共查询到20条相似文献,搜索用时 15 毫秒
1.
Akon Higuchi Yasunari Takanashi Takeshi Ohno Tetsuo Asakura Chong-Su Cho Toshihiro Akaike Mariko Hara 《Cytotechnology》2002,39(3):131-137
The fibroblast cells from normal human skin were cultured on Langmuir-Blodgett (LB) and cast membranes prepared using extracellular
matrix proteins (e.g., collagen, fibronectin, laminin and vitronectin). The cell density of the fibroblast cells cultured
on the cast membranes was found to be higher than that on the cast membranes made of fibronectin, vitronectin and collagen-blended
membranes. This indicates that not only the primary structure of proteins but the preparation methods of the membranes, i.e.,
casting and LB methods, are a strong factor affecting cell growth. The concentration and production of interferon-β per unit
cell were found to be higher on the LB membranes than on the cast membranes made of the same proteins except in the case of
collagen. However, the cell density on the cast membranes was higher than that on the LB membranes. These results appear to
result from the suppressed growth of NB1-RGB cells on the LB membranes leading to the enhanced production of interferon-β
on the LB membranes. The highest production of interferon-β per unit cell was observed for the NB1-RGB cells on the collagen-blended
membranes with fibronectin and vitronectin. The collagen-blended membranes appear to offer a more natural and appropriate
environment for NB1-RGB cells to produce interferon-β.
This revised version was published online in September 2006 with corrections to the Cover Date. 相似文献
2.
Mariko Hara Yasunari Takanashi Nobuya Tuzuki Hiroko Kawakami Kazunori Toma Akon Higuchi 《Cytotechnology》2003,42(1):13-20
Cell growth and production of interferon-β (IFN-β) were investigated for normal human skin fibroblast cells (NB1-RGB) cultured
on membranes prepared from peptide-lipids containing the arginine-glycine-aspartic acid [Arg-Gly-Asp] (RGD), tyrosine-isoleucine-glycine-serine-arginine
[Tyr-Ile-Gly-Ser-Arg] (YIGSR) and arginine-glutamic acid-aspartic acid-valine [Arg-Glu-Asp-Val] (REDV) peptides. Cell density
was found to be approximately the same on various peptide-lipid membranes, whereas production of IFN-β depended significantly
on the peptide-lipid membranes on which NB1-RGB cells were cultured. The highest production of IFN-β was observed for NB1-RGB
cells on REDV-lipid membranes prepared by a casting method (REDV-cast membranes) after 24 hr of cultivation. Specific binding
between REDV of REDV-cast membranes and the receptor on the NB1-RGB cells may have caused the specific cell response for the
production of IFN-β.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
3.
The Bombyx mori 30Kc gene is known to have anti-apoptotic activity and can enhance the cell growth and expression of recombinant proteins in anchorage-dependent CHO cell cultures. In this study, an interferon-β (IFN-β)-producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed to investigate the effect of 30Kc6 expression on the production of IFN-β in serum-free suspension culture. The 30Kc6 expressing cell line showed lower apoptotic activity and prolonged cell viability under apoptotic conditions induced by the addition of sodium butyrate, staurosporine, or the removal of serum. The 30Kc6 expressing cell line also suppressed the loss of mitochondrial membrane potential induced under these conditions. It was observed that viability, and production of IFN-β were also enhanced by 30Kc6 expression in serum-free suspension cultures. These results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines. 相似文献
4.
Using high-cell-density culture of Escherichia coli under the control of an l-arabinose promoter (ParaB), several factors affecting the production of recombinant protein and the formation of inclusion bodies were studied. The
inducer, l-arabinose, showed a maximal induction level above 10.7 mM in the final concentration. The concentration of inducer also affected
the partition of interferon-α (IFN-α) into the soluble form and inclusion bodies. Induction kinetics of the rate of accumulation
of IFN-α on the ParaB promoter showed a slower rate than those of other promoter systems, for example T7, lac or tac. These innate characteristics of ParaB enabled cells to grow continuously in spite of the metabolic burden induced by the expression of foreign protein. The duration
time of induction could control the expression of both soluble and insoluble protein. The ratio of yeast extract to glycerol
(N/C ratio) in feeding media significantly affected both the production level of recombinant protein and inclusion body formation.
The reason for decreasing specific bioactivity during induction can be explained by the increased proportion of inclusion
bodies in the total expressed IFN-α.
Received: 21 May 1999 / Received last revision: 16 August 1999 / Accepted: 2 September 1999 相似文献
5.
