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1.
Tadashi Mio Yuichi Adachi Debra J. Romberger Ronald F. Ertl Stephen I. Rennard 《In vitro cellular & developmental biology. Animal》1996,32(7):427-433
Summary Fibroblastsin vivo reside in a three-dimensional (3-D) matrix. The 3-D culture method using collagen gels provides valuable information, but
is also has some practical difficulties. In particular, the changes caused by the contraction of gels and the occasional abrupt
detachment from the underlying surface have made extended culture difficult. In this study, the 3-D culture method was modified
in order to observe the cells with minimal change of substrata for longer periods. The proliferation characteristics of fibroblasts
cultured in gels in response to fetal calf serum (FCS), to two defined growth factors, insulin and platelet-derived growth
factor (PDGF), and to a growth inhibitory factor, prostaglandin E2 (PGE2), were evaluated with this system in comparison with monolayer cultured fibroblasts. The DNA content of fibroblasts cultured
both in gels and on dishes increased in response to FCS in a concentration-dependent manner. The proliferation of gel-cultured
fibroblasts, however, was lower than that of dish-cultured cells, and higher concentrations of serum were necessary for proliferation.
The response of gel-cultured cells to PDGF was also less than that of dish-cultured cells. In addition, fibroblasts cultured
in gel culture did not respond to insulin, while the fibroblasts on dishes responded to insulin in a concentration-dependent
manner. In contrast to the reduced response to growth stimulators, PGE2 inhibited proliferation in gel culture and in monolayer culture similarly. The reduced responsiveness to growth stimulation
but equivalent response to growth inhibition may account for reduced proliferation of fibroblasts in 3-D culture. 相似文献
2.
3.
Lucie Gregoire Adnan Munkarah Raja Rabah Robert T. Morris Wayne D. Lancaster 《In vitro cellular & developmental biology. Animal》1998,34(8):636-639
Summary The objective of this work was to establish an in vitro multidimensional culture system for human ovarian surface epithelial (HOSE) cells as a model for ovarian carcinogenesis.
The epithelial origin of cell outgrowth from cells obtained from the ovarian surface was confirmed by keratin staining. Two
cultures from two different patients were established, HOSE-A and HOSE-B. Cultures were infected with a retrovirus expressing
human papillomavirus genes E6 and E7 to extend their life span. HOSE cells were seeded onto collagen gels containing NIH3T3-J2
fibroblasts as feeder cells and grown to confluence submerged in growth medium. The collagen bed was then raised to the air-medium
interface for 7 d (organotypic culture). Microscopically, fixed cultures revealed a single layer of flat cells growing on
the collagen surface, reminiscent of HOSE cells in vivo. Infected HOSE-A and HOSE-B cells exhibited aberrant growth because they stratified. In addition, established ovarian cancer
lines grown in this fashion stratified and showed malignant phenotypes. Thus, cells grown in organotypic culture resemble
their in vivo counterparts, providing a basis for establishing a system to study growth, proliferation, differential gene expression, and
perhaps malignant transformation of HOSE cells. 相似文献
4.
Reconstituted human oral and esophageal mucosa in culture 总被引:14,自引:0,他引:14
Dolphine Oda Christopher E. Savard Lydia Eng John Sekijima Geoffrey Haigh Sum P. Lee 《In vitro cellular & developmental biology. Animal》1998,34(1):46-52
Summary We have successfully established monolayer and organotypic culture techniques for growing human oral and esophageal epithelial
cells. Cells in monolayer culture were grown in serum-free medium, modified from techniques previously reported by our group.
The organotypic cultures were grown in a defined medium supplemented with 10% fetal calf serum. Oral and esophageal cells
were maintained in keratinocyte basal medium with pituitary extract and other supplements, and 0.05 mM calcium for 7–9 and 9–11 passages, respectively. Both cell types had similar morphology by phase contrast microscopy. When
confluent, the cells were predominantly small, basaloid, and uniform and interspersed with larger, differentiated cells. By
immunohistochemistry, both cell types in monolayer were positive to AE1, AE3, and 34BE12 antibodies to keratins of stratified
epithelia. Oral epithelial cells in monolayer also were positive to 35BH11, representative of simple epithelial keratins,
while esophageal cells were not. The esophageal cells were focally positive to K13, while the oral cells were negative. Both
were negative for K19. When comparing monolayer to organotypic cultures and to in vivo specimens, there was a significant difference in the expression of keratins.
