Enrichment of epidermal stem cells by rapid adherence and analysis of the reciprocal interaction of epidermal stem cells with neighboring cells using an organotypic system

Keratinocytes have the ability to adhere to extracellular matrix rapidly. With this in mind, in this study we isolated keratinocytes known as rapidly adhering (RA) cells. To compare epidermal regenerative abilities, skin substitutes were reconstructed by adding keratinocytes or RA cells to two groups of bioengineered dermis made by fibroblasts and hair follicle dermal cells respectively. After transplantation, the results illustrated that the skin substitutes including RA cells were integrated into the host tissue. Furthermore, with hair follicle dermal cells' influences, the RA cells could form structures very similar to normal hair follicles. These results indicate that RA cells are predominately comprised of epidermal stem cells. The results also demonstrated that besides the reciprocal interaction of epidermal stem cells with dermal cells, the interaction of epidermal stem cells with keratinocytes were critical in epidermis morphogenesis and self-renewal, and application of RA cells could optimize engineering of skin substitutes.     

蝘蜓消化道5-羟色胺细胞的免疫组织化学定位

应用5-羟色胺(5-Hydroxytryptamine 5-HT)特异性抗血清对蝘蜓(Sphenomorphus iudieus)消化道5-HT细胞的分布及形态进行了免疫组织化学研究。结果表明,5-HT阳性细胞在堰蜓消化道各段均有分布,其中以胃幽门部位分布密度最高,食道与直肠部位其次,空肠部位分布密度最低。消化道各段5-HT细胞形态多样,有圆形、椭圆形、梭形、楔形、不规则形,其中梭形细胞多具有胞突。文中对堰蜓消化道5-HT细胞的分布、形态与功能相适应的特点进行了讨论。     

5-氨基γ-酮戊酸及其己酯衍生物对几种细胞株的光动力作用

5 氨基γ 酮戊 (ALA)及其己酯 (He ALA)具有内源生成光敏剂的特点 ,在肿瘤光动力探测及治疗中显示出了优势。ALA及He ALA对神经母细胞瘤、肝癌细胞及成纤维细胞的光动力作用被研究比较。由特征荧光光谱证实 ,经ALA或He ALA培养后 ,三种细胞内均可生成原卟啉 (PpIX)产物。激光扫描荧光显微镜显示 ,在经ALA或He ALA培养后的神经母细胞瘤中 ,PpIX均以弥散方式分布在细胞质中。PpIX在三种细胞中的积聚动力学过程不同 ,随着ALA或He ALA培育时间的增长 ,PpIX在肝癌细胞及成纤维原细胞中的积累增加 ,而在神经母细胞瘤中PpIX在 8h后已达到饱和。此外 ,在同样的培育条件下 ,神经母细胞瘤中PpIX的生成浓度明显高于肝癌细胞及成纤维细胞。经ALA培养及光照射后 ,可使近 90 %的神经母细胞瘤失活 ;而在同样条件下却只能杀伤 5 0 %左右的肝癌细胞及成纤维细胞。揭示了神经母细胞瘤对ALA光动力作用有极高的敏感性 ,并适于光动力治疗。与ALA相比 ,He ALA可在三种细胞内造成与ALA相近的杀伤率 ,但所用的药物浓度却比ALA低 10倍 ,显示He ALA具有极高的光动力灭活效率。因此在内源光动力治疗中 ,He ALA是一种极具开发前景的新药物。     

Quantification of chemotactic response of quiescent and proliferating fibroblasts in Boyden chambers by computer-assisted image analysis

The chemotactic response of human gingival fibroblasts to platelet-derived growth factor (PDGF) was investigated in 48-well modified Boyden chambers. Results were quantified using computer-assisted image analysis of propidium iodidestained cells and were compared with results obtained using the conventional method of quantification by direct counting. Quantification by image analysis was found to be rapid and accurate, and correlated closely with results obtained by direct counting. Bromodeoxyuridine (BrdU)-labeled proliferating cells were double stained with propidium iodide and fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antiserum. The cycling cells showed a markedly reduced chemotactic response to PDGF, whereas cells in S-phase of the cell cycle did not show any response. This method of quantification of results of Boyden chamber assays is simple and reliable, and allows the use of double labels to investigate the chemotactic response of subpopulations of cells within a heterogeneous population of cells.     

