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1.
The relationship between cell size, [3H]thymidine incorporation capacity, and cell surface property of human diploid fibroblasts was investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. Small cells in late passage populations adsorbed RBCs well with the RBC coating method (in which ConA-coated RBCs are adsorbed to fibroblasts) as did large cells of this population, while small cells in early passage populations did not. The RBC adsorption capacity of rapidly dividing cells with this method differed among young, middle-aged and old cell populations. The results suggest that temporal cell size and [3H]thymidine incorporating capacity is not a measure of the division age of human diploid cells at the individual cell level. On the other hand, RBC adsorption with the fibroblast coating method (in which RBCs are adsorbed to ConA-coated fibroblasts) occurred to non-dividing cells of the populations. Thus, the increase in RBC adsorption with this method is considered to be a reflection of the increase in non-dividing cells at phase III. Our results support the hypothesis that RBC adsorption with the RBC and fibroblast-coating methods represents a cell surface marker for division age and senescence of human diploid cells, respectively, at the individual cell level.  相似文献   

2.
We have described previously how concanavalin A (conA)-coated red blood cell (RBC) adsorption to human diploid fibroblasts could serve as a marker of the in vitro aging of these cells. Since the heterogeneity in RBC adsorption and the proliferative property of young and old cell populations was observed, the correlation of this cell surface property with the proliferative behavior of individual clones was examined as a function of cell age. The RBC adsorption capacity of cells in both large and small colonies changed with the aging of the parent cell populations; the cells from early passage populations did not adsorb RBCs, but those from late passage populations adsorbed them well. Thus, the amount of RBC adsorption was not a function of colony size, but was related to the age of the culture.  相似文献   

3.
Age-related changes in cell surfaces of human diploid fibroblasts (TIG-1) were investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. When ConA-coated RBCs were adsorbed to fibroblasts (RBC coating method), the amount of RBCs adsorbed per mg of fibroblast protein increased continuously from the early phases of cell passage up through cell senescence. On the other hand, when RBCs were adsorbed to ConA-coated fibroblasts (fibroblast coating method), RBC adsorption did not occur throughout phase II and increased with the advance of phase III. [3H]ConA binding to fibroblasts, however, did not change with aging to the extent that could explain the observed changes in RBC adsorption. These age-related characteristics in RBC adsorption and [3H]ConA binding were also observed for WI38 and IMR-90 cells. In addition, SV40- and 60Co-transformed WI38 cells showed a close resemblance in their RBC adsorption capacity to early phase III cells.RBC adsorption with both the RBC and fibroblast coating methods was not a function of cell cycle phase and time spent in culture (metabolic time). Co-culturing of young cells with old or transformed cells did not affect the RBC adsorption capacity of respective cells. These results suggest that RBC adsorption with the RBC and fibroblast coating methods may represent cell surface markers for division age and senescence of aging human diploid cells.  相似文献   

4.
Aging in vitro : Growth of cultured cells from the Galapagos tortoise   总被引:6,自引:0,他引:6  
Skin biopsies from two growing and two fully-grown Galapagos tortoises, a species that lives twice as long as man, were explanted in vitro. Cellular outgrowth was more vigorous from explants of young tortoises but epithelial cells stopped dividing early in the history of all cultures, even at the optimum temperature of 30 °C. Serially subcultured fibroblasts from young tortoises divided more rapidly and achieved longer lifespans than fibroblasts from old tortoises in terms of mean population doublings and to a lesser extent, calendar time. For all cultures the lifespans exceeded those reported for human diploid fibroblasts. The results indicate that a proportionality exists between the potential (remaining) lifespan in vivo and the mitotic capacity of cultured diploid cells in vitro.  相似文献   

