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1.
Summry— The aim of this study was to localise two ionic S-phase markers in tissue sections using SIMS microscopy: aluminium as a potential endogenous marker and bromine as an exogenous marker after in vivo injection of bromodeoxyuridine (BrdU). This study was performed in an experimental model of hyperplastic proliferation after partial hepatectomy in rat. Aluminium was never detected in nuclei which were positive or negative for tritiated thymidine uptake, as determined by autoradiography in tissue prepared by cryotechniques. In contrast, bromine of BrdU was found in hepatocyte nuclei. However, there was a discrepancy between SIMS bromine images and BrdU immunohistochemistry detection which appears more sensitive. This is probably due to problems of stereology intrinsic to the correlation method which requires serial sections for this multi-instrumental approach.  相似文献   

2.
BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled anti-BrdU and counterstained with To-Pro-3 are scanned using confocal laser scanning microscopy (CLSM). For every point in the image, the nucleus density of BrdU-labeled nuclei and the total nucleus density of the neighborhood of that point are calculated from the BrdU and the To-Pro-3 signal, respectively. The ratio of these densities gives an indication of the amount of cell proliferation at that point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manual count. RESULTS: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calculating the topographical cell proliferation using the automated method is faster and does not suffer from interobserver variability. CONCLUSIONS: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissue. This can be visualized by a topographical map that corresponds to the tissue under study.  相似文献   

3.
The standard method for assessment of cell proliferation in paraffin-embedded tissue sections is 5-bromodeoxyuridine (BrdU) immunohistochemistry (IHC). BrdU can be administered to laboratory animals via IP injections, is readily incorporated into nuclei during the DNA synthetic phase of the cell cycle, and is detected with an anti-BrdU antibody. This method has several disadvantages, and an accurate method for evaluation of proliferative activity that can substitute for BrdU IHC, when necessary, is of great interest to investigators. Alternative methods for detection of proliferating cells in tissue sections are proliferating cell nuclear antigen (PCNA) IHC, Ki-67 IHC, and in situ hybridization (ISH) for histone mRNA. To determine the optimal choice, we analyzed the correlation of anti-PCNA, anti-Ki-67(MIB-5), and histone mRNA labeling indices (LIs) with anti-BrdU LI in rat highly replicative (renewing) tissues. The correlation between anti-BrdU and histone mRNA LIs, as well as the correlation between anti-BrdU and anti-Ki-67 LIs, was statistically significant. There was no significant correlation between anti-BrdU and anti-PCNA LIs. These results suggest that both ISH for histone mRNA and IHC with MIB-5 are preferable techniques for assessment of cell proliferation in rat paraffin-embedded renewing tissues compared to PCNA IHC. They can substitute for BrdU IHC when necessary.  相似文献   

4.
Immunohistochemical assessment of 5-bromo-2-deoxyuridine (BrdU) in tissue sections is a widely used method to evaluate cell proliferation in vivo. However, this method requires time-consuming preparation of paraffin sections and laborious counting of BrdU-labeled nuclei on multiple sections. Here, we report the development of a rapid and reliable method to quantitate BrdU incorporation in intestinal and liver tissues using a dot-blot method. In vivo models of colon or liver proliferation were used to analyze the usefulness and reliability of this new method. Mice were killed after BrdU injection, and genomic DNA was isolated from the tissues, denatured, and dot-blotted onto a nitrocellulose membrane. The incorporated BrdU was detected with a BrdU monoclonal antibody, and the signal intensity was densitometrically quantified. Results were compared with BrdU index determined by conventional immunohistochemistry on the same tissue samples. The patterns of colonic BrdU incorporation during fasting and refeeding, measured by the dot-blotting method and the immunohistochemical method, were similar. The BrdU incorporation in the regenerating liver after partial hepatectomy, evaluated by these two different methods, showed a strong correlation (R(2) = 0.91, P < 0.01). In addition, the inhibition of colon proliferation by the phosphoinositol 3-kinase inhibitor wortmannin was demonstrated by this dot-blotting method. In conclusion, the dot-blotting technique described in this report provides an accurate, more efficient, and possibly more reliable method for the assessment of in vivo proliferation compared with conventional immunohistochemical determination of BrdU-labeling index.  相似文献   

