体外培养细胞的显微注射

借助玻璃微吸管进行体细胞显微注射技术是由Graessmann(1968)和Liacumakos等(1970)发展起来。这种技术已成功地将DNA、mRNA、tRNA和蛋白质等显微注射进细胞内,并研究其生物效应。目前已经能将一个体外培养的体细胞作为“活试管”,通过显微注射技术,把DNA等生物大分子注射进细胞内来研究复杂的生物学现     

外周血树突状细胞的体外培养

本文用塑料贴壁的外周血单个核细胞(PBMNCs)在含自体血浆、rhGM-CSF、rhIL-4和TNFα的RPMI1640培养基,37℃,5%CO2湿化空气培养树突状细胞(DCs).经10天培养,可获得大量具DCs形态学特征的细胞,其有很强的刺激同种淋巴细胞增殖功能,约30%的细胞表达HLA-DR和CD1a.健康志愿者每1×107PBMNCs可收获细胞约5×105.本文培养方法产率高,培养体系简单,用自体血浆代替小牛血清,培养过程简便,这些均适应临床治疗要求.     

有的人在睡眠时为什么会"磨牙"?

问 :有的人在睡眠时为什么会“磨牙”?答 :夜间出现“磨牙”以青少年和儿童为多 ,主要原因有以下几方面 :1)睡前吃得过多。睡前若是吃得太饱 ,胃排空的时间相对较长 ,那么胃就在一定程度上一直处于兴奋状态 ,即便是已熟睡也是如此。所以胃壁感受器受到刺激会把兴奋传入中枢 ,进而引起三叉神经的兴奋 ,导致咀嚼肌的收缩 ,出现“磨牙”。2 )肠道寄生虫的影响。肠道内的某些寄生虫 ,比如蛔虫 ,是在夜间活动 ,在它们蠕动时或者是它们分泌的一些毒素 ,刺激了肠道壁内感受器 ,同样会引起中枢的兴奋 ,导致“磨牙”。3)胃肠疾病的影响。胃肠内的某些…     

鸡X期胚盘细胞体外培养

为证实经遗传修饰的鸡X期胚盘细胞具有参与受体胚胎发育和形成嵌合体的能力,本研究将由鸡X期胚盘制成的细胞悬液与经脂质体包埋的抗鸡传染性支气管炎病毒基因重组质粒PGS1共孵育后,直接显微注入同期受体胚盘（140枚）;或对转染后供体细胞进行G418抗性筛选性后显微注入同期受体鸡胚盘（140枚）;或将供体细胞体外培养48h,再与脂质体-P^GSI复合物共孵育后显微注入同期受体鸡胚盘（190枚）,制备转基因嵌合体鸡,并应用PCR和RAPD方法,对鸡胚和雏鸡不同组织或血液中的DNA进行检测。结果表明：直接注射组孵化率（5.7%)显著（P＜0.01)高于G418筛选处理组（1.4%)和培养48h处理组（2.1%);G418筛选处理组不同胚龄鸡胚组织、器官中外源DNA的PCR检测阳性率均高于其它二个组。实验结果证明,体外培养48h并经遗传修饰的胚盘细胞仍然具有形成嵌合体的能力,利用早期胚盘细胞途径制备转基因鸡是可行的。     

硬脑膜细胞体外培养模型的建立

目的：建立兔硬脑膜缺损的体外培养模型,观察不同细胞外基质对其愈合过程的影响,研究硬脑膜修复的机制,为以后检测硬脑膜移植物提供理论基础。方法：兔硬脑膜体外培养,再用免疫细胞化学方法和扫描电镜进行观察。结果：预先铺过胶原的培养板中培养8～10d后有细胞从硬脑膜缺损的边缘逸出、增殖,13～15d硬脑膜缺损愈合;而以多聚赖氨酸及层粘连蛋白铺底的培养板中没有看到细胞逸出的现象。硬脑膜细胞对波形蛋白抗体呈强阳性反应,对第Ⅶ因子抗体则为阴性反应;在电镜下可以看到新生胶原的生成。结论：成功建立一种硬脑膜缺损的体外培养模型;成纤维细胞从硬脑膜创缘的逸出、增殖是硬脑膜缺损愈合的重要机制。     