Using organotypic cultures, AE1, AE3, and 34BE12 were strongly positive in both oral and esophageal cells, similar to in vivo tissues. In contrast to monolayers, both were also focally positive for K19. Esophageal cells were strongly positive for
K13, while the oral cells were middly but uniformly positive. Both were negative for keratins of simple epithelia. These two
cell culture techniques offer unique opportunities to study the pathobiology, including carcinogenesis, of stable cell systems
from the oral and esophageal epithelia. 相似文献
5.
K Takeuchi Y Nagata B A Sandow G D Hodgen 《Molecular reproduction and development》1992,32(3):236-242
We have established a monolayer culture system for human fallopian tube epithelial cells. The cells were isolated from tubes using collagenase digestion, and were cultured in Ham's F-10 supplemented with 15% fetal calf serum. The epithelial cells derived from culture were characterized using immunocytochemical staining and electron microscopy. These cells were stained with antikeratin and anti-epithelial membrane antigen, but showed no staining after treatment with antivimentin. Electron microscopy showed many microvilli on the cell surface and tight junctions or desmosomes at areas of cell-cell contact. Cell proliferation was enhanced by epidermal growth factor, but not by fibroblast growth factor, insulin, transferrin, estradiol-17 beta, or progesterone. The 2-cell ICR mouse pre-embryos were co-cultured for 4 days with tubal epithelial cells (A) (n = 98), in cell-conditioned medium (B) (n = 83), or in medium alone (C) (n = 72). During the first 24 h in culture, for groups A and B, the rates of cleavage to the 4-cell stage were 90.9% and 81.9%, respectively. Cleavage rates in these two groups were significantly higher (P = 0.0012, P less than 0.00001) than in group C (56.9%). After 72 h in culture, the rates of development to the blastocyst stage were significantly higher for groups A and B compared to group C (89.6% and 73.5% vs. 54.5%, P less than 0.00001, P = 0.0002). These results suggest that factor(s) from tubal epithelial cells may facilitate the development of mouse pre-embryos throughout the pre-implantation stages. 相似文献
6.
Properties of a polarized primary culture from rat renal inner medullary collecting duct (IMCD) cells 总被引:1,自引:0,他引:1
Birgit Ruhfus H. Günther Bauernschmitt Rolf K. H. Kinne 《In vitro cellular & developmental biology. Animal》1998,34(3):227-231
Summary A primary culture from rat renal IMCD cells was established to investigate the permeability characteristics of the luminal
and contraluminal plasma membranes of the papillary collecting duct in vitro. Freshly isolated IMCD cells were grown on filters in a special “epithelial cell” medium. Confluency was proved with an epithelial
volt/ohm meter. After 7 d of culture the transepithelial resistance reached more than 1000 Ω×cm2. A polarization of the cells with regard to a basolateral localization of a lactate efflux system, and an l-alanine transport system was achieved. The hypotonicity-activated release systems for the organic osmolytes sorbitol and
betaine were also located basolaterally, whereas taurine, glycerophosphorylcholine, and myo-inositol left the cells at both
cell poles but with different capacity. Morphological observations revealed also that the monolayer was well differentiated.
Thus, a model of a renal collecting duct epithelium was established which can be used to analyze polarized and differentiated
transport processes across the epithelial cells and their plasma membranes. 相似文献
7.
Derivation of the clonal‐cell lines from Siberian sturgeon (Acipenser baerii) head‐kidney cell lines and its applicability to foreign gene expression and virus culture
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This study was conducted to establish and characterize the clonal‐cell lines from Siberian sturgeon Acipenser baerii head‐kidney tissues and to evaluate its applicability as a research tool. From the culture of A. baerii head‐kidney derived cells, 10 cell lines were established first and then eight clonal‐cell lines were derived from clonal growth and colony expansion of two cell lines that showed significant high colony‐forming ability. All eight clonal‐cell lines were morphologically similar and grew stably under monolayer culture but their growth rates were significantly different. They possessed diploid DNA contents, expressed epithelial cell‐related genes and showed strong anchorage dependency to substrates. When a clonal‐cell line was transfected separately with three plasmid vectors including fluorescent reporter genes driven by cytomegalovirus, marine medaka Oryzias dancena β‐actin or A. baerii β‐actin promoter, the cell lines expressed fluorescent signals regardless of promoter types. The cells harbouring foreign genes could be expanded to stable cell lines under drug selection and then they additionally could form the extensively proliferating colonies at low‐density culture. Finally, the clonal‐cell lines showed the susceptibility to viral haemorrhagic septicaemia virus (VHSV). Collectively, the clonal‐cell lines from A. baerii head kidney were established and these cell lines will be able to provide an excellent in vitro system for various biological studies in this fish species. 相似文献
8.
