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1.
目的:建立分离培养小鼠原代主动脉血管平滑肌细胞(VSMC)的方法并检测其生长特性。方法:剥离小鼠主动脉中膜层,分别采用组织块培养法及胶原酶消化法分离培养小鼠主动脉来源的原代VSMC,免疫荧光法检测细胞的纯度和分化状态;3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑蓝(MTT)法测定小鼠主动脉VSMC传代细胞的生长、增殖特性。结果:组织块培养法培养组织块8d后,细胞从组织块边缘爬出,18 d后细胞汇合度达到80%以上后传代;胶原酶消化法分离培养的细胞生长7 d后,汇合度可达80%,此时进行传代;2种方法获得的细胞进行免疫荧光染色,结果显示细胞传至第3代时纯度在95%以上,传至第8代时分化状态并没有改变;MTT法显示细胞生长3~5 d时处于指数生长期。结论:本研究建立了2种可靠稳定的分离和培养小鼠主动脉VSMC的方法,VSMC纯度高,多次传代后细胞特征稳定。 相似文献
2.
Benjamins JA Nedelkoska L Lisak RP Hannigan JH Sokol RJ 《Neurochemical research》2011,36(9):1677-1686
To characterize immunomodulatory mechanisms that affect oligodendroglia (OL) and white matter following ethanol exposure during
early CNS development, we investigated the direct effects of ethanol and cytokines on glia. Mixed glial cultures from newborn
rat brain were exposed to 6.5–130 mM ethanol for 1–3 days. OL were sensitive to ethanol, with death ranging from 32 to 88%
with increasing time and ethanol concentrations. Little cell death occurred in astroglia or microglia. Mixtures of cytokines
representative of those produced by pro-inflammatory Th1 and monocyte/macrophage (M/M) cells as well as those produced by
anti-inflammatory Th2 cells were all protective. Three of the cytokines in the Th1 mixture, IL-2, TNF-α and IFN-γ, were protective
individually, although no single cytokine was as effective as the mixture. The protective effects of the Th1 mixture and of
IL-2 were reversed by inhibition of both MAP kinase and PI-3 kinase signaling pathways. We conclude that cytokines can act
either directly on OL or indirectly through effects on astroglia or microglia to protect OL from ethanol toxicity. 相似文献
3.
Heidi Andrea Declercq Leo Isabelle De Ridder Maria Jozefa Cornelissen 《Cytotechnology》2005,49(1):39-50
Selection of appropriate cultures having an osteogenic potential is a necessity if cell/biomaterial interactions are studied
in long-term cultures. Osteoblastic cells derived from rat long bones or calvaria have the disadvantage of being in an advanced
differentiation stage which results in terminal differentiation within 21 days. In this regard, less differentiated periosteum-derived
osteoprogenitors could be more suitable.
Periosteum-derived cells were isolated from the tibiae of adult Wistar rats (n = 12). The osteogenic potential with regard to alkaline phosphatase activity, morphology, nodule formation and mineralization
was studied by culturing them in an osteogenic medium for up to 4 months.
Seventy-five percent of the cultures (n = 9) did not show any increase in alkaline phosphatase activity nor nodule formation during long-term culture for up to 4
months. Nevertheless, in 25% of the cultures, alkaline phosphatase activity started from negligible (<5 mM pNP/mg protein)
and increased towards approximately 50 mM pNP/mg protein. Three-dimensional nodule formation was observed at passages 3–5.
In further passages (P5–P7), nodule formation capacity decreased and a diffuse mineralization pattern was observed.
Suitable cultures with osteogenic capacity, can be selected at early passages based on the presence of cuboidal cells. These
cells have the advantage of retaining their osteogenic potential even after prolonged cultivation (6–7 passages) before final
differentiation occurs. Although periosteal cells are suitable for long term in vitro evaluation of biomaterials, the isolation and selection is time consuming. Hence, a more appropriate source to study cell/biomaterial
interactions should be more convenient. 相似文献
4.
