转染超抗原SEA肝癌细胞的构建及鉴定

目的用基因重组的方法将葡萄球菌肠毒素A(SEA)基因转染至肝癌细胞.方法先从产SEA标准菌株中提取并扩增SEA全长基因,将SEA基因克隆及亚克隆至真核表达载体pLXSN构建pLXSN-SEA重组质粒,再将重组质粒转染至人肝癌细胞系BEL-7402细胞,用RT-PCR和ELISA法鉴定.结果从产SEA标准菌株ATCC13565中提取并扩增出SEA基因全长片段;SEA基因克隆和亚克隆至真核表达载体pLXSN,经测序证实所得序列与GenBank中的标准序列完全一致;将pLXSN-SEA质粒转染肝癌细胞BEL-7402,经筛选获得稳定表达的抗性单克隆.RT-PCR扩增出约800bp的基因片段,经ELISA分析细胞培养上清中SEA蛋白的含量在皮克的水平.结论成功构建了重组超抗原SEA基因的克隆载体及真核表达载体,将SEA基因转染至肝癌细胞株BEL-7402细胞后癌细胞能够表达并持续分泌SEA蛋白.     

人IL-8正义和反义真核表达载体的构建与表达

目的:构建人IL- 8正义和反义真核表达载体.方法:RT-PCR扩增正义和反义IL- 8基因片段后, 将其克隆入真核表达载体pcDNA3.1(+), 通过菌落PCR、双酶切及测序进行鉴定后, 采用脂质体法分别瞬时转染至人卵巢癌细胞系A2780和SKOV3细胞中, 并用RT-PCR和ELISA法检测细胞转染后IL- 8的表达水平.结果:经验证, 重组人IL- 8正义/反义真核表达载体构建正确.pcDNA3.1(+)-ssIL- 8转染A2780细胞后, 其IL- 8 mRNA及蛋白表达水平均显著增加;而pcDNA3.1(+)-asIL- 8转染SKOV3细胞后, 其IL- 8分泌水平降低.结论:成功构建了人IL- 8正义/反义真核表达质粒pcDNA3.1(+)-ssIL- 8/pcDNA3.1(+)-asIL- 8, 为后续研究IL- 8在卵巢癌及其他肿瘤中的作用打下基础.     

转染超抗原SEA肝癌细胞的构建及鉴定

目的用基因重组的方法将葡萄球菌肠毒素A（SEA）基因转染至肝癌细胞．方法先从产SEA标准菌株中提取并扩增SEA全长基因，将SEA基因克隆及亚克隆至真核表达载体pLXSN构建pLXSN—SEA重组质粒，再将重组质粒转染至人肝癌细胞系BEL-7402细胞，用RT—PCR和ELISA法鉴定．结果从产SEA标准菌株ATCCl3565中提取并扩增出SEA基因全长片段；SEA基因克隆和亚克隆至真核表达载体pLXSN，经测序证实所得序列与GenBank中的标准序列完全一致；将pLXSN—SEA质粒转染肝癌细胞BEL-7402，经筛选获得稳定表达的抗性单克隆．RT—PCR扩增出约800bD的基因片段，经ELISA分析细胞培养上清中SEA蛋白的含量在皮克的水平．结论成功构建了重组超抗原SEA基因的克隆载体及真核表达载体，将SEA基因转染至肝癌细胞株BEL－7402细胞后癌细胞能够表达并持续分泌SEA蛋白．     

人VMAT_2基因RNA探针制备及其在COS-7细胞中的表达

本研究目的在于制备人 型囊泡单胺转运体 (VMAT2 )基因的反义和正义 RNA探针 ,以检测 VMAT2 基因能否在真核细胞表达。从携带 p GEM-Easy-T-VMAT2 克隆载体中通过限制性酶切反应得到 VMAT2 基因 ,与 p BK-RSV载体重组构建真核表达载体 p BK-RSV-VMAT2 ;分别以 T7和 T3聚合酶制备反义和正义探针 ,通过斑点杂交检测探针浓度。转染猴肾成纤维细胞COS-7,原位杂交及免疫荧光细胞化学检测 VMAT2 的表达。结果证明 ,反义和正义探针浓度分别为 80 ng/μl及 12 0 ng/μl;原位杂交及免疫组织化学证实重组的真核表达载体 p BK-RSV-VMAT2 在 COS-7的表达阳性率为 (10 .6± 1.2 ) %。本研究结果表明获得 VMAT2 基因反义链及正义链 RNA探针 ,并检测到 VMAT2 基因在真核细胞中表达     

携带增强绿色荧光蛋白基因的Id2真核表达载体构建

目的：构建大鼠Id2基因真核荧光表达载体，为骨骼肌的组织工程研究提供有效的分子工具。方法：利用RT-PCR的方法扩增出Id2全长cDNA，利用T4 DNA连接酶将载体pGEM-T和Id2 cDNA进行连接，构建克隆载体，经限制性内切酶EcoR1酶切pGEM-Id2克隆载体和pEGF-C2真核表达载体，构建出重组真核表达载体pEGFP-C2-Id2，经酶切分析、PCR鉴定及DNA测序证实cDNA片段大小和序列的正确性；并通过细胞转染技术将Id2基因导入L6成肌细胞中。结果：经酶切分析和序列测定证实pEGFP-C2-Id2含大小正确的正向Id2 cDNA片段，获取了转染外源性Id2基因的L6细胞。结论：我们成功构建了同时携带有G418筛选位点和增强绿色荧光蛋白的Id2真核表达载体？     

