利用流式细胞光度术鉴定苹果倍性的研究

利用流式细胞光度术测定了苹果１２个二倍体,５个三倍体细胞ＤＮＡ含量。结果表明：二倍体细胞核ＤＮＡ含量平均为２．２７ｐｇ,三倍体细胞核ＤＮＡ含量平均为３．１３ｐｇ。     

Ploidy class-dependent variations during 24 h of glucose-6-phosphate and succinate dehydrogenase activity and single-stranded RNA content in isolated rat hepatocytes

The time-dependent variations over 24 h of glucose-6-phosphate dehydrogenase (G6PDH) activity, succinate dehydrogenase (SDH) activity and single-stranded RNA (ssRNA) content have been investigated by cytophotometric analysis of cytochemically stained isolated hepatocytes of different ploidy classes from adult male rats. A marked variation of 48 % over the day in G6PDH activity of the mononuclear diploid cells was revealed, but no significant variation in the binuclear tetraploid cells. The cells of the inbetween ploidy classes showed an amplitude of variation of 38 % (binuclear diploid cells) and 24% (mononuclear tetraploid cells), respectively. All cells showed a maximum activity of the enzyme at the middle of the day and a minimum during the night. The relative enzyme activity per mononuclear diploid cell was significantly higher than the relative activity in the other cells, especially at its maximum. The variation of the SDH activity in hepatocytes isolated from the same rats was similar in all cells, irrespective of their ploidy class. The activity was highest at the end of the activity phase of the animals. The SDH activity per cell was directly proportional to the quantity of genome copies. The ssRNA content of the hepatocytes showed a time-dependent variation with a maximum during the resting phase of the animals and a minimum during their activity phase. The variation was larger in the mononuclear diploid cells than in the cells of other ploidy classes and the ssRNA content was also significantly higher in these cells than in the hepatocytes of other ploidy classes when calculated on the basis of genome copies. It is concluded that the large amplitude of variation over the day and the high relative amount of G6PDH activity and ssRNA content in mononuclear diploid cells is related to the function of these cells as stem cells of the liver parenchyma.     

Growth patterns of rat hepatocytes during postnatal development.

During postnatal growth in the liver of the rat, a characteristic shift towards binuclear cells and cells of higher ploidy class occurs. When the protein content of individual isolated hepatocytes of different ploidy classes is analysed cytophotometrically using the specific protein stain Naphthol Yellow S, it appears that the growth in mass in the period 30-99 days is due mainly to increase of protein content of binuclear diploid (BD) and mononuclear tetraploid (MT) cells. The mononuclear diploid (MD) cells play a quickly diminishing role in the parenchymal population after the initial growth phase and cells of highest ploidy degree remain unimportant quantitatively. The quickly growing BD and MT cells only reach a Naphthol Yellow S protein value twice that of MD cells after a certain period of growth, whereas changes in protein content are slight or absent from 99 days onwards in all cell types investigated.     

Peculiarities of liver regeneration in Chinese hamster <Emphasis Type="Italic">Cricetulus griseus</Emphasis>

With the aid of cytofluorimetry and interference microscopy, the ploidy level and the hepatocyte ploidy class distribution were studied and the dry mass of hepatocytes was measured in hepatocytes in liver of Chinese hamsters Cricetulus griseus and of Balb/c mice before and one month after partial hepatectomy. The mean ploidy level in hepatocytes of the Chinese hamster normal liver amounted to 2.35 ± 0.03 c. The modal class was mononuclear hepatocytes with diploid nuclei (82.4 ± 1.3%). The mean dry mass of hepatocytes amounted to 605.2 ± 4.8 pg. In the process of liver regeneration in the Chinese hamsters, the ratio of ploidy classes and the hepatocyte dry mass did not change. After a similar liver resection in the mice, a significant polyploidization of liver parenchyma occurred. The mean ploidy level in hepatocytes rose by 32%. Instead of 4cx2-hepatocytes, the modal class became mononuclear octaploid cells the relative portion of which increased, on average, by five times. The portion of binuclear hepatocytes with octaploid nuclei in mouse liver rose by more than five times. Thus, in the Chinese hamsters Cricetulus griseus, unlike mice, regeneration of liver occurred exclusively at the expense of proliferation of hepatocytes.     