Differences in the response of early and mid or late G1 WI38 cells treated with cyclohexamide to HeLa inducers of DNA synthesis

The inducibility of DNA synthesis after treatment with cyclohexamide (CHM) during mitosis and the G1 phase of WI38 cells has been studied in the heterokaryons following fusion with HeLa cells in S phase. Synchronized mitotic cells treated for up to 5 h with CHM were not delayed in the initiation of DNA synthesis in the heterokaryons. The G1 cells treated with CHM for 3-24 h were slow in responding to inducers of DNA synthesis generated by HeLa cells in the heterokaryons. The results suggest that there is a specific point in early G1 that regulates the entry of cells into a cycling state. In the presence of CHM, mitotic cells divide, but the daughter cells fail to enter G1 leading to DNA synthesis, and CHM treatment of G1 cells results in their transient entry into a G0 state.     

The behaviour and function of bottle cells during gastrulation of Xenopus laevis

The behaviour of bottle cells in normal and microsurgically altered gastrulae and in cultured explants of Xenopus laevis was analysed, using time-lapse micrography, scanning electron microscopy (SEM) and cell tracing with fluorescein dextran amine (FDA). The results shed new light on the function of bottle cells. Bottle cells forming in vivo show a predominantly animal-vegetal apical contraction and a concurrent apical-basal elongation, whereas those forming in cultured explants show uniform apical contraction and remain rotund. Bottle cells forming in embryos with fewer subblastoporal cells contract more uniformly than those in normal embryos and release of normal bottle cells from supra- and subblastoporal cells results in immediate loss of the bottle shape. These results, and an analysis of the effects of bottle cell formation on the shapes and movements of surrounding tissues, show that unique shape of bottle cells and their probable function in development are not intrinsic properties but result from a modulation of the effect of a uniform and intrinsic apical contraction by the geometric and mechanical properties of the surrounding tissue. Mechanical simulations of bottle cell formation, using the finite element method, suggest how the site of bottle cell formation and the thickness and stiffness of adjacent tissues might change the effects of their formation. These results and FDA marking of prospective bottle cells and the adjacent deep mesodermal cells suggest that bottle cells function during their formation to initiate the involution of the prospective mesodermal mantle. Later they respread to deepen the archenteron and to form its peripheral wall.     

The production of terminal deoxynucleotidyl transferase (TdT) positive cells in cultures of human pluripotent hemopoietic progenitor cells

A transient increase in terminal deoxynucleotidyl transferase positive (TdT+) cells was observed during the early phase of (less than or equal to day 5) cultures supporting the growth of pluripotent myeloid progenitor cells (CFU-mix). T-cell growth-promoting medium and erythropoietin were not required. The rapidity with which TdT+ cells appeared in cultures and the results of cultures where TdT+ cells were high initially (greater than 800 cells/culture) were not consistent with their having been produced by proliferation of pre-existing TdT+ cells from the bone marrow inoculum. The results suggest production of TdT+ cells from a TdT-negative precursor either by altered enzyme expression or by production of TdT+ progeny.     

Effect of R-plasmid RP1 on surface hydrophobicity of Proteus mirabilis

The presence of R-plasmid RP1, as well as the conditions of growth, affected the surface hydrophobicity of a clinical isolate of Proteus mirabilis. However, results depended upon the method of assessment. Stationary phase plasmid-containing cells appeared to be less hydrophobic than plasmid-free cells when hydrophobicity was measured by the contact angle method, but more hydrophobic when measured by bacterial adherence to hydrocarbons or hydrophobic interaction chromatography. Cells growing in a chemostat differed in hydrophobicity from stationary phase cells and results varied with the growth rate. Plasmid-mediated effects were greatest in iron-depleted cells, and differences between plasmid-containing and plasmid-free cells were virtually eliminated by pre-treatment with antiserum.     

Disruption of focal adhesion kinase slows transendothelial migration of AU-565 breast cancer cells

Transendothelial migration of cancer cells from the vasculature into tissue stroma is a final step in the metastatic cascade, prior to formation of secondary tumors. Due to its role in 2-dimensional migration of cells on extracellular matrix proteins, we hypothesized that focal adhesion kinase (FAK) promotes transendothelial migration of cancer cells. AU-565 cells are weakly invasive metastatic breast adenocarcinoma cells that migrate through bovine lung microvessel endothelial cell monolayers. Electric cell-substrate impedance sensing detects a significant decrease in monolayer resistance upon addition of AU-565 cells. Immunofluorescence microscopy and filter-based migration assays demonstrate that this drop in resistance correlates with transendothelial migration. Transfection of AU-565 cells with FAK siRNA results in significantly diminished transendothelial migration of AU-565 cells within 15h. Expression of the dominant negative FAK inhibitor FAK-related non-kinase (FRNK) also results in delayed AU-565 transendothelial migration, whereas over-expression of wildtype FAK does not impact transendothelial migration substantially. These results demonstrate that FAK affects the rate of a key step in the metastatic cascade.     

