磁激活细胞富集孕妇外周血中胎儿有核红细胞行产前诊断

目的：研究磁激活细胞分离富集孕妇外周血胎儿有核红细胞进行产前诊断的可行性。方法：２１名孕１３－２９周孕妇及５名未妊娠妇女外周血４－６ｍｌ,经单密度梯度离心和激活细胞分离,收集阳性组分抽提ＤＮＡ,经ＰＣＲ扩增预测胎儿性别,并以出生后婴儿性别验证预测性别的准确性。     

Exploiting T cell receptor genes for cancer immunotherapy

Adoptive antigen-specific immunotherapy is an attractive concept for the treatment of cancer because it does not require immunocompetence of patients, and the specificity of transferred lymphocytes can be targeted against tumour-associated antigens that are poorly immunogenic and thus fail to effectively trigger autologous T cell responses. As the isolation and in vitro expansion of antigen-specific lymphocytes is difficult, 'conventional' adoptive T cell therapy can only be carried out in specialized centres in small numbers of patients. However, T cell receptor (TCR) genes isolated from antigen-specific T cells can be exploited as generic therapeutic molecules for 'unconventional' antigen-specific immunotherapy. Retroviral TCR gene transfer into patient T cells can readily produce populations of antigen-specific lymphocytes after a single round of polyclonal T cell stimulation. TCR gene modified lymphocytes are functionally competent in vitro, and can have therapeutic efficacy in murine models in vivo. TCR gene expression is stable and modified lymphocytes can develop into memory T cells. Introduction of TCR genes into CD8(+) and CD4(+) lymphocytes provides an opportunity to use the same TCR specificity to produce antigen-specific killer and helper T lymphocytes. Thus, TCR gene therapy provides an attractive strategy to develop antigen-specific immunotherapy with autologous lymphocytes as a generic treatment option.     

Magnetic labeling and cell sorting

Magnetic labeling by a magnetic-antibody conjugate has been combined with magnetic filtration (high gradient magnetic separation) to effect a rapid and efficient separation of a selected cell population from a suspension of single cells. Samples of more than 10⁸ cells could be fractionated in about 5 min with complete recovery. The system has been applied to a model system using red blood cells (sheep or chicken) and commercial antibodies against species-determined cell surface antigens. Enrichments of labeled cells by factors of up to 37-fold were observed. The approach was relatively insensitive to details in the experimental protocol and the number of unlabeled cells which were in the sample. Thus, the method was easy to use and can readily be scaled up to handle large samples containing 10⁸ labeled cells in a total of 10¹¹ or more. It should be useful as a pre-enrichment scheme for suspensions in which cells of interest are rare and, consequently, very inefficiently sorted by fluorescence-activated instruments.     

Single cell sorting and cloning

Cell sorters now allow the selection of cells and other bodies according to a range of quite diverse criteria. The additional refinement that allows the sorting of individual cells based on these criteria has seen application in many fields of research. Single cells may be sorted for microscopy, for culture and for genetic analysis by way of single cell PCR (polymerase chain reaction). In practical terms, in the setting up of an instrument for single cell sorting, there are additional requirements to ensure that each detected event is indeed a single cell or body, that this cell can be reliably sorted via saline droplet, separate from its fellow travelers, that the aiming of the droplet deflection is sufficiently precise to find the target vessel and that the cell will be undamaged on arrival. Among the diverse reported applications of the technique, two fields which have benefited greatly are lymphocyte development and haemopoiesis. In the former case, the analysis of gene rearrangements in lymphocytes, both in the pre- and post-antigenic phases of development, has been enabled by the combined technologies of single cell sorting and PCR. It is argued that such experiments could not have been done without that partnership. In a similar way, the single cell sorting technique has been found to be the perfect way to demonstrate precursor/progeny relationships between haemopoietic cells and, further, to demonstrate rigorously the effects of particular cytokines on the haemopoietic system.     

