人胸腺上皮细胞培养及其分泌产物

本文通过降低培养基中血清含量,向RPMI 1640培养基中补加三碘甲腺原氨酸而获得一种人胸腺网状上皮细胞占优势生长的培养物。在此培养基中细胞经传代培养长达90天,仍维持正常形态特征。胸腺组织在培养14天后,新生细胞的突起形成网状结构,细胞化学检查和电镜观察表明具有丰富的分泌颗粒,囊泡及张力原纤维束和桥粒等上皮细胞特征。收集合并细胞培养液,经部分纯化后检查其生物活性,表现出具有促进玫瑰花结形成和降低胸腺细胞TdT活性的作用,说明培养细胞的分泌产物具有胸腺激素活性。根据形态学,细胞化学和生物活性检测结果,我们倾向于认为该培养物主要为网状上皮细胞。     

Chorologische phänomene in Wasserpflanzengesellschaften Mitteleuropas

On the basis of three selected examples chorological phenomena in waterplant communities of central, east and southern Europe are represented. Species of the Atlantic-oceanic floral element of the order Juncetalia bulbosi, like Pilularia globulifera, Eleocharis multicaulis and Deschampsia setacea are growing in markedly secondary sites near their eastern border, i.e. in the Lusatian district of central Europe, especially in sand, gravel and clay pits as well as in residue lakes of opencast mines. As a result of intensive recent human activities numerous new sites are being established now causing an expansion of their distribution area. With decreasing oceanity from the central part to the eastern border of the area a change in the floristic-sociological structure of the relevant plant communities and in the hydrochemical composition of the respective water bodies takes place. In western Europe the species grow in soft water poor in minerals, in the eastern part (central Europe) in mineral-rich, hard water with calcium and sulphate.At present Ceratophyllum submersum has a high frequency in the northeastern area in Mecklenburg. The sites are morainic lakes (kettle holes) and small waters and ponds near settlements, which are enriched in minerals and nitrales in consequence of the intensive agricultural use of inorganic fertilizers. Moreover the waters are alkaline and rich in minerals, especially bicarbonate and possess a high range of total hardness. The example of eight different sites of Aldrovanda vesiculosa illustrates a striking change of its diagnostic value in its disjunct north-south distribution. In the northern part of central Europe Aldrovanda is found in the communities of the Utricularietea intermedio-minoris, both in waters with a low acidity rich in sulphate and in alkaline waters rich in bicarbonate (Sphagno-Utricularion and Scorpidio-Utricularion respectively). In southern central Europe and in southeastern Europe Aldrovanda occurs in Lemnetea and Potametea communities. In northeastern Poland Aldrovanda is found together with Hydrilla verticillata, Chara mentosa, Myriophyllum verticillatum and Scorpidium scorpioides.

Die Sippennomenklatur richtet sich nach Rothmaler et al. (1970).     

钙调素抑制剂——三氟拉嗪对肿瘤细胞增殖和微管组装的影响

本文用钙调素抑制剂——三氟拉嗪处理人胃癌MGC-803细胞,用免疫荧光细胞化学方法,放射免疫法和速流荧光分析等方法研究了钙调素对细胞增殖,环核苷酸代谢及微管组装,有丝分裂等细胞功能的调节作用。实验结果表明,TFP明显地抑制了人胃癌细胞的增殖,这种抑制增殖的作用,具有剂量和时间依赖关系,细胞群体中G_1期细胞增多,S期细胞下降,DNA合成明显地受到抑制。TFP处理的胃癌细胞仅在短时间内(5'-30')cAMP含量升高,cGMP浓度降低,cAMP/ ??cGMP比值比对照组高4.4倍,但此后环核苷酸含量又很快恢复到对照组水平。本实验还观察到TFP处理后的MGC-803细胞胞质铺展,细胞形态的改变与胞质微管的分布有密切联系,实验结果表明TFP加强了人胃癌细胞MTOC对微管的组装能力,使微管分布得到恢复,微管纤维呈放射状延伸到细胞边缘,充满胞浆,使细胞呈现出展平的多边形,趋向于正常上皮细胞形态的变化,本实验结果表明TFP抑制癌细胞增殖及使微管组装加强可能是通过对CaM活性的抑制作用。此结果有助于说明转化细胞内钙调素的变化,可能是与转化细胞增殖失控和胞质微管消退有关。     

人骨髓基质细胞的长期培养及其对病毒的敏感性

为了长期培养骨髓基质细胞和研究其对病毒的敏感性,我们采用静置贴壁培养法,体外长期培养了胎儿、儿童和成人骨髓基质细胞,并将传至５代以上的肌样骨髓基质细胞采用微量细胞病变（ＣＰＥ）法,开展了对５种病毒的敏感性试验。结果表明,人骨髓基质肌样细胞对滤泡性口腔炎病毒,脊髓灰质炎病毒、Ⅰ型和Ⅱ型单纯疱疹病毒均敏感,能产生明显的ＣＰＥ,其效价（ＴＣＩＤ５０）可达１０＾－３～１０＾－４,其中胎儿骨髓基质肌样细胞对     