5.
We previously reported that mice implanted with mammary tumors show a progressive thymic involution that parallels the growth of the tumor. The involution is associated with a severe depletion of CD4+8+ thymocytes. We have investigated three possible mechanisms leading to this thymic atrophy: 1) increased apoptosis, 2) decreased proliferation, and 3) disruption of normal thymic maturation. The levels of thymic apoptosis were determined by propidium iodide and annexin V staining. A statistically significant, but minor, increase in thymic apoptosis in tumor-bearing mice was detected with propidium iodide and annexin V staining. The levels of proliferation were assessed by in vivo labeling with 5'-bromo-2'-deoxyuridine (BrdU). The percentages of total thymocytes labeled 1 day following BrdU injection were similar in control and tumor-bearing mice. Moreover, the percentages of CD4-8- thymocytes that incorporated BrdU during a short term pulse (5 h) of BrdU were similar. Lastly, thymic maturation was evaluated by examining CD44 and CD25 expression among CD4-8- thymocytes. The percentage of CD44+ cells increased, while the percentage of CD25+ cells decreased among CD4-8- thymocytes from tumor-bearing vs control animals. Together, these findings suggest that the thymic hypocellularity seen in mammary tumor bearers is not due to a decreased level of proliferation, but, rather, to an arrest at an early stage of thymic differentiation along with a moderate increase in apoptosis.  相似文献   

6.
Radiation-induced apoptosis in the neonatal and adult rat spinal cord   总被引:5,自引:0,他引:5  
Li YQ  Wong CS 《Radiation research》2000,154(3):268-276
This study was designed to characterize radiation-induced apoptosis in the spinal cord of the neonatal and young adult rat. Spinal cords (C2-T2) of 1-, 2- and 10-week-old rats were irradiated with a single dose of 8, 18 or 22 Gy. Apoptosis was assessed histologically according to its specific morphological features or by using the TUNEL assay. Cell proliferation was assessed immunohistochemically using BrdU. Identities of cell types undergoing apoptosis were assessed using immunohistochemistry or in situ hybridization using markers for neurons, glial progenitor cells, microglia, oligodendrocytes and astrocytes. The time course of radiation-induced apoptosis in 1- or 2-week-old rat spinal cord was similar to that in the young adult rat spinal cord. A peak response was observed at about 8 h after irradiation, and the apoptosis index returned to the levels in nonirradiated spinal cords at 24 h. The neonatal rat spinal cord demonstrated increased apoptosis compared to the adult. Values for total yield of apoptosis over 24 h induced by 8 Gy in the neonatal rat spinal cord were significantly greater than that in the adult. Immunohistochemistry studies using Leu7, galactocerebroside, Rip and adenomatous polyposis coli tumor suppressor protein indicated that most apoptotic cells were cells of the oligodendroglial lineage regardless of the age of the animal. No evidence of Gfap or factor VIII-related antigen-positive apoptotic cells was observed, and there was a small number of apoptotic microglial cells (lectin-Rca1 positive) in the neonatal and adult rat spinal cord. In the neonatal but not adult rat spinal cord, about 10% of the apoptotic cells appeared to be neurons and were immunoreactive for synaptophysin. Labeling indices (LI) for BrdU in nonirradiated 1- and 2-week-old rat spinal cord were 20.0 and 16.3%, respectively, significantly greater than the LI of 1.0% in the 10-week-old rat spinal cord. At 8 h after a single dose of 8 Gy, 13.4% of the apoptotic cells were BrdU-positive in 10-week-old rat spinal cord, whereas 62.4 and 44.1% of the apoptotic cells showed BrdU incorporation in 1- and 2-week-old rat spinal cord, respectively. Regardless of the age of the animal, the apoptosis indices in BrdU-positive cells were greater than those in BrdU-negative cells. We conclude that the neonatal spinal cord demonstrates a greater level of apoptosis after exposure to ionizing radiation than the young adult spinal cord. This increase in apoptosis may be associated in part with the greater percentage of proliferating cells in the neonatal spinal cord, which demonstrate a greater level of radiation-induced apoptosis than nonproliferating cells.  相似文献   

