Activation of the alternative pathway by pneumococcal cell walls.

The present studies were performed in order to identify the pneumococcal subcellular component responsible for activating the alternative pathway. Purified pneumococcal cell walls were able to activate the alternative pathway at a concentration as low as 5 mug/ml and were more active than crude cell walls, which in turn were more active than the whole organism. Purified pneumococcal cell membranes also were able to activiate the alternative pathway but had less than 10% of the activity of the purified walls. Thus, the cell wall appears to play a major role in pneumococcal activation of the alternative pathway. Pneumococcal cell walls containing ethanolamine were as effective as cell walls containing choline in activating the alternative pathway. Since C-reactive protein binds specifically to the phosphorylcholine residue of pneumococcal C-polysaccharide, it is unlikely that pneumococcal cell walls must combine with C-reactive protein in order to activate the alternative pathway.     

Cell wall proteins of flax phloem fibers

A cell wall has been isolated from single-type cells, phloem fibers of flax (Linum usitatissimum L.) being at the stage of the active formation of a thick secondary cell wall. Weakly bound proteins of the cell wall of phloem fibers were extracted and separated, and their mass spectra were recorded. The identification of the proteins and their assignment to a particular cell compartment were performed using a variety of bioinformatics methods. In all, 93 proteins were identified of which many proteins were defined as predicted, putative, and hypothetical. Twenty one proteins were identified as cell-wall proteins. The absence of the marker proteins of primary cell walls such as xyloglucan endotransglycosylase and expansins indirectly confirms the predominance of the secondary cell wall in a sample for protein extraction.     

EBV转化的人乳头瘤病毒感染者B淋巴母细胞系的建立及应用

建立EB病毒(EBV)转化的B淋巴母细胞系(LCL),应用此细胞系作为抗原递呈细胞筛选抗原特异性的T细胞克隆。收集人乳头瘤病毒(HPV)感染者外周血20份,分离外周血单个核细胞(PBMC),利用Pan T细胞试剂盒去除PBMC中的T细胞,获得non-T淋巴细胞,EBV病毒转化non-T淋巴细胞。应用转化的LCL细胞作为抗原递呈细胞,ELISPOT筛选可能的HPV抗原特异性的T细胞克隆。成功建立18个LCLs,建株成功率为90%。转化成功的LCL细胞体积增大,聚集成团状或簇状。建株失败的两份标本,用于转化的non-T淋巴细胞数少于2×106。应用LCL细胞作为抗原递呈细胞筛选出64个HPV抗原特异性的T细胞克隆。成功建立LCLs,此细胞系细胞可作为体外研究HPV特异性免疫反应的刺激细胞。     

Ultrastructures of the Egg Apparatus and the Central Cell of Calystegia hederacea (Convolvulaceae) Before and After Fertilizati on

Ultrastructures of the mature embryo sac of Calystegia hederacea Wall. and its changes after fertilization are described. The positional organization of the egg cell, the two synergids and the central cell, as well as their interrelationships were studied. Some regions of the cell boundaries between the egg cell and the central cell, as well as between the egg cell and the synergids were devoid of typical cell wall before fertilization, displayed a feature quite similar to the characteristic absence of the cell wall in the fertilization target zone occurred in most angiosperms. Besides the genera ultrastructural characteristics of the egg apparatus and the central cell, there were several unusual aspects in C. hederacea, such as the egg nucleus located above the large vacuole near the chalazal end of the cell, many polyribosomes in the cytoplasm of the egg cell and wall ingrowths on both sides of the hooks of the central cell. All these unusual characteristics seemed to be closely associated with the short duration of the fertilization and the absence of antipodal cells in the mature embryo sac. It is concluded that the female germ unit of C. hecleracea is considered to be a topographical and physiological unit to realize their functions for successful double fertilization.     

Silencing LINC00504 inhibits cell proliferation,invasion as well as migration and promotes cell apoptosis in lung cancer cells via upregulating miR-876-3p

