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1.
Human diploid cells (CF-1) derived from newborn foreskin tissue were maintained in a non-mitotic state for as long as 177 days by reducing the serum concentration of the incubation medium to 0.5%. The cells could be returned to the proliferative state by subcultivation with normal growth medium containing 10% serum. Cells treated in such a manner reached passage levels equivalent to controls that had been continuously cultured on growth medium, but they took a proportionately longer calendar time to achieve the equivalent passage levels. Also, by using 3H-thymidine incorporation, cells held in the non-mitotic conditions showed a longer ‘predictable life span’ than control cultures. During 21-day maintenance periods there was a 10–20% cell loss and ca 30% loss of protein per cell. The finite life span of these human diploid cells was clearly related to the number of cumulative population doublings rather than to the total calendar time in vitro. 相似文献
2.
Chick embryo fibroblasts were maintained at confluency for up to 35 days in medium containing 0.5% or 0.75% fetal bovine serum or 2.5% or 5.0% horse serum. At weekly intervals cells were subcultured and serially propagated in medium containing 10% FBS until their replicative lifespans were completed. The results showed that the replicative lifespan of embryonic chick fibroblasts was dependent on the cumulative number of population doublings undergone by the culture and was not related to the calendar time cells were in culture. Further characterization of 0.75% FBS maintained chick cells returned to 10% FBS medium showed that cells had protein contents and incorporated 3H-thymidine into DNA at a rate that resembled that of young cells, despite an advanced chronological age. 相似文献
3.
W. H. J. Douglas W. L. Whittle R. T. Dell'orco 《In vitro cellular & developmental biology. Plant》1976,12(1):74-82
Summary Human diploid fibroblasts can be maintained in vitro in an arrested, essentially nonmitotic state for extended periods of
time by reducing the serum concentration in the medium from 10 to 0.5%. Arrested cells can be induced to re-enter the proliferative
state by subcultivation in medium containing 10% serum. Fine structure, acid phosphatase, cytochrome oxidase, and extracellular
carbohydrates in arrested cells were examined and compared to cultures growing in 10% serum and to cells transferred to 10%
serum after 21 days in 0.5% serum. Cells in 10% serum possessed a well-developed Golgi complex, extensive rough endoplasmic
reticulum, mitochondria containing transverse cristae, and many free ribosomes in the cytoplasm. In arrested cells, Golgi
complexes were rarely observed, the number of both free and membranebound ribosomes was reduced, the number of cristae per
mitochondrion was decreased and the amount of demonstrable cytochrome oxidase activity was diminished. There was an accumulation
of intercellular carbohydrate components. After subcultivation with medium containing 10% serum, arrested cells regained the
ultrastructural characteristics of cells continuously cultured at this serum level; however, the amount of intercellular carbohydrate
remained elevated. These results indicate that distinct yet reversible changes occur in the subcellular morphology and organization
of cells maintained in an essentially nonmitotic state. This arrested state may be a close approximation to the situation
as it occurs in vivo in expanding cell populations. 相似文献
4.
Human diploid fibroblasts can be maintained in vitro in an arrested, essentially nonmitotic state for extended periods of time by reducing the serum concentration in the medium from 10 to 0.5%. Arrested cells can be induced to re-enter the proliferative state by subcultivation in medium containing 10% serum. Fine structure, acid phosphatase, cytochrome oxidase, and extracellular carbohydrates in arrested cells were examined and compared to cultures growing in 10% serum and to cells transferred to 10% serum after 21 days in 0.5% serum. Cells in 10% serum posessed a well-developed Golgi complex, extensive rough endoplasmic reticulum, mitochondria containing transverse cristae, and many free ribosomes in the cytoplasm. In arrested cells, Golgi complexes were rarely observed, the number of both free and membrane-bound ribosomes was reduced, the number of cristae per mitochondria was decreased and the amount of demonstrable cytochrome oxidase activity was diminished. There was an accumulation of intercellular carbohydrate components. After subcultivation with medium containing 10% serum, arrested cells regained the ultrastructural characteristics of cells continuously cultured at this serum level; however, the amount of intercellular carbohydrate remained elevated. These results indicate that distinct yet reversible changes occur in the subcellular morphology and organization of cells maintained in an essentially nonmitotic state. This arrested state may be a close approximation to the situation as it occurs in vivo in expanding cell populations. 相似文献
5.