Arun M. Khurad Min-Juan Zhang Chanchal G. Deshmukh Ravindra S. Bahekar Ashish D. Tiple Chuan-Xi Zhang 《In vitro cellular & developmental biology. Animal》2009,45(8):414-419
A new continuous cell line from ovarian tissue of commercial variety “Kolar Gold” of silkworm, Bombyx mori, was established and designated as DZNU-Bm-12. The tissue was grown in MGM-448 insect cell culture medium supplemented with
10% fetal bovine serum (FBS) and 3% heat-inactivated B. mori hemolymph at 25 ± 1°C. The migration of partially attached small round refractive cells from the fragments of ovarioles began
from the beginning of explantation. The cells multiplied partially attached in the primary culture initially, and some of
them become freely suspended after 20 passages. The cells were adapted to MGM-448 and TNM-FH media each with 10% FBS and the
population doubling time of cell line was about 36 and 24 hr, respectively. The chromosome number was near diploid at initial
passages and slightly increased at 176th passage, but a few tetraploids and hexaploids were also observed. DNA profiles using
simple sequence repeat loci established the differences between DZNU-Bm-12 and DZNU-Bm-1 and most widely used Bm-5 and BmN
cell lines. The cell line was found susceptible to B. mori nucleopolyhedrovirus (BmNPV) with 85–90% of the cells harboring BmNPV and having an average of 3–17 OBs/infected cell. We
suggest the usefulness of this cell line in BmNPV-based baculoviral expression system and also for studying in vitro virus
replication. 相似文献
5.
Saulius Mikalauskas Laura Mikalauskiene Helge Bruns Arash Nickkholgh Katrin Hoffmann Thomas Longerich Kestutis Strupas Markus W. Büchler Peter Schemmer 《Amino acids》2011,40(4):1139-1150
Hepatotoxic side effects of neoadjuvant chemotherapy for colorectal liver metastases increase perioperative morbidity and
mortality. Glycine protects liver from injury in various animal models. Thus, this study was designed to assess its effect
on liver after chemotherapy. Sprague–Dawley rats (200–220 g) were fed a synthetic diet containing 5% glycine for 5 days. Subsequently,
chemotherapy (FOLFIRI: irinotecan, folinic acid and fluorouracil, or FOLFOX: oxaliplatin, folinic acid and fluorouracil) was
administered at standard doses. Transaminases, histology, immunohistochemistry and in vivo microscopy were used to index liver
injury, to monitor intrahepatic microperfusion and activation of Kupffer cells. Glycine significantly decreased transaminases
after chemotherapy to 25–50% of control values (p < 0.05). Microvesicular steatosis was significantly reduced from 18.5 ± 3.4 and 57.1 ± 8.6% in controls to 9.5 ± 1.8 and
37.7 ± 4.4% after FOLFIRI and FOLFOX, respectively. Furthermore, phagocytosis of latex beads was reduced by about 50%, while
leukocyte adherence in central and midzonal subacinar zones decreased to 60–80% after glycine (p < 0.05). Glycine significantly reduced expression of inducible nitric oxide synthase after chemotherapy, while hepatic microcirculation
was increased (p < 0.05). This study shows for the first time that glycine reduces chemotherapy-induced liver injury. The underlying mechanisms
most likely include Kupffer cells and an improved intrahepatic microperfusion. 相似文献
6.
We investigated relationship between the maturity and density of muscle cells and developed a rapid isolation method to acquire
stem cells from skeletal muscle. Mononuclear cells were isolated from the lower hind-limb muscles of 7-d-old male Sprague–Dawley
rats and separated by Percoll density gradient centrifugation. After centrifugation, the cells were layered in the interfaces
between each Percoll density layer. Flow cytometry was used to investigate the Sca-1, Pax7, CD34, CD45, M-cadherin, and myosin
expression of the cells in each density layer. We found that CD45-positive cells were not present in freshly isolated muscle
cells. CD34-, Pax7-positive cells were mainly observed at the interface between the 15% and 25% Percoll layers and had a density
of 1.0235–1.0355 g/ml. Cells positive for M-cadherin were at the 25–35% Percoll density interface and had a density of 1.0355–1.0492 g/ml.
We conclude that because there appears to be a correlation between maturity and density, muscle-derived stem cells may be
isolated successfully from the 15–25% Percoll interface. 相似文献
7.
水牛皮肤成纤维细胞的分离与体外培养 总被引:7,自引:0,他引:7
探讨水牛成纤维细胞的分离与传代培养方法。组织块培养法培养的成纤维细胞原代生长较慢,需12天左右方可汇合形成单层,而酶消化法培养的成纤维细胞原代生长相对生长快,仅需8天便可汇合形成单层。两种方法传代细胞的生长速度相似,仅需4-5天就可汇合形成单层。通过体细胞的核型分析发现,成纤维细胞在传代培养过程中的核型变化不大,66.67%~81.67%的细胞具有正常的二倍体核型,各代之间无显著差异。结果表明,水牛成纤维细胞均能稳定地进行传代培养。 相似文献
8.
