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1.
Nucleotide pool sizes, DNA polymerizing enzymes, and DNA synthesis were studied in mouse thymocytes over a 24-hr period of culture in Marbrook chambers. The initial rate of cell division was high with an average mitotic index of 1.6%/hr for 12 hr, followed by a decline to 0.14%/hr at 24 hr. The decline in DNA synthesis was not closely correlated with the activities of DNA-dependent DNA polymerases-α or -β or with the activity of terminal deoxynucleotidyl transferase. The amounts of several ribo- and deoxyribonucleotides per thymocyte were measured using high performance liquid chromatography and DNA polymerase. Pool sizes of ATP, GTP, dATP, and dTTP were less than 10% of pool sizes commonly observed in mammalian cells. The rates of DNA and RNA synthesis in thymocytes may be critically affected by minor changes in the availability of nucleotides. Cultures of thymocytes serve as useful experimental systems for investigation of nucleotide and nucleic acid metabolism during lymphoid differentiation.  相似文献   

2.
The in-vitro proliferation kinetics of young rabbit articular chondrocytes were compared in primary culture and at the first passage. The growth curves labelling and mitotic indices, percentage labelled mitosis (PLM) curves and DNA content distributions by flow-microfluorometric analysis during a 7-day growth period were determined in both cases. The length of the cell cycle and the doubling time calculated from the exponential part of the growth curve were quite similar: Tc = 19 hr and Td = 20 hr for the primary culture, Tc = 17 X 3 hr and Td = 20 hr for the first passage. However, the growth curve and the DNA distribution during the 7-day period showed some differences. The duration of the lag period studied by the growth curve was longer in the primary culture than at the first passage. This phenomenon was also observed using the FCM analysis. The growth fraction determination on the second day of culture was in accordance with the lower proliferation capacity of the cells in primary culture. These data suggest that it would be better to study growth kinetics and drug modifications in articular chondrocytes at the first passage than in primary culture.  相似文献   

3.
Mitotic activity and cell proliferation of newt ( Triturus pyrrhogaster ) embryo were examined with special reference to primary induction.
Mitotic activity of gastrula ectoderm gradually decreases during gastrulation. The ectoderm, which is isolated from mid-gastrula (stage 12b) and cultured in vitro , also shows gradual decrease in mitotic activity during cultivation and the mitotic activity steeply decreases after 48 hr.
The ectoderm cultured with heterologous inductor (GPL-extract) shows a temporal suppression in mitotic activity. The ectoderm of the whole gastrula also shows a regional suppression where it is in contact with the chorda-mesoderm.
The number of the ectodermal cells increases about 2 times after 24 hr culture and to more than 3 times after 48 hr culture. Accordingly it is certain that the majority of the ectodermal cells divides at least one time in the course of 48 hr.
Histological examination of the ectoderm cultured together with the inductor reveals that differentiation of undifferentiated ectoderm to neural tissues is accomplished at least within 48 hr after cultivation with the inductor.
The present examination shows the possibility that the mitotic activity of the ectoderm may be temporarily suppressed by the inductor and that it then decreases along with neural cell differentiation after recovery of the activity.
The results also suggest that the determination of undifferentiated ectoderm to neural tissues occurs before the second cell division after the contact with the inductor and the events occurring during the first cell cycle after activating by the inducing stimulus are critical for the primary induction.  相似文献   

4.
A single subcutaneous injection of the active androgen DHT (5 alpha-androstan-17 beta-ol-3-one or 5 alpha-dihydrotestosterone; 5 mg/Kg body weight) into rats 7 days after orchidectomy stimulated DNA synthesis in the seminal vesicle, which began between 30 and 35 hr after injection and peaked at 48 hr. Mitotic activity began between 35 and 45 hr after DHT injection and peaked around 55 hr. These proliferative responses appeared to be confined mainly to the epithelial cells of the seminal vesicles. The DHT injection did not rapidly (between 5 min and 2 hr) increase the cyclic AMP level, but it did result in two small later surges of the cyclic nucleotide; the first of which peaked between 18 and 28 hr, but well before the onset of DNA synthesis, and the second of which peaked between 48 and 60 hr, when both the DNA-synthetic and mitotic activities were near or at their peak values.  相似文献   

