雪旺氏细胞内NGF含量的定量测定方法研究

目的：建立一套测定单个细胞内蛋白含量的新方法。方法：纯化培养乳鼠雪旺氏细胞，施加实验因素，通过间接免疫荧光法标记细胞内ＮＧＦ蛋白，而后用流式细胞仪（适用于细胞悬液样品）或粘附细胞仪（适用于粘附细胞样品）进行测定。结果：流式细胞仪及粘附细胞仪均可得到每一实验组的精确测定结果，并能进行统计分析，提供统计图表和各种数据。结论：应用流式细胞仪或粘附细胞仪可对单个雪旺氏细胞内的ＮＧＦ含量进行定量研究     

Cell cycle analysis using bromodeoxyuridine: comparison of methods for analysis of total cell transit time

Results are presented supporting the study of cellular proliferation utilizing 5-bromodeoxyuridine (BrdU) incorporation followed by sister chromatid differential staining. In order to determine the relative accuracy of this method in estimating total cell transit time (TC), we utilized a thermosensitive rat embryonic cell line to compare measurement of TC based on the percent differentially labeled (PDLM) technique, with cell cycle measurements using [3H]-thymidine [( 3H]-TdR) incorporation and the percent labeled mitoses (PLM) technique. Results of PLM and PDLM analysis were shown to be highly concordant, indicating the utility of the BrdU method for analysis of Tc. Results are presented suggesting the general application of the PDLM method for estimations of TC in cultures of human tumors.     

Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number

Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components—a finding resulting from the biological follow-up of unbiased human genetic studies.     

Multiparametric image analysis of lung‐branching morphogenesis

Background: Lung‐branching morphogenesis is a fundamental developmental process, yet the cellular dynamics that occur during lung development and the molecular mechanisms underlying recent postulated branching modes are poorly understood. Results: Here, we implemented a time‐lapse video microscopy method to study the cellular behavior and molecular mechanisms of planar bifurcation and domain branching in lung explant‐ and organotypic cultures. Our analysis revealed morphologically distinct stages that are shaped at least in part by a combination of localized and orientated cell divisions and by local mechanical forces. We also identified myosin light‐chain kinase as an important regulator of bud bifurcation, but not domain branching in lung explants. Conclusions: This live imaging approach provides a method to study cellular behavior during lung‐branching morphogenesis and suggests the importance of a mechanism primarily based on oriented cell proliferation and mechanical forces in forming and shaping the developing lung airways. Developmental Dynamics 242:622–637, 2013. © 2013 Wiley Periodicals, Inc.     

Two-dimensional and three-dimensional time-lapse microscopic magnetic resonance imaging of Xenopus gastrulation movements using intrinsic tissue-specific contrast.

The amphibian embryo undergoes radical tissue transformations during blastula and gastrula stages, but live observation of internal morphogenetic events by optical microscopy is not feasible due to the opacity of the early embryo. Here, we report on the use of microscopic magnetic resonance imaging (MRI) to directly follow morphogenetic movements during blastula and gastrula stages of the Xenopus laevis embryo. We compare three different MRI modalities that take advantage of the intrinsic contrast present in embryonic tissues: three-dimensional (3D) fat-imaging, 3D water-imaging, and 2D high-speed high-resolution imaging of early embryonic stages. We show that the features revealed by the intrinsic contrast correlate with the histological structure of the embryo. Using this tissue specific intrinsic contrast, the main embryonic tissues and internal tissue movements as well as archenteron invagination can be differentiated without cell labeling. We present 2D and 3D time-lapse sequences of early Xenopus embryonic development, spanning the stages from early blastula to the end of gastrula, which show the complex internal rearrangements of gastrulation in essentially real-time.     

Location and possible developmental cycle of mycoplasmas and bacterial L forms in cell cultures

With the use of the vital dye Chlorazol Black E the location and behavior of five species of mycoplasmas and a stable L form of group A -hemolytic streptococcus were studied in various continous cell lines. The mycoplasmas and L forms were shown to pass through a definite developmental cycle in the cells; initally they were located extracellularly and on the cell surface, later intracellularly when they multiplied intensively; later still they were again found on the cell surface and extracellularly. This cycle was shown to depend on the type of infection. The character of localization was shown to depend both on the species of both agent and culture.N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. D. Timakov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 4, pp. 439–440, April, 1976.     