LINC00504 acts as an oncogene and associates with unfavorable prognosis in patients with lung cancer. Silencing LINC00504 may be a promising strategy for treatment of lung cancer and its effects were firstly investigated in lung cancer cells this study. The gene expression level of miR-876-3p as well as LINC00504 were measured via PCR assay. The cell proliferation was investigated through Cell Counting Kit-8 (CCK-8) assay and colony formation assay. Flow cytometry was applied for detection of cell apoptosis. Wound healing and transwell assay were performed for measurement of cell migration and invasion respectively. The apoptosis related protein expressions were measured by western blot. Luciferase report assay was conducted for verification the target gene. LINC00504 was higher expressed in five types of lung cancer cells studied herein when compared with the control normal cells. LINC00504 knockdown exerted inhibitory effects on cell apoptosis, cell migration as well as cell invasion and promoted cell apoptosis. All the effects mentioned above were counteracted by miR-876-3p inhibitor. Silencing LINC00504 possessed anti-proliferation, repression of cell invasion as well as migration and pro-apoptosis effects via targeting up-regulation of miR-876-3p in lung cancer cells, proving the new therapeutic targets and highlighting the potential application in future diagnosis and treatment in lung cancer.     

CHO细胞株开发技术策略探讨

主要介绍了单克隆抗体药物工业生产中宿主细胞选择、表达载体构建、转染方法、筛选技术、细胞培养工艺技术方法以及最后选定细胞株的标准等,结合单抗药物CHO细胞株开发和培养工艺的经验,对当前我国单抗CHO细胞株开发技术策略进行了探讨。     

Effect of Differentiation and Cell Density on Glycosphingolipid Class and Molecular Species Composition of Mouse Neuroblastoma NB2a Cells

The effects of cell density and retinoic acid-induced differentiation on the class and molecular species composition of mouse neuroblastoma NB2a cell glycosphingolipids were examined under conditions where the period of culture was controlled. The total amount of neutral glycosphingolipids per cell decreased both with differentiation and as the cells became confluent. The relative amount of the neutral glycosphingolipid classes was not affected by differentiation, whereas there were small but significant changes in the relative amount of the neutral glycosphingolipid classes as the cells became confluent. The total amount of the gangliosides was unaffected by either differentiation or cell density, but there were significant changes in the ganglioside class composition as a result of both cell density and differentiation, and the effects were additive. The molecular species of all the major neutral glycosphingolipid and ganglioside classes were essentially identical, and were altered only slightly by either differentiation or cell density.     

太空环境改变生物工程细胞CHO(dhfr-

目的 :了解太空诱变对生物工程细胞CHO(dhfr^----     

Changes in surface relief of suspended cells are morphological signs of the initial stage of neoplastic transformation in fibroblastic monolayer cultures.

The percentages of cells with different types of cell surface relief were determined in cell suspensions derived from monolayer cultures. Primary cultures of rat embryo fibroblasts (REF) and cell lines REF (LT) and REF-1, immortalized cells of which preserved normal phenotypic characteristics of the initial primary culture REF, as well as morphologically transformed tumorigenic lines REF (LT) ras and REF-2EJ were studied. In REF suspensions the cells with the blebbed type of surface relief were shown to be predominant as compared with those with microvillus relief whereas cell suspensions derived from both immortalized and fully transformed cultures display the reverse ratio of cells with those types of surface relief. Therefore, the pattern of cell surface relief in cell suspensions derived from fibroblastic monolayer cultures may serve as a morphological marker of the initial stage of neoplastic transformation-immortalization when typical morphological signs of cell transformation are not yet manifested in monolayer cultures.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

Cell wall proteins of plant fibers

The cell wall of the same type - phloem fibers (Linum usitatissimum L.), active forming the thick secondary cell wall, - was obtained. Weakly bound cell wall proteins of phloem fibers were extracted and it subsequent separation and obtaining mass spectra was carried out. For identification and attachment of identified proteins to a specific cell compartments a variety ofbioinformatics methods was used. Were identified 93 proteins, many of which were defined as predicted, putative or hypothetical. At the same 21 proteins were identified as cell-wall protein. The absence of such marker proteins of the primary cell wall as xyloglucan-endotransglycosilase, expansins indirectly confirms that in the sample for extraction of proteins dominated the secondary cell wall.     

New approach for the establishment of mouse early embryonic stem cells and induction of their differentiation