5.
The response of thymic lymphocytes of the pig to phytohemagglutinin was studied with H3 thymidine in cultures, from 0–72 hours. At the beginning of the culture period 6–18% of lymphocytes were in DNA synthesis. during the first 24 hours a sharp decrease in the number of DNA synthesizing cells was observed in both pha and control cultures, although pha cultures consistently showed small but significantly greater numbers of DNA synthesizing cells. this was followed by a definite peak in DNA synthesis and mitotic response of a minority of the cells in pha cultures between 48–54 hours, whereas in control cultures activity ceased. in addition, a small proportion of the progeny of initially DNA synthesizing medium sized lymphocytes was apparently stimulated by pha and found in mitosis by 48 hours. It was concluded that the thymus contains a fraction of lymphocytes, not in the mitotic cycle, which are capable of being transformed by pha to mitotic activity. the data also suggests some stimulation of cells already in the mitotic cycle.  相似文献   

6.
When deprived of exogenous nutrients some amoebas of Dictyostelium discoideum do continue to progress through the cell cycle. There are two distinct periods when mitotic cell division occurs. Labeling studies show that during the first period, which begins at the onset of development and ceases at the first visible signs of aggregation (rippling), only those cells which are beyond a certain point in G2 at the initiation of development divide. The second period of mitotic activity begins at tip formation, reaches maximum activity at the grex stage, and ceases during early culmination. Significantly, examination of the development of amoebas harvested when in the stationary phase of growth (and thus arrested in G2) shows that these cells still undergo mitotic cell division during the second period but do not show any such division during the preaggregation phase. The extent to which increases in cell number can be taken to be indicative of mitotic cell division varies from one culture to another due to the presence of variable numbers of multinucleate cells which become mononucleate during the first 10 hr of development. However, when due allowance has been made for the existence of these cells in axenically growing amoebal populations, our data show that by completion of fruiting body construction there has been a doubling in cell number as a direct result of mitotic cell division. Nuclear DNA synthesis also occurs at two distinct periods during development, these coinciding with the periods of mitotic activity. However, since no more than 35% of the cells have undergone nuclear DNA synthesis by the end of the developmental phase, our results are inconsistent with the conclusion that all cells accumulate at a position in G2 at the time of aggregation. Our results do suggest, however, that mitotic cell division of a fraction of the cells may be an integral part of the developmental phase.  相似文献   

7.
Isolated cells from the siliceous sponge Geodia cydonium as well as small primary aggregates (diameter: 70 mum) consisting of them show no increase in rates of programmed syntheses and mitotic activity with time. After addition of a highly purified aggregation factor to a culture with primary aggregates which subsequently form secondary aggregates (diameter: larger than 1000 mum), a dramatic increase of DNA, RNA and protein synthesis occurs. Together with this increase, the cells show a high mitotic activity. The values for the mitotic coefficient reach a first maximum 8 h after the beginning of the secondary aggregation process. The stimulation of the mitotic activity of cells during the aggregation factor induced secondary aggregation process can be suppressed by inhibitors of RNA and protein synthesis as well as by a blocker of DNA synthesis. This finding may indicate that cells from the G0-population enter the proliferating cell pool via the G1-phase.  相似文献   

8.
DNA synthesis of adult mammalian cardiac muscle cells in long-term culture   总被引:1,自引:0,他引:1  
A C Nag  M Cheng 《Tissue & cell》1986,18(4):491-497
Adult rat cardiac ventricular muscle cells were isolated and cultured in monolayer for 30-45 days. Most of the cardiac muscle cells undergo external and internal structural alterations, resembling embryonic/neonatal cardiac muscle cells in culture (Nag and Cheng, 1981; Nag et al., 1983). These cultured cells underwent DNA synthesis and mitosis as revealed by autoradiography studies that involved the exposure of the cells to [3H]-thymidine for 24 hr prior to the termination of the culture at selected intervals. During the first week of culture, cardiac muscle cells showed less than 5% labeled cells. The labeling index of myocytes attained a peak in the second week of culture, exhibiting approximately 23% labeled cells. The labeling indices of cardiac muscle cells declined over the period of 30 days of culture. During the end of the incubation period, approximately 4% of the myocytes were labeled. When the extent of the total cell population involved in DNA synthesis was examined by exposing the cells to [3H]-thymidine continuously for long periods of time, it was observed that approximately 26% of the cardiac muscle cells regained the capacity for DNA synthesis during 1-10 days of culture. From day 1 to day 14, approximately 29% of the total muscle cell population was labeled. When the cells were exposed to the radioactive isotope continuously for 30 days, approximately 31% of the cells incorporated radioactive isotope, showing their capacity for DNA synthesis. Approximately 90% of the cardiac muscle cells in long-term culture contained more than one nucleus. The nuclei were often observed in multiples of two. Labeled mitotic apparatus was observed in cardiac myocytes, indicating the replication of DNA, followed by karyokinesis.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