Bacillus subtilis MinC destabilizes FtsZ-rings at new cell poles and contributes to the timing of cell division

Division site selection in rod-shaped bacteria depends on nucleoid occlusion, which prevents division over the chromosome and MinCD, which prevent division at the poles. MinD is thought to localize MinC to the cell poles where it prevents FtsZ assembly. Time-lapse microscopy demonstrates that in Bacillus subtilis transient polar FtsZ rings assemble adjacent to recently completed septa and that in minCD strains these persist and are used for division, producing a minicell. This suggests that MinC acts when division proteins are released from newly completed septa to prevent their immediate reassembly at new cell poles. The minCD mutant appears to uncouple FtsZ ring assembly from cell division and thus shows a variable interdivisional time and a rapid loss of cell cycle synchrony. Functional MinC-GFP expressed from the chromosome minCD locus is dynamic. It is recruited to active division sites before septal biogenesis, rotates around the septum, and moves away from completed septa. Thus high concentrations of MinC are found primarily at the septum and, more transiently, at the new cell pole. DivIVA and MinD recruit MinC to division sites, rather than mediating the stable polar localization previously thought to restrict MinC activity to the pole. Together, our results suggest that B. subtilis MinC does not inhibit FtsZ assembly at the cell poles, but rather prevents polar FtsZ rings adjacent to new cell poles from supporting cell division.     

The priming/completion paradigm to explain growth factor-dependent cell cycle progression

Approximately 50 years ago, researchers established conditions to maintain cells in tissue culture Likely et al. (1952) [Further studies on the proliferation in vitro of single isolated tissue cells. J Natl Cancer Inst 13, 177–184]; Scherer et al. (1953) [Studies on the propagation in vitro of poliomyelitis viruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J Exp Med 97, 695–710]; Eagle (1955a) [The specific amino acid requirements of a mammalian cell (strain L) in tissue culture. J Biol Chem 214, 839–852]; (1955b) [Nutrition needs of mammalian cells in tissue culture. Science 122, 501–514]. This simple model system set the stage for discovery of growth factors and the signaling systems that they engage to mediate cellular responses such as proliferation. The purpose of this review is to present the original view of how growth factors regulate cell cycle progression and an updated (priming/completion) version of how growth factors advance resting cells through the cell cycle.     

siRNA对卵巢癌细胞周期及EGFR表达的影响

目的体外构建编码人类表皮生长因子受体（EGFR）的小干扰RNA（siRNA）的质粒表达载体,观察其对卵巢癌Skov-3细胞EGFR的特异性抑制作用及对细胞凋亡和周期的影响。方法构建pSilencer-EGFR siRNA真核基因表达载体,采用脂质体介导的方法将其转染到卵巢癌细胞株Skov-3,实验分为以下3组：空白对照组（未经转染的人卵巢癌Skov一3细胞）、非特异性转染组（转染非特异性质粒载体）及特异性转染组。用RT-PCR的方法检测mRNA水平的变化,用免疫荧光法检测EGFR蛋白水平的变化,流式细胞仪检测其对卵巢癌细胞周期及凋亡的影响。结果成功构建pSilencer—EGFR siRNA真核基因表达载体。psiRNA—EGFR质粒明显下调Skov-3细胞中EGFR的表达,阻断细胞周期在G1期,促进细胞凋亡。结论重组质粒psiRNA—EGFR能有效地抑制Skov-3细胞内EGFR的表达、抑制细胞增殖,其抑制机制可能与引起细胞周期再分布、降低S期细胞比例和促进细胞凋亡有关。     

Skp2 contributes to cell cycle progression in trophoblast stem cells and to placental development

All trophoblast subtypes of the placenta are derived from trophoblast stem cells (TSCs). TSCs have the capacity to self‐renew, but how the proliferation of these cells is regulated in the undifferentiated state has been largely unclear. We now show that the F‐box protein Skp2 regulates the proliferation of TSCs and thereby plays a pivotal role in placental development in mice on the C57BL/6 background. The placenta of Skp2^?/? mouse embryos on the C57BL/6 background was smaller than that of their Skp2^+/+ littermates, with the mutant embryos also manifesting intrauterine growth retardation. Although the Skp2^?/? mice were born alive, most of them died before postnatal day 21, presumably as a result of placental defects. Depletion of Skp2 in TSCs cultured in the undifferentiated state resulted in a reduced rate of proliferation and arrest of the cell cycle in G₁ phase, indicative of a defect in self‐renewal capacity. The cell cycle arrest apparent in Skp2‐deficient TSCs was reversed by additional ablation of the cyclin‐dependent kinase inhibitor (CKI) p57 but not by that of the CKI p27. Our results thus suggest that Skp2‐mediated degradation of p57 is an important determinant of the self‐renewal capacity of TSCs during placental development, at least in mice of certain genetic backgrounds.     