Guo F Liu Y Chen Y Tang T Jiang W Li Y Li J 《Applied microbiology and biotechnology》2011,90(4):1277-1283
A new rapid and continuous procedure was developed for purifying magnetosomes from Magnetospirillum gryphiswaldense MSR-1 cells on a large scale. The procedure included these steps: disruption of cells with a high-pressure homogeniser, isolation
of magnetosomes with a continuous magnetism isolation system accompanied by low-power ultrasonication and urea treatment,
removal of adsorbed and surface proteins with proteinase K, removal of nucleic acids with electro-elution, and replacement
of the PBS buffer with distilled water by a magnetically stirred system. The purified magnetosomes were stored at −20 °C after
lyophilized and treated with γ-rays. The time required for purification was reduced from 20–30 to 2–5 days. Evaluation of
the purity of the resulting magnetosomes was carried out with SDS-PAGE, PCR, and Fourier-transform infrared spectroscopy.
The overall data suggest that the method presented here is a simple, rapid, continuous, and highly efficient procedure for
large-scale purification of magnetosomes. 相似文献
9.
In order to develop a simplified method for long-term primary culture of highly-pure rat embryonic hippocampal neurons of
low-density (103 cells/cm2), we optimized and modified conventional culturing methods. The modifications of our simplified method include: (1) combinational
application of two growth substrates, tail collagen and poly-L-lysine, to coat plastic culture dishes and coverslips for a
better neuronal attachment; (2) dissociation of hippocampal tissues with combinational use of two milder enzymes (collagenase
and dispase) and trypsin of a lower concentration to minimize enzymatic damages to cultured neurons; (3) a cell pre-plating
step to preliminarily eliminate the contaminating non-neuronal cells; (4) a modified culture medium as a critical step to
promote highly pure neurons of low-density for a long term; and (5) appropriately reduced frequency and volume of refreshment
of the culture medium. Using our modified method, the β-tubulin III-immunostained and Hoechst 33342 counterstained neurons
harvested a steady and healthy growth with a longer culture time of over 35 days, and a clear distinction between TAU-1- and
MAP2-immunoreactive neurites was apparent at the early culturing period. In addition, the purity of neurons was over 95% at
the different time points in comparison with the control culture using conventional serum-free method in which most neurons
degenerated and died within 5 days. Thus, our modified method proved to be a simple, feasible as well as time- and resource-saving
approach for a long-term survival of pure rat embryonic hippocampal neurons of low-density. 相似文献
10.
Primary cell cultures (n = 16) were initiated from tissues of embryonic and neonatal larval Ornithodoros moubata following methods developed for hard ticks. After maintenance for 20–25 months in vitro, cell multiplication commenced in
surviving cultures, leading to the establishment of six cell lines designated OME/CTVM21, 22, 24, 25, 26 and 27. All lines
are maintained at 28°C, with subculture at 2–8 week intervals. The cultures comprise heterogeneous populations of large cells
of 15–100 μm in diameter, often with finger-like protrusions and/or intracellular crystals, rarely attached, predominantly
floating and forming clumps or hollow multicellular vesicles up to 1 mm in diameter. Attempts to cryopreserve the cells are
described. Tick-borne encephalitis virus has been serially passaged ten times in OME/CTVM21 cells without significant decrease
in virus production and with no change in its biological properties as shown by the size and morphology of plaques produced
in porcine kidney cells. 相似文献
11.