小鼠睾丸支持细胞体外培养特性

支持细胞是睾丸的重要组成部分,其主要功能是为生精细胞提供适宜的生长环境。从幼鼠睾丸中分离得到支持细胞,并通过苏木精-伊红和Fas-L免疫组化染色对分离得到的细胞进行了鉴定。通过对原代小鼠睾丸支持细胞的贴壁、生长和培养液中葡萄糖、谷氨酰胺、氨基酸等营养底物及其副产物乳酸、铵根离子等的代谢以及培养液渗透压和pH的研究,发现支持细胞的贴壁时间主要集中在接种后2～4h;当培养液中氨根离子浓度高于2.3mmol/L,渗透压高于326mosm/kg,pH≤6.8时支持细胞生长进入衰亡期;在氨基酸代谢方面,发现培养过程中丙氨酸和谷氨酸浓度迅速增加,缬氨酸、亮氨酸和异亮氨酸浓度略有降低,丝氨酸、精氨酸和甘氨酸浓度基本保持不变。因此培养液中铵根离子浓度的过量积累、渗透压和pH的异常和贴壁面积不足是限制支持细胞静态生长的主要因素。研究结果为支持细胞大规模培养及工艺优化奠定了基础。     

体外培养哺乳类细胞去核的研究

为了研究细胞核和质这两个主要部分的各自独立又相互协调的作用,人们常设想把细胞核从细胞质中分离出来,或者用再重新加以组合的方法来进行此项研究。这样就需要一种在生理条件下进行核质分离的技术。自从1939年Commandon等最先成功地进行去核和核移植实验以来,一直是倚仗显微操作术进行这样的工作。这种去核方法所能进行的工作是非常有限的。     

体外培养细胞的加力实验装置

机械力对细胞生物学行为的影响是目前细胞生物学和细胞力学研究的一个重要课题。体外分离细胞和加载培养技术是细胞力学的基础之一和目前细胞力学研究的重要方法。大致可分为离心力加载、流体加载、单细胞加载、压力传导加载和基底形变加载技术。     

鸡Ⅹ期胚盘细胞体外培养

为证实经遗传修饰的鸡X期胚盘细胞具有参与受体胚胎发育和形成嵌合体的能力 ,本研究将由鸡X期胚盘制成的细胞悬液与经脂质体包埋的抗鸡传染性支气管炎病毒基因重组质粒PGS1共孵育后 ,直接显微注入同期受体胚盘 (14 0枚 ) ;或对转染后供体细胞进行G418抗性筛选后显微注入同期受体鸡胚盘 (14 0枚 ) ;或将供体细胞体外培养 4 8h ,再与脂质体 PGS1复合物共孵育后显微注入同期受体鸡胚盘 (190枚 ) ,制备转基因嵌合体鸡 ,并应用PCR和RAPD方法 ,对鸡胚和雏鸡不同组织或血液中的DNA进行检测。结果表明 :直接注射组孵化率(5 7% )显著 (P <0 0 1)高于G418筛选处理组 (1 4 % )和培养 4 8h处理组 (2 1% ) ;G418筛选处理组不同胚龄鸡胚组织、器官中外源DNA的PCR检测阳性率均高于其它二个组。实验结果证明 ,体外培养 4 8h并经遗传修饰的胚盘细胞仍然具有形成嵌合体的能力 ,利用早期胚盘细胞途径制备转基因鸡是可行的。     

体外培养主动脉细胞钙化作用研究

目的：初步探讨主动脉钙化的机制。方法：外殖块法分离培养家兔主动脉平滑肌细胞,进行vonKossa染色以显示钙化,生化法检测细胞内外不溶性钙。放兔法测定培养上清骨钙素的含量。RT-PCR方法检测X型胶原mR-NA的表达。结果：25-羟基胆固醇（A组）和β-甘油磷酸盐（B组）分别培养的传代细胞均可见多个细胞结节,且细胞结节von kossa染色阳性;而对照组（C组）无细胞结节形成,von kossa染色阴性,前二者每孔不溶性钙沉积量,以及培养上清中骨钙素含量明显高于后者,且A、B两组X型胶原mRNA的表达为阳性。结论：25-羟基胆固醇、β-甘油磷酸盐均可促进主动脉中膜细胞发生钙化,主动脉中膜在钙化过程中与成骨细胞相似,骨钙素分泌增多且有X型胶原mRNA的表达。     

