共查询到20条相似文献,搜索用时 15 毫秒
1.
Robert D. Greenleaf Robert J. Mason Mary C. Williams 《In vitro cellular & developmental biology. Plant》1979,15(9):673-684
Summary Centrifugal elutriation (counterflow centrifugation) was used to develop a reproducible method for obtaining a nearly pure
population of isolated alveolar type II cells. Lung was dissociated into individual cells with recrystallized trypsin, and
the type II cells were partially purified by centrifugation on a discontinuous density gradient. The alveolar type II cells
were finally purified by centrifugal elutriation. Cells were collected from the elutriator rotor by stepwise increases in
flow rates. Cells obtained at flow rates of 7 and 14 ml per min were lymphocytes, other small cells, a few type II cells and
cell debris; cells collected at flow rates of 18 and 22 ml per min were mainly type II cells; and cells collected at flow
rates of 28, 34 and 43 ml per min were macrophages, some type II cells, other lung cells and cell aggregates. At flow rates
of 18 and 22 ml per min, 1.9±1.0×106 cells per rat lung (mean±S.D.,n=30) were recovered of which 86±6% were type II cells. At these flow rates, 94% of the cells excluded the vital dye erythrosin
B from their cytoplasm. They consumed oxygen at a rate of 101±21 nmol per hr·106 cells (mean±S.D.,n=4), and their oxygen consumption increased only 10% after 10mm sodium succinate was added. The cells incorporated [14C]leucine into protein and lipid for 4 hr. Electron micrographs of the cells collected at flow rates of 18 and 22 ml per min
show a high percentage of morphologically intact alveolar type II cells. We conclude that centrifugal elutriation is a reproducible
method for obtaining nearly pure, metabolically active alveolar type II cells.
Postdoctoral trainee supported by Grants HL-05251 and HL-07192 from the National Heart, Lung and Blood Institute.
This work was supported by U.S. Public Health Service Grants Program-Project HL-06285 and Pediatric Pulmonary SCOR HL-19185,
and by a grant-in-aid from the American Heart Association (77-1098). 相似文献
2.
The activities of several enzymes which metabolize xenobiotics were measured and compared in freshly isolated rabbit Clara cells (50–70% purity) and alveolar type II cells (80–95% purity) or microsomal preparations from the isolated cell fractions. The presence of 1 mM nicotinamide in protease and cell isolation buffers increased significantly 7-ethoxycoumarin (7-EC) deethylase and epoxide hydrolase activities in the isolated Clara and type II cells. Isolated Clara cell fractions metabolized 7-EC to umbelliferone at a rate of 241 ± 27 pmoles/mg prot/min (mean ± S.E., N=5), while the 7-EC deethylation rate in type II cells was 111 ± 15 pmoles/mg prot/min. Coumarin hydroxylation activity, however, was more than ten times greater in the Clara cells than in the type II cells on a per mg cellular protein basis. N-oxidation of N,N-dimethylaniline, catalyzed by a flavin monooxygenase, was about 2 times as great in microsomes of Clara cells as in microsomes of type II cells. Epoxide hydrolase activity with benzo(a)pyrene 4,5-oxide as substrate was about 10 times higher in Clara cells than in type II cells. Because of the greater cellular, structural and functional heterogeneity in lung, differential distribution of enzymes responsible for xenobiotic metabolism in this tissue may contribute to cell selective chemical toxicity and carcinogenesis.Abbreviations 7-EC
7-ethoxycoumarin
- DMA
N,N-dimethylaniline 相似文献
3.
Carl G. Figdor Jack M. M. Leemans Willy S. Bont Jan E. de Vries 《Cell biochemistry and biophysics》1983,5(2):105-118
Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation
of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution
capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed.
Experiments carried out with human red blood cells (rbc) indicated that aselective losses of rbc from the rotor caused by
the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In
addition, high cell loads (5×108 rbc) resulted in better separations than low cell loads (5×107 rbc).
Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the
same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing
the rotor speed. In all cases similar results were obtained.
These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly
in sedimentation velocity. 相似文献
4.