Modulation of the biologic activities of IgE-binding factors. III. Switching of a T cell hybrid clone from the formation of IgE-suppressive factor to the formation of IgE-potentiating factor

Incubation of rat-mouse T cell hybridoma cells, 23B6, with rat immunoglobulin E (IgE) results in the formation of the 15,000-dalton IgE-suppressive factor and the 30,000-dalton IgE-binding factor, which has neither potentiating activity nor suppressive activity on the IgE response. Another T cell hybridoma, 23A4 cells, produces the 30,000-dalton "inactive" IgE-binding factor upon incubation with IgE. Both the 15,000-dalton IgE-suppressive factor and the 30,000-dalton IgE-binding factor lacked affinity for lentil lectin but bound to peanut agglutinin. When the 23B6 cells were incubated with IgE in the presence of lysolecithin, the majority of the 15,000-dalton IgE-binding factor formed by the cells gained affinity for lentil lectin, and this factor selectively potentiated the IgE response. The glycosylation-enhancing factor, which was formed by stimulation of normal spleen cells with lymphocytosis-promoting factor (LPF or pertussigen), also switched 23B6 cells from the formation of IgE-suppressive factor to the formation of IgE-potentiating factor. It was also found that the 30,000-dalton "inactive" IgE-binding factor, formed by both 23B6 and 23A4 cells, gained the ability to potentiate the IgE response, when the cells were cultured with IgE in the presence of glycosylation-enhancing factor. The results indicate that IgE-potentiating factor and IgE-suppressive factor share common precursors, and that biologic activities of IgE-binding factors are decided by their carbohydrate moieties. Incubation of the two hybridoma cells with lysolecithin or glycosylation-enhancing factor results in an increase in the proportion of FC epsilon R+ cells, suggesting that the assembly of N-linked oligosaccharide to precursor molecules is intrinsic for the expression of FC epsilon R.     

Comparative evaluation of seven cell collection devices for cervical smears

OBJECTIVE: To compare the most commonly used cervical sampling devices. STUDY DESIGN: We examined seven cytology sampling devices (Cytobrush, Cervex brush, Szalay spatula, Papex spatula, WrGKK spatula [main social security agency in Vienna], cotton swab and loop). Eight hundred smears were assessed for even distribution of cells, percentage of slide surface covered with cells, and presence and number of endocervical cells. RESULTS: Even distribution of cells was best with the WrGKK spatula. Percentage of slide surface covered with evaluable cells was best with the Cytobrush. Highest ranking for the presence of endocervical cells was found for the Cytobrush. Cotton swabs and loop showed inferior results in all categories. CONCLUSION: The use of cervical cell sampling devices showing the best cytologic results improves the interpretation and validity of cervical smears. Our results suggest that cotton swabs and loops should not be used for cervical cell sampling.     

Nonuniform behavior of multiple isoactins in the same cell is a cell-dependent phenomenon

The functional significance of multiple isoactins in the same cell is still not understood. To address this question, we examined the response of smooth muscle and cardiac muscle alpha-isoactins to a serial extraction procedure applied to both muscle and nonmuscle cell types. We compared these extraction results with results obtained with the beta- and gamma-nonmuscle actin isoforms from the same cells. In differentiated BC3H1 nonfusing muscle cells (smooth muscle alpha-isoactin), in human rhabdomyosarcoma cells (cardiac alpha-isoactin), and in chick skeletal muscle cells (cardiac alpha-isoactin), different fractions were found selectively enriched in either the nonmuscle or the muscle-specific actin isoforms compared with their relative abundance in whole cell extracts. Conversely, when these same isoactins were examined either in undifferentiated BC3H1 cells or in mouse nonmuscle cells stably transfected with a cardiac alpha-isoactin gene, no enrichment of these isoforms above their relative abundance in whole cell extracts was observed. These results indicate that within the muscle or muscle-like cells examined, the different actin isoforms were either selectively utilized or localized. These results further show that isoactin-specific responses observed were apparently related to the cell type in which they were found and not to differences in inherent physical properties such as solubility of the different isoactins examined.     