Population doublings versus time in culture were compared in human postnatal skin fibroblasts from normal donors, a cancer patient, and from donors suffering from Cockayne syndrome, Ataxia telangiectasia, and Fanconi's anemia (FA). Confluent cultures were maintained in a nonproliferating state for 14 to 27 d in 0.5% serum medium. The results show that the ability of cells to resume division after a resting stage can be influenced by pathologic conditions. In arrested FA cell populations an increase of the population doublings and of the calendar time were observed. It is possible that in some cell populations the resting stage favors the expression of growth potentialities related to instability of the cells. 相似文献
6.
C. Diatloff-Zito A. Macieira-Coelho 《In vitro cellular & developmental biology. Plant》1982,18(7):606-610
Summary Population doublings versus time in culture were compared in human postnatal skin fibroblasts from normal donors, a cancer
patient, and from donors suffering from Cockayne syndrome, Ataxia telangiectasia, and Fanconi’s anemia (FA). Confluent cultures
were maintained in a nonproliferating state for 14 to 27 d in 0.5% serum medium. The results show that the ability of cells
to resume division after a resting stage can be influenced by pathologic conditions. In arrested FA cell populations an increase
of the population doublings and of the calendar time were observed. It is possible that in some cell populations the resting
stage favors the expression of growth potentialities related to an instability of the cells.
This work was supported by contract CRL 802030 from the I.N.S.E.R.M. and by Euratom. 相似文献
7.
Chick embryo fibroblasts serially propagated in media containing division ratelimiting amounts of fetal bovine serum underwent premature culture senescence as illustrated by accelerated declines in the number of cells incorporating 3H-thymidine, increased population doubling times, reduced cell densities at subcultivation, and reduced replicative life-spans compared to cells grown in medium containing non-rate-limiting amounts of serum. Low serum serially propagated “senescent” cultures returned to 10% serum containing medium had proliferative rates, incorporated 3H-thymidine, and attained saturation densities at confluency similar to younger cells. “Senescent” cells serially propagated in low serum and returned to 10% serum achieved life-spans similar to cells continuously grown in the presence of 10% serum. The results of these and other studies show that cells serially propagated in the presence of division rate-limiting amounts of fetal bovine serum, or at high inoculation densities, accumulate a substantial number of cells in the population during exponential growth conditions that are not senescent but are prevented from entering DNA synthesis becuase of mitogen limitations. Our results indicate that the amount of serum mitogen in the growth medium affects only the rate at which cells express their genetically predetermined replicative potential and not the replicative lifespan per se. These results are discussed in relation to the techniques that should be employed for studying cellular aging and the mechanism of senescent cell formation. 相似文献
8.
When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state. 相似文献
9.
Confluent cultures of human diploid fibroblasts were maintained for 28 days with medium containing 0.5% serum. Periodically during this time cells were exposed to 3H-thymidine for 72 h; harvested; and analysed by flow microfluorometric, ‘cell sorting’, and autoradiographic techniques. The results showed that cells cultured under these conditions maintain a stable population distribution similar to that occurring when a population reaches confluency in medium containing 10% serum. Low labeling indices, sparce grain densities, and the presence of some mitotic cells indicated that a limited amount of cell-cycle traverse did occur but that both the S and G2 phases were prolonged. This new state of reduced mitotic activity with prolonged cell-cycle times may mimic the long-term inhibition of cell-cycle traverse of expanding tissues in vivo. 相似文献
10.
A saline extract was prepared from Drosophila eggs. When diluted to a concentration of 1% with Drosophila tissue culture medium, it did not support growth of cells from the Drosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time four Drosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonic Drosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. 相似文献
11.
It has been postulated that superoxide dismutase (SOD) protects cells from free radical-induced damage. In these experiments SOD specific activity was measured as established human diploid cell lines from various donor ages progressed through their in vitro lifespan. Significant elevations in activity occurred during the in vitro lifespans of cells from fetal and newborn donors, but no change in activity was detected during the lifespan of cells from an adult donor. In addition, a direct relationship between enzyme activity and donor age was detected with the following relative activities: adult greater than newborn greater than fetal. The possible relationship between these findings and the free radical theory of aging is discussed. 相似文献
12.