María Asunción Bosch Cristina Risco Angel Martin-Municio 《Molecular and cellular biochemistry》1990,93(2):167-172
Summary Alterations in pulmonary surfactant are partly responsible for the respiratory insufficiency seen under septic shock process. We have used an experimental model of LPS-induced shock in rats to examine the cells responsible for the pulmonary surfactant synthesis and its relationship to lung injury. (14C)Choline incorporation into phosphatidylcholine was significantly reduced in lung homogenates or type II cells obtained from LPS-treated animals. Addition of LPS in vitro fails to increase (14C)choline incorporation in type II cells obtained from LPS-treated animals. We suggest that this depression of pulmonary phosphatidylcholine synthesis may partly explain the occurrence of respiratory failure with septic shock. 相似文献
5.
R. A. Romanov N. V. Kabanova S. L. Malkin S. S. Kolesnikov 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2009,3(1):71-80
In cells of different types outward voltage-gated (VG) ion currents are generally carried by potassium ions. However, in mouse type II taste cells these currents persist when K+-selective ion channels are inhibited. In this study, we examined the ion channels that provide a pathway for atypical VG outward currents in type II taste cells. These channels are found to be weakly selective and permeabile to large molecules such as NMDG, gluconate, and ATP. According to non-stationary fluctuation analysis, single channel conductance is about 200 pS. The data obtained suggest that the nonselective ion channels are similar to hemichannels formed by connexins, the gap-junction proteins, in the plasma membrane of vertebrate cells. 相似文献
6.
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8.
Background
Bone physiology is increasingly appreciated as an important contributor to metabolic disorders such as type 2 diabetes. However, progress in understanding the role of bone in determining metabolic health is hampered by the well-described difficulty of obtaining high quality RNA from bone for gene expression analysis using the currently available approaches. 相似文献9.
Lysosomal and mitochondrial pathways in H2O2-induced apoptosis of alveolar type II cells 总被引:1,自引:0,他引:1
Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (AT II) cell apoptosis remain poorly defined. Here we investigated the role of lysosomes as modulators of oxidant-mediated AT II cell apoptosis using an in vitro model of H(2)O(2)-stress. H(2)O(2) stress led to time-dependent increases in intracellular oxidants, mitochondrial membrane polarization, cytochrome c release, lysosomal rupture, and AT II cells apoptosis. Increased apoptosis was prevented by specific inhibition of the caspase cascade using the broad-spectrum caspase inhibitor z-VAD-fmk or a caspase 3 inhibitor, or by using functional inhibitors for cathepsin D (pepstatin A) or cathepsin B. Inhibition of cathepsin D also prevented mitochondrial permeabilization and cythocrome c release suggesting that lysosomal rupture precedes and is necessary for the activation of the mitochondrial pathway of cell death. 相似文献
10.
Background
LysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.Methods
Mouse lung cells were sorted on the basis of CD45negCD31negEpCAMposLysoTrackerpos expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay. To track AT2 cell differentiation, lung epithelial stem and progenitor cells were cultured in a CFU-Epi assay with LysoTracker-supplemented media.Results
The purity of sorted AT2 cells as determined by SP-C staining was 97.4% and viability was 85.3%. LysoTrackerpos AT2 cells generated SP-Cpos alveolar epithelial cell colonies in culture, and when added to the CFU-Epi culture medium, LysoTracker marked the differentiation of stem/progenitor-derived AT2 cells.Conclusions
This study describes a novel method for isolating AT2 cells from mouse lungs. The high purity and viability of cells attained by this method, makes them suitable for functional analysis in vitro. The application of LysoTracker to live cell cultures will allow better assessment of the cellular and molecular mechanisms that regulate AT2 cell differentiation. 相似文献11.
An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung 总被引:11,自引:0,他引:11
Jin Ning Lou Nabil Mili Christine Decrind Yves Donati Sylvie Kossodo Anastase Spiliopoulos Bara Ricou Peter M. Suter Denis R. Morel Philippe Morel Georges E. Grau 《In vitro cellular & developmental biology. Animal》1998,34(7):529-536
12.