S. W. Hayward R. Dahiya G. R. Cunha J. Bartek N. Deshpande P. Narayan 《In vitro cellular & developmental biology. Animal》1995,31(1):14-24
Summary This report describes the development and characterization of an epithelial cell line (BPH-1) from human prostate tissue obtained
by transurethral resection. Primary epithelial cell cultures were immortalized with SV40 large T antigen. One of the isolated
clones was designated BPH-1. These cells have a cobblestone appearance in monolayer culture and are non-tumorigenic in nude
mice following subcutaneous injection or subrenal capsule grafting. They express the SV40 large T antigen and exhibit increased
levels of p53, as determined by immunocytochemistry. Cytogenetic analysis by G-banding demonstrated an aneuploid karyotype
with a modal chromosome number of 76 (range 71 to 79,n=28) and 6 to 8 marker chromosomes. Some structurally rearranged chromosomes were observed, but the Y chromosome was normal.
The expressed cytokeratin profile was consistent with a prostatic luminal epithelial cell. This profile was the same as that
of primary prostatic epithelial cultures from which the BPH-1 cells were derived. In serum-free culture in plastic dishes
epidermal growth factor (EGF), transforming growth factor (TGF)-α, fibroblast growth factor (FGF) 1 (aFGF), and FGF 7 (KGF) induced increased proliferation in these cells whereas FGF 2 (bFGF),
TGF-β1, and TGF-β2 inhibited proliferative activity. Testosterone had no direct effect on the proliferative rate of BPH-1 cells. 5α-Reductase, 3α-hydroxysteroid oxidoreductase, and 17β-hydroxy-steroid oxidoreductase activities were detected in BPH-1 cells. Expression of androgen receptors and the secretory
markers, prostate specific antigen and prostatic acid phosphatase, were not detectable by immunocytochemistry, biochemical
assay, or RT-PCR analysis. 相似文献
9.
Oliver Baylis Paul Rooney Francisco Figueiredo Majlinda Lako Sajjad Ahmad 《Journal of cellular physiology》2013,228(5):1025-1030
Limbal stem cell deficiency is a blinding disease which affects the cornea at the front of the eye. The definitive cure involves replacing the corneal epithelial (limbal) stem cells, for example by transplanting cultured limbal epithelial cells. One method of performing cultures is to grow a sheet of epithelial cells from a limbal explant on human amniotic membrane. The growth of limbal tissue can be variable. The aim of this study is to investigate how different donor and culture factors influence the ex vivo growth of cadaveric limbal explants. Limbal explant cultures were established from 10 different cadaveric organ cultured corneo‐scleral discs. The growth rate and the time taken for growth to be established were determined. Statistical analysis was performed to assess correlation between these factors and donor variables including donor age, sex, time from donor death to enucleation, time from enucleation to organ culture storage and duration in organ culture. Growth curves consistently showed a lag phase followed by a steeper linear growth phase. Donor age, time between death and enucleation, and time between enucleation and organ culture were not correlated to the lag time or the growth rate. Time in organ culture had a significant correlation with the duration of lag time (P = 0.003), but no relationship with the linear growth rate. This study shows that an important factor correlating with growth variation is the duration of corneo‐scleral tissue in organ culture. Interestingly, donor age was not correlated with limbal explant growth. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc. 相似文献
10.
11.