Optic tectum morphogenesis: a step-by-step model based on the temporal-spatial organization of the cell proliferation. Significance of deterministic and stochastic components subsumed in the spatial organization

Background: Cell proliferation plays an important morphogenetic role. This work analyzes the temporal–spatial organization of cell proliferation as an attempt to understand its contribution to the chick optic tectum (OT) morphogenesis. Results: A morphogenetic model based on space‐dependent differences in cell proliferation is presented. Step1: a medial zone of high mitotic density (mZHMD) appears at the caudal zone. Step2: the mZHMD expands cephalically forming the dorsal curvature and then duplicates into two bilateral ZHMDs (bZHMD). Step3: the bZHMDs move toward the central region of each hemitectum. Step4: the planar expansion of both bZHMD and a relative decrement in the dorsal midline growth produces a dorsal medial groove separating the tectal hemispheres. Step5: a relative caudal displacement of the bZHMDs produces the OT caudal curvature. Numerical sequences derived from records of mitotic cells spatial coordinates, analyzed as stochastic point processes, show that they correspond to 1/f^(β) processes. The spatial organization subsumes deterministic and stochastic components. Conclusions: The deterministic component describes the presence of a long‐range influence that installs an asymmetric distribution of cell proliferation, i.e., an asymmetrically located ZHMD that print space‐dependent differences onto the tectal corticogenesis. The stochastic component reveals short‐range anti‐correlations reflecting spatial clusterization and synchronization between neighboring cells. Developmental Dynamics 241:1043–1061, 2012. © 2012 Wiley Periodicals, Inc.     

Translational Mini-Review Series on B cell subsets in disease. Reconstitution after haematopoietic stem cell transplantation - revelation of B cell developmental pathways and lineage phenotypes

Haematopoietic stem cell transplantation (HSCT) is an immunological treatment that has been used for more than 40 years to cure a variety of diseases. The procedure is associated with serious side effects, due to the severe impairment of the immune system induced by the treatment. After a conditioning regimen with high-dose chemotherapy, sometimes in combination with total body irradiation, haematopoietic stem cells are transferred from a donor, allowing a donor-derived blood system to form. Here, we discuss the current knowledge of humoral problems and B cell development after HSCT, and relate these to the current understanding of human peripheral B cell development. We describe how these studies have aided the identification of subsets of transitional B cells and also a robust memory B cell phenotype.     

Microarray analysis of striatal embryonic stem cells induced to differentiate by ensheathing cell conditioned media.

The mammalian central nervous system contains well-defined regions of plasticity in which cells of the aldynoglia phenotype promote neuronal growth and regeneration. Only now are the factors that regulate the production of new cells from multipotential neural precursors (MNP) starting to be identified. We are interested in understanding how differentiation towards the aldynoglia phenotype is controlled, and to study these events we have induced the differentiation of embryonic MNP towards this phenotype in vitro. Accordingly, we have used microarrays to analyze gene expression in three different cell populations: olfactory bulb ensheathing cells (EC), a prototypic aldynoglia cell type; undifferentiated MNP; and MNP differentiated in vitro for 24 hr in EC-conditioned media. The expression profiles identified support the idea that the EC are more closely related to Schwann cells and astrocytes than to oligodendrocytes. Following MNP differentiation, more strongly expressed genes define a neuroglial cell phenotype. RT-PCR confirms that S100a6, Mtmr2, and Col5a were highly expressed by EC, whereas Pou3f3 were more strongly expressed in MNP than in EC, and SafB1 and Mash1 expression were induced in MNP by EC-conditioned media. The profile of gene expression after differentiation suggests that Wnt signaling may be inactivated during this process, while activation of the BMP pathway may be elicited through the BMPr1A. These results provide us with a starting point to study the genes involved in the induction of aldynoglia differentiation from MNP.     

Activated human CD4+ T cells induced by dendritic cell stimulation are most sensitive to transforming growth factor-beta: implications for dendritic cell immunization against cancer.

The secretion of immunosuppressive factors like transforming growth factor-beta (TGF-beta) by tumor cells has been recognized as one of the mechanisms involved in tumor immunological escape. This study aimed to examine whether dendritic cell (DC) immunization could reverse TGF-beta-induced immunosuppression by simulating the in vivo interaction among infused DCs, host T cells, and tumor-secreted TGF-beta in an in vitro study. We found that both immature and mature DCs were relatively resistant to TGF-beta. The addition of TGF-beta to naive human CD4+ T cells, which are required by genetically modified DC to elicit antitumor immunity, resulted in their hyporesponsiveness to DC stimulation in a dose-dependent manner. When activated by allogeneic DCs in the presence of TGF-beta, CD4+ T cells displayed a reduced capacity to proliferate. More importantly, activated CD4+ T cells induced by DC stimulation were very sensitive to TGF-beta, and this susceptibility was enhanced by their previous exposure to TGF-beta. The underlying mechanism was linked to TGF-beta-induced apoptosis of activated T cells. However, the presence of stimulation from DC or antibodies to CD3 plus CD28 could partly reverse the immunosuppressive effect of TGF-beta on activated CD4+ T cells. Taken together, our results indicate that the efficacy of DC immunization may be impaired by tumor-derived TGF-beta.     