Carolyn M. King John G. Innes Dianne Gleeson Neil Fitzgerald Tom Winstanley Barry O’Brien Lucy Bridgman Neil Cox 《Biological invasions》2011,13(10):2391-2408
Reinvasions provide prime examples of source-sink population dynamics, and are a major reason for failure of eradications
of invasive rats from protected areas. Yet little is known about the origins and population structure of the replacement population
compared with the original one. We eradicated eight populations of ship rats from separate podocarp-broadleaved forest fragments
surrounded by open grassland (averaging 5.3 ha, scattered across 20,000 ha) in rural landscapes of Waikato, New Zealand, and
monitored the- re-establishment of new populations. Rats were kill-trapped to extinction during January to April 2008, and
then again after reinvasion in April–May (total n = 517). Rats carrying Rhodamine B dye (n = 94), available only in baits
placed 1–2 months in advance in adjacent source areas located 170–380 m (average 228 m edge to edge) away, appeared in 7 of
the 8 fragments from the first day of the first eradication. The distribution of age groups, genders and proportions of reproductively
mature adults (more immature juvenile males and fewer fully mature old females) was different among marked rats compared with
all other rats (P = 0.001, n = 509); in all rats caught on days 7+ of the first eradication compared with on days 1–6 (P = 0.000); and in the total sample collected in fragments by trapping to and after local extinction compared with in brief,
fixed-schedule sampling of populations in continuous forests (P = 0.000). Genotyping of 493 carcases found no significant population-level differentiation among the 8 fragments, confirming
that the rats in all fragments belonged to a single dynamic metapopulation. Marked rats of both genders travelled up to 600 m
in a few days. Conservation of forest fragments is compromised by the problem that ship rats cannot be prevented from rapidly
reinvading any cleared area after eradication. 相似文献
12.
Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture
Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of time of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can be reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the BV formed and released between 48 and 96 hpi are most likely from FP mutant infected cells. 相似文献
13.
目的对经典的原代视网膜小胶质细胞体外纯化培养方法进行简单的改良以提高细胞产量。方法在视网膜小胶质细胞原代培养过程中以McCarthy等的经典方法为基础,寻找适当的初始种植密度,并在初次振荡分离后继续培养以获得多次产出,使用免疫细胞化学染色方法进行小胶质细胞纯度鉴定。结果视网膜小胶质细胞原代培养最宜初始种植密度为1×106/mL,多次分离纯化获得的视网膜小胶质细胞均达到97%以上的纯度。结论视网膜小胶质细胞原代培养过程中,以适当的初始种植密度和多次振荡分离方法可在保证有效纯度的基础上提高细胞产量。 相似文献
14.
The aim of the present study was to produce astrocyte cultures of high purity from mouse hippocampal neural stem cells and
to compare their in vitro properties with those isolated from enriched mixed glial cultures prepared from mouse hippocampus,
which are commonly contaminated by microglia. We produced primary cultures of newborn mouse hippocampal neural stem cells,
which have the potential to differentiate into astrocytes, neurons, and oligodendrocytes. We produced monoclonal neural stem
cell colonies by limiting dilution. We induced astrocyte differentiation by plating the colonies on poly-l-lysine and culturing them in induction medium consisting of minimum essential medium/F12 supplemented with 10% fetal bovine
serum and 100 ng/ml ciliary neurotrophic factor. We then further purified the cells by differential adherence and shaking
at a constant temperature, followed by a second round of limiting dilution. Immunocytochemistry for glial fibrillary acidic
protein showed that our method yielded 99.4 ± 0.5% pure astrocytes, whereas traditionally enriched mixed glial cultures yielded
94.2 ± 2% pure astrocytes. Induced cells resembled primary astrocyte cultures in functional properties such as cell proliferation
rates and lack of tumorigenicity and p53, and expression of epidermal growth factor receptor, bystin, and nitric oxygen synthase.
Our novel method of culture and purification of neural stem cells can therefore be used routinely for the primary culture
of highly purified astrocytes from mouse hippocampus. 相似文献
15.
16.
Theodosia Kazazoglou Eleni Fleischer-Lambropoulos Taxiarchis Geladopoulos Susan Kentroti Costas Stefanis Antonia Vernadakis 《Neurochemical research》1996,21(5):609-614
In this study, we were interested to compare the responsiveness to growth factors, NGF, b-FGF and EGF and cytokines, IL1β,
and TNF-α, in late passages (74–79) C6 glial cells committed astrocytes and astrocytes of advanced passages (26–28) in cultures
derived from aged mouse cerebral hemispheres (MACH). Cultures were grown in either DMEM or chemically defined medium (CDM/TIPS)
in order to test the effects of growth factors or cytokines. The activity of glutamine synthetase (GS), a marker for astrocytes,
was used as a test parameter. We found that treatment with growth factors increased GS activity in both glial cell culture
systems with the exception of EGF in C-6 glial cells. Treatment with cytokines markedly decreased GS activity in the late
passage C6 glial cells whereas only TNF-α had a similar effect on MACH astrocytes. In view of the generally opposite effects
of growth factors and cytokines on GS activity, we-speculate that these molecules which are also endogenously present in glial
cells may play a role in the maintenance of cellular homeostasis. 相似文献
17.