Eleven early embryonic stem (EES) cell lines were established using a new novel method. Two cell stage embryos from the ddY mouse strain were cultured in alpha-MEM supplemented with 10% fetal calf serum (FCS) and embryotrophic factors (ETFs) and allowed to develop to the trilaminal germ disc embryonic stage. Only small round cells (EES cells) were isolated by the colony isolating technique and subsequently cultured in the same medium containing the ETFs and leukemia inhibitory factors (LIF-10 ng/ml). The newly established embryonic stem (ES) cells isolated from inner cell mass of blastocysts differentiated from two cell stage embryo in culture. The EES and ES cell lines were maintained in an undifferentiated state using Ham's F12 medium supplemented with 10% FCS and 1 ng/ml of LIF. The EES cells maintained their normal genetic and morphological features as well as their potential to differentiate into a broad spectrum of cell types as well as their ability to contribute to all cell lineages in chimeric mice. Moreover, these cell lines changed and differentiated into various kinds of cells by removing LIF and by the addition of ETFs to the vitro culture system. All 11 EES cell lines and 3 ES cell lines formed embryoid bodies; however, cell line EES-4 formed tube-like structures which extended, anastomosed with each other, and finally formed networks when the LIF were absent. Primitive germ organ-like structures composed of 3 germ layers were recognized in the cultures following the administration of ETFs. In conclusion, the new method devised by us is a novel, easy and reliable technique for establishing EES cell lines.     

Stable expression of the human 5alpha-reductase isoenzymes type I and type II in HEK293 cells to identify dual and selective inhibitors

A eucaryotic cell assay was established to identify novel, dual and selective inhibitors of human 5alpha-reductase. For this purpose the cDNAs encoding 5alpha-reductase type I and type II were inserted into a pRcCMV vector and expressed in human embryonic kidney (HEK293) cells. Single cell clones with substantially high enzymatic activity were selected and established as permanent cell lines. KM values were determined for both isozymes. The inhibitory potency of several steroidal and non-steroidal compounds synthesized in our group, as well as finasteride and 4MA as controls, were tested by measuring the conversion of [3H]androstenedione. Reaction products were quantified by a HPLC reversed phase technique. Using the new cell assays, selective as well as novel dual 5alpha-reductase inhibitors with IC50 values between 1.0 and 2.5 microM were identified.     

Flow microfluorometric studies of lectin binding to mammalian cells. II. Estimation of the surface density of receptor sites by multiparameter analysis

A flow-system multiparameter cell analyzer that simultaneously measures and processes fluorescence and cell volume signals from single cells was used to study the binding of fluorescein-conjugated Concanavalin A (Con A–F) to the cell surface. Cells reacted with Con A–F were passed through a flow chamber where sensors measured both cell volume and fluorescence of each individual cell. Sensor signals were electronically processed by first converting the cell volume signals to two-thirds power (proportional to surface area) and then forming the fluorescence-to-surface area ratio. These ratios, which were considered as estimates of the surface density of binding sites, were displayed as frequency distribution histograms using a multichannel pulse-height analyzer for various cell populations differing in cell size. Comparisons between cell lines showed characteristic differences in binding site density. Cell cycle dependent changes were not found for CHO cells synchronized by mitotic selection. An important benefit of this analysis method was the ability to quantitate very weak cell surface fluorescence.     

Integrins alpha5beta1, alphavbeta1, and alphavbeta6 collaborate in squamous carcinoma cell spreading and migration on fibronectin

The expression of alphavbeta6 fibronectin/tenascin receptor integrin is induced in malignant transformation of oral epithelium. In this study, we demonstrate the contribution of alphavbeta6 as well as other fibronectin receptor integrins in squamous cell carcinoma (SCC) cell adhesion and migration. Of 11 SCC cell lines isolated from the head and neck area, 8 (73%) expressed alphavbeta6 integrin on the cell surface. Three cell lines were chosen for further functional experiments: 1 with relatively high, 1 with moderate, and 1 with minimal surface expression of alphavbeta6 integrin. In addition to alphavbeta6, all 3 cell lines expressed alpha5beta1 and alphavbeta1 fibronectin receptor integrins. Function-blocking experiments with inhibitory anti-integrin antibodies showed that all these three integrins were functional in SCC cell spreading on fibronectin. Integrin alphavbeta6, however, was not used as a primary but as an alternative fibronectin receptor by SCC cells, as the inhibitory anti-beta6 integrin antibody alone had no effect on spreading. In migration, however, alphavbeta6, alpha5beta1, and alphavbeta1 integrins were all used in cooperation. The presence of alphavbeta1 integrin in SCC cells is a novel finding as is its contribution to SCC cell migration. When one or two of these three receptors were blocked, the cells demonstrated an adaptive ability to remain migratory using integrins that were not targeted by antibodies. Utilization of a combination of receptors of different affinities may be beneficial for SCC cell migration versatility.     

