MCF-7细胞瞬时基因表达转染模式的建立

目的　建立一种高效、简便、低毒、价廉的瞬时基因表达细胞转染模式。　方法　选择人乳腺癌细胞 系MCF 7,采用Polyethylenimine(PEI)作为转染剂,对细胞密度、载体DNA量、PEI氮与DNA磷的比率(PEI N∶DNA P) 以及PEI DNA混合物与细胞共存的无血清培养时间等对转染效率的影响进行了比较分析。　结果　转染时的24 孔板每孔接种的细胞以2×105为宜,PEI N∶DNA P的最优比率不是固定的,它与转染时DNA的量有关,每孔转染1 μgDNA时其最优PEI N∶DNA P比率为33∶1左右,而每孔转染4μgDNA时其最优PEI N∶DNA P比率为9∶1左右。 另外,PEI DNA混合物与细胞共存的无血清培养时间至少需要3h,最佳时间为5～7h。　结论　利用转染剂PEI, 控制合适的细胞密度、DNA质量、PEI N∶DNA P比率和无血清培养的时间,可成为一种高效、简便、低毒、价廉的瞬时 基因表达的细胞转染模式。     

新型非病毒载体聚乙烯亚胺介导基因转染参数的研究

聚乙烯亚胺是一种新型阳离子多聚物基因释放载体,可结合浓缩DNA,通过粘附内吞,进入细胞,使携带的质粒表达。本研究目的通过对聚乙烯亚胺各种转染参数的测定,为合成以聚乙烯亚胺为骨架的人工载体积累数据。方法:本研究利用聚乙烯亚胺分别结合含β半乳糖甙酶报告基因的pSVβ表达质粒、含绿色荧光蛋白报告基因的pEGFP质粒转染Cos-7细胞,通过组织化学法测定细胞抽提产物中β半乳糖甙酶的表达量、流式细胞仪法测定绿色荧光蛋白阳性细胞的表达比例,来测定影响转基因效率的各种参数。结果:在培养液中,6μg/ml聚乙烯亚胺作用24h,NIH 3T3细胞生存率为64.2%,7μg/ml聚乙烯亚胺细胞生存率为54.4%。电泳阻滞试验,聚乙烯亚胺在N/P比在3.0以上方可完全结合DNA。溶酶体抑制剂氯喹可增加聚乙烯亚胺的转染效率。培养液中的白蛋白、血清可降低转染效率。作为配制聚乙烯亚胺/DNA复合物的溶媒,HEPES缓冲液优于生理盐水,生理盐水优于5%葡萄糖。配制聚乙烯亚胺/DNA复合物的溶媒中加入Mg2+降低转染效率。聚乙烯亚胺转染效率优于SuperFectTM(断裂型树突状多聚物),而毒性低于SuperFectTM。结论:本研究首次报道了聚乙烯亚胺与DNA结合配伍的N/P比计算公式,N/P=7.75×b/c,这里b是PEI的质量(μg),c是质粒的质量(μg);PEI的工作终浓度应≤6μg/ml。通过体外细胞试验证明,聚乙烯亚胺是一种有效的真核细胞转染剂和人工合成基因载体的骨架。     

聚乙烯亚胺介导三质粒共转染HEK293细胞的条件优化

本研究用聚乙烯亚胺(PEI)介导表达绿色荧光蛋白(GFP)的rAAV包装系统(pAAV-CMV-GFP、pAAVRC和pHelper)转染人胚肾293细胞(HEK293细胞)。用ImagePro Plus6.0图像分析软件计算表达GFP的细胞比例来测定1、2、3、4、5、6μg/孔不同质粒剂量,1∶1、3∶1、5∶1、7∶1不同PEI/质粒比值及转染12、24、36、48、60、72h不同时间三个变量对转染效率的影响,以优化PEI介导三质粒共转染HEK293细胞的条件。结果显示,质粒剂量为4μg/孔、PEI/质粒比值为3∶1~5∶1时转染效率维持较高水平;转染效率-转染时间曲线为S型增长曲线,转染效率于60h达到平台期。优化PEI转染条件能增加三质粒共转染HEK293细胞的转染效率。     