Induction of B cell responsiveness to growth factors by Epstein-Barr virus conversion: comparison of endogenous factors and interleukin-1.

Immortalized B lymphocytes produce a factor(s) that stimulates growth of B cell lines carrying Epstein-Barr virus (EBV). Stimulatory supernatants derived from B cells also exhibit interleukin-1 (IL-1) activity in costimulator assays with the D10.G4.1 helper T cell line. Experiments with purified macrophage-derived IL-1 and recombinant IL-1 beta demonstrate that IL-1 stimulates proliferation of the cell lines that respond to the factors from B lymphocyte lines. One B cell line, Ramos, an EBV-Burkitt's lymphoma, contrasts with other B cell lines in that it is refractory to the growth enhancing effects of B cell conditioned medium and macrophage-derived IL-1. When EBV was introduced into Ramos cells, growth was enhanced by the factor(s) in B cell conditioned medium (six out of seven lines); growth of EBV-converted Ramos lines (six out of seven lines) also was enhanced by IL-1. These findings demonstrate that infection of a non-responsive transformed B lymphocyte by EBV induces cellular responsiveness to factor-mediated growth stimulation.     

Virus-induced non-specific signals cause cell cycle progression of primed CD8(+) T cells but do not induce cell differentiation.

In this report the significance of virus-induced non-specific T cell activation was re-evaluated using transgenic mice in which about half of the CD8(+) T cells expressed a TCR specific for amino acids 33-41 of lymphocytic choriomeningitis virus glycoprotein I. This allowed tracing of cells with known specificity and priming history in an environment also containing a normal heterogeneous CD8(+) population which served as an intrinsic control. Three parameters of T cell activation were analyzed: cell cycle progression, phenotypic conversion and cytolytic activity. Following injection of the IFN inducer poly(I:C), proliferation of memory (CD44(hi)) CD8(+) T cells but no phenotypic or functional activation was observed. Following injection of an unrelated virus [vesicular stomatitis virus (VSV)], naive TCR transgenic cells did not become significantly activated with respect to any of the parameters investigated. In contrast, memory TCR transgenic cells were found to proliferate extensively early after VSV infection (day 0-3), whereas limited proliferation was observed later (day 3-6) when proliferation of non-transgenic CD8(+) T cells is maximal. This aborted response did not result from anergy to TCR stimulation, as memory TCR transgenic cells proliferated vigorously upon stimulation with their nominal peptide. Despite the massive proliferation of memory cells observed early after VSV infection, no phenotypic or functional activation was observed. Together these findings indicate that both non-specific and antigen-specific signals contribute to the initial virus-induced proliferation of CD8(+) T cells, but for further proliferation and differentiation to take place, TCR-ligand interaction is required. The implications for maintenance of T cell memory is discussed.     

A light microscopic and morphometric analysis of the Sertoli cell during the spermatogenic cycle of the rat

Summary Testes from 8 adult rats were perfusion-fixed with buffered glutaraldehyde and semi-thin sections of the seminiferous epithelium at all stages of the spermatogenic cycle were subjected to morphometric analysis at the light microscope level. Methods are presented to derive the volume occupied by the Sertoli cells within the total volume of the seminiferous epithelium at each stage of the cycle for a single testis. The total number of Sertoli cells present in each stage for the whole testis was calculated from a measurement of Sertoli cell numerical density and the total volume of the seminiferous tubule in the testis at each stage of the cycle. Average volume of a single Sertoli cell for each stage was derived by dividing the first set of data by the latter data. Average Sertoli cell volume exhibited a cyclic variation in relation to the stages of the spermatogenic cycle. Sertoli cells were smallest during stages VI–VIII (5300–5500 m³) increased to maximum volume during stages XII–XIV (7700–8000 m³) and thereafter during stages I–V, gradually contracted in volume to complete the cycle. Stage-dependent cyclic variations in Sertoli cell volume offers evidence that the morphology of the Sertoli cell undergoes structural modifications to accommodate changes in the shape and volume of the developing germ cells. Furthermore these volume changes implicate the Sertoli cells in cyclic metabolic, absorptive and secretory functions which possibly direct the maturation of germ cells during the spermatogenic cycle.