利用体细胞核移植技术制作人胰岛素原转基因牛 （英文）

通过体细胞核移植技术制作了人胰岛素原转基因牛。在CMV启动子指导下以内部核糖体进入位点序列(IRES)连接的新霉素抗性基因和绿色荧光蛋白基因组成了双重标记基因的筛选系统,用于转基因细胞的富集以及细胞和植入前胚胎的筛选。转基因通过电穿孔的方法(900V/cm,5ms)转入体外培养的牛胎儿成纤维细胞,基因转染细胞在添加G418 (800μg/mL)的培养基中培养10天以富集转基因细胞。选择表达绿色荧光蛋白的转基因细胞作为核供体进行体细胞核移植,重构胚经体外培养至囊胚阶段,选择表达绿色荧光蛋白的囊胚进行胚胎移植。为比较基因转染以及供体细胞所处周期对转基因细胞核移植胚胎发育的影响,用作核移植供体的转基因细胞或非转基因细胞先饥饿培养2—4天(0.5 ?S) ,然后恢复培养(10?S) 10 h使细胞同步化于G1期,以正常培养的细胞作为对照进行核移植。结果表明,转基因细胞作为核供体得到的核移植胚胎的体外囊胚发育率低于以非转基因细胞为核供体的对照组(23.2% VS 35.2 %,P<0.05) ;转基因细胞周期同步化处理与否对其克隆胚囊胚发育率无显著影响(23.2% VS 18.9 %,P>0.05)。胚胎移植后2个月直肠检查发现7头受体牛(每头移植2—4枚胚胎)中有一头妊娠,并最终发育足月产下一头小牛。聚合酶链反应(PCR)检测和DNA测序分析表明其为转人胰岛素原基因的转基因克隆牛。     

艾滋病病毒蛋白酶高效表达载体及体外活性检测系统的构建

艾滋病是本世纪80年代初发现的一种烈性传染病,5年病死率为100％,致病因子为人免疫缺陷病毒,该病毒的蛋白酶在病毒复制和成熟中具有决定性的意义。由于目前国内外尚未获得艾滋病病毒蛋白酶的高效表达的重组子及活性检测系统,限制了它的研究与应用。本文利用PCR技术修饰了艾滋病病毒蛋白酶的基因,使其具有便于克隆及表达用的限制酶切位点及转录终止码,井在其C末端设置了一个可用于检验该酶活性的特殊序列。DNA序列分析揭示上述突变策略成功,将修饰后的艾滋病病毒蛋白酶基因克隆入大肠杆菌表达系统,并获得高效表达(>30％),Western-Bolt鉴定结果表明所表达的蛋白为艾滋病病毒所特有,并具有较好的生物活性。     

Next generation organoids for biomedical research and applications

Organoids are in vitro cultures of miniature fetal or adult organ-like structures. Their potentials for use in tissue and organ replacement, disease modeling, toxicology studies, and drug discovery are tremendous. Currently, major challenges facing human organoid technology include (i) improving the range of cellular heterogeneity for a particular organoid system, (ii) mimicking the native micro- and matrix-environment encountered by cells within organoids, and (iii) developing robust protocols for the in vitro maturation of organoids that remain mostly fetal-like in cultures. To tackle these challenges, we advocate the principle of reverse engineering that replicates the inner workings of in vivo systems with the goal of achieving functionality and maturation of the resulting organoid structures with the input of minimal intrinsic (cellular) and environmental (matrix and niche) constituents. Here, we present an overview of organoid technology development in several systems that employ cell materials derived from fetal and adult tissues and pluripotent stem cell cultures. We focus on key studies that exploit the self-organizing property of embryonic progenitors and the role of designer matrices and cell-free scaffolds in assisting organoid formation. We further explore the relationship between adult stem cells, niche factors, and other current developments that aim to enhance robust organoid maturation. From these works, we propose a standardized pipeline for the development of future protocols that would help generate more physiologically relevant human organoids for various biomedical applications.     

A DNA Vaccine Encoding a Codon-Optimized Human Papillomavirus Type 16 E6 Gene Enhances CTL Response and Anti-tumor Activity

Summary The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8⁺ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8⁺ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.     

人结核分枝杆菌特异性核酸探针制备及结核病PCR检测技术的初步临床应用

采用人结核分枝杆菌(Mycobacterium tuberculosis TB)染色体DNA为模板,选择位于插入片段IS6110中884～865和568～588碱基对处的两个片段为引物,扩增出317bp的特异性片段．将其克隆进pUCl9载体。酶切图谱分析和DNA序列测定证实为目的片段。该片段经DIG标记,分别与11种分枝杆菌DNA进行Southern杂交,结果证明只与人型复合分枝杆菌发生杂交反应。利用该对引物建立的PcR检测拄术对74份结核病痰液标本进行检测,并与临床细菌快速培养结果相比较,发现48份临床阳性均为PcR阳性,在26份临床阴性标本中亦发现11份PCR检测阳性。将标本PCR产物与克隆探针进行杂交,显示两者结果完全一致。说明PCR检测体系结果可靠,其灵敏度明显高于目前临床所采用的方法,可作为一种常规技术用于结核病的临床检测。     

The genetics of complex diseases

Genetic factors influence virtually every human disorder, determining disease susceptibility or resistance and interactions with environmental factors. Our recent successes in the genetic mapping and identification of the molecular basis of mendelian traits have been remarkable. Now, attention is rapidly shifting to more-complex, and more-prevalent, genetic disorders and traits that involve multiple genes and environmental effects, such as cardiovascular disease, diabetes, rheumatoid arthritis and schizophrenia. Rather than being due to specific and relatively rare mutations, complex diseases and traits result principally from genetic variation that is relatively common in the general population. Unfortunately, despite extensive efforts by many groups, only a few genetic regions and genes involved in complex diseases have been identified. Completion of the human genome sequence will be a seminal accomplishment, but it will not provide an immediate solution to the genetics of complex traits.