Studies on the mechanism of antibody-mediated enhancement of Getah virus infectivity

Inoculation on BHK-21 cells with Getah virus sensitized with hyperimmune homologous mouse antiserum resulted in higher infective titers than those obtained with non-sensitized control virus. This phenomenon was not observed with Vero cells. Experiments were carried out on the mechanism of this enhancement with the following results. When BHK-21 cells were pretreated with a mixture of UV-irradiated virus and antiserum before inoculation, the enhancement of infectivity of Getah virus was markedly decreased. IgG antibody against Getah virus which had been digested with 1--2% of pepsin did not show any enhancing activity. Sensitized sheep erythrocytes adhered to monolayered cultures of BHK-21 cells. These results indicate that the appearance of enhancing activity of a complex of the virus and antibody is closely related with the existence of a receptor for the Fc part of IgG on ordinary tissue culture cells, BHK-21 cells.     

桔梗胚乳吸器的细胞化学研究

对桔梗的胚乳吸器进行了细胞化学研究,结果显示,胚乳吸器的细胞质、胚乳吸器周围解体的珠心细胞和珠被细胞均呈强ＰＡＳ正反应。随着胚乳吸器的发育,吸器附近的珠心细胞和珠被细胞中贮存的大量淀粉粒逐渐减少和消失。胚乳吸器的细胞质,尤其是与胚乳本体细胞交界处的细胞质富含蛋白质。在球形胚前期,胚乳细胞中已积累大量的蛋白质颗粒。结果表明胚乳吸器起营养物质的吸收和转运作用,向胚乳提供养料。     

Intermediate cells in the adult human pancreas. Contribution to the transformation of differentiated cells in vertebrates

Problems associated with the transformation of differentiated cells in vertebrate organisms are discussed based on electron microscopical results of intermediate cells (i.e. cells with morphological characteristics of exocrine acinar cells and endocrine cells of Langerhans' islets) in the pancreas of human adults with chronic insulin-dependent diabetes mellitus. In this context, reference is made to experimental results of Scarpelli, Rao, and coworkers relating to the occurrence of hepatocyte-like cells in the pancreas of Syrian golden hamsters (Rao and Scarpelli 1980; Scarpelli and Rao 1981; Rao et al. 1983). These observations show that exocrine acinar cells of the pancreas may, even beyond the neonatal period, become transformed, depending upon different triggering stimuli, into different endocrine islet cells, or into hepatocytes, this being accomplished either directly or by new formation of cells (regeneration) with abnormal differentiation (metaplasia). Obviously, transformation is effected through a change in the activation of gene loci: the normally stably blocked genes are partially or completely deblocked for the functions of different endocrine islets cells or hepatocytes, and the original genetic expression of exocrine pancreatic functions is blocked either partially or completely. The results presented and quoted in this paper suggest that in all differentiated cells derived from the endoderm of the foregut, such as duct cells, exocrine and endocrine pancreatic cells, and hepatocytes, functional programs are retained which can be modified in the manner quoted to enable partial or complete transformation into one or another of these differentiated cells in the adult organism.     

Effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells investigated by atomic force microscopy

Cell mechanics plays an important role in cellular physiological activities. Recent studies have shown that cellular mechanical properties are novel biomarkers for indicating the cell states. In this article, temperature-controllable atomic force microscopy(AFM) was applied to quantitatively investigate the effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells. First, AFM indenting experiments were performed on six types of human cells to investigate the changes of cellular Young's modulus at different temperatures and the results showed that the mechanical responses to the changes of temperature were variable for different types of cancer cells. Second, AFM imaging experiments were performed to observe the morphological changes in living cells at different temperatures and the results showed the significant changes of cell morphology caused by the alterations of temperature. Finally, by co-culturing human cancer cells with human immune cells, the mechanical and morphological changes in cancer cells were investigated. The results showed that the co-culture of cancer cells and immune cells could cause the distinct mechanical changes in cancer cells, but no significant morphological differences were observed. The experimental results improved our understanding of the effects of temperature and cellular interactions on the mechanics and morphology of cancer cells.     

Distribution of anti-histone-antibody-secreting cells in NZB/NZW mice

Using a histone-specific plaque assay, we examined anti-histone-antibody (AHA) production at the organ level in the autoimmune NZB/NZW strain. The spleen had the highest absolute numbers of AHA-secreting cells. High percentages of immunoglobulin-secreting cells producing AHA were characteristic of spleen and bone marrow but not lymph node. AHA-secreting cells were detected in NZB/NZW mice with elevated serum activity but not in mice with normal serum levels. Serum AHA activity correlated with the number of AHA-secreting cells in the spleen but not with the total number of immunoglobulin-secreting cells in the spleen nor with the total serum immunoglobulin level. These findings concerning the organ distribution of AHA-secreting cells contrast with results of other investigators studying autoantibodies of other specificities. Furthermore, our results suggest that AHA production does not solely result from a generalized increase in total immunoglobulin synthesis present in NZB/NZW mice.     

