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1.
J. venom. anim. toxins incl. trop. dis ; 27: e20200105, 2021. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1180822

ABSTRACT

Amphibians inhabit the terrestrial environment, a conquest achieved after several evolutionary steps, which were still insufficient to make them completely independent of the aquatic environment. These processes gave rise to many morphological and physiological changes, making their skin (and cutaneous secretion) rich in bioactive molecules. Among the tree frogs, the secretion is composed mainly of peptides; but alkaloids, proteins and steroids can also be found depending on the species. The most known class of biologically active molecules is the antimicrobial peptides (AMPs) that act against bacteria, fungi and protozoans. Although these molecules are well-studied among the hylids, AMPs ontogeny remains unknown. Therefore, we performed peptidomic and proteomic analyses of Pithecopus nordestinus (formerly Phyllomedusa nordestina) in order to evaluate the peptide content in post-metamorphosed juveniles and adult individuals. Methods: Cutaneous secretion of both life stages of individuals was obtained and analyzed by LC-MS/MS after reduction and alkylation of disulfide bonds or reduction, alkylation and hydrolysis by trypsin. Results: Differences in the TIC profile of juveniles and adults in both treatments were observed. Moreover, the proteomic data revealed known proteins and peptides, with slight differences in the composition, according to the life stage and the treatment. AMPs were identified, and bradykinin-potentiating peptides were observed in trypsin-treated samples, which suggests a protein source of such peptide (cryptide). Conclusion: In general, skin secretion contents were similar between juveniles and adults, varying in quantity, indicating that the different stages of life are reflected in the number of molecules and not on their diversity.(AU)


Subject(s)
Animals , Female , Peptides , Trypsin , Proteomics , Amphibians , Bodily Secretions , Hydrolysis
3.
Chinese Journal of Biotechnology ; (12): 2435-2442, 2020.
Article in Chinese | WPRIM | ID: wpr-878499

ABSTRACT

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.


Subject(s)
Amino Acid Sequence , Chromatography, Liquid , Chymotrypsin/metabolism , Digestion , Humans , Membrane Proteins , Tandem Mass Spectrometry , Trypsin , Vitamin K Epoxide Reductases
4.
Chinese Journal of Biotechnology ; (12): 741-748, 2019.
Article in Chinese | WPRIM | ID: wpr-771336

ABSTRACT

Proteomics is a fast-growing discipline that aims at systematic identification, quantification of proteins and their post-translational modifications in cells. Mass spectrometry-based shotgun proteomics technology is currently one of the mainstream methods for proteomics research. With this method, proteins need to be digested to peptides by site-specific proteases before they can be detected with mass spectrometry. Therefore, site-specific proteases played key roles in this process and so far, a variety of specific proteases have been developed and used in proteomics study. Particularly, the identification, characterization and development of proteases that cleave at the N-termini of corresponding amino acid residues, which are just mirrors to those of typical C-termini proteases, provide novel tools for proteomics analysis. In this review, we summarized the proprieties of LysargiNase, a most recently identified mirror trypsin, and its applications in proteomics research to promote its more widespread usage.


Subject(s)
Mass Spectrometry , Metalloproteases , Chemistry , Metabolism , Protein Processing, Post-Translational , Proteomics , Trypsin , Chemistry
5.
Article in English | WPRIM | ID: wpr-760343

ABSTRACT

In canine acute pancreatitis (AP), inappropriate release and activation of zymogen proteases within the pancreas results in the consumption of serum antiproteases. The aim of this study was to examine whether the serum concentrations of α₂-macroglobulin (A2MG), α₁-antitrypsin (A1AT), and C-reactive protein (CRP) differ between dogs with AP and healthy dogs. Twenty healthy dogs and 20 dogs with AP were included in this study. Concentrations of A2MG, A1AT, and CRP were measured in the sera of healthy dogs and dogs diagnosed with AP. Serum A2MG and A1AT concentrations were significantly lower in dogs with AP than in healthy dogs, whereas the serum CRP concentration was significantly higher. In addition, the concentrations of A2MG and A1AT were significantly higher in AP survivors than in AP non-survivors, while the CRP concentration was significantly lower. However, in both AP survivors and non-survivors, the CRP concentrations showed a negative correlation with A2MG concentrations but not with A1AT. These findings indicate that serum antiproteases and CRP concentrations might be associated with the mortality rate of AP in dogs.