Isolation and identification of clara cells from rabbit lung 总被引:6,自引:0,他引:6
Theodora R. Devereux James R. Fouts 《In vitro cellular & developmental biology. Plant》1980,16(11):958-968
Summary A procedure has been developed for the isolation of nonciliated bronchiolar epithelial cells (Clara cells) from rabbit lung.
Following pulmonary lavage to eliminate macrosphages, cells (5% Clara cells) were released by digestion with 0.1% Protease
I in HEPES-buffered balanced salt solution containing 0.5 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid instilled through the trachea. These cells were then separated on the basis of size using the Beckman JE-6
elutriator rotor. The fourth fraction collected from the elutriator contained about 30% Clara cells. This fraction was then
layered on a two-polymer aqueous phase system consisting of 5% dextran T500 (DT) and 3.8% polyethylene glycol 6000 (PEG) in
sodium phosphate buffer. A cell fraction was obtained from the PEG phase, which included approximately 70% Clara cells. These
cells were found to be greater than 90% viable by trypan blue dye exclusion.
Identification of isolated Clara cells was confirmed by light microscopic observation of nitro blue tetrazolium staining and
by ultrastructural characteristics as observed by electron microscopy.
This research was supported in part by the U.S. Environmental Protection Agency under an interagency agreement relating to
the Federal Interagency Energy/Environmental Research and Development Program. 相似文献
13.
Salil K. Das Pampa Chakrabarti Francis H. C. Tsao Tultul Nayyar Shyamali Mukherj 《Molecular and cellular biochemistry》1992,115(1):79-84
A new group of calcium-regulating proteins, called annexins or Ca++-dependent phospholipid-binding proteins (PLBP), have been detected in different species, organs and cell types. In the present study, we have identified and quantitated PLBP from guinea pig lung, lavage fluid and alveolar type II cells to elucidate the possible role of PLBP in lung surfactant biogenesis and secretion. Lungs were lavaged and type II cells from lavaged lung were isolated by elastase digestion and purified by centrifugal elutriation. For the quantitative identification of PLBP, we performed ELISA assays and Western blot analysis by using an antiserum raised in guinea pigs against a pure rabbit lung 36 kDa PLBP. The lavage fluid, cytosol from lung and type II cells contained 784,167 and 435 ng per mg protein, respectively, of PLBP. The SDS-PAGE electrophoretic pattern and Western blot confirmed that all lung samples have band corresponding to a 36 kDa protein. This indicates that both alveolar type II cells and lavage fluid have higher levels of PLBP than whole lung cytosol. 相似文献
14.
Alka Khera Thungapathra MuthuKumarappa Dheeraj Dumir Poonam Kanta Gaurav Kumar Jaswinder Kalra 《International Journal of Biochemistry and Molecular Biology》2022,13(3):17
Background: RNA isolation from ossified bone is a difficult and time-consuming process which often results in poor recovery of RNA. The yield is limited and might not be suitable for gene quantification studies by real time PCR. Methodology: The present study demonstrates RNA extraction from rat femur utilizing the silica column along with the trizol reagent. Quality of RNA was assessed by agarose gel analysis and its suitability for real-time PCR analysis was determined by β-actin Ct values. Results: The RNA isolated using silica columns in conjugation with trizol reagent resulted in higher yield of RNA and purity (A260/280=2.04; yield =1545.73 µg/ml) compared to the trizol method alone (A260/280=1.85; yield =571.2 µg/ml). Ct value of β actin obtained from RNA isolated by trizol method was higher than the Ct value obtained by trizol in conjugation with the column method (31.41 and 15.41 respectively). Conclusion: Combination of trizol along with silica column resulted in better quality and improved yield of RNA suitable for gene quantification by Real time PCR. 相似文献
15.