The relationship between growth and interferon- (IFN-) production in the recombinant cell line CHO 320 was studied by varying the foetal calf serum (FCS) concentration. The specific growth rate varied with the initial FCS concentration in a manner which could be well fitted by the Monod model. TheK
s and
max
values were found to be 0.771% (v/v) serum and 0.031 h–1 respectively. The average specific IFN- production rates during the exponential phase increased with increasing FCS concentration. A good correlation between specific production rate and specific growth rate was found in all phases of the culture except the lag phase and it was clearly demonstrated that IFN- production was growth associated. Specific glucose and glutamine utilisation rates were inversely related to specific growth rates. 相似文献
6.
Tamio Mizukami Yoshinori Komatsu Noriko Hosoi Seiga Itoh Tetsuo Oka 《Biotechnology letters》1986,8(9):605-610
Summary Unusually low culture temperature, such as 20°C, was shown to be preferable for the synthesis of active human interferon- (IFN-) inE.
coli harboring a recombinant plasmid. TheE.
coli cells cultured at 20°C gave 8.6-fold higher IFN- activity than those cultured at 37°C. However, almost the equal amounts of IFN- protein were accumulated in both cells cultured at 20°C and at temperature higher than 20°C, suggesting that IFN- might exist as an active form in the cells cultured at 20°C, while as a rather denatured form in the cells cultured at higher temperature. 相似文献
7.
Takayoshi Tokiwa Kazuhisa Taketa Jiro Sato 《In vitro cellular & developmental biology. Plant》1987,23(12):830-836
Summary When primary cultures of fetal human liver cells established on type I collagen gels were compared to sister cultures developed
on tissue culture plastic, the cells in contact with type I collagen secreted albumin at a higher rate than those without
contact. The albumin secretion was dependent on the presence of hydrocortisone (HC) in the medium. Also, α-fetoprotein (AFP),
of which the level decreased gradually and became undetectable after 6 d regardless of the presence or absence of HC in the
cells cultured on plastic, was maintained for longer periods of time by plating the cells on type I collagen gels in the presence
of HC. Different secretion rates of albumin and AFP were observed after Day 13 and Day 16, respectively, between cells maintained
on type I collagen gels and those on film plastic. The cells secreted larger amounts of both albumin and AFP in plates coated
with type IV or I collagens than with fibronectin after Day 10. The cells cultured on type I collagen gels were cuboidal in
shape, whereas those on plastic were flattened in cultures with HC. These data indicate that the secretion of human albumin
and AFP is facilitated by synergies between HC and collagenous substrata. 相似文献
8.
9.
Yoshida M Nishikawa Y Omori Y Yoshioka T Tokairin T McCourt P Enomoto K 《Cell and tissue research》2007,329(2):273-282
Embryonic development of the liver is closely associated with vascular organization. However, little is known about the mechanisms
of vascular differentiation during liver development. Our previous study showed that the maturation of sinusoidal endothelial
cells (SECs) occurred during embryonic day 13.0 (E13.0) to E15.0. To improve our understanding of SEC differentiation, we
examined here the expression of maturation markers, SE-1 and stabilin-2, in fetal livers and also attempted to establish an
in vitro SEC differentiation system by culturing E13.5 fetal liver cells. Immunohistochemical examination of SE-1 and stabilin-2
expression during fetal rat liver development revealed that these differentiation markers were co-expressed in SECs in the
late stage of liver development, although stabilin-2 was expressed in almost all vascular endothelial cells in the early stage.
Liver cells from the E13.5 rat fetus were cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), transforming
growth factor β1 (TGF-β1) and VEGF plus SB-431542 (an inhibitor of the TGF-β1 receptor, activin receptor-like kinase 5 [ALK-5]).
In vitro SEC differentiation, as indicated by the appearance of cells co-expressing SE-1 and stabilin-2 and of cells with
cytoplasmic fenestrae in endothelial sheets, was induced by the addition of both VEGF and SB-431542, an inhibitor of the phosphorylation
of Smad2/3 but not that of Smad1/5/8 in the cultured cells. These results indicate for the first time that both VEGF signaling
and the blocking of the ALK-5-Smad2/3 signal pathway are important for the fetal differentiation of SECs. 相似文献
10.
The importance of cell cycle analysis in cell culture development has been widely recognised. Whether such analysis is useful
in indicating future performance of high cell density culture is uncertain. Using flow cytometric approach to address this
question, we utilised the fraction of cells in the S phase to control specific growth rate and productivity in spin filter
perfusion cultures and found a significant increase in the accumulated interferon-γ over that obtained from the nutrient-based
controlled fed culture. While a general decrease with time exists in both percentage of S phase cells and specific growth
rate, a clear oscillatory behaviour of both parameters is found in perfusion cultures.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
11.