Subject(s)
Animals , C-Reactive Protein , Dogs , Humans , Mortality , Pancreas , Pancreatitis , Peptide Hydrolases , Protease Inhibitors , Survivors , Trypsin
6.
Article in Korean | WPRIM | ID: wpr-716123

ABSTRACT

BACKGROUND: As nonsurgical interventions for vitiligo are not always successful, various surgical modalities have been used in patients with refractory vitiligo. Of these, non-cultured epidermal suspension transplantation (NCES) was recently introduced to treat large recipient sites using cells from small donor tissue. OBJECTIVE: We assessed the effectiveness and safety of NCES as a surgical treatment for patients with refractory vitiligo. METHODS: We retrospectively reviewed 20 cases in 17 patients (11 females; median age 25 years) who underwent NCES from July 2015 through March 2018. Suction blisters (20 mm in diameter) were collected from the patient's inner thigh at a donor-to-recipient area ratio of 1:5. After the addition of 5 mL recombinant trypsin solution to the suction blisters, followed by incubation at 37℃ for 60 min, epidermal cells were manually scraped off the blister surface, and epidermal cell suspension was obtained by centrifugation at 1,500 RPM for 5 min. The suspension was applied to the vitiligo regions after epidermal ablation of those regions. Phototherapy resumed 1 month later. Treatment success was defined as ≥75% repigmentation of the surgical site, and all adverse events were noted. RESULTS: Overall, 85.0% of cases (17/20) exhibited treatment success. Adverse events included hyperpigmentation (20%) and surgical site infection (5%), but the treatment was tolerable in all cases. CONCLUSION: NCES is a reliable surgical option for patients with vitiligo refractory to nonsurgical treatment. Large areas of vitiligo can be treated by NCES, and use of this technique should be encouraged in Korea.


Subject(s)
Blister , Centrifugation , Female , Humans , Hyperpigmentation , Korea , Phototherapy , Retrospective Studies , Suction , Surgical Wound Infection , Thigh , Tissue Donors , Transplantation , Trypsin , Vitiligo
7.
Article in English | WPRIM | ID: wpr-715802

ABSTRACT

A 28-year-old man with a history of alcohol abuse was diagnosed with acute pancreatitis based on clinical symptoms, laboratory findings and computed tomography findings. On the second day of hospitalization, he complained of sudden visual disturbance. The ophthalmologic examination showed Purtscher's-like retinopathy. Two weeks after initial presentation, his vision was significantly improved along with epigastric pain. Retinal arteriolar occlusion by complement-mediated leukoembolization is the proposed pathogenic mechanism of Purtscher's-like retinopathy. Increased activity of proteases such as trypsin, associated with acute pancreatitis might be linked with the production of complement C5a. We report a rare case of Purtscher's-like retinopathy associated with acute pancreatitis.


Subject(s)
Adult , Alcoholism , Complement C5a , Hospitalization , Humans , Pancreatitis , Peptide Hydrolases , Retinaldehyde , Trypsin
8.
Chinese Journal of Traumatology ; (6): 323-328, 2018.
Article in English | WPRIM | ID: wpr-771648

ABSTRACT

PURPOSE@#Early application of protease inhibitors through the intestinal lumen could increase survival following experimental shock by blocking the pancreatic digestive enzymes. Hence, it was hypothesized that two-route injection (intraintestinal + intravenous) of ulinastatin (UTI), a broad-spectrum protease inhibitor, could better alleviate intestinal injury than single-route injection (either intravenous or intraintestinal).@*METHODS@#A sepsis model induced by lipopolysaccharide on rats was established. The rats were randomly divided into five groups: sham, sepsis, UTI intravenous injection (Uiv), UTI intraintestinal injection (Uii), and UTI intraintestinal + intravenous injection (Uii + Uiv) groups. The mucosal barrier function, enzyme-blocking effect, levels of systemic inflammatory cytokines, and 5-day survival rate were compared among groups. The small intestinal villus height (VH), crypt depth (CD), and two components of mucosal barrier (E-cadherin and mucin-2) were measured to evaluate the mucosal barrier function. The levels of trypsin and neutrophil elastase (NE) in the intestine, serum, and vital organs were measured to determine the enzyme-blocking effect.@*RESULTS@#Compared with the single-route injection group (Uiv or Uii), the two-route injection (Uii + Uiv) group displayed: (1) significantly higher levels of VH, VH/CD, E-cadherin, and mucin-2; (2) decreased trypsin and NE levels in intestine, plasma, and vital organs; (3) reduced systemic inflammatory cytokine levels; and (4) improved survival of septic rats.@*CONCLUSION@#Two-route UTI injection was superior to single-route injection in terms of alleviating intestinal injury, which might be explained by extensive blockade of proteases through different ways.