7.
Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling   总被引:4,自引:0,他引:4  
We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.  相似文献   

8.
The proliferative activities of 98 biopsied specimens of advanced gastric cancers were measured by the in vitro bromodeoxyuridine (BrdU) labelling method. BrdU labelling indices (LIs) varied from 4.8 to 45.1%. There was no correlation of BrdU LIs and histological type, distant metastasis, serosal invasion, macroscopic type or tumor size. However, BrdU LIs of tumors with lymph node metastases were significantly higher than those without them. Patients with tumors showing high BrdU LIs had a three-fold relative risk of death, as compared with those showing low BrdU LIs. When the BRdU LIs and all the clinicopathological parameters were entered simultaneously into the Cox model, BrdU LIs, peritoneal dissemination and serosal invasion emerged as independent prognostic parameters. These results indicate that the in vitro BrdU labelling method using biopsied materials may be useful in predicting lymph node metastasis preoperatively and designing operative procedures for individual patients.  相似文献   

9.
5'-Bromodeoxyuridine (BrdU) is a thymidine analogue which can be detected by monoclonal antibodies (MAb). We have developed a method for the simultaneous visualization of BrdU and a wide range of neural antigens in paraformaldehyde-fixed brain sections. Pregnant mice were injected intraperitoneally with a single pulse of BrdU. Young adult offspring were processed for immunocytochemistry following a double immunoperoxidase sequence. BrdU was detected using diaminobenzidine (DAB) intensified with nickel ammonium sulfate and neural antigen-containing elements were visualized with DAB alone. BrdU-positive nuclei and tissue antigen-immunoreactive cells were easily differentiated. Furthermore, double-labeled cells characterized by the presence of a black immunoreactive nucleus surrounded by a brown immunopositive cytoplasm were unambiguously recognized. Satisfactory results were obtained using either MAb or polyclonal antibodies against a variety of cell antigens, including neuropeptides, CA++ binding proteins, and cytoskeletal components of the glial cells. The method reported here permits analysis of the neurogenesis and proliferation of subsets of neurons and glial cells, identified by immunocytochemical markers.  相似文献   

10.
BACKGROUND: Measurement of vascular cell proliferation in animal models of hypertension is currently accomplished by demonstrating [(3)H]-thymidine ([(3)H]-dT) incorporation into DNA using autoradiography. This method, however, is labor intensive, requires radioactivity, and is limited by the inherent difficulty in discriminating labeled and unlabeled cells. To address these limitations, a flow cytometric-based method is described utilizing incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA of nuclei isolated from blood vessels. METHODS: Pulmonary hypertension was induced in rats by exposure to 10% O(2) (hypoxia) for varying periods of time. Pulmonary arteries and aorta from rats injected with BrdU prior to sacrifice were isolated, fixed with 10% formalin, and digested with Protease XIV. The intact nuclei liberated by this treatment were successively treated with HCl/Triton X-100 and sodium borate. Processed nuclei were probed with a BrdU-specific fluorescein-conjugated antibody, and the percentage of BrdU staining cells was determined using flow cytometry. RESULTS: An approximately 20-fold increase in BrdU-positive cells at 3 days of hypoxia in pulmonary arteries (relative to control) with no change in aorta was observed. These results were similar to previous studies using [(3)H]-dT labeling. CONCLUSIONS: Flow cytometric determination of cell proliferation in blood vessels is a simple, objective technique that may facilitate measurement of cell proliferation in animal models of vascular disease.  相似文献   