M. F. Frosolono Y. Kress M. Wittner R. M. Rosenbaum 《In vitro cellular & developmental biology. Plant》1976,12(10):708-717
Summary Type II cell enriched fractions were isolated from rabbit and rat lungs using density gradient centrifugation. Cultures established
from these fractions contained predominantly cells similar in most morphological respects to type II pneumocytes. These were
in continuous replicating culture for 1 year and still exhibited contact inhibition. Membrane-bound structures reminiscent
of, but no longer strictly identical to, type II cell lamellar cytosomes were seen in cells from these long-term cultures
although their numbers were reduced in comparison to lamellar bodies in freshly isolated cells. Mitochondrial numbers and
sizes, determined morphometrically, were reduced after culture in comparison to freshly isolated type II cells and those in
situ. Phosphatidylcholine was synthesized by these cells and released into the extracellular medium. Application of laser
activated electronic sizing data, confirmed by direct micrometry, demonstrated a significant increase in cell size as a function
of culture. This sizing data, after prior confirmation by electron microscopy, was used as an aid in identifying type II cells
and macrophages in dispersion, especially with those cells derived from rabbit lungs. 相似文献
16.
Michiko Horiguchi Shinichi Hata Yuya Tsurudome Kentaro Ushijima 《Journal of cellular and molecular medicine》2021,25(9):4298-4306
Regenerative therapeutic approaches involving the transplantation of stem cells differentiated into insulin-producing cells are being studied in patients with rapidly progressing severe diabetes. Adipose-derived mesenchymal stem cells have been reported to have varied cellular characteristics depending on the biological environment of the location from which they were harvested. However, the characteristics of mesenchymal stem cells in type II diabetes have not been clarified. In this study, we observed the organelles of mesenchymal stem cells from patients with type II diabetes under a transmission electron microscope to determine the structure of stem cells in type II diabetes. Transmission electron microscopic observation of mesenchymal stem cells from healthy volunteers (N-ADSC) and those from patients with type II diabetes (T2DM-ADSC) revealed enlarged nuclei and degenerated mitochondrial cristae in T2DM-ADSCs. Moreover, T2DM-ADSCs were shown to exhibit a lower expression of Emerin, a constituent protein of the nuclear membrane, and a decreased level of mitochondrial enzyme activity. In this study, we successfully demonstrated the altered structure of nuclear membrane and the decreased mitochondrial enzyme activity in adipose-derived mesenchymal cells from patients with type II diabetes. These findings have contributed to the understanding of type II diabetes-associated changes in mesenchymal stem cells used for regenerative therapy. 相似文献
17.
J. Van Kapel B.G. Loef J. Lindemans J. Abels 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,676(3):307-313
25 mg of human holo-transcobalamin II with a specific cobalamin-binding capacity of 0.95 mol cobalamin/mol TC II was purified from 122 kg Cohn fraction III with a yield of 73% and a purification factor of 9.34 · 105. Consecutive purification steps comprised CM-Sephadex batchwise ion-exchange chromatography, affinity chromatography, using cyanocobalamin as a ligand, thermolabilly attached to 3.3′-diaminodipropylamine-substituted CH-Sepharose, and gel filtration. The high yield of the purification procedure was achieved by improving the stability of apo-transcobalamin II in the eluate of the CM-Sephadex, and by a few other modifications of a former procedure. In the latter, rapid denaturation of apo-transcobalamin II prohibited the use of long term affinity chromatography, which is obligatory for processing large amounts of Cohn fraction. In addition, subfractionation of transcobalamin II into smaller fragments which occurred in SDS-polyacrylamide gel electrophoresis in previous studies, was now reduced, indicating that proteolysis in the CM-Sephadex eluate had been prevented effectively. 相似文献
18.
19.
After many unsuccessful attempts to obtain biologically active mRNAs from spruce (Picea abies) tissues using available protocols, we have adapted a procedure for the isolation of RNAs from needles, shoots, and callus
ofPicea species. Our modifications permit the recovery of and an average of 300 μg RNA per g of needles that is suitable for translationin vitro, northern hybridizations, and the construction of cDNA libraries. 相似文献
20.
Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both
a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions
and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic
data indicated that fractions containing ≥97% G1 cells, ≥80% S cells, and 70–75% G2 cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number
of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle
was a constant 60–70%, which was comparable to the 60–80% usually found for asynchronous 9L cells. The percentage of cells
in the G1, S, and G2 phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of
the DNA histogram obtained from flow cytometry. In general, the degree of synchrony was related to the coefficient of variation
(CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the
≥97% G1 population was allowed to progress to S and G2, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in
the S or G2 phases was direct elutriation with the long collection method. 相似文献