Subject(s)
Animals , Cadherins , Metabolism , Cytokines , Metabolism , Disease Models, Animal , Glycoproteins , Pharmacology , Inflammation Mediators , Metabolism , Injections, Intralesional , Injections, Intravenous , Intestinal Diseases , Drug Therapy , Metabolism , Intestinal Mucosa , Metabolism , Pathology , Intestines , Leukocyte Elastase , Metabolism , Male , Mucin-2 , Metabolism , Rats, Wistar , Sepsis , Trypsin , Metabolism , Trypsin Inhibitors , Pharmacology
9.
Mycobiology ; : 204-208, 2017.
Article in English | WPRIM | ID: wpr-729293

ABSTRACT

Nosema ceranae is an obligate intracellular fungal parasite that causes mortality in honey bees and enhances the susceptibility of honey bees to other pathogens. Efficient purification of Nosema spores from the midgut of infected honey bees is very important because Nosema is non-culturable and only seasonably available. To achieve a higher yield of spores from honey bees, in this study, we considered that the initial release of spores from the midgut tissues was the most critical step. The use of 2 mm beads along with enzymatic treatment with collagenase and trypsin enhanced the homogenization of tissues and the yield of released spores by approximately 2.95 times compared with the use of common 3 mm beads alone. The optimal time for the enzyme treatment was determined to be 1 hr as measured by the yield and viability of the spores. A one-step filtration using a filter paper with an 8–11 µm pore size was sufficient for removing cell debris. This method may be useful to purify not only N. ceranae spores but also other Nosema spp. spores.


Subject(s)
Bees , Collagenases , Filtration , Honey , Methods , Mortality , Nosema , Parasites , Seasons , Spores , Trypsin
10.
Article in English | WPRIM | ID: wpr-633449

ABSTRACT

BACKGROUND: Asthma chornic obstructive pulmonary disorder(COPD) overlap syndrome (ACOS) was formally described by the joint project of global initiative for asthma (GINA) and global Initiative for chronic obstructive lung disease (GOLD) as persistent airflow limitation with several features usually associated with both asthma and COPD. ACOS is identified by the features shared with both asthma and COPD.The underlying cause though remains unknown,hence the project did not offer current formal definition.CASE: This is a case of a 29-year-old male, asthmatic with an eight-pack year smoking history who presented with chronic obstructive respiratory symptoms with non significant improvement on control of exacerbation despite standard maximal  therapy.Diagnostic tests such as pulmonary function Tests,2D Echo,chest CT scan and even assay for alpha 1 anti trypsin were done to rule out for other disease entities and prognosticate the patient's condition leading to the diagnosis of asthma COPD overlap syndrome (ACOS). CONCLUSION: ACOS as a disease entity is still under debate and still has no current formal definition due to lack of specific biomarkers and lack of defining characteristics.Despite this,management should not be compromised since these patients often present with higher rates of exacerbations,hospitalization,associated co morbid illness and mortality.Treatment therefore is individualized.


Subject(s)
Humans , Male , Adult , Asthma , Trypsin , Smoking , Diagnostic Tests, Routine , Pulmonary Disease, Chronic Obstructive , Respiratory Function Tests , Biomarkers
11.
An. acad. bras. ciênc ; 89(3,supl): 2155-2165, 2017. tab, graf
Article in English | LILACS | ID: biblio-886808

ABSTRACT

ABSTRACT Leaves of Psidium guajava L. (guava) have been widely used in the popular way for prevention and treatment of various diseases. Thus, the objective of this study was to evaluate the inhibitory potential of leaves aqueous extract from three cultivars of P. guajava (Pedro Sato, Paluma and Século XXI) on α-amylase, α-glycosidase, lipase, and trypsin enzymes, in the presence or not of simulated gastric fluid and to determine the content of phenolic compounds by high performance liquid chromatography. All cultivars presented the same composition in phenolic compounds, but in different proportions. The compounds identified are gallic acid, epigallocatechin gallate, syringic acid, o-coumaric acid, resveratrol, quercetin, and catechin (which was the major compound in all the cultivars evaluated). In the absence of simulated gastric fluid, it was observed different inhibitions exercised by the leaves aqueous extracts from three cultivars of P. guajava on each enzyme. In presence of simulated gastric fluid, all cultivars showed increase in the inhibition of lipase and α-glycosidase, and decrease in inhibition of α-amylase and trypsin enzymes. These results indicate that P. guajava leaves aqueous extracts from all cultivars evaluated possess potential of use as an adjuvant in the treatment of obesity and other dyslipidemias.


Subject(s)
Plant Extracts/pharmacology , Plant Extracts/chemistry , Plant Leaves/chemistry , Enzyme Inhibitors/pharmacology , Antioxidants/pharmacology , Obesity/drug therapy , Phenols/analysis , Water/analysis , Trypsin/pharmacology , Chromatography, High Pressure Liquid , Psidium/chemistry , alpha-Amylases/pharmacology , alpha-Glucosidases/pharmacology , Lipase/pharmacology
12.
Genomics & Informatics ; : 142-146, 2017.
Article in English | WPRIM | ID: wpr-192018