欢迎订阅2008年《生物学教学》杂志

北京市第22中学的生物学教师陈珊问：动物细胞培养时为什么会出现贴壁现象？ 答：第1,绝大部分细胞在机体内的环境下是与其他细胞或者细胞外基质结合的,不是孤立存在的。第2。细胞与细胞或者细胞外基质结合的物质基础以及在体外培养中贴壁的物质基础是什么。[第一段]     

Attachment of Agrobacteria to Grape Cells

The presence of the Ti plasmid favorably influences the attachment of agrobacteria to grape callus cells, especially during the early stages of a 2-h incubation. Agrobacterium strains attached to a similar extent to both the crown gall-resistant cultivar (Catawba), Vitis labruscana, and the crown gall-susceptible cultivar (Chancellor), Vitis sp. Attachment of the virulent strain to grape callus cells is blocked by the avirulent strain HLB-2 in both the tissue culture cell suspension and the seedling root systems.     

The Role of Glycosaminoglycan Binding of Staphylococci in Attachment to Eukaryotic Host Cells

Attachment of microorganisms to host cells is believed to be a critical early step in microbial pathogenesis. The aim of the study was to determine the role of the known glycosaminoglycan (GAG) binding activity of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in their attachment to six different eukaryotic cell lines. Three staphylococcal species expressing GAG binding capacity—S. aureus, S. epidermidis, and S. hemolyticus—were chosen for investigation. Six different eukaryotic cell lines, endothelial HUVEC and EA. hy 926 cells, epithelial A549 and HeLa S3 cells, fibroblasts HEL Sp 12 and macrophages J774.A1, were included. A modified ELISA with biotinylated bacteria was used for estimating the adhesion of staphylococci to each of the cell lines. Our results showed that staphylococci adhered to each of the cell lines studied, although the binding of CoNS strains to epithelial cells was lower than to the other cells. The attachment to all cell types could be partially decreased by pretreatment of the bacteria with various polysulfated agents (highest inhibition was 60%), as well as by chlorate and heparitinase treatment of the cells. These observations may suggest that at least one mode of staphylococcal attachment utilizes GAG chains present on the surface of virtually all adherent cells. Received: 6 September 2000 / Accepted: 29 December 2000     

Filoviruses Utilize Glycosaminoglycans for Their Attachment to Target Cells

Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelope glycoprotein (GP), responsible for both receptor binding and fusion of the virus envelope with the host cell membrane, has been demonstrated to interact with multiple molecules in order to enhance entry into host cells. Here we have demonstrated that filoviruses utilize glycosaminoglycans, and more specifically heparan sulfate proteoglycans, for their attachment to host cells. This interaction is mediated by GP and does not require the presence of the mucin domain. Both the degree of sulfation and the structure of the carbohydrate backbone play a role in the interaction with filovirus GPs. This new step of filovirus interaction with host cells can potentially be a new target for antiviral strategies. As such, we were able to inhibit filovirus GP-mediated infection using carrageenan, a broad-spectrum microbicide that mimics heparin, and also using the antiviral dendrimeric peptide SB105-A10, which interacts with heparan sulfate, antagonizing the binding of the virus to cells.     

Microbial Competition in Reactors with Wall Attachment

Competition for nutrient and the ability of bacteria to colonize the gut wall are factors believed to play a role in the observed stability of the indigenous microbiota of the mammalian large intestine. These factors were incorporated into the two-strain continuous-stirred tank reactor (CSTR) model formulated and numerically investigated by Freter et al. In their model simulations, the reactor is parameterized using data for the mouse intestine. An invading bacterial strain is introduced into a CSTR that has already been colonized by a resident strain. The two strains compete for a single growth-limiting nutrient and for limited adhesion sites on the wall of the reactor. The mathematical model described in this paper is motivated in part by the CSTR model, but is based on the plug flow reactor (PFR). Parameter values and initial conditions are chosen so that the numerical performance of the PFR can be compared to that of the CSTR. In simulations bearing a remarkable qualitative and quantitative resemblance to those of the CSTR it is found that the invader is virtually eliminated, despite the fact that it has uptake rate and affinity for the wall identical to those of the resident. The PFR model is then parametrized using data for the human large intestine, and the two-strain simulations are repeated. Though obvious quantitative differences are noted, the more important qualitative outcome is preserved. It is also found that when three strains compete for a single nutrient and for adhesion sites there exists a steady-state solution characterized by the segregation of the bacterial strains into separate nonoverlapping segments along the wall of the reactor.     