兔胚胎神经干细胞的分离、培养和鉴别 总被引:2,自引:0,他引:2
目的:研究兔胎脑神经干细胞体外生长特性,为探讨神经干细胞的临床应用及神经系统的发育奠定基础。方法:采用含碱性成纤维细胞生长因子(bFGF)和表皮细胞生长因子(EGF)的N2无血清培养技术,取18天龄兔胚胎脑组织,分离神经干细胞,并观察分离的细胞体外培养、增殖、分化潜能,免疫组化鉴定。结果:从18天龄兔胎脑皮质和纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈半贴壁状态生长,形成神经球,可传代。细胞呈Nestin免疫反应阳性;在含血清培养基中培养时则分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论:来自兔胎脑神经干细胞能在体外培养、增殖并保持传代能力。无血清N2EGF、bFGF培养基有利于兔胎脑神经干细胞的存活和增殖,含血清培养基能诱导兔胎脑神经干细胞分化。 相似文献
13.
Oxygen concentration regulates the proliferative response of human fibroblasts to serum and growth factors 总被引:5,自引:0,他引:5
This report demonstrates that oxygen concentration within the physiologic range of 2.5 to 20% controls the pattern of proliferation of human diploid fibroblasts by modulating their response to serum and purified growth factors. Reducing oxygen concentration from 20 to 2.5% increased the division rate and final density of fibroblasts cultured in serum-containing medium. DNA synthesis in response to serum, as well as to EGF and PDGF, was enhanced significantly. Exposing quiescent cells to reduced oxygen enhanced serum-induced DNA synthesis in a time-dependent manner. The stimulatory effect persisted when the oxygen concentration was raised to ambient levels before the addition of serum. These results suggest that oxygen concentration within the physiologic range may control proliferation indirectly by altering the activity of a stable intermediate that regulates the cellular response to growth factors. 相似文献
14.
L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts 总被引:1,自引:0,他引:1
Telomere is the repetitive DNA sequence at the end of chromosomes, which shortens progressively with cell division and limits the replicative potential of normal human somatic cells. L-carnosine, a naturally occurring dipeptide, has been reported to delay the replicative senescence, and extend the lifespan of cultured human diploid fibroblasts. In this work, we studied the effect of carnosine on the telomeric DNA of cultured human fetal lung fibroblast cells. Cells continuously grown in 20 mM carnosine exhibited a slower telomere shortening rate and extended lifespan in population doublings. When kept in a long-term nonproliferating state, they accumulated much less damages in the telomeric DNA when cultured in the presence of carnosine. We suggest that the reduction in telomere shortening rate and damages in telomeric DNA made an important contribution to the life-extension effect of carnosine. 相似文献
15.
Yao Dang Yongpan An Jinzhao He Boyue Huang Jie Zhu Miaomiao Gao Shun Zhang Xin Wang Baoxue Yang Zhengwei Xie 《Aging cell》2020,19(1)
Although aging and senescence have been extensively studied in the past few decades, however, there is lack of clinical treatment available for anti‐aging. This study presents the effects of berberine (BBR) on the aging process resulting in a promising extension of lifespan in model organisms. BBR extended the replicative lifespan, improved the morphology, and boosted rejuvenation markers of replicative senescence in human fetal lung diploid fibroblasts (2BS and WI38). BBR also rescued senescent cells with late population doubling (PD). Furthermore, the senescence‐associated β‐galactosidase (SA‐β‐gal)‐positive cell rates of late PD cells grown in the BBR‐containing medium were ~72% lower than those of control cells, and its morphology resembled that of young cells. Mechanistically, BBR improved cell growth and proliferation by promoting entry of cell cycles from the G0 or G1 phase to S/G2‐M phase. Most importantly, BBR extended the lifespan of chemotherapy‐treated mice and naturally aged mice by ~52% and ~16.49%, respectively. The residual lifespan of the naturally aged mice was extended by 80%, from 85.5 days to 154 days. The oral administration of BBR in mice resulted in significantly improved health span, fur density, and behavioral activity. Therefore, BBR may be an ideal candidate for the development of an anti‐aging medicine. 相似文献
16.