Magnetic cell sorting using colloidal protein-magnetite

Magnetic filtration of labelled cells as a way of separating leucocyte subpopulations was tested with a very simple and easy filtration device, using colloidal magnetite as the labelling reagent. In order to quantitate cell enrichment, a double label (both fluorescent and magnetic) was used, under conditions which labelled less than 10% of the cells in the initial sample. Up to 20 million cells were simply passed through a small magnetic filter with a hand-held syringe. Depletion of labelled cells in the suspension that passed through was threefold, and enrichment of labelled cells in the wash of the filter after its removal from the magnet was approximately fivefold. Factors which limited the quality of separation are discussed. Other, more preliminary, experiments found enrichments of 15- to 30-fold with the same colloidal magnetite and hand-held apparatus when the cell labelling system was more selective.     

血红细胞在渗透实验中的变形分析

目的数值模拟血红细胞在渗透实验中的变形具体全过程并寻找渗透压临界值。方法建立细胞的壳体模型,采用Neo-Hookean应变能形式的超弹性本构关系,采用文献提供的细胞膜剪切模量实测数据,使用有限元软件ABAQUS进行计算。结果得到了在血红细胞渗透实验中细胞膜受渗透压作用而逐渐变形的全过程,发现当渗透压增加到50～60 mPa时,细胞膜由双凹形迅速变成椭球形。该渗透压临界值落在文献给出的计算结果范围内。同时得到了在该临界值两侧细胞膜的Mises应力分布图。结论血红细胞由双凹形变成椭球形的临界渗透压为50～60 mPa。采用壳体模型以及Neo-Hookean形式的超弹性材料本构关系进行血红细胞渗透实验的数值模拟,可以较好地展示血红细胞在渗透实验中的变形全过程。     

The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients

BACKGROUND: Before clinical application, the feasibility and safety of autologous testicular stem cell transplantation should be explored. Apart from limitations in their numbers, spermatogonial stem cells may also be contaminated by malignant cells. Therefore, both enrichment and decontamination before transplantation may be necessary. This study aimed at evaluating the decontaminating potential of magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) for both murine and human testicular cell suspensions. In the mouse, the effectiveness of the transplantation technique after cell sorting was also assessed. METHODS: Murine testicular cells were contaminated with 5% EL4 cells. Fresh and frozen-thawed suspensions were sorted using MACS (CD49f (+)) and FACS (CD49f(+), H-2Kb(-)) and evaluated by FACS, cell culture and transplantation into W/W(v) mice. Human testicular cells were contaminated with 5 or 0.05% CCRF-SB (SB) cells. Frozen-thawed suspensions were sorted using FACS (HLA class I(-)) and evaluated by FACS, cell culture and PCR for the B-cell receptor. RESULTS: In the mouse, the sorted fractions contained 0.39% H-2K(b)-positive and 76.55% CD49f-positive cells. After transplantation, 1 in 20 recipient mice developed a malignancy. In the human experiments, an average of 0.58% SB cells was detected after sorting. In only 1 of 11 samples, there were no SB cells observed. CONCLUSION: MACS and/or FACS are insufficient for completely depleting testicular tissue of malignant cells. Although more research on alternative decontamination techniques is necessary, developing a reliable method to screen a priori testicular tissue for malignant cells may be equally important.     

利用磁性细胞分选技术阴性选择法分离外周血嗜酸性粒细胞

目的 :从健康献血者和哮喘等变态反应性疾病病人的外周血中分离出高纯度、高存活率的嗜酸性粒细胞。方法 :采用磁性细胞分选技术的阴性选择法分离外周血嗜酸性粒细胞 ,并检测分离后嗜酸性粒细胞的纯度、存活率 ,计算其回收率。结果 :①健康献血者外周血嗜酸性粒细胞的回收率为 6 9.7%± 6 .8% ,存活率 95 .4%± 1.8% ,纯度为 90 .7%± 4.3% ;②哮喘等变态反应性疾病病人外周血嗜酸性粒细胞的回收率为 6 0 .4%± 5 .4% ,存活率为 96 .1%± 0 .7% ,纯度为 92 .6 %± 5 .1%。结论 :采用磁性细胞分选技术的阴性选择法 ,能从外周血中分离出高纯度、高存活率的嗜酸性粒细胞 ,因此该方法是一种高效的分离外周血嗜酸性粒细胞的免疫学方法。     

Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations

Neural cells differentiated in vitro from human embryonic stem cells (hESC) exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. Here, we describe standard conditions for the use and discovery of markers for analysis and cell selection of hESC undergoing neuronal differentiation. To generate better-defined cell populations, we established a working protocol for sorting heterogeneous hESC-derived neural cell populations by fluorescence-activated cell sorting (FACS). Using genetically labeled synapsin-green fluorescent protein-positive hESC-derived neurons as a proof of principle, we enriched viable differentiated neurons by FACS. Cell sorting methodology using surface markers was developed, and a comprehensive profiling of surface antigens was obtained for immature embryonic stem cell types (such as stage-specific embryonic antigen [SSEA]-3, -4, TRA-1-81, TRA-1-60), neural stem and precursor cells (such as CD133, SSEA-1 [CD15], A2B5, forebrain surface embryonic antigen-1, CD29, CD146, p75 [CD271]), and differentiated neurons (such as CD24 or neural cell adhesion molecule [NCAM; CD56]). At later stages of neural differentiation, NCAM (CD56) was used to isolate hESC-derived neurons by FACS. Such FACS-sorted hESC-derived neurons survived in vivo after transplantation into rodent brain. These results and concepts provide (a) a feasible approach for experimental cell sorting of differentiated neurons, (b) an initial survey of surface antigens present during neural differentiation of hESC, and (c) a framework for developing cell selection strategies for neural cell-based therapies.     

Purification of cybrids by fluorescence-activated cell sorting

A general method to isolate and purify substantial numbers of viable cybrids from cultured mammalian cells immediately following cytoplast-cell fusion is described. This method uses cytoplasts whose mitochondria are selectively stained in vivo by the cationic fluorescent rhodamine dye, rhodamine 123. Large numbers of highly purified, rhodamine-stained cytoplasts are fused to appropriate recipient cell lines and then the fusion mixture is sorted based on forward angle scatter and fluorescence parameters. Plating the positively sorted population in culture for as short as 12 h eliminates contaminating cytoplasts which, lacking a nucleus, are unable to adhere or survive. The resultant population, based on an analysis of genetic markers, is 75–100% cybrids, an enrichment of 1000- to 10,000-fold over the initial fusion mixture. Cybrids purified by cell sorting may be useful for detailed molecular studies of mitochondrial DNA gene expression and in the specific induction of new mitochondrial DNA mutants.     

免疫磁珠法分离人外周血CD4+CD25+调节性T细胞

目的 建立人外周血单个核细胞中CD4 CD25 调节性T细胞(regulatery T cells,Treg)免疫磁性细胞分离力(megnetic activated cell sorting,MACS),并鉴定其分离效率.方法 采用免疫磁珠两步法(即阴性分选和阳性分选2步)分离人周血单个核细胞中的CD4 CD25 调节性T细胞,首先采用生物素标记的鸡尾酒抗体和抗生物素标记的磁珠阴性分选CIM细胞,再用抗CD25 的磁珠阳性分选CD4 CD25 T细胞.分离后的细胞经抗体染色后再通过流式细胞仪检测其分离纯度;内因子染色检测其转录因子FOXV3的表达频率;台盼蓝染色检测细胞的存活率;3H-TdR掺入法检测其对CD4 CD25-T细脆殖抑制效应.结果 阴性分选CD4 T细胞的纯度为(92.2±1.7)%,阳性分选后CD4 CD25 Treg细胞的纯度(95.1±1.2)%.胞内因子染色FOXF3在CD4 CD25 Treg细胞中的表达率为(80.4±1.2)%,台盼蓝染色细胞存活率为(95.6±3.3)%.3H-TdR掺入法检测其对CIM CD25-T细胞具有明显的抑制作用.结论 采用免疫磁性细胞分离技术能够高效、快地得到一群纯度高并且细胞活力好的CD4 CIY25 Treg,为进一步研究其功能提供了方便.     

Exploiting bacterial peptide display technology to engineer biomaterials for neural stem cell culture

Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering.     