5.
M T Pérez  M Pinilla  P Sancho 《Life sciences》1999,64(24):2273-2283
In order to explore possibilities of using erythrocytes as carrier systems for delivery of pharmacological agents, we have studied the in vivo survival of murine carrier red blood cell populations enriched in young or old cells. Hypotonic-isotonic dialysis has been used to modify the cells as carrier systems and Percoll/albumin density gradients or counter-current distribution in aqueous polymer two-phase systems to separate them according to age. Hypotonic-isotonic dialysis produces a decrease in the red blood cell populations in vivo survival rate (from 9.5 to 7.8 days). Among the cells modified as carriers, the enriched young red blood cell populations show a higher in vivo survival (half-life 6.5-7.4 days) than populations made up of predominantly old red blood cells (half-life 4.7-6.2 days). Half-life of young or old circulating red blood cells was approximately one day longer when these cells were separated by counter-current distribution rather than by Percoll density gradients. Based on these results, hypotonic-isotonic dialysis of whole and enriched young or old red blood cell populations, with higher or lower survival rates, can be considered as a useful tool for modification of these cells as carriers. The final outcome of such changes can be translated into better control of plasma drug delivery during therapy.  相似文献   

6.
The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning 51Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.  相似文献   

7.
Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were generated against partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associated, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectin molecule were not accessible in the intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, as well as purified human plasma fibronectin. These antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro senescence. This may enable us to investigate directly the relationship between cellular aging and aging of the individual.  相似文献   

8.
The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning51Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.  相似文献   

9.
Rhodamine 123 fluorescence in populations of young and old human fibroblasts was analysed and quantified with a fluorescence-activated cell sorter (FACS). Old fibroblasts exhibited a higher mean relative fluorescence than young fibroblasts. Moreover, two distinct subpopulations were evident in the fluorescence distributions of old cells--but not in those of young cells.  相似文献   

10.
Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have not found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, countercurrent distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter. These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.  相似文献   

11.
Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have now found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, counter-current distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter.These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.  相似文献   

12.
The relationship between human aging and cell replication has been investigated using two complementary approaches: in vitro studies of human fibroblasts derived from young and old volunteer members of the Baltimore Longitudinal Study and in vivo examinations of bone marrow cell populations from young and old mice and rats. Total proliferative capacity measured as either the onset of cell culture senescence or as in vitro life span was significantly diminished in cell cultures derived from old human donors when compared to parallel cultures established from young donors. Acute replicative abilities as measured by percent replicating cells, cell pupulation doubling time, cell number at confluency, and colony size distribution were also significantly decreased in human old cell populations. An in vivo cytogenetic technique for measuring cell replication was developed utilizing the differential staining properties of metaphase chromosomes of cells that have replicated in the presence of bromodeoxyuridine. With this technique, cell cycle times have been derived in vivo as well as in vitro. Preliminary in vivo results in both mice and rats indicate that cell replication is slowed in old animal cell populations. Further research will be directed both in vitro and in vivo at discerning the mechanisms for this impairment of cellular replication with aging.  相似文献   

13.
Glycophorins extracted from membranes of young and old human red blood cells have within an error of +/- 1.5% the same sialic acid content when referred to a relative measure of the number of glycophorins. The degree of surface iodination in glycophorins, which was shown to be the same in young and old cells, served as this relative measure. This finding implies that senescent human red blood cells hardly reveal desialylated surface proteins (less than or equal to 3%). However, the sialic acid content per cell was repeatedly reported to be 10 to 15% lower in old than in young cells. Therefore, we conclude 1) that human red blood cells lose intact glycophorin together with membrane during red blood cell senescence, and 2) that removal of desialylated and senescent red blood cells from the circulation proceeds by different routes.  相似文献   