Co-culture of primary pulmonary cells to model alveolar injury and translocation of proteins 总被引:2,自引:0,他引:2
James B. Mangum Jeffrey I. Everitt James C. Bonner Lynne R. Moore Arnold R. Brody 《In vitro cellular & developmental biology. Plant》1990,26(12):1135-1143
Summary Primary rat alveolar type II cells and early passage rat lung fibroblasts were co-cultured on opposite sides of a collagen-coated
polycarbonate filter. This is an approach to “model”, in part, an alveolar wall to study mechanisms of cytotoxicity and translocation
of bioactive materials from the alveolar space to the lung interstitium. Type II cells were recovered from adult rat (Fischer
344) lungs by enzyme digestion and “panning”. Lung fibroblasts were separated from the same species, cultured initially in
10% fetal bovine serum and used in the co-culture system at early passage. The type II cells formed a monolayer of defferentiated
epithelium which provided a barrier on the upper side of the collagen (human type IV)-coated filter. The fibroblasts on the
bottom of the filter replicated logarithmically in the presence of serum, could be rendered quiescent in defined medium and
then returned to rapid growth phase with the reintroduction of serum. The intact epithelial monolayer excluded trypan blue,
albumin, platelet-derived growth factor, and alpha2-macroglobulin from the lower compartment of the culture chamber. Altering the integrity of the monolayer by a variety of
means allowed translocation of these materials through the collagen-coated filters. Particularly interesting was the effect
of taurine chloramine which caused subtle changes in the alveolar epithelium and allowed subsequent translocation of albumin.
In addition, we showed that rat alveolar macrophages remain viable with some spreading on the surface of the epithelial monolayer.
This co-culture system will have future application in the study of how reactive oxygen species might affect the epithelial
barrier, and whether macrophage-derived growth factors can influence fibroblast proliferation if the monolayer is intact or
injured. 相似文献
12.
Aimei Zhong Guohua Wang Jie Yang Qijun Xu Quan Yuan Yanqing Yang Yun Xia Ke Guo Raymund E. Horch Jiaming Sun 《Journal of cellular and molecular medicine》2014,18(7):1257-1266
True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co‐culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non‐macromastic epithelial cells when co‐cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia‐derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co‐culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co‐cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy. 相似文献
13.
Dolphine Oda Christopher E. Savard Toan D. Nguyen Eric R. Swenson Sum P. Lee 《In vitro cellular & developmental biology. Animal》1998,34(3):211-216
Summary Attempts to grow human pancreatic duct epithelial cells in long-term culture have proven difficult. We have developed a system
of growing these cells for several passages by adapting methods used to culture dog pancreatic duct cells. Epithelial cells
were enzymatically dissociated from the main pancreatic duct and plated onto collagen-coated culture inserts suspended above
a human fibroblast feeder layer. After primary culture, the cells were either passaged onto new inserts or plastic tissue
culture plates in the absence of collagen. Cells grown on the latter plates were maintained in a serum-free medium. Primary
pancreatic duct epithelial cells grow steadily to confluence as a monolayer in the feeder layer system. After primary culture,
cells passaged onto new inserts with fresh feeder layer or plastic plates and fed with serum-free medium continued to develop
into confluent monolayers for up to four passages. The cells were columnar with prominent apical microvilli, sub-apical secretory
vesicles, and lateral intercellular junctions resembling the morphology of normal in vivo epithelial cells. These cells were also positive for cytokeratin 19, 7, and 8 and carbonic anhydrase II, as measured by immunohistochemistry.
Metabolically, these cells synthesized and secreted mucin, as measured by incorporation of tritiated N-acetyl-d-glucosamine. In conclusion, we demonstrated that human pancreatic epithelial cells from the main duct can be successfully
grown in culture and repeatedly passaged using a feeder layer system, with serum-free medium, and in organotypic cultures. 相似文献
14.
Our laboratory had developed a methodology to expand epithelial cells in culture by growing keratinocyte monolayers, under large volumes of medium that produces large numbers of keratinocytes that leave the monolayer and move into suspension. The cells have been defined as epithelial Pop Up Keratinocytes or ePUKs cells and appear to be highly suitable for clinical applications. In this publication we extend the characterization of the cells with a detailed analysis of the capabilities of the monolayer of a single culture flask to produce, over time, ePUK cells. The cells were characterized using standard epithelial markers for proliferation and differentiation. Analysis of morphology of the monolayer formed and total number of cells produced is presented for a variety of human epithelial cell strains. These keratinocytes provide an additional controlled human cell system for investigation of the mechanisms regulating epithelia cell growth and differentiation and since they are produced in large numbers, they are highly suitable for use in epithelial cell banking. 相似文献
15.
Continuous multiplication of rabbit tracheal epithelial cells in a defined,hormone-supplemented medium 总被引:16,自引:0,他引:16
Summary An improved Ham’s F12 nutrient medium supplemented with epidermal growth factor (EGF), insulin (INS), and transferrin (TF)
was developed for continuous proliferation and clonal growth of primary rabbit tracheal epithelial (TE) cells in culture.