Midori Hosobuchi Martha R. Stampfer 《In vitro cellular & developmental biology. Plant》1989,25(8):705-713
Summary Normal human mammary epithelial cells (HMEC) from different individual reduction mammoplasty specimens were all growth inhibited,
and showed a flattened, elongated morphology in response to human recombinant transforming growth factor β1 (TGFβ). The degree
of growth inhibition varied among specimens, but none of the normal HMEC maintained growth in the continued presence of TGFβ.
The degree of growth inhibition also varied with cell age in vitro, cells closer to senescence being more sensitive. TGFβ
sensitivity was additionally assayed in two established cell lines derived from one of the reduction mammoplasty specimens
after exposure to benzo(a)pyrene. Although varying degrees of growth inhibition and morphologic changes were observed in the
cell lines, both lines contained populations that maintained active growth in the presence of TGFβ. Subclones of these lines
demonstrated a great plasticity in their growth response to TGFβ, with individual clones ranging from strongly growth inhibited
to nearly unaffected. These results suggest that multiple factors influence the extent of TGFβ-induced growth effects on both
normal and transformed mammary epithelial cells, and that some of these factors may act through epigenetic mechanisms.
This work was supported by CA24844 from the National Institutes of Health, Bethesda, MD, and the Office of Energy Research,
Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-AC03-76SF00098. 相似文献
12.
Proteolytic cleavage of recombinant human interferon- (IFN-) expressed in Chinese hamster ovary (CHO) cells during batch fermentation has been monitored by mass spectrometric peptide mapping. IFN- was purified from cell-free culture supernatant by immunoaffinity chromatography and cleaved by endoprotease Asp-N. Peptide fragments were resolved by reverse-phase HPLC and identified by a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and automated N-terminal peptide sequencing. Using this approach, a peptide was identified as the C-terminal fragment of the IFN- polypeptide. Analysis of this peptide by MS indicated that the recombinant IFN- polypeptide secreted by CHO cells was truncated by at least ten amino acids, initially at Gln133-Met134. No full length (143 amino acids) polypeptide molecules were observed at any stages of the fermentation. Additional proteolytic cleavages at basic amino acids N-terminal of Gln133 occurred during the later stages of the culture resulting in a heterogeneous IFN- polypeptide population with 'ragged' C-termini. 相似文献
13.
Hiroshi Fukamachi John A. McLachlan 《In vitro cellular & developmental biology. Animal》1991,27(12):907-913
Summary The effects of different substrata and estradiol-17β(E2) on proliferation and differentiation of mouse uterine epithelial cells was examined in a serum-free primary culture system.
When cultured on rat-tail collagen gels, the epithelial cells rapidly increased in number to form a simple squamous cell layer
that exhibited a relatively undifferentiated state (a few short microvilli, no secretory granules, and poorly developed endoplasmic
reticulum). Addition of E2 into the culture medium did not affect the proliferation of epithelial cells on collagen gel. Uterine epithelial cells grown
on a reconstituted basement membrane-like substratum (Matrigel) formed a simple columnar/cuboidal cell layer exhibiting fully
developed characteristics (many long microvilli, many secretory granules, and fully developed endoplasmic reticulum). Examination
of epithelial proliferation by counting substratum-attached cell number revealed only a slow increase in cell growth on Matrigel,
and E2 did not significantly affect it. However, measurement of proliferating cells by labeling cells with 5-bromo-2′-deoxyuridine
revealed that cells on Matrigel were replicating and that E2 (10−7 to 10−11
M) actually significantly suppressed epithelial proliferation. However, there was not an effect of E2 on total cell number, indicating that the cells in control medium replicate faster and detach more readily from the substratum
than those in E2-supplemented medium on Matrigel. Thus, it is probable that E2 significantly reduces the rate of cell detachment from the substratum, which may mimic the in vivo condition where significant
decrease in apoptosis or cell death is induced by E2. 相似文献
14.
Kazuo Fushimi Kakuji Torigoe Hiroshi Yamauchi Shouji Furusako Masashi Kurimoto Masayoshi Namba 《In vitro cellular & developmental biology. Animal》1998,34(6):463-467
Summary To develop a new gene therapy model for cancer, a clonal cell line (KMST-6/TNF) which produces human tumor necrosis factor
α (hTNF-α) has been developed by introducing hTNF-α cDNA into a human immortal fibroblast cell line (KMST-6). The conditioned
medium (CM) of KMST-6/TNF cells inhibited the growth of various malignant human cell lines, but not that of normal human fibroblasts.