11.
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.  相似文献   

12.
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.  相似文献   

13.
The use of bromodeoxyuridine (BrdU) and subsequent immunocytochemical visualization for studying cell proliferation in plant meristems was investigated in Allium cepa L. root-tips. We describe the optimization of an indirect immunoperoxidase method for detecting incorporation of this DNA precursor in pulse-labeled cells. The basic object of this study is to quantify the extent to which the fraction of S-phase cells can reliably be estimated in asynchronous populations. A matrix of parallel labeling schedules with tritiated-thymidine or BrdU was developed, and the labeling indices provided by autoradiography or immunocytochemistry were compared. Thus, 0.5 mM BrdU assured saturation S-phase labeling after an exposure time of 30 min, and the mean length of the S-phase determined under such conditions was similar to that previously reported for this plant system. Interestingly, Feulgen staining did not interfere with subsequent detection of the BrdU probe. This allowed comparative evaluations of the nuclear DNA content by Feulgenmicrodensitometry and the position of a given cell in G1, S or G2 compartments. We also explored the possibility of quantifying BrdU-incorporation in single nuclei by densitometry measurement of the peroxidase label.  相似文献   

14.
The use of bromodeoxyuridine (BrdU) and subsequent immunocytochemical visualization for studying cell proliferation in plant meristems was investigated in Allium cepa L. root-tips. We describe the optimization of an indirect immunoperoxidase method for detecting incorporation of this DNA precursor in pulse-labeled cells. The basic object of this study is to quantify the extent to which the fraction of S-phase cells can reliably be estimated in asynchronous populations. A matrix of parallel labeling schedules with tritiated-thymidine or BrdU was developed, and the labeling indices provided by autoradiography or immunocytochemistry were compared. Thus, 0.5 mM BrdU assured saturation S-phase labeling after an exposure time of 30 min, and the mean length of the S-phase determined under such conditions was similar to that previously reported for this plant system. Interestingly, Feulgen staining did not interfere with subsequent detection of the BrdU probe. This allowed comparative evaluations of the nuclear DNA content by Feulgenmicrodensitometry and the position of a given cell in G1, S or G2 compartments. We also explored the possibility of quantifying BrdU-incorporation in single nuclei by densitometry measurement of the peroxidase label.  相似文献   

15.
We describe here a new technique for isolating nuclei from long-term label-retaining cells (LRCs), a subpopulation enriched with stem cells from colon, and for measuring their proliferation rates in vivo. A double-label approach was developed, combining the use of bromodeoxyuridine (BrdU) and 2H2O. Male Fisher 344 rats were administered BrdU in drinking water continuously for 2–8 wk. BrdU was then discontinued (BrdU washout), and animals (n = 33) were switched to 2H2O in drinking water and killed after 2, 4, and 8 wk. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percentages of LRCs were 7 and 3.8% after 4 and 8 wk of BrdU washout, respectively. Turnover rates of LRCs were measured on the basis of deuterium incorporation from 2H2O into DNA of LRC nuclei, as determined by mass spectrometry. The proliferation rate of the LRCs collected was 0.33–0.90% per day (half-life of 77–210 days). Significant contamination from other potentially long-lived colon cells was excluded. In conclusion, this double-labeling method allows both physical isolation of nuclei from colon epithelial LRCs and measurement of their in vivo proliferation rates. Use of this approach may allow better understanding of mechanisms by which agents induce or protect against colon carcinogenesis. carcinogenesis; deuterated water; long-term label-retaining cells; stable isotopes  相似文献   