磁转联合脂质体实现高效细胞转染

目的优化体外培养细胞的外源基因转染方案。方法采用磁性微粒联合脂质体，将重组人绿色荧光蛋白表达载体（pAAV-IRES-GFP）外源DNA定向转染COS-7细胞，荧光显微镜下观察外源基因的表达，比较不同转染条件及DNA载量的细胞转染效果。结果DNA与Combimag按1：1的比例混合转染可以得到很高的转染效率，过高的DNA载量使转染效率减低。结论利用磁转结合脂质体可使低剂量的DNA达到快速、简便和高水平的转染。     

N-亚甲基磷酸化壳聚糖基因纳米粒子的体外细胞毒性及基因转染实验研究

目的 考察N-亚甲基磷酸化壳聚糖( NMPCS)基因纳米粒子的体外细胞毒性及基因转染效率.方法 采用均相反应法制备了NMPCS,用复凝聚法制备了NMPCS/DNA纳米粒子;通过MTT实验考察了NMPCS及其与DNA复合物对HeLa细胞的细胞毒性,以荧光索酶质粒为报告基因考察了NMPCS及NMPCS-CaZ+载体介导的体外基因转染效率.结果 NMPCS及其与DNA的复合物在体外表现出很小的细胞毒性,远远低于同等浓度时聚乙烯亚胺(PEI)的毒性.通过对壳聚糖进行亚甲基磷酸化修饰后,可大幅提高载体的基因转染效率.结论 N-亚甲基磷酸化壳聚糖有望成为一种新型、安全、高效的非病毒基因载体.     

肝靶向DNA载体乳糖化多聚乙烯基亚胺及体外转染试验

目的 研制肝靶向的DNA载体,用于干扰素的肝细胞内表达或抗病毒反义核酸的肝靶问给药。方法 以还原胺化法制备了肝靶向基因载体乳糖化多聚乙烯基亚胺（L－PEI）,并进行了体外转染试验和稳定性试验。结果 靶向载体L－PEI与DNA形成的复合物,对受体阳性的Huh－7细胞具有较高的转染效率;靶向载体乳糖化白蛋白能够竞争拮抗这种转染。靶向载体／DNA复合物在大鼠血清中具有较好的稳定性。结论 乳糖化多聚乙烯基亚胺能将DNA选择的投放于肝细胞,具有较好的应用前景。     

聚乙烯亚胺用作基因转染的研究进展

 基因载体问题以及与载体相关的免疫反应、细胞毒性和安全性等问题，是基因治疗领域亟待解决的关键问题之一。聚乙烯亚胺（PEI）是阳离子聚合物非病毒载体的典型代表^[1]，是一种很早便为人所知并予以应用的有机大分子。目前，以 PEI 阳离子聚合物与 DNA 形成的 PEI/DNA 复合物已成为非病毒基因载体的研究热点。本文就近年来这方面的研究进展作简要综述。 1 PEI的特性 PEI 每 3 个原子中有 1 个胺基原子，使其具有较高正电荷密度。根据 pH 与质子作用之间的对应关系可得出：自由 PEI 的结构在生理条件下有 1/6 至 1/5 胺基发生质子化反应，从而使溶酶体肿胀破裂，从而起到“质子海绵”作用，使 PEI/DNA 复合物得以释放入胞质，很大程度上减少了 DNA 在吞噬泡内富集并进而被降解的作用，因而可以提高转染效率^[2]。     