Conditions for the Attachment of Calystegia Cells to Solid Substrates

Mechanically isolated mesophyll cells of Calystegia sepium L.incubated in thin layers of non-agitated liquid media readilyattach to their substrate (culture vessel). Conditions for thisattachment of living plant cells are: a medium consisting ofa low concentration of at least one macroelement and one sugar,a pH value between 5 and 7, and a substrate with a hydrophiliccharacter (e.g. glass, polystyrene). Calystegia sepium, mesophyll cells, cell attachment     

The Use of Double Staining Techniques for Investigating Bacterial Attachment to Mucopolysaccharide-Coated Epithelial Cells

Double staining techniques were devised to study Escherichia coli attachment to mucopolysaccharide-coated urinary tract epithelial cells. In addition, vital stains were used to distinguish between viable and nonviable epithelial cells. Alcian blue was used to confirm the presence of glycosaminoglycans and periodic acid Schiff was used to distinguish a further group of polysaccharides, proteoglycans, neutral mucosubstances and glycolipids. The staining methods enabled investigations to be carried out concerning the possible importance of mucopolysaccharides in the attachment of bacteria to mucosal surfaces. Staining techniques were also used to investigate whether the presence of a mucopolysaccharide coat is related to epithelial cell viability. The combinations of vital and mucopolysaccharide stains were found to give reproducible results when cell preparations were evaluated by three individuals. Both in vivo and in vitro certain populations of epithelial cells have been found with a large number of bacteria preferentially attached. The double staining techniques described here may help to reveal the nature of these target cells.     

层粘连蛋白糖肽对实体型瘤细胞粘着于层粘连蛋白基质的影响

 本文报告由小鼠EHS瘤提取的层粘连蛋白(Laminin,LN)经链霉蛋白酶(Pronase)消化,再经Sephadex-G50层析分离得到LN总糖肽。它可显著抑制黑色素瘤细胞B16-MBK及S180肉瘤细胞与LN基质的识别及粘着,并具有明显剂量依赖性。与五肽(YIGSR),卵清蛋白及其糖肽,胎球蛋白及其糖肽比较,LN总糖肽的抑制效果显著高于YIGSR及胎球蛋白糖肽,而其它三者均无抑制作用。因而提示:LN分子中一定结构的糖链特异地参与了LN对肿瘤细胞表面LN受体的识别与结合。     

Selective Attachment of Oligonucleotides to Interleukin-1β and Targeted Delivery to Cells

Abstract

Conjugates between oligodeoxyribonucleotides and an interleukin-1β mutant protein have been constructed using a heterobifunctional cross-linker. These protein-DNA conjugates had conserved binding activity to the interleukin-1 receptor. The oligonucleotide hybridization properties were unchanged.     

牛胎儿成纤维细胞的组织块贴附法分离培养与电穿孔法基因转染

为获得转基因克隆牛的供体细胞,采用组织块贴附培养的方法分离培养牛胎儿皮肤成纤维细胞,经2～3次传代纯化,绘制生长曲线,分别分析体外传代培养10代以内和20代以上细胞的核型特征。分别采用800、900、1000V／cm和1、5、10、15和20ms的参数组合,将线性化的带有新霉素抗性和绿色荧光蛋白双重筛选标记的人胰岛素原乳腺特异表达载体pNEI电穿孔转入体外培养的牛胎儿成纤维细胞,经800μg／mLG418筛选2周,继续以300μg／mLG418扩大培养2～3代,取部分筛选后的细胞进行PCR检测结果表明,体外培养的牛胎儿成纤维细胞生长旺盛,体外传代20次后核型未发生改变;转染后24～48h在荧光镜下检测各组均可观察到绿色荧光表达,筛选后各组克隆形成数以900V／cm和5ms组最多;PCR检测得到了预期条带,说明目的基因已经成功导入。分离得到的牛胎儿耳成纤维细胞有可能作为体细胞核移植的供体,进行转基因克隆研究。