Age-related alterations in the size of human hepatocytes

Age-related alterations in the size of human hepatocytes (both mononuclear and binucleate forms), were studied in histological sections and in separated cells and nuclei using cytophotometrical and microspectrophotometrical methods. The following results were obtained: 1. The volume of nuclear DNA increased in proportion to nuclear size. The increase occurred in a group pattern reflecting nuclear polyploidization. 2. Cell size increased in proportion to nuclear size. Tetraploid cells (4C) were roughly two times greater than diploid cells (2C). 3. In most of the binucleate cells examined, the ploidy class of the two nuclei in a binucleate cell was observed to be equal. Heterogeneity of the ploidy class among the nuclei of a binucleate cell was present in less than 1% of total binucleate cells examined. The nuclear DNA volume of individual nuclei in binucleate cells appeared to be the same as that of mononuclear cells. 4. The cell size of binucleate cells corresponded with that of mononuclear cells whose ploidy class was the same as the sum of the ploidy classes of two nuclei of a binucleate cell. 5. The incidence of binucleate cells in the lobular periphery was about 4 to 6% in the third decade, and increased slightly with age up to 5 to 7% in the tenth decade. 6. The incidence of binucleate cells in the liver at different ages followed a similar pattern to that observed in mononuclear cells whose ploidy class was half of the sum of ploidy classes of the two nuclei of the binucleate cell.     

Establishment of a diploid reference value for DNA ploidy analysis by image cytometry in mouse cells.

OBJECTIVE: To establish a diploid reference value for DNA ploidy analysis of mouse cells (Mus musculus) by image cytometry using the CAS 200, an analysis system suitable for DNA content studies in human cells. STUDY DESIGN: To establish this standard, we used spleen imprints from 26 normal animals. A minimum of 150 lymphocytes present in each imprint was counted. The mean DNA content (pg/cell) of the G0/G1 peak and the DNA index observed in all samples were statistically analyzed. Cytospins with peritoneal cells from the same animals were then analyzed with this reference DNA value to confirm the diploid range. RESULTS: The DNA diploid reference value was determined by the mean DNA content of all spleen samples, which was 6.42 +/- 0.234 pg/cell, and the diploid range, defined as the diploid value +/- 10%, was 5.78-7.06 pg/cell. All the peritoneal samples showed a DNA diploid histogram, with a mean value for the G0/G1 peak DNA content of 6.742 +/- 0.15. CONCLUSION: The diploid reference value found in this study differs from those reported for other species, including the human being, and should be used in further studies of mouse pathology.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Summary Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

A cytophotometric and karyometric study of the cardiac muscle cells of young rats exposed to intermittent high altitude hypoxia

Thirty-day-old rats were exposed to intermittent high altitude in barochamber (7000 m, 4 h per day, 4 exposures) and number of mono- and binucleated myocytes as well as DNA content per nucleus were determined in the right ventricular myocardium. The data indicate enhancement of mitotic division of mono- and binuclear diploid cells in hypoxic animals. Simultaneously, an increase in mononuclear and binuclear tetraploid cells occurred; the latter is probably due to DNA synthesis not followed by mitosis of some binuclear diploid cells.     

Binucleation and polyploidization patterns in developmental and regenerative rat liver growth

The hepatocellular binucleation rate, measured as the percentage of binuclear cells amongst newly formed bromodeoxyuridine-labelled and immunostained collage-nase-isolated rat hepatocytes, decreased from 12% to 4% between days 30 and 40 after birth, rose to 20% between days 50 and 60, and then declined again to the adult rate of about 10% at day 80. During regenerative growth following a two-thirds partial hepatectomy, the rate of binucleation declined to about 3%, causing the fraction of binuclear cells to fall from 27% (before hepactectomy) to 5% (at 45 h after hepactectomy) as pre-existing binuclear cells replicated and formed mononuclear daughter cells. Essentially all (97%) hepatocytes replicated at least once, starting their DNA synthesis at around 13 h and reaching a peak at 30 h, irrespective of ploidy and nuclearity. At later time points, the diploid hepatocytes had a higher labelling index than the polyploid cells, suggesting a greater tendency to go through several cell cycles.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry

The technique of DNA flow cytometry was used to study variation in DNA content among different ploidy levels, as well as among diploid species, of Vaccinium section Cyanococcus. In a sample of plants of varying ploidy level, the relative fluorescence intensity (RFI) of nuclei stained with propidium iodide was a function of the number of chromosome sets (x), as represented by the linear equation RFI=3.7x-2.3 (r²=95%). The data indicated that DNA flow cytometry could be useful for the determination of ploidy level at the seedling stage in blueberry. They also suggest that conventional polyploid evolution has occurred in this section of the genus Vaccinium with an increase in nuclear DNA content concurrent with the increase in chromosome number. The nuclear DNA content of diploid species of Vaccinium section Cyanococcus was estimated from the relationship of the observed RFI to an internal known DNA standard (trout red blood cells). A nested analysis of variance indicated significant variation among species, as well as among populations within species, in nuclear DNA content, although this variation was small compared to the variation among ploidy levels. The variation in nuclear DNA content corresponded to the phylogenetic relationships among species determined from previous studies.     

Stable ploidy levels in long-term callus cultures of loblolly pine

Ploidy levels were calculated for callus cultures of loblolly pine (Pinus taeda L.), based on nuclear DNA content measured by Feulgen cytophotometry. The nuclear DNA content of initial stem explants showed a predominant 2C condition with less 3C and 4C, in ratios approximating those expected from diploid cells as they replicate DNA in the mitotic cell cycle. Cells with higher ploidy were produced during callus initiation, as indicated by a sharp reduction in the 2C population and a concomitant increase in higher DNA levels up to 8C. A gradual decrease in the higher ploidy levels occurred in subsequent subculture intervals, so that by 18 weeks the diploid nuclear DNA distribution was again observed, with complete elimination of DNA levels greater than 4C. Established callus cultures derived from stem or embryo explants and cultured on three different nutrient media for 48–76 weeks also showed the diploid nuclear DNA distribution with no indication of polyploid cells.Abbreviations BAP benzylaminopurine - NAA -naphthaleneacetic acid - BL Brown and Lawrence's medium - BLG modified BL medium - LM Litvay's medium Paper No. 11952 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh NC 27695-7643, USA     

Noninvasive determination of genome size and ploidy level in fishes by flow cytometry: detection of triploid Poecilia formosa

BACKGROUND: In order to understand the evolutionary significance of single triploids among the mostly diploid Poecilia formosa we have developed a simple, noninvasive technique for DNA content and ploidy determination. METHODS: From dorsal fin clips of 14 different fish species single cell suspensions were obtained by chopping the material in 2.1% citric acid/0.5% Tween20, passing it through a 0. 6-gauge needle and incubating it for 20 min at room temperature (RT) with gentle agitation. After overnight fixation in 70% ethanol, the cells were treated with 1ml 0.5% pepsin/0.1 M HCl for 15 min at RT before adding DAPI to a final volume of 2 ml. The cells were stained for 1-3 h and then analyzed by flow cytometry. RESULTS: We obtained good measurements with CVs ranging from 1.23% to 3.36%. The poeciliid species measured contain from 1.6 to 2.0 pg/nucleus, Oryzias latipes (Medaka) exhibits a nuclear DNA content of 2.2 pg, Danio rerio (zebrafish) 4.6 pg, Tetraodon fluviatilis (freshwater fugu) 0.70 pg. All values except zebrafish are in good agreement with the literature. CONCLUSIONS: The identification of living specimens of different ploidy for breeding experiments, behavioral studies and tissue transplantations is now made possible. With slight modifications the method can be extended to a field technique, providing therefore a useful tool for a variety of researchers.     

The systematic value of nuclear DNA content for all species of Narcissus L. (Amaryllidaceae)