9.
The in vitro life span of murine spleen lymphocytes stimulated by endotoxin (LPS) was determined. Lymphocytes synthesizing DNA spontaneously in culture and those stimulated to DNA synthesis early (24 hr) and later (48 hr) in culture by LPS had half-lives of approximately 24 hr. The continuing presence of LPS in culture did not prolong cell longevity nor did free LPS have to be present to allow successive rounds of DNA synthesis in committed cells. Once activated to DNA synthesis, blast cells and lymphoblast-like cells did not revert to small lymphocytes.  相似文献   

10.
Using the computer-assisted method of smoothed spatial averaging, spatial and temporal patterns of cell distribution and mitotic activity were analyzed in the cranial mesenchyme underlying the mesencephalic neural folds of mouse embryos maintained in roller tube culture. Total cell density increased in central and medial mesenchymal regions after 12 hr in culture, decreased after 18 hr, and showed a further decrease after 24 hr when the neural folds of the embryos had elevated, converged, and were fusing or fused. Mitotic activity, as measured by the ratio of 3H-thymidine-labeled cells to unlabeled cells, was highest in the central mesenchyme at all culture times. Embryos were also cultured in the presence of diazo-oxo-norleucine (DON), which inhibits glycosaminoglycan and glycoprotein synthesis. After 24 hr in culture, neural folds of DON-treated embryos had failed to elevate. Total cell density increased in central and medial regions of the mesenchyme of DON-treated folds at 12 hr but showed no significant decrease in these regions with further culture. Mitotic activity was highest in the central mesenchyme of these treated embryos. These results suggest that cell distribution patterns observed in the cranial mesenchyme during neural fold elevation in normal cultured embryos are not produced by regional differences in mitotic activity. Rather, we propose that cell distribution patterns in the central and medial regions of the mesenchyme result from expansion of a glycosaminoglycan-rich extracellular matrix that disperses cells from these regions and decreases their density. In DON-treated embryos, in which expansion of the mesenchyme is prohibited by the decreased glycosaminoglycan and glycoprotein content of the extracellular matrix, mitotic activity apparently determines these patterns.  相似文献   

11.
Intravenous infusions of hydroxyurea were established in mice and maintained for periods up to 48 hr. The influence of different rates of hydroxyurea infusion on the number of viable cells gathered in S phase was studied in eight different mouse tissues. An infusion rate which was sufficiently slow not to block thymidine incorporation completely, resulted in gathering of cells in S phase while offering some protection against hydroxyurea-induced cell death. The duration of the period of DNA synthesis following release from hydroxyurea inhibition appeared to be shortened in some tissues. After the release of hydroxyurea blockades maintained for 12-24 hr, each of the tissues showed sharp increases in mitotic activity and peak mitotic index values were as much as twenty times greater than values found in tissues of control animals. An important finding was that the time of maximal mitotic activity for different tissues after release of blockade could differ by many hours.  相似文献   

12.
Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ~80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ~70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G0 phase, but that when they formed monolayers, they progressed to the G1 phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions. © 1993 Wiley-Liss, Inc.  相似文献   

13.
The growth of embryonic chick cardiac myocytes and fibroblasts in tissue culture was evaluated by the kinetics of nuclear labeling during continuous exposure to [3H]thymidine. The fraction of mitotically active cells, the mean intermitotic period and the population doubling times were determined in each cell type during 3 weeks in culture. After 24 hr in culture, 90% of the muscle cells were mitotically active with minimal population doubling times of 65 hr. By 17 days in culture only 5% of the myocytes continued to divide with population doubling times greater than 3000 hr. Primarily, the lengthening of doubling times was due to a withdrawal of cells from the mitotic cycle and much less to a lengthening of the intermitotic period. Growth of cardiac muscle cells from embryonic hearts from 4 to 10 days of development was also compared. Muscle cells from younger hearts displayed greater mitotic activity than those from older hearts at equivalent times in culture.  相似文献   

14.
In cultures of duck anterior pituitaries, the synthesis and renewal of the specific secretory protein prolactin and of total newly synthesized tissue proteins were studied. As concerns prolactin, assay of the tissue and culture media hormone content demonstrates de novo synthesis of prolactin in vitro at a constant rate during at least 2 wk. The prolactin content after 1 wk and after 2 wk of culture is the same and is similar to the initial content. The renewal time of this prolactin can be estimated at 28 or 48 hr. As concerns total proteins, the use of a chase after a short pulse of 5 min in the presence of tritiated L-leucine demonstrated that newly synthesized proteins are excreted into the culture medium from 30 min to 1 hr after the beginning of the chase. Therefore, the synthesis and excretion of proteins are two discontinuous phenomena. The migration rate of the total proteins was slower than that of prolactin, indicating that this hormone does not represent more than about half of the newly synthesized proteins. These conclusions are in good agreement with those based on high resolution radioautographic data previously obtained on the same material.  相似文献   