Lack of infectivity of the endogenous avian leukosis virus-related genes in the DNA of uninfected chicken cells.

The infectivity of the avian leukosis virus-related genes in the DNA of four genetically distinct types of chicken cells was determined. Infectious DNA of Rous-associated virus-O(RAV-O) was obtained from V- chicken cells which were experimentally infected with RAV-O and from V+tvbs chicken cells, which spontaneously produced RAV-O and were sensitive to exogenous RAV-O infection. However, infectious DNA of RAV-O was not obtained from uninfected V- chicken cells or from V+tvbr chicken cells, which spontaneously produced a low titer of RAV-O but were resistant to exogenous RAV-O infection. No detectable amplification of the RAV-O related DNA sequences in the V+tvbs cells was found by hybridization of RAV-O 125I-labeled RNA to the DNAs of V+tvbs and uninfected V- cells. These results indicate that the endogenous avian leukosis virus-related genes in uninfected V- and V+tvbr cells differ from the RAV-O proviruses in RAV-O-infected V- and V+tvbs cells. The lack of infectivity of the DNA of V+tvbr cells is consistent with the hypothesis that the endogenous RAV-O genome in V+tvbr cells is linked to a cis-acting control element, which results in its inefficient expression.     

miR-125a-5p通过靶向调控GAB2抑制乳腺癌细胞的迁移能力

miR-125a-5p可负性调节GAB2表达,抑制胶质瘤细胞的侵袭和转移。本研究旨在证明miR-125a-5p抑癌作用的普遍性,即miR-125a-5p是否可通过靶向抑制GAB2抑制乳腺癌细胞的迁移。荧光素酶实验结果显示,miR-125a-5p可特异识别GAB2的3′-UTR,抑制报告酶的表达。荧光定量PCR结果揭示,与正常乳腺上皮细胞MCF-10A比较,miR-125a-5p在乳腺癌细胞MDA231和MCF-7中的表达明显降低;与迁移能力相对较低的MCF-7细胞比较,miR-125a-5p在迁移能力较高的MDA231细胞中的表达量更低。Western 印迹结果证明,与空载体（对照）和anti-miR125a 5p转染细胞比较,转染miR-125a-5p明显抑制GAB2蛋白在乳腺癌细胞中的表达。Transwell结果显示,与空载体转染的对照细胞比较,转染miR-125a-5p的乳腺癌细胞穿过基质胶的细胞数明显减少;相反,转染anti-miR125a-5p的细胞穿过基质胶的细胞数却明显增多。上述结果提示,miR-125a-5p在正常的乳腺细胞中高表达,而在乳腺癌细胞中低表达,其表达水平与癌细胞的迁移能力和GAB2表达呈反向关系。本研究结果还提示,miR-125a-5p通过靶向负调控GAB2抑制乳腺癌细胞的迁移能力。总之,本研究证明,miR-125a-5p在肿瘤中发挥抑癌作用。     

Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose

In order to study the effect of glucose on the differentiation of cultured human colon cancer cells, a subpopulation of HT-29 cells was selected for its capacity to grow in the total absence of sugar. These cells (Glc-cells) exhibit, after confluency, an enterocytic differentiation, in contrast to cells grown with glucose (Glc+ cells), which always remain undifferentiated. The differentiation is characterized by a polarization of the cell layer with apical brush borders and tight junctions, and by the presence of sucrase-isomaltase. The differentiation of Glc- cells is reversible: the addition of glucose to postconfluent cultures of Glc- cells results in an inhibiting effect on the expression of sucrase-isomaltase; switching growing cultures of Glc- cells to the Glc+ medium for several passages results in a progressive reversion to the undifferentiated state, which is completed after seven passages. The dedifferentiation process is associated with a parallel, passage-related, increase in the rates of glucose consumption and lactic acid production, and decreases of intracellular glycogen content, which return to the values of the undifferentiated original Glc+ cells. The values of these metabolic parameters are correlated, at each passage, with the degree of dedifferentiation of the cells. When these dedifferentiated cells, after having been cultured in Glc+ medium for 20 passages, are switched back to the Glc- medium, they readily grow without mortality, and reexpress the same enterocytic differentiation as the parent Glc- cells. These results show that the capacity of this subpopulation to grow and differentiate in the absence of sugar is a stable characteristic. They further suggest that glucose metabolism interferes with the program of differentiation of HT-29 cells.