The taxonomy of all species of Narcissus (Amaryllidaceae), an important horticultural crop, has not been investigated recently. As a new approach, genome size was determined by flow cytometry with propidium iodide from 375 accessions. The somatic nuclear DNA contents (2C) were shown to range from 14 to 38 pg for the diploids. Narcissus assoanus and N. gaditanus are, based on their nuclear DNA content, removed from section Apodanthi and placed in a new section Juncifolii. The different ploidy levels and species involved were entangled for N . “fernandesii” s.l. and a new allotetraploid form is named here. Section Pseudonarcissus was much more heterogeneous in nuclear DNA content than expected. Sixty-five accessions of N. pseudonarcissus possessed, with 23.7 pg, similar amounts of DNA. However, several species from this section were clearly distinctive in nuclear DNA content. It runs from the diploid N. primigenius with 21.7 pg to the also diploid N. nevadensis with 38.2 pg. Also N. abscissus and N. moleroi are with about 26 pg clearly different from N. pseudonarcissus. For the first time, in 11 accessions, hexaploidy was found in N. pseudonarcissus ssp. bicolor. A new section Nevadensis with 30–39 pg of nuclear DNA was split off from the section Pseudonarcissus with now 21–27 pg. A nonoploid N. dubius with 96.3 pg has by far the highest amount of nuclear DNA and can be calculated to have the highest ploidy ever reported in Narcisssus. The total number of Narcissus species was determined as 36, nine more than in Flora Europaea and they were divided up in two subgenera and 11 sections. Flow cytometry is shown to produce easily obtainable and original systematic data that lead to new insights. Genome size or C-value turns out to be one of the most salient features to define the status of the species in the genus Narcissus.     

I. Quantitative determination of the amount of DNA per nucleus by interference microscopy

A method for the determination of the DNA content of isolated nuclei of different ploidy has been developed. It is based on measurement of the nuclear dry mass, with an integrating microinterferometer, before and after DNase treatment. The values found are slightly low, because, as indicated by biochemical determinations, consistently 5% to 8% of DNA is not extracted by DNase under these conditions. The average DNA values thus obtained for diploid and tetraploid nuclei of adult rat liver are 7.7 and 15.6 pg (10^-12 g), respectively. Definite advantages of this procedure are: i) comparisons with biochemical determinations to give DNA values for each class of ploidy, ii) comparisons with histophotometry of the Feulgen dye-DNA complex to give absolute values instead of arbitrary units.     

Nuclear DNA content variation among Central European Koeleria taxa

BACKGROUND AND AIMS: Polyploidization plays an important role in the evolution of many plant genera, including Koeleria. The knowledge of ploidy, chromosome number and genome size may enable correct taxonomic treatment when other features are insufficient as in Koeleria. Therefore, these characteristics and their variability were determined for populations of six central European Koeleria taxa. METHODS: Chromosome number analysis was performed by squashing root meristems, and ploidy and 2C nuclear DNA content were estimated by flow cytometry. KEY RESULTS: Three diploids (K. glauca, K. macrantha var. macrantha and var. pseudoglauca), one tetraploid (K. macrantha var. majoriflora), one decaploid (K. pyramidata) and one dodecaploid (K. tristis) were found. The 2C nuclear DNA content of the diploids ranged from 4.85 to 5.20 pg. The 2C DNA contents of tetraploid, decaploid and dodecaploid taxa were 9.31 pg, 22.89 pg and 29.23 pg, respectively. The DNA content of polyploids within the K. macrantha aggregate (i.e. within K. macrantha and K. pyramidata) was smaller than the expected multiple of the diploid genome (K. macrantha var. macrantha). Geography-correlated variation of DNA content was found for some taxa. Czech populations of K. macrantha var. majoriflora had a 5.06% smaller genome than the Slovak ones. An isolated eastern Slovakian population of K. tristis revealed 8.04% less DNA than populations from central Slovakia. In central and north-west Bohemia, diploid and tetraploid cytotypes of K. macrantha were sympatric; east from this region diploid populations, and towards the west tetraploid populations were dominant. CONCLUSIONS: Remarkable intra-specific inter-population differences in nuclear DNA content were found between Bohemian and Pannonian populations of Koeleria macrantha var. majoriflora and between geographically isolated central and eastern Slovakian populations of K. tristis. These differences occur over a relatively small geographical scale.     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Intoxication of rats with CCl4 (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS+) hepatocytes were reestablished. However, while the GS+ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS+ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS+ and GS- hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS+ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS- hepatocytes. Immediately after liver damage by CCl4 and during regeneration small but significant changes in the nuclear pattern were noted for GS- hepatocytes. However, the first GS+ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS- hepatocytes. These results indicate that new GS+ hepatocytes may be derived from formerly GS- cells which are induced to express GS if they have reached the border of the central veins.     

A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis

A method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone. The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two-thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S-phase cells.