Although the growth inhibitory effects of KMST-6/TNF CM were neutralized to a considerable degree by anti-TNF-α antibody,
its inhibitory effects were more marked than the purified human natural TNF-α itself in the same units, suggesting that KMST-6/TNF
CM contains some growth inhibitory substances other than TNF-α. However, interferons α, β, and γ were undetectable in the
KMST-6/TNF CM. 相似文献
15.
Felicia L. Cianciarulo Paul D. Phillips Vincent J. Cristofalo 《In vitro cellular & developmental biology. Animal》1993,29(8):656-660
Summary Tumor necrosis factor-α (TNF) and various interferons (IFN) have potent cytostatic or cytotoxic effects on a variety of human
tumor-derived cell lines. Their effects on normal cells are more controversial. We have examined the effects of TNF and IFN-β
on the proliferation of WI-38 cells in a serum-free, growth factor-supplemented medium and in serum-containing medium. These
cells respond to the combination of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and dexamethasone
by DNA synthesis at a rate and extent equivalent to serum-stimulated cells. TNF has no effect on this growth factor-stimulated
proliferation. However, it is stimulatory in serum-containing medium. IFN-β inhibits DNA synthesis 60 to 70% in both young
and senescent cells. TNF and IFN-β together have a synergistic effect and completely inhibit growth factor-stimulated DNA
synthesis in young cells. No synergism was observed with senescent cells. TNF stimulated an increase in the number of EGF
specific binding sites two- to threefold in 24 h in both young and senescent cells. This seems to result from a proportional
increase in a very high affinity binding site. IFN-β has little or no effect on EGF binding either alone or in combination
with TNF. 相似文献
16.
Yifan Zhu Svetlana Karakhanova Xiaolun Huang Shao ping Deng Jens Werner Alexandr V. Bazhin 《Experimental cell research》2014
The cytokine interferon-α (IFNα) belongs to the group of type I interferons already used in cancer therapy. This drug possesses radio- and chemo-sensitizing, and shows anti-angiogenic properties. Cancer stem cells (CSC) are a unique population of tumor cells that initiate secondary tumors, and are responsible for metastasis formation. Patients with pancreatic ductal adenocarcinoma (PDAC) have an especially poor prognosis, with 5-year survival rates of only ~1% and median survival of 4–6 months. PDAC is characterized by the presence of CSC. In this work we demonstrate for the first time that IFNα up-regulates the expression of the CSC markers CD24, CD44 and CD133 in in vitro and in vivo models of PDAC. We showed the IFNα effects on the migration and invasion of PDAC cells, which is associated with the level of the CSC marker expression. In vivo, this drug inhibits tumor growth but promotes metastasis formation in the early stage of tumor growth. We propose that IFNα may enhance the enrichment of CSC in PDAC tumors. Additionally we also suggest that in combination therapy of solid tumors with IFNα, this drug should be given to patients prior to chemotherapy to achieve the CSC activation. 相似文献
17.
The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA
bovine serum albumin
- CHO
chinese hamster ovary
- DHFR
dihydrofolate reductase
- FCS
foetal calf serum
- IFN-
human interferon-gamma
-
q
IFN
specific interferon production rate
-
specific growth rate
- 2N
doubly-gycosylated
- 1N
singly-glycosylated
- ON
non-glycosylated 相似文献
18.
19.
20.
George I. Malinin Francis J. Hornicek Theodore I. Malinin 《Molecular and cellular biochemistry》1990,95(2):177-182
Interleukin-1 (IL-1) production by periodic acid (H5IO6)-oxidized human peripheral blood mononuclear (PBMN) cells was assessed by the thymocyte co-mitogenesis assay. Maximum IL-1 levels ( 1.2 U/ml) in the conditioned media of PBMN cells were registered within the first 24 hrs post-oxidation, whereas no IL-1 was detected in the media from 24 hrs control cultures. Thymocyte proliferation, driven by periodic acid-induced IL-1, was abolished by an antibody to IL-1alpha and IL-1. Quantitative analysis of IL-1-containing medium by radioimmunoassay (RIA) indicated that IL-1 comprised about 80% of total IL-1. Partial characterization of H5IO6-induced IL-1 indicated that it was identical to IL-1 produced by lipopolysaccharide-stimulated macrophages. It is concluded that oxidation of human PBMN cells by H5IO6 triggers synthesis and release of IL-1, most of which was in its IL-1 form. 相似文献