ABSTRACT

More effective production of human insulin is important, because insulin is the main medication that is used to treat multiple types of diabetes and because many people are suffering from diabetes. The current system of insulin production is based on recombinant DNA technology, and the expression vector is composed of a preproinsulin sequence that is a fused form of an artificial leader peptide and the native proinsulin. It has been reported that the sequence of the leader peptide affects the production of insulin. To analyze how the leader peptide affects the maturation of insulin structurally, we adapted several in silico simulations using 13 artificial proinsulin sequences. Three-dimensional structures of models were predicted and compared. Although their sequences had few differences, the predicted structures were somewhat different. The structures were refined by molecular dynamics simulation, and the energy of each model was estimated. Then, protein-protein docking between the models and trypsin was carried out to compare how efficiently the protease could access the cleavage sites of the proinsulin models. The results showed some concordance with experimental results that have been reported; so, we expect our analysis will be used to predict the optimized sequence of artificial proinsulin for more effective production.


Subject(s)
Computer Simulation , DNA, Recombinant , Humans , Insulin , Molecular Dynamics Simulation , Proinsulin , Protein Sorting Signals , Trypsin
13.
Int. braz. j. urol ; 42(4): 817-824, July-Aug. 2016. tab, graf
Article in English | LILACS | ID: lil-794669

ABSTRACT

ABSTRACT Purpose: In a previous study the vaccine was effective against bladder cancer in a mouse model. However, a small portion of tumors regrew because the vaccine could not eliminate bladder cancer stem cells (CSCs). In this study, we showed a modified method for the isolation of bladder CSCs using a combination of differential adhesion method and serum-free culture medium (SFM) method. Materials and Methods: Trypsin-resistant cells and trypsin-sensitive cells were isolated from MB49, EJ and 5637 cells by a combination of differential adhesion method and SFM method. The CSCs characterizations of trypsin-resistant cells were verified by the flow cytometry, the western blotting, the quantitative polymerase chain reaction, the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay. Results: Trypsin-resistant cells were isolated and identified in CSCs characters, with high expression of CSCs markers, higher resistance to chemotherapy, greater migration in vitro, and stronger tumorigenicity in vivo. Conclusion: Trypsin-resistant cells displayed specific CSCs properties. Our study showed trypsin-resistant cells were isolated successfully with a modified method using a combination of differential adhesion method and SFM method.


Subject(s)
Animals , Mice , Neoplastic Stem Cells/cytology , Urinary Bladder Neoplasms/pathology , Trypsin/pharmacology , Cell Adhesion/drug effects , Cell Separation/methods , Cell Culture Techniques/methods , Neoplastic Stem Cells/chemistry , Biomarkers, Tumor , Cell Differentiation , Culture Media, Serum-Free , Cancer Vaccines/immunology , Cell Line, Tumor , Real-Time Polymerase Chain Reaction , Flow Cytometry , Mice, Nude
14.
Chinese Journal of Biotechnology ; (12): 306-316, 2016.
Article in Chinese | WPRIM | ID: wpr-337413

ABSTRACT

Proteomics is a powerful subject focusing on large-scale study of protein structures and functions. A complete enzymatic digestion of protein complexes is the key step in modern high-resolution and high-throughput mass spectrometry (MS)-based identification and quantification. To achieve MS analysis, both peptide sample pretreatment and data acquisition are prerequisite in proteomic studies. In this paper, we summarized both the enzymatic proprieties of three common proteolytic enzymes, Trypsin, Lys-C and Glu-C, the optimization of multi-enzyme combination and an advanced sample pretreatment in proteomics research.


Subject(s)
Enzymes , Chemistry , Mass Spectrometry , Proteins , Chemistry , Proteomics , Methods , Trypsin , Chemistry
15.
Article in English | WPRIM | ID: wpr-812586

ABSTRACT

It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival.


Subject(s)
Amino Acid Sequence , Animals , Base Sequence , Female , Kinetics , Male , Mice , Molecular Sequence Data , Protease Inhibitors , Chemistry , Toxicity , Scorpion Venoms , Chemistry , Genetics , Toxicity , Scorpions , Chemistry , Genetics , Trypsin , Chemistry
16.
Article in English | WPRIM | ID: wpr-201376

ABSTRACT

Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH₂ and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH₂-induced PAR2 activation resulting in decreased mobilization of intracellular Ca²⁺ in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH₂ and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH₂-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH₂ downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH₂ in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis.


Subject(s)
Animals , Chemokine CCL17 , Dermatitis, Atopic , Epidermis , GTP-Binding Proteins , Homeostasis , Humans , Inflammation , Interleukin-8 , Keratinocytes , Kinetics , Mice , Mice, Hairless , Necrosis , Permeability , Receptor, PAR-2 , Skin , Trypsin
17.
Article in English | WPRIM | ID: wpr-200501

ABSTRACT

BACKGROUND: This study aimed to identify pathogenic variants of PRSS1, SPINK1, CFTR, and CTRC genes in Korean patients with idiopathic pancreatitis. METHODS