改进的培养体系在小鼠体细胞核移植及重构胚ES细胞分离培养中的应用

取8周后的雌性昆明小鼠进行超排,取卵母细胞用作核受体,收集卵母细胞周围的卵丘细胞作核供体,进行体细胞核移植。核移植重构胚经SrCl2激活处理6h后,与改良的M16培养液和小鼠输卵管上皮细胞共培养;将发育到早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加含心肌细胞培养液的ES细胞培养液;把孵出的ICM进行消化接种培养,对孵出的ES细胞集落进行鉴定培养。结果显示,以小鼠卵丘细胞为核供体,体细胞核移植重构胚激活率为65．23％,囊胚发育率为11．69％;9个核移植重构囊胚中分离出ES细胞集落,分离率为2．77％;分离出的核移植ES细胞集落具有岛屿状团状隆起结构、碱性磷酸酶染色呈阳性,体外分化可形成类胚体,并能分化成上皮样或梭形细胞。ES细胞集落经常规冻存和复苏后,显示出同冻存前相似的集落形态,并具有较强的增殖能力。实验证实小鼠输卵管上皮细胞、改良的M16培养液及含心肌细胞培养液的ES细胞培养液可以更为成功地运用于小鼠的体细胞核移植及ES细胞的分离培养研究。     

单细胞测序方法研究进展

近年来,高通量测序技术(Next-generation sequencing,NGS)快速发展,已广泛应用于生命科学各个领域,但传统的混合细胞测序(Bulk cell sequencing)检测的是细胞群体的总平均反应,无法反应每个细胞的真实情况,这会影响研究者对细胞功能认知的准确性。单细胞测序技术(Single cell sequencing,sc-Seq)的出现,从一定程度上解决了传统测序固有的缺陷。单细胞测序是针对单个细胞的RNA或DNA进行测序,能够准确测出单个细胞的基因结构和表达状态,从而分析相同表型细胞的异质性。本文首先介绍单细胞测序的原理、测序类型和测序平台,有助于理解单细胞测序和在进行科研项目时设计合适的项目方案。进一步介绍单细胞转录组测序的分析流程和各种常用的分析工具或软件,并重点阐述单细胞转录组测序分析中的细胞聚类和拟时序分析的原理和研究进展,为进行单细胞转录组测序数据分析提供参考。最后,本文简述了单细胞测序研究热度、单细胞测序的应用、挑战和展望等,有助于更全面地认识单细胞测序。     

Eigenstrain as a mechanical set-point of cells

Cell contraction regulates how cells sense their mechanical environment. We sought to identify the set-point of cell contraction, also referred to as tensional homeostasis. In this work, bovine aortic endothelial cells (BAECs), cultured on substrates with different stiffness, were characterized using traction force microscopy (TFM). Numerical models were developed to provide insights into the mechanics of cell–substrate interactions. Cell contraction was modeled as eigenstrain which could induce isometric cell contraction without external forces. The predicted traction stresses matched well with TFM measurements. Furthermore, our numerical model provided cell stress and displacement maps for inspecting the fundamental regulating mechanism of cell mechanosensing. We showed that cell spread area, traction force on a substrate, as well as the average stress of a cell were increased in response to a stiffer substrate. However, the cell average strain, which is cell type-specific, was kept at the same level regardless of the substrate stiffness. This indicated that the cell average strain is the tensional homeostasis that each type of cell tries to maintain. Furthermore, cell contraction in terms of eigenstrain was found to be the same for both BAECs and fibroblast cells in different mechanical environments. This implied a potential mechanical set-point across different cell types. Our results suggest that additional measurements of contractility might be useful for monitoring cell mechanosensing as well as dynamic remodeling of the extracellular matrix (ECM). This work could help to advance the understanding of the cell-ECM relationship, leading to better regenerative strategies.     

The effect of the cell separation procedure on neutrophilic granulocyte function. I. Research on the morphology and phagocytosis behavior of isolated granulocytes

The investigations were aimed at detecting the impact of various cell separating techniques on the morphology and function of neutrophilic granulocytes. Methodically simple cell separating techniques were used, such as spontaneous sedimentation of ACD-AG blood and mechanically defibrinated blood and a flotation technique which makes use of cell isolation by means of a separating solution and centrifugation. Granulocytes which were isolated by the cell separator Haemonetics M 30 for therapeutic purposes and which, according to data in literature, are not damaged by cell separating techniques were used as reference cells. The electronic-optical evaluation of granulocyte morphology and the in vitro investigation of the phagocytic capacity for rice starch (ingestion rate) of differently isolated granulocytes were made after cell separation.