16.
F. Pera  P. Mattias 《Chromosoma》1976,57(1):13-18
A method of labelling DNA in vivo with 5-bromodeoxyuridine (BrdU) is described. After 6 h permanent subcutaneous infusion of BrdU in rodents (adult Microtus agrestis, pregnant NMRI-mice), cell nuclei which have undergone DNA synthesis during the BrdU treatment can be differentiated from the nuclei of other cycle stages by means of their altered staining behaviour after Giemsa. 24 h after the BrdU treatment, mitoses from both bone marrow of the adult animals and tissues from the fetuses showed a differential sister chromatid staining. In male M. agrestis, sister chromatid exchanges were most frequently found in the euchromatic part of the X and in the constitutive heterochromatin of both sex chromosomes.  相似文献   

17.
Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to invesitgate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidiumiodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.  相似文献   

18.
We studied the use of acute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detection of DNA-synthesizing cells in the liver and kidney of B6C3F1 male mice. Six-week-old mice were exposed to BrdU either acutely with a single-pulse (IP) injection 1 hr before sacrifice or chronically with the use of slow-release pellets or osmotic minipumps at one of four BrdU dose rates. Pellets (2.5, 10, 25, and 50 mg) and minipumps (2.5 and 10 mg equivalents) were implanted subcutaneously on the backs of the animals 4 or 7 days before sacrifice). BrdU incorporation into DNA was determined by immunohistochemistry using an anti-BrdU antibody. Mice chronically exposed to BrdU demonstrated increased levels of nuclear labeling compared with those receiving a single-pulse injection. No time-related increases in nuclear labeling were detected in hepatocytes or renal tubule cells of mice exposed to BrdU pellets and in the kidneys of mice receiving BrdU minipumps at the 7-day compared with the 4-day time point. In some cases, the labeling indices at 7 days were significantly decreased compared with those at 4 days. In contrast, a time-related increase in nuclear labeling was seen in hepatocytes and Kupffer cells of mice exposed to BrdU minipumps. Therefore, the method used to administer BrdU chronically to the animal appears to play an important role in presenting the true proliferative scenario in cell kinetic studies. Our findings also provide evidence for an effect of BrdU on normal proliferation rates in these tissues.  相似文献   

19.
惊厥后大鼠海马神经再生与凋亡的动态变化   总被引:1,自引:0,他引:1  
探讨惊厥持续状态(status convulsion,SC)后大鼠海马神经再生与凋亡的动态变化。建立成年Wistar鼠30minSC模型,在SC后1天至56天的6个时间点上处死动物,处死前1天均腹腔注射5-溴2-脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine,BrdU);采用免疫组织化学方法动态检测BrdU、nestin的表达,确定神经干细胞增殖水平;双重荧光染色标记nestin/TUNEL,确定新生神经干细胞存活时间。与对照组相比,BrdU阳性细胞数目于SC后第7天在CA1区达增殖高峰,28天降至正常水平;于SC后第28天在齿状回达增殖高峰,56天降至正常水平;在SC后第7天,CA3区有大量的BrdU阳性细胞;BrdU和nestin阳性细胞数目无统计学差异。在SC后的前3天,CA1区新增殖的神经细胞呈TUNEL阳性;齿状回新增殖细胞始终表现TUNEL阴性。上述结果提示:SC后能激活自体神经干细胞原位增殖,并且部分新生细胞向损伤区域迁移。  相似文献   

20.
Effects of angiotensins II (AngII), angiotensin IV (AngIV, 3-8 fragment of angiotensin II) and losartan (an antagonist of angiotensin receptor type 1) on the proliferation of adrenocortical cells in ovariectomized rats have been studied. The incorporation of bromodeoxyuridine (BrdU) into cell nuclei was used as an index of cell proliferation. AngIV decreased BrdU labeling index mainly in the reticularis zone and losartan (Los) was able to partially reverse this inhibitory effect of AngIV. AngII had no effect on the adrenocortical cell proliferation when given alone, however Los given simultaneously diminished BrdU incorporation into nuclei of glomerulosa and reticularis zones as compared with AngII. These findings suggest that AngII and AngIV modulate adrenocortical cell proliferation in ovariectomized rats.  相似文献   

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