The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measurable stimulation
on TE cell growth and plating efficiency. However, serum levels higher than 0.1% inhibited cell growth and also masked the
growth stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented
medium, and in the presence of a trace amount of serum (0.01%), 10 to 20% of the protease-dissociated TE cells attached to
the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal growth occurred at a seeding
density of 17 cells/cm2 with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a
0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of
cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron
microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture
for extended periods of time without the aid of a fibroblast feeder layer or explant tissue. This system could be useful for
the study of cell differentiation of tracheal epithelium. 相似文献
16.
Chengwei Chai Shuaiyu Zheng Jiexiong Feng Xiaojuan Wu Jixin Yang Mingfa Wei 《In vitro cellular & developmental biology. Animal》2010,46(10):820-823
Culture of extrahepatic bile duct epithelial cells is a useful model to investigate physiology of extrahepatic bile duct epithelia
and hepatobiliary disease mechanisms. The aim of this work was to establish and characterize a primary murine extrahepatic
bile duct epithelial cell culture. Epithelial cells were isolated from extrahepatic bile ducts of BALB/c mice that were intraperitoneally
injected with newborn bovine serum to induce the proliferation of extrahepatic bile ducts’ epithelial cells and cultured on
rat tail type I collagen-coated plastic culture flask containing DMEM/HamF12 with 10% FBS and 10 ng/ml epidermal growth factor
at 37°C in an incubator with 5% humidified CO2. The cells showed typical morphologic characteristics of epithelial phenotypes with cobblestone appearance in monolayer within
5–6 d after culture; they were positive against anticytokeratin-19 immunostaining. Transmission electron microscopy showed
typical bile duct epithelia with microvilli on the cytomembrance, Golgi complex, massive mitochondria, and rough endoplasmic
reticulum in the cytoplasmic. The growth curve of the epithelial cells was determined by a MTT assay which showed a normal
sigmoidal growth curve. This culture technique might be a reliable method for isolation, purification, and primary culture
of extrahepatic bile duct epithelial cells that can serve as a model for in vitro studies on the pathophysiology of hepatobiliary
diseases as well as pharmacological and toxicological targets relevant to hepatobiliary diseases. 相似文献
17.
This study investigated the effects of the in vitro co-culture of mouse embryos with non-polarized or polarized uterine epithelial cells, using sequential culture media, on their development to blastocysts, blastocyst quality (blastocyst diameter and cell number), apoptosis, Bcl-2 and Bax gene expression. There were three treatments, all of which used sequential culture media. The treatments were no co-culture (control), non-polarized or polarized epithelial cell monolayer co-culture in 24-well tissue culture plates. Mouse uterine epithelial cells were isolated enzymatically and were seeded either on the surface of the culture plate (non-polarized monolayer) or on a Millipore filter insert coated with extra-cellular matrix extract (polarized monolayer) that was then placed in the culture plate. Two-cell mouse embryos were cultured in G-1 ver3 medium to the eight-cell stage when they were randomly assigned to the treatments. The culture medium was G-2 ver3 during the treatment phase of the study. Significances of differences were evaluated by the one-way analysis of variance for continuous data. The epithelial cells cultured on Millipore filters became polarized and their morphology compared favorably with those cultured on the surface of the culture plate and in vivo uterine epithelial cells. After 96 h on the treatments, the polarized monolayer had supported the development of significantly more hatched blastocysts (80.0%; P<0.05) than the non-polarized monolayer (63.4%) or the control (61.4%) culture treatments. Co-culture resulted in the production of blastocysts with significantly more cells (non-polarized monolayer 56.7+/-2.1, polarized monolayer 61.9+/-2.1) than the control culture (42.8+/-2.6; P<0.05) but the diameter and shape of the blastocysts were not significantly different. The proportion of blastocysts with apoptotic blastomere was higher for the control culture (94.4%) than for the non-polarized (68.2%) or polarized (66.7%) co-culture systems (P<0.05). Moreover, the apoptotic index was significantly higher in control blastocysts (5.6+/-0.9; P<0.05) than in non-polarized (1.7+/-0.3) or polarized (1.5+/-0.3) co-culture. In the control, Bax mRNA was strongly expressed when compared to co-culture treatments (P<0.05), whereas, the relative abundance of Bcl-2 mRNA to the beta-tubulin was lower than co-culture treatments (P<0.05). It is concluded that a co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method of producing mouse embryos. 相似文献
18.
Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes 总被引:5,自引:0,他引:5
Summary
In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration
of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated
mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20°
C in 5% CO2:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered
to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets
within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d
after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive
nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus,
cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA
synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of
epithelium in bivalves. 相似文献
19.
Arvind K. Virmani Bashoo Naziruddin Vinay C. Desai Jon P. Lowry Donald C. Graves Goverdhan P. Sachdev 《In vitro cellular & developmental biology. Animal》1992,28(2):120-127
Summary The purpose of this investigation was to provide evidence for the secretion of high molecular weight mucins, CTM-A and CTM-B,
in primary culture of canine tracheal epithelial (CTE) cells. The cells were isolated from tracheas of mongrel dogs by pronase
treatment. Primary cultures of the epithelial cells were established using ICN cellagen inserts in Dulbecco’s modified Eagle’s/F12
medium supplemented with growth factors and could be maintained for up to 23 days. The evidence for the mucin secretion in
culture medium and their localization in the cells was established by a) positive immunocytochemical staining using specific
antibodies developed against purified native as well as deglycosylated CTM-A and CTM-B; b) incorporation of labeled amino
acids, followed by electrophoresis and autoradiography detection of glycoconjugates purified from the culture medium; c) comparison
of the amino acid compositions of mucin purified from canine tracheal pouch secretions and that purified from the culture
medium; and d) Western blot analyses using specific polyclonal antibodies directed against deglycosylated CTM-A and CTM-B.
Immunoaffinity purified secreted labeled glycoconjugates were resistant to hyaluronidase treatment. The effects of cyclic
AMP (1 × 10−5
M), dibutyryl cyclic AMP (1 × 10−5
M), 8-bromocyclic AMP (1 × 10−5
M), and prostaglandin E1 (1 × 10−6
M) on mucin secretion by CTE cells were also investigated. Secretion of mucins by CTE cells in culture was considerably more
enhanced by 8-bromocyclic AMP than that observed for other secretagogues used in this study. 相似文献
20.
Characterization of differentiated syrian golden hamster pancreatic duct cells maintained in extended monolayer culture 总被引:4,自引:0,他引:4
S. Hubchak M. M. Mangino M. K. Reddy D. G. Scarpelli 《In vitro cellular & developmental biology. Plant》1990,26(9):889-897
Summary Epithelial cells isolated from fragments of hamster pancreas interlobular ducts were freed of fibroblast contamination by
plating them on air-dried collagen, maintaining them in serum-free Dulbecco's modified Eagle's (DME):F12 medium suppleneted
with growth factors, and selecting fibroblast-free aggregates of duct cells with cloning cylinders. Duct epithelial cells
plated on rat type I collagen gel and maintained in DME:F12 supplemented with Nu Serum IV, bovine pituitary extract, epidermal
growth factor, 3,3′, 5-triodothyronine, dexamethasone, and insulin, transferrin, selenium, and linoleic acid conjugated to
bovine serum albumin (ITS+), showed optimal growth as monolayers with a doubling time of about 20 h and were propagated for as long as 26 wk. Early
passage cells consisted of cuboidal cells with microvilli on their apical surface, complex basolateral membranes, numerous
elongated mitochondria, and both free and membrane-bound ribosomes. Cell grown as monolayers for 3 mo. were more flattened
and contained fewer apical microvilli, mitochondria, and profiles of rough surfaced endoplasmic reticulum; in addition, there
were numerous autophagic vacuoles. Functional characteristics of differentiated pancreatic duct cells which were maintained
during extended monolayer culture included intracellular levels of carbonic anhydrase and their capacity to generate cyclic
AMP (cAMP) after stimulation by 1×10−6
M secretin. From 5 to 7 wk in culture, levels of carbonic anhydrase remained stable but after 25 to 26 wk decreased by 1.9-fold.
At 5 to 7 wk of culture, cyclic AMP increased 8.7-fold over basal levels after secretin stimulation. Although pancreatic duct
cells cultured for 25 to 26 wk showed lower basal levels of cAMP, they were still capable of generating significant levels
of cAMP after exposure to serretin with a 7.0-fold increase, indicating that secretin receptors and the adenyl cyclase system
were both present and functional. These experiments document that pancreatic duct monolayer cultures can be maintained in
a differentiated state for up to 6 mo. and suggest that this culture system may be useful for in vitro physiologic and pathologic
studies.
This research was supported by grant CA34051 from the National Cancer Institute, Bethesda, MD. 相似文献