syk真核表达载体的构建及其对人乳腺癌细胞MHC-I类分子表达的影响

目的：构建非受体蛋白酪氨酸激酶Syk的真核表达载体，转染人乳腺癌细胞，使其在细胞中稳定表达，以研究Syk对人乳腺癌细胞MHC—Ⅰ类分子表达的影响。方法：以RT-PCR法从人乳腺癌细胞株MDA-MB-468扩增出叼击编码序列基因片段，将其克隆到真核表达载体pcDNA3-1D／V5-His—TOPO中。对重组质粒进行酶切、PCR及测序鉴定后，以脂质体介导法转染Syk缺失的人乳腺癌细胞MDA-MB-231，经G418筛选，构建syk基因稳定表达的细胞株，通过Westernblot、RT-PCR和流式细胞术检测转染乳腺癌细胞Syk、MHC-Ⅰ及ICAMⅠ的表达。结果：构建了hsyk真核表达载体pcDNA3-1D／V5-His-TOPO／hsyk，并在人乳腺癌细胞MDA—MB-231中获得稳定表达。表达Syk的MDA—MB-231细胞同时可以高表达MHC-Ⅰ和ICAMⅠ。结论：成功地构建了syk真核表达载体并在真核细胞中表达，为进一步的肿瘤免疫研究奠定了实验基础。     

神经生长因子基因重组逆转录病毒载体的构建与鉴定

目的构建神经生长因子(NGF)基因重组逆转录病毒表达载体,研究NGF在神经干细胞(NSC)中的表达情况。方法从大鼠海马组织提取总RNA,利用RT-PCR的方法获得编码大鼠β-NGF的基因片段,应用基因重组技术,将大鼠β-NGF基因片段克隆到逆转录病毒表达载体pLEGFP-N1中,通过脂质体Lipofectamine2000转染包装细胞PT67,经G418筛选后,收集阳性克隆病毒上清,用于感染神经干细胞(NSC),观察该NSC表达的NGF对PC12细胞突起生长的作用。结果限制性内切酶酶切分析鉴定表明为正确重组子,β-NGF基因在NSC中获得表达,该NSC的培养上清液可以促进PC12细胞突起生长。结论重组逆转录病毒表达载体pLEGFP-NGF构建成功,β-NGF基因可在NSC中表达并具有生物学活性。     

人α-防御素-1(α-HNP-1)基因真核表达载体的构建及在大鼠骨髓间充质干细胞中的转染表达

目的构建人α-防御素-1(α-HNP-1)基因的真核表达载体,并且转染大鼠骨髓间充质干细胞。方法提取人外周血粒细胞中的总RNA,反转录为cDNA作为模板,采用PCR的方法扩增得到人α-防御素-1(α-HNP-1)的基因片断。将扩增产物连接入pGEM-T载体,转化大肠杆菌DH5α感受态细胞,蓝白筛选,对PCR及酶切鉴定含有目的片断的克隆进行测序。经测序证实无误后,将获得的pGEM-T-HNP-1重组质粒上的α-HNP-1基因亚克隆到真核表达载体pcDNA3.1(-)上,构建HNP-1的真核表达载体pcDNA3.1-HNP-1。将真核表达载体pcDNA3.1-HNP-1用脂质体法转染原代大鼠骨髓间充质干细胞,用免疫组化法检测α-HNP-1的表达。结果获得预期大小为303bp的RT-PCR产物;经PCR、酶切鉴定和DNA测序分析证实重组质粒载体pcDNA3.1-HNP-1构建正确;免疫组化法显示转染细胞呈阳性反应。结论成功构建HNP-1基因的真核表达载体,并且在大鼠骨髓间充质干细胞中能成功表达。     

EGFP-Sam68融合蛋白在HeLa细胞的表达和定位

目的克隆有丝分裂Src相关蛋白p68(Sam68)基因cDNA全长,构建Sam68真核绿色荧光蛋白表达载体,并确定其在HeLa中的表达和定位。方法提取HeLa细胞总RNA,RT-PCR扩增Sam68基因片段,经酶切鉴定及测序正确后,克隆到真核绿色荧光蛋白表达载体(pcDNA3.0-EGFP)上,并转染HeLa细胞,Western blot法检测Sam68表达,免疫荧光细胞化学染色观察其细胞定位。结果酶切和测序证实Sam68正确插入pcDNA3.0-EGFP载体中,且载体正确表达了EGFP-Sam68融合蛋白,该蛋白定位在HeLa细胞核内。结论成功构建了真核表达载体pcDNA3.0-EGFP-Sam68。     

人Gax基因真核表达载体的构建及在血管平滑肌细胞中的表达

目的:构建人Gax基因真核表达载体,并观察在兔血管平滑肌细胞中的表达。方法:通过PCR从pCMV-SPORT6-Gax质粒中扩增出人Gax cDNA片段,经双酶切后装入到有绿色荧光蛋白报告基因的真核表达载体pEGFP-N1中,经限制性内切酶酶切分析和DNA测序鉴定后通过梭华-Sofast转染试剂介导重组质粒转染至兔血管平滑肌细胞中进行表达,通过荧光显微镜观察转染细胞的绿色荧光蛋白表达和RT-PCR扩增转染细胞的cDNA来鉴定Gax在兔血管平滑肌细胞中的表达。结果:琼脂糖凝胶电泳检测PCR扩增产物人Gax基因片段约915bp,与预期分子量相符;酶切分析和测序鉴定证明人Gax真核表达载体pEGFP-N1-Gax连接正确;荧光显微镜观察到重组质粒转染细胞中有绿色荧光蛋白表达,及RT-PCR证明转染细胞有人GaxmRNA表达。结论:成功构建人Gax基因的重组真核表达载体pEGFP-N1-Gax,并证实在兔血管平滑肌细胞中表达,为进一步研究Gax基因在心血管病中的作用提供了实验基础。     

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.