Development of mRNA quantification after fluorescence activated cell sorting

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. These cells can be separated from other cells by fluorescence-activated cell sorting (FACS) using antibodies that bind to surface antigen; however, the majority of these cells do not have specific surface antigens. Furthermore, the cells must be kept alive throughout the procedure to analyze their biological characteristics. To improve these limitations, we have established a novel laboratory test named mRNA quantification after fluorescence-activated cell sorting(FACS-mQ). Using this method, cells are sorted by a specific gene expression pattern, and the gene expression profile in the sorted cells can be easily analyzed. In this feature, we will introduce the typical applications of FACS-mQ targeting intracellular mRNA and intracellular nuclear antigens. These methods will hopefully contribute to the accumulation of knowledge regarding human stem cells, cancer stem cells, and small populations of cells, the biological characteristics of which are mostly unknown.     

Non-linear osmosis

1. The relation between osmotic gradient and rate of osmotic water flow has been measured in rabbit gall-bladder by a gravimetric procedure and by a rapid method based on streaming potentials. Streaming potentials were directly proportional to gravimetrically measured water fluxes.2. As in many other tissues, water flow was found to vary with gradient in a markedly non-linear fashion. There was no consistent relation between the water permeability and either the direction or the rate of water flow.3. Water flow in response to a given gradient decreased at higher osmolarities. The resistance to water flow increased linearly with osmolarity over the range 186-825 m-osM.4. The resistance to water flow was the same when the gall-bladder separated any two bathing solutions with the same average osmolarity, regardless of the magnitude of the gradient. In other words, the rate of water flow is given by the expression (O(m) - O(s))/[R(o)' + (1/2)k' (O(m) + O(s))], where R(o)' and k' are constants and O(m) and O(s) are the bathing solution osmolarities.5. Of the theories advanced to explain non-linear osmosis in other tissues, flow-induced membrane deformations, unstirred layers, asymmetrical series-membrane effects, and non-osmotic effects of solutes could not explain the results. However, experimental measurements of water permeability as a function of osmolarity permitted quantitative reconstruction of the observed water flow-osmotic gradient curves. Hence non-linear osmosis in rabbit gall-bladder is due to a decrease in water permeability with increasing osmolarity.6. The results suggest that aqueous channels in the cell membrane behave as osmometers, shrinking in concentrated solutions of impermeant molecules and thereby increasing membrane resistance to water flow. A mathematical formulation of such a membrane structure is offered.     

Mechanisms of cell polarity and aquaporin sorting in the nephron

The kidneys participate in whole-body homeostasis, regulating acid–base balance, electrolyte concentrations, extracellular fluid volume, and regulation of blood pressure. Many of the kidney’s functions are accomplished by relatively simple mechanisms of filtration, reabsorption, and secretion, which take place in the nephron. The kidneys generate 140–180 l of primary urine per day, while reabsorbing a large percentage, allowing for only the excretion of approximately 2 l of urine. Within the nephron, the majority of the filtered water and solutes are reabsorbed. This is mainly facilitated by specialized transporters and channels which are localized at different segments of the nephron and asymmetrically localized within the polarized epithelial cells. The asymmetric localization of these transporters and channels is essential for the physiological tasks of the renal tissues. One family of these proteins are the water-permeable aquaporins which are selectively expressed in cells along the nephron and localized at different compartments. Here, we discuss potential molecular links between mechanisms involved in the establishment of cell polarity and the members of the aquaporin family. In the first part of this review, we will focus on aspects of apical cell polarity. In the second part, we will review the motifs identified so far that are involved in aquaporin sorting and point out potential molecular links.     

Exploiting extracellular matrix-stem cell interactions: a review of natural materials for therapeutic muscle regeneration

Myopathies of skeletal muscle are prevalent diseases worldwide. To address this, regenerative therapies are being developed to restore perfusion to ischemic muscle and to reverse muscle wasting. There are adult stem cell populations that inherently possess these therapeutic properties; however, cell transplantation trials in the clinic have shown modest results at best, being limited by poor cell persistence and viability post-transplantation, and by cell relocation to non-target sites. Many materials exist that can elicit and enhance beneficial cell responses - these materials can be applied directly, or used as stem cell delivery vehicles, for regenerative therapies. In particular, components of the body's extracellular matrices may be advantageous for therapeutic application because cells already have a pre-disposition for recognizing them, and also because their usage carries a low probability of inducing negative immune responses. This review will survey the major components of the extracellular matrix and their interactions with relevant stem cell populations for the regeneration of muscle. Future material-based therapies will benefit from a more precise control over therapeutic cell populations implicated in the regenerative response.