14.
We have used two Chinese hamster ovary subclones whose surface phenotype has been extensively investigated with regard concanavalin A-mediated cell-cell agglutination and concanavalin A-induced receptor site clustering to investigate what changes in membrane composition, if any, can be correlated with the concanavalin A-detected changes in surface phenotype. These cell clones are uniquely disposed for this purpose since maintenance of the cells under different growth conditions produces changes in agglutinability and receptor site mobility in one cell clone (H-7W) but not the other (K-1). After extensive characterization of the surface membranes of these two subclones we have been unable to identify any change in the membrane peptides, glycopeptide, cholesterol, or fatty acid composition which can be directly correlated with the concanavalin A-detected surface phenotypes. It is of particular interest to note that we have been unable to correlate the presence or absence of the large external transformation-sensitive glycoprotein with the relative mobility of the lectin receptors or with the degree of concanavalin A-mediated cell agglutination. Furthermore we have been unable, in this system, to corroborate earlier data suggesting a role for cholesterol in determining the relative mobility of the lectin receptors. Thus using a cell system consisting of genetically matched cell clones, we have been unable to identify any changes in the biochemical composition of the plasma membrane which might be associated with the surface phenotypes detected by concanavalin A.  相似文献   

15.
Some membrane characteristics of normal and Rauscher leukemia virus (RLV)-infected mouse red blood cells (RBC) were compared, both with regard to total populations and young and old groups of cells. Osmotic fragility, density distribution of cells and agglutinability by poly- -lysine (pLys), concanavalin A (ConA), phytohemagglutinin (PHA) and soybean agglutinin (SBA), were examined. RBC from RLV-infected mice were agglutinated at a higher rate and to a higher degree than normal mice RBC by pLys and by the lectins PHA and ConA. These RBC were generally osmotically more resistant and contained a young cell population of unusually high specific gravity. Comparison of RBC from RLV-infected mice with old RBC from normal mice showed some common membrane characteristics. Similarly to old RBC, RBC from RLV-infected mice have a high specific gravity and high agglutinability by pLys. However, they differ in that the RBC from RLV-infected mice are osmotically more resistant and are agglutinated by ConA; they are also agglutinated at a higher rate by PHA.  相似文献   

16.
The B subunit of cholera toxin, a protein which binds specifically to cell surface ganglioside GM1, has been shown to have a bimodal effect on DNA synthesis in Swiss 3T3 fibroblasts. The B subunit induced cellular proliferation of confluent and quiescent cells while it inhibited the growth of the same cells when they were sparse and rapidly dividing. The amount of cell surface GM1 increased when the cells reached confluency. To examine the hypothesis that the variation in levels of GM1 was responsible for the bimodal effect, we increased GM1 levels in rapidly dividing cells by insertion of exogenous GM1 or by treatment of the cells with neuraminidase to convert polysialogangliosides to GM1. Even after the level of GM1 was increased to levels similar to those found in confluent cells, the B subunit still inhibited, rather than stimulated, their growth. Therefore, this result indicates that the bimodal response to the B subunit is not solely a function of the concentration of cell surface GM1; rather it is the growth stage that determines the fate of the signal transduced by the interaction of the B subunit and ganglioside GM1.  相似文献   

17.
The capability of the bone marrow (BM) to generate new B cells in aging was studied in vitro. BM cells from old (26 to 30 mo) or young (3 mo) BALB/c and (C3H/eB x C57BL/6)F1 mice were depleted of mature B cells and these surface Ig (sIg) BM cells were incubated in culture for 3 days. The frequency of newly generated B cells in these cultures was determined by assessing the frequency of slg+ cells and of B cells forming colonies in agar and by assaying the proliferative capacity of these newly generated B cells after stimulation by LPS. We found that BM cells from aged mice are significantly inferior to young ones in their capability to generate new B cells in culture. By mixing old and young slg- BM cells, we found that, in general, this reduction was not caused by a suppressive effect of T cells or of any other cells, but rather to lack of some sort of supportive cell or factor in the aged BM. In addition, we found that the frequency of cells expressing the B220 surface molecule, a B lineage-specific marker, is significantly reduced in aged BM. These results indicate that a quantitative decrease in B cell progenitors combined with changes in cell populations that are important in supporting B cell generation contribute to the age-related decrease in the capacity to generate B cells.  相似文献   