流式细胞仪分选非染色S期CHO细胞有助于提高聚乙烯亚胺的转染效率

目的利用流式细胞仪(flow cytometry,FCM)分选获得非染色、同步化的中国仓鼠卵巢(Chinese hamsterovary,CHO)细胞,观察聚乙烯亚胺(polyethylenimine,PEI)在不同周期CHO细胞的瞬时转染和稳定转染效率。方法悬浮培养CHODG44细胞,应用流式细胞仪的光学参数-前向角和侧向角,在细胞主群中设置12个门,然后进行分选。分选出的各门内细胞经碘化丙啶(PI)染色进行细胞周期检测。以PEI为载体分别向含G0/G1、S、G2/M比例较高的细胞亚群和未经分选的细胞转染含绿荧光蛋白(Green Fluorescence Protein,GFP)质粒,利用流式细胞仪检测瞬时转染、稳定转染效率。结果细胞DNA含量与侧向角相关,各细胞亚群G0/G1期细胞所占比例总体上随着侧向角的增大而减小,G2/M细胞所占比例随着侧向角的增大而增多。低侧向角的细胞亚群P2内G0/G1期细胞比例可达80.49%,中侧向角的细胞亚群P7内S期细胞比例可达61.14%,高侧向角的细胞亚群P13内G2/M期细胞比例可达49.88%。转染48h,GFP阳性细胞在G2/M期细胞比例较高的细胞亚群所占比例最高,但瞬时转染效率,S期细胞比例较高的细胞群大于G2/M期细胞比例较高的细胞群。转染2w,稳定转染效率仍为S期细胞比例较高的细胞亚群大于其它细胞亚群及未分选的混合细胞群。结论应用流式细胞仪的光学参数可以有效分选非诱导、无染色分别含有较高比例各周期时相的CHO细胞;PEI的转染效率与转染时细胞所处的细胞周期时相相关,且S期细胞亚群的瞬时和稳定转染效率均高于其它细胞周期亚群。     

PSMA-CAR慢病毒表达载体的包装研究

目的前列腺特异性膜抗原-嵌合抗原受体(PSMA-CAR)慢病毒表达载体的包装方法研究。方法采用慢病毒三质粒病毒包装系统(Del8.9+VSVG、PsAX2+OG)及包装细胞(HEK-293T、HEK-293FT)在转染试剂[聚乙烯亚胺(PEI)、Lipofectamine 2000、磷酸钙]的介导下,包装慢病毒颗粒。通过检测转染效率筛选最佳包装细胞、最佳包装质粒组合、最佳转染试剂和剂量。结果 HEK-293FT细胞的转染效率高于HEK-293T细胞的转染效率。PsAX2+OG组合的转染效率高于Del8.9+VSVG组合的转染效率。PEI方法转染时,包装质粒相同,空载体的转染效率明显要高很多;目的质粒相同,采用包装质粒为PsAX2+OG组合时,转染效率更高;数据统计表明,质粒总量∶PEI的比例在1∶9时转染效率更高。Lipofectamine 2000方法转染时,包装质粒和包膜质粒相同,空载体的转染效率更高;目的质粒和包装、包膜质粒相同,质粒总量∶Lipofectamine 2000的比例在1.0∶2.0和1.0∶2.5时转染效率显著高于其他两组比例。磷酸钙转染法时,随着加入的磷酸钙的增多,转染效率呈递减趋势。结论采用包装质粒PsAX2和包膜质粒OG时转染效率相对更高。质粒总量和加入的PEI量比例在1∶9时转染效率更高;质粒总量和加入的Lipofectamine 2000量比例在1.0∶2.0和1.0∶2.5时转染效率更高。PEI包装法和Lipofectamine 2000包装法转染效率相当,Lipofectamine 2000包装法相对高一些,但在考虑包装成本的前提下后续大量包装PSMA-CAR慢病毒颗粒时,应选择PEI包装法。     

脂质体介导转染N2a细胞的优化方法

背景:阳离子脂质体介导的细胞转染具有结果可靠、可重复性强的特点,但面临一个共同的缺点,就是转染效率常较低。目的:探讨阳离子脂质体转染N2a细胞(小鼠神经胶质瘤细胞)的优化方法。方法:采用24孔培养板,1.5μL阳离子脂质体Lipofectamine?LTX介导,通过贴壁法和悬浮法,将500 ng带有绿色荧光蛋白(GFP)基因的重组质粒pcDNA3-GFP转入N2a细胞,倒置荧光显微镜观察细胞内绿色荧光蛋白表达情况,并比较二者转染效率。采用悬浮转染法,将500 ng质粒DNA分别用1.0,1.5,2.0,2.5μL Lipofectamine?LTX进行转染,探讨最适脂质体和DNA比例。结果与结论:1.5μL脂质体/500 ng DNA,悬浮法转染效率显著高于贴壁法转染效率(P0.01);1.0,1.5,2.0,2.5μL Lipofectamine?LTX对500 ng质粒DNA的转染效率分别为(76.60±3.85)%,(80.00±4.17)%,(88.00±5.89)%,(54.96±4.23)%,提示脂质体2.0μL与质粒DNA 500 ng的转染效率最高。     

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.     

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.