15.
Summary Secondary platings of chick embryo fibroblast cells were found to be capable of subsisting in the absence of serum supplements for periods exceeding 1 week. No significant mitotic activity was detected in these serumdeprived cultures. When serum was added, a coordinated mitotic burst occurred between 12 and 14 hr after the serum addition had been made. A complete mitotic burst could also be obtained with an abbreviated serum exposure of only 6 hr. Patterns of DNA and RNA synthesis were investigated following serum addition to serum-deprived cultures or serum removal from normal cultures. The results obtained are commensurate with mitotic-specific serum stimulations which fall within the G1 growth phase. This investigation was supported by USPHS research grants CA 04774 and CA 05619 from the National Cancer Institute.  相似文献   

16.
The requirement for DNA synthesis in the induction of cytolytic T lymphocytes (CTL) by alloantigens has been investigated. C57BL/6 splenic T cells purified by passage on nylon wool columns were stimulated in vitro in mixed leukocyte culture (MLC) and assayed for cytotoxicity against 51Cr-labeled target cells. With this system, CTL activity was detectable after 24 hr of MLC and reached high levels after 48 hr. Addition of cytosine arabinoside (ARA-C) or hydroxyurea to such cultures at concentrations that were sufficient to inhibit DNA synthesis by greater than 98% did not reduce CTL activity measured after 24 hr; however, the increase in activity that occurred between 24 and 48 hr in control cultures was strongly reduced (or abolished) by these drugs. Velocity sedimentation analysis of MLC cells activated for 48 hr in the presence of ARA-C further revealed that CTL precursor lymphocytes had enlarged into medium- to large-sized CTL under these conditions. These studies provide direct evidence that the primary induction of CTL by alloantigens can be dissociated into a differentiation step, which occurs within 24 hr in the absence of DNA synthesis and is accompanied by blast transformation, and a subsequent proliferation.  相似文献   

17.
The action of cyclohexamide (in doses of 1 and 10 mkg/ml in the course of 24 hours) on porcine embryo kidney cells in culture is accompanied by a powerful suppression of protein and DNA synthesis, mitotic activity, a weaker suppression of RNA synthesis, a lowering of the activity of succinate-, lactate- and alpha-glycerophosphate-dehydrogenase, which leads to disorders in the ultrastructure of the cells. After incubation of cells in a fresh medium (in the course of 18, 24 hours) there occurs a total restoration of the ultrastructure of the nuclei, granular endoplasmatic reticulum, mitochondria, due to the repairing and strong intensification of synthetic processes, respiration and glycolysis, mitotic activity of the cells.  相似文献   

18.
Hepatocyte DNA synthesis, initiated by epidermal growth factor (EGF), is reversibly inhibited by 2% dimethyl sulfoxide (DMSO). At that concentration, both the survival of the cells in culture and the expression of differentiated functions are prolonged. DMSO does not affect thymidine uptake or EGF receptor binding. Moreover, EGF receptor binding is maintained at 84% of initial 12 hr binding when cells are cultured for several days in the presence of DMSO, whereas specific receptor binding declines to 49% of initial binding under standard culture conditions without DMSO. Studies of hepatocyte functional activity indicate that, during early culture, total cellular export protein synthesis, specific albumin synthesis, and glycogen synthesis are enhanced in the presence of DMSO. Dexamethasone is required for the effect of DMSO on survival, and although dexamethasone alone enhances hepatocyte DNA synthesis in the presence of EGF, it does not reverse the inhibitory effect of 2% DMSO on DNA replication. The correlation of prolonged survival with growth inhibition supports the hypothesis that hepatic growth and differentiated functional activity may be reciprocally regulated.  相似文献   

19.
《Biochemical medicine》1976,15(3):246-253
Spontaneous DNA synthesis observed in freshly prepared tonsil lymphocytes dropped to zero in about 6 hr during culture. This stop of DNA synthesis was followed by a decrease in the mean diameter of cells and by the disappearance of large lymphocytes and blasts. Thus, the originally heterogeneous tonsil lymphocyte population was converted into homogeneous, resting cells, which could be stimulated with PHA. The difference in cell diameter between the control and PHA-stimulated cultures was statistically significant. Both the spontaneous and the mitogen-induced DNA syntheses were fully inhibited by 10−5m arabinosyl cytosine. The [3H]thymidine incorporation was correlated with the DNA polymerase activity of both freshly prepared and stimulated cells. In PHA-stimulated cells there was a fourfold increase in DNA polymerase activity.  相似文献   

20.
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