18.
We have investigated the relationship of concanavalin. A binding to the cell surface of normal and transformed cells and the subsequent agglutination of the transformed cells. At room temperature almost no differences could be detected in agglutinin binding between transformed and untransformed cells. At 0°C, however, where endocytosis was negligible, the transformed cells bound three times more agglutinin. However, transformed cells and trypsin-treated normal cells do not agglutinate at 0°C although the amounts of agglutinin bound at 0°C are sufficient to permit agglutination when such cells are shifted up to room temperature. Both transformed and trypsin-treated normal cells show a marked increase in agglutination at 15°C as compared to agglutination at 0°C. From this, as well as the observation that mild glutaraldehyde fixation of the cell surface inhibited agglutination but not agglutinin binding, it was concluded that concanavalin A-mediated cell agglutination requires free movement of the agglutinin receptor sites within the plane of the cell surface.  相似文献   

19.
The roles of ornithine decarboxylase (ODC, EC 4.1.1.17) and polyamines in cellular aging were investigated by examining serum-induced changes of these parameters in quiescent IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL) and in human progeria fibroblasts. Serum stimulation caused increases of ODC and DNA synthesis in IMR-90 human diploid fibroblasts, with maximal values occurring, respectively, 10 hr and 22 hr after serum stimulation. Both serum-induced ODC activity and DNA synthesis in IMR-90 cells were found to be inversely related to their PDL. Maximal ODC activity and DNA synthesis in young cells (PDL = approximately 18-22) were, respectively, five-fold and six-fold greater than that in old cells (PDL = approximately 50-55), which in turn were comparable or slightly higher than that in progeria fibroblasts. Polyamine contents (putrescine, spermidine, and spermine) in quiescent IMR-90 cells did not show significant PDL-dependency. The putrescine and spermine contents in quiescent progeria cells were comparable to those in quiescent IMR-90 cells. The spermidine content in quiescent progeria cells, however, was extremely low, less than half of that in quiescent IMR-90 cells. Serum stimulation caused a marked increase in putrescine content in young cells but not in old cells or in progeria cells. The spermidine and the spermine content in IMR-90 cells, either young or old, and in progeria cells did not change significantly after serum stimulation. Our study indicated that aging of IMR-90 human diploid fibroblasts was accompanied by specific changes of polyamine metabolism, namely, the serum-induced ODC activity and putrescine accumulation. These changes were also observed in progeria fibroblasts derived from patients with Hutchinson-Gilford syndrome.  相似文献   

20.
Energy metabolism in cultured human fibroblasts during aging in vitro   总被引:1,自引:0,他引:1  
To explore the relationship between energy metabolism and the limited replicative life span of cultured human fibroblasts, we studied several bioenergetic parameters in normal fibroblasts at early passage (young cells) and at late passage (old cells) and early passage cells from a subject with the Hutchinson-Gilford (progeria) syndrome. Old cells consumed more glucose and produced more lactate during growth, but O2 consumption, both basal and following maximum uncoupling of oxidative phosphorylation by SF-6847, was the same as in young cells. Progeria cells produced the most lactate but did not consume more glucose, while their basal and uncoupled O2 consumption was similar to that of young and old cells during both log and confluent states. Consumption of glutamine, a source of both oxidative energy and lactate, was approximately the same in all three cell types as was 14CO2 production from 2- 14C-pyruvate and 5- 14C-glutamate. ATP and ADP concentrations were similar in all cell types with a rise in the ATP/ADP ratio during growth from log to confluent state. Thus, old and progeria cells, in contrast to young cells, produce more lactate during growth consistent with a rise in energy demand and/or inefficiency of oxidative phosphorylation. Although limitations in total energy output do not appear to be causal to the loss of replicative capacity in normal cells after serial passage, they could play a role in the curtailed replicative capacity of progeria cells.  相似文献   

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