Human skin fibroblasts, both postnatal and embryonic, were cultured in the stationary phase of growth for 6-10 days in the DMEM with bovine serum (BS), 0.1-0.5% fetal calf serum (FCS) or 1% human serum (HS). On the day 4 of culturing, a considerable increase was observed in the synthesis and secretion of protein by postnatal fibroblasts in the Eagle medium with 0.1-0.5% FCS, or with 0.5% BS, and in medium 199 with 0.1-0.5 BS, or with 0.1 FCS. Maximum synthesis and secretion of 14C-proline labeled protein was observed on day 2 of culturing of cells in the DMEM medium with 1% HS. In the DMEM medium with low serum content, protein synthesis being virtually unchanged, 75-80% of protein was secreted by cells into the culture medium with BS on days 2-4; in the medium with FCS such a high secretion of protein was observed only on day 4. High synthesis of protein by fetal fibroblasts in the DMEM medium with 0.1% BS and high protein secretion in all the media with 0.1% BS or 0.5% FCS were observed. The maximum level of secretion of protein by fibroblasts coincided with a considerable increase in both RNA and DNA syntheses. The data obtained suggest that cells in deep resting state actively react to the composition of the medium as well as to the quality and quantity of the serum. It may also be suggested that the mechanism of protein secretion has an important role in maintenance of the constant level of intracellular proteins in resting cells. 相似文献
17.
It has been reported that the replicative lifespan of human fibroblasts can be substantially extended by supplementing the growth medium with hydrocortisone or increased levels of serum proteins. These observations have been made only on cell populations transferred many times at high cell density, and cumulative population doublings have been recorded, rather than a more direct measure of cell division potential. We have measured the replicative potential of human fibroblasts cultured so as to avoid conditions of high cell density, medium depletion, and departure from exponential growth. Two fetal lung and two newborn foreskin fibroblast strains were serially passaged in the presence or absence of hydrocortisone (HC), epidermal growth factor (EGF), and fibroblast growth factor (FGF) until they senesced. At each passage cells were plated at densities sufficiently low that colony-forming efficiency could be calculated. We determined cumulative population doublings and also estimated the number of cell generations attained under each condition. FGF caused small but possibly significant changes, while HC and EGF failed to substantially alter replicative lifespan. The reported effect of HC on the doubling potential of fetal lung fibroblasts is therefore not an inevitable action of this hormone on the senescence mechanism, but may instead depend for its apparent activity on the passage regimen used. The fibroblast's insensitivity to EGF as a modulator of replicative potential, as compared with the keratinocyte, whose lifespan can be tripled by EGF, implies that the mechanisms limiting the replicative potential of these two cell types are not identical. 相似文献
18.
The ability of purified basic somatomedin to reinitiate cell division in nondividing cultures of human embryonic lung fibroblasts (WI-38) maintained in serum-free medium was determined in order to assess the direct mitogenic effect of this substance on mammalian tissue. Resting cultures were prepared by incubation of the cells in serum-free medium for 48–72 hours. Addition of partially purified somatomedin resulted in cellular hypertrophy, DNA synthesis and cell division with a time course similar to that seen when serum was added. Although cells divided in response to physiological concentrations of somatomedin, doses up to 100x in excess of this did not produce as much cell division as 10% fetal calf serum. Addition of fresh medium containing somatomedin to cells previously stimulated by somatomedin failed to induce further cell division. A highly purified somatomedin preparation also stimulated cell division. 相似文献
19.
Spontaneous cell transformation is a common feature of all murine cell cultures grown in vitro for extended periods of time. During early passages, these cultures show either progressively reduced growth or complete cessation of growth; after such a 'crisis' they display increasing growth rates and unlimited lifespan. Here we use a novel bromodeoxyuridine/Hoechst flow-cytometric technique to examine the growth response of diploid and transformed cells of the murine species Micromys minutus under a variety of growth conditions. After spontaneous transformation, growth factor exposure results in increased G0/G1 cell recruitment and higher growth rates than shown by the nontransformed diploid cell fraction. Despite clonal heterogeneity, this difference is seen at all fetal calf serum (FCS) concentrations, although it is most pronounced with low serum. Epidermal growth factor and insulin are shown to act synergistically and promote growth equal to exposures of transformed cultures to 10% FCS. The observed differences in growth factor response between diploid and aneuploid cells could explain the reported lack of a classical growth crisis in growth factor-supplemented media during the spontaneous in vitro transformation of primary cell cultures. 相似文献
20.
S. Goldstein 《Experimental cell research》1974,83(2):297-302
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