Ravinder Kaur Grewal Monika Lulsdorf Janine Croser Sergio Ochatt Albert Vandenberg Thomas D. Warkentin 《Plant cell reports》2009,28(8):1289-1299
This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture
using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for
Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15–20 days of culture initiation with
2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress
treatments were used: (1) flower clusters were treated at 4°C for 4 days, (2) anthers were subjected to electric shock treatment
of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω resistance, (3) anthers were centrifuged
at 168–1,509g for 2–15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers
were then transferred to a solid embryo development medium and, 15–20 days later, embryo development was observed concomitant
with a small amount of callus growth of 0.1–3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation
treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological
studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10–30-day-old calli. Flow cytometric
analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during
early regeneration stages. 相似文献
18.
The study examines the relationship between sudden changes in weather conditions in summer, represented by (1) sudden air
temperature changes, (2) sudden atmospheric pressure changes, and (3) passages of strong atmospheric fronts; and variations
in daily mortality in the population of the Czech Republic. The events are selected from data covering 1986–2005 and compared
with the database of daily excess all-cause mortality for the whole population and persons aged 70 years and above. Relative
deviations of mortality, i.e., ratios of the excess mortality to the expected number of deaths, were averaged over the selected
events for days D−2 (2 days before a change) up to D+7 (7 days after), and their statistical significance was tested by means
of the Monte Carlo method. We find that the periods around weather changes are associated with pronounced patterns in mortality:
a significant increase in mortality is found after large temperature increases and on days of large pressure drops; a decrease
in mortality (partly due to a harvesting effect) occurs after large temperature drops, pressure increases, and passages of
strong cold fronts. The relationship to variations in excess mortality is better expressed for sudden air temperature/pressure
changes than for passages of atmospheric fronts. The mortality effects are usually more pronounced in the age group 70 years
and above. The impacts associated with large negative changes of pressure are statistically independent of the effects of
temperature; the corresponding dummy variable is found to be a significant predictor in the ARIMA model for relative deviations
of mortality. This suggests that sudden weather changes should be tested also in time series models for predicting excess
mortality as they may enhance their performance. 相似文献
19.
Yu-Chuan Juang Sunil S. Adav Duu-Jong Lee Juin-Yih Lai 《Applied microbiology and biotechnology》2009,85(2):383-388
Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and
tumor cells, which may possibly be used as an antimicrobial agent. We report here the application of small ubiquitin-related
modifier (SUMO) fusion technology to the expression and purification of cationic antibacterial peptide ABP-CM4. The fusion
protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography and 112 mg protein of interest
was obtained per liter of fermentation culture. After the SUMO–CM4 fusion protein was cleaved by the SUMO protease at 30 °C
for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 24 mg recombinant CM4 was obtained from 1 l fermentation
culture with no less than 96% purity and the recombinant CM4 had similar antimicrobial properties to the synthetic CM4. Thus,
the SUMO-mediated peptide expression and purification system potentially could be employed for the production of recombinant
cytotoxic peptides. 相似文献
20.
We have previously estimated the productivity and photosynthetic efficiency of the microalga Chlorella sp. grown in an outdoor open thin-layer photobioreactor under climate conditions typical of the Middle European region, i.e.
with many days unsuitable for intensive growth of algae (cloudy and rainy days, low air temperature, low solar PAR input).To
estimate the real potential productivity of the bioreactor, we collected data on algae yields obtained during clear summer
day periods. Cultivation was performed in fed-batch cycles in a bioreactor with a 224 m2 culture area (length 28 m, slope 1.7%), and a 6–7 mm-thick layer of algal culture. The suspension volume in the bioreactor
was 2,000 L. The mean values found for Třeboň (49°N), Czech Republic, as an average of several sunny summer cultivation periods
in July, were: net areal productivity, P
net = 38.2 g dry weight (DW) m-2 day-1; net volumetric productivity, Pvol, = 4.3 g algal DW L-1 day-1, photosynthetic efficiency (based on PAR), ηnet = 7.05%. The peak values were: P
net about 50 g (DW) m-2 day-1, ηnet about 9%. Algal growth rate was practically linear up to high biomass densities (40–50 g DW L-1, corresponding to an areal density of 240–300 g DW m-2), at which point the culture was harvested. The concentration of dissolved oxygen increased from about 10 mg L-1 at the beginning to about 23 mg L-1 at the end of culture area at noon. Use of the above-described technology for economical production of bioethanol is proposed. 相似文献