-PAGE 及 Tricine-SDS-PAGE 对纯化产物进行鉴定。利用 MTT 法、吖啶橙/溴化乙锭(AO/ EB)荧光染色法、小鼠 H22腹水转移型肝癌实体瘤抑瘤实验,研究30肽的抗肿瘤活性。结果成功重组并表达肿瘤抑素30肽。体外实验显示,30肽具有抑制 HGC-27胃癌细胞、 HUVEC 人脐静脉细胞增殖和促进这两种细胞凋亡的作用。体内实验显示,30肽对小鼠 H22腹水型肝癌抑瘤率达43.18 ％ 。结论重组的肿瘤抑素30肽具有较强的抗肿瘤活性。     

肿瘤抑素抗血管活性相关肽的表达及活性研究

目的利用大肠杆菌表达系统表达肿瘤抑素抗血管活性相关肽-21肽,检测其生物学活性。方法人工合成21肽基因序列(肿瘤抑素75～95位氨基酸),克隆到表达载体pTYB2上,转化大肠杆菌BL-21(DE_3),酶切和测序鉴定后,用IPTG诱导表达融合蛋白。经几丁质亲和层析柱纯化21肽。通过MTT实验,细胞生长曲线测定,小鼠肝癌转移瘤模型的大体抑瘤实验及组织病理学切片研究其活性。结果获得的可溶性21肽对人脐静脉内皮细胞的生长具有抑制作用。小鼠肝癌转移瘤模型的大体抑瘤实验抑瘤率达42.86%。组织病理学切片结果可见H22小鼠肝癌细胞死亡,血管数量减少。结论经初步检测获得具有抗血管活性的21肽,为进一步研究肿瘤抑素的作用机制和肿瘤的临床治疗奠定了基础。     

人内皮抑素抗肿瘤相关肽基因的克隆表达及活性研究

目的克隆并表达人内皮抑素抗肿瘤相关肽,检测其生物活性。方法人工合成人内皮抑素1~30位氨基酸(30肽,序列25~31由RGIRGAD改为RGDRGD)所对应的核苷酸序列,连接到质粒pTYB2中,再转化至大肠埃希菌BL21(DE3)中表达,几丁质亲和层析树脂一步纯化30肽。通过MTT法、鸡胚绒毛尿囊膜(CAM)实验、小鼠体内抑瘤实验比较30肽和内皮抑素抗肿瘤活性。结果MTT证实30肽体外对人脐静脉内皮细胞(HUVEC)、胃癌7901细胞(SGC-7901)半数抑制浓度IC50为36μg/ml、47μg/ml,显著低于内皮抑素IC50179μg/ml、202μg/ml。CAM实验中30肽对血管的抑制作用更强。30肽在小鼠体内抑瘤率47.8%,效果优于内皮抑素28.7%。结论30肽具有更强抗肿瘤活性,有可能成为治疗肿瘤的一种新药物。     

IL-21基因治疗小鼠H22细胞皮下移植肝癌模型的效果评价

用已构建的mIL-21/pcDNA3.1重组质粒对H22细胞建立的小鼠肝癌模型进行基因治疗,观察IL-21对小鼠体内抗肿瘤免疫应答的影响及对小鼠生存的影响。采用BALB/c小鼠左腋皮下注射腹水型肝癌细胞株H22细胞建立小鼠移植肝癌模型,给荷瘤小鼠瘤体内注射mIL-21/pcDNA3.1进行基因治疗,MTT比色法检测IL-21对荷瘤小鼠T细胞增殖水平及NK细胞杀伤活性的影响,观察治疗后荷瘤小鼠生存情况及肿瘤生长情况的改变。病理检测结果显示,成功建立了小鼠移植型肝癌模型,MTT比色法显示基因治疗后小鼠T细胞增殖水平及NK细胞杀伤活性显著升高,荷瘤小鼠肿瘤生长速度减慢,生存期显著延长。IL-21基因治疗肝癌荷瘤小鼠可显著提高荷瘤小鼠体内抗肿瘤免疫应答水平,抑制肿瘤生长,延长荷瘤小鼠生存期。     

人肿瘤抑素相关T21RGD肽的表达与纯化

目的 构建引入RGD序列人肿瘤抑素相关21肽（T21RGD）―内含肽―几丁质结合蛋白融合表达载体，实现目的肽的几丁质珠亲和层析一步纯化。方法 人工设计合成T21RGD肽基因， 经重组定向克隆插入到原核表达载体pTYB2，酶切和测序鉴定正确后转化入大肠杆菌BL21（DE3），IPTG诱导其融合表达，对表达蛋白进行几丁质珠亲和层析，DTT诱导柱上裂解以获得T21RGD肽。经Trcine-SDS-PAGE和反相高效液相色谱(RP-HPLC)对纯化产物进行鉴定。结果 质粒酶切和测序结果显示，含T21RGD肽基因按正确方向和序列插入质粒,融合蛋白表达量达菌体蛋白总量的50％以上，Trcine-SDS-PAGE显示了T21RGD肽目的条带与预期相符，反相高效液相色谱测定峰纯度达97％。结论 成功构建T21RGD肽融合表达载体，实现了融合蛋白在大肠杆菌中高效表达和T21RGD肽几丁质珠亲和层析一步纯化，为进一步研究T21RGD肽抗肿瘤活性和作用机理奠定基础。     

IL-21 gpi和sGM-CSF共同修饰的瘤苗构建及其抗肿瘤效应

目的:构建膜锚定IL-21和分泌性GM-CSF(sGM-CSF)双表达瘤苗,并对其抗肿瘤效应及其机制作初步探讨.方法:用分子生物学方法构建双表达IL-21 gpi和sGM-CSF重组质粒,将鉴定过的重组质粒以脂质体转染B16F10细胞制成瘤苗,用流式细胞仪检测转染瘤苗IL-21 gpi和sGM-CSF的表达.以瘤苗治疗荷瘤鼠,经观察小鼠肿瘤体积、生存率来分析瘤苗的抗瘤性,并检测了瘤苗治疗鼠的细胞免疫活性.结果:正确构建了pRSC/IL-21 gpi-sGM-CSF重组质粒,转染细胞可很好地表达膜锚定IL-21和分泌性GM-CSF,制备的瘤苗能有效地发挥抗肿瘤效应,其机制与瘤苗治疗鼠的脾细胞增殖活性、NK细胞及CD8+细胞细胞毒活性增强有关.结论:成功构建了具有抗肿瘤活性的膜锚定IL-21和分泌性GM-CSF双表达瘤苗,为进一步抗肿瘤免疫治疗研究奠定了基础.     

糖基化磷脂酰肌醇修饰的小鼠IL-21瘤苗抗肿瘤效应及其机制初探

目的 构建糖基化磷脂酰肌醇(glycosyl phosphafidylinositol,GPI)修饰的小鼠IL-21瘤苗,并对此瘤苗的抗肿瘤效应及其机制作初步探讨.方法 通过重叠PCR方法获得IL-21-GPI融合基因并将其插入空载体pcDNA3.1.将鉴定过的重组载体以脂质体法转染B16F10细胞制成瘤苗,细胞间接免疫荧光法及流式细胞仪检测转染瘤细胞膜表面IL-21的表达,通过对小鼠脾细胞的增殖作用鉴定表达的IL-21的生物学活性.将瘤苗接种小鼠后,通过观察小鼠肿瘤体积和生存率分析瘤苗的抗瘤性,并检测了瘤苗免疫鼠的细胞免疫活性.结果 正确构建了pcDNA3.1/IL-21-GPI重组载体,膜表达的IL-21有良好的生物学活性,制备的瘤苗能发挥抗肿瘤效应,其机制与免疫鼠细胞免疫活性增强有关.结论 成功构建了具有抗肿瘤活性的GPI修饰的IL-21瘤苗,为其进一步抗肿瘤免疫治疗研究奠定了基础.     

TAT-凋亡素基因重组质粒的构建、蛋白的表达纯化及体内活性实验

目的 克隆构建TAT-凋亡素质粒并提取融合蛋白，为进一步研究该蛋白功能奠定基础。方法 PCR合成TAT-凋亡素基因，与pTYB2质粒连接后转入Rosetta菌，经IPTG诱导表达，几丁质亲和层析一步纯化目的蛋白，用昆明小鼠H22动物模型检测活性。结果 克隆载体经过PCR筛选、测序鉴定，其大小和核苷酸序列正确，诱导后融合蛋白出现在上清，纯化出的TAT-凋亡素蛋白具有明显的抗肿瘤活性。结论 本实验所构建的重组质粒pTYB2/TAT-apoptin经诱导表达出了可溶性目的蛋白TAT-凋亡素，纯化后具有明显的生物活性，为研究TAT-凋亡素蛋白的进一步研究奠定了基础。     

小鼠白细胞介素21 cDNA的克隆及真核表达质粒的构建

目的：克隆小鼠自细胞介素2l(IL-21)基因，构建真核表达质粒，用以进行肿瘤的基因治疗。方法：用RT-PCR法。从ConA活化的小鼠T细胞中扩增IL-21 cDNA。克隆人哺乳动物细胞高效表达质粒pcDNA3．1中，构建重组mIL-21真核表达质粒。重组体用载体上的通用引物和PCR下游引物为测序引物，鉴定克隆的正确性。将已鉴定的重组质粒用脂质体法转染Sp2／0细胞，用RT-PCR法鉴定转染细胞中IL-21基因的表达，用MTT比色法检测表达的mIL-21诱导的NK细胞杀伤活性的增强。结果：正确构建了重组真核表达质粒pcDNA3．1／mIL-21，并在转染的细胞中检测出IL-2l的表达，表达的mIL-21可在体外增强NK细胞的杀伤活性：结论：成功地构建了重组真核表达质粒pcDNA3．1／mIL-21，为进一步在肿瘤动物模型中进行IL-21基因治疗及疗效观察奠定了基础.     

重组内皮抑素联合靶向性preS2反义RNA对肝癌细胞的体内抑制作用

单纯抗血管生成治疗肿瘤 ,仅使肿瘤处于休眠状态 ,容易出现复发。如何将抗血管生成与其他肿瘤治疗方法有效结合 ,是目前肿瘤基因治疗中的研究热点。本研究首次提出重组内皮抑素联合靶向性preS2反义RNA抑制肝癌细胞的策略 ,并进行体内实验研究。构建肝癌细胞特异性HBVpreS2反义RNA表达载体 ,并将其与针对表皮生长因子受体(EGFR)的 16肽配体寡肽和流感病毒血凝素HA2 0寡肽混合 ,制备肝癌靶向性HBV反义RNA转移系统 ,即AFP增强型四元复合体。同时 ,以自行构建的毕赤酵母菌表达重组人内皮抑素蛋白 ,纯化后 ,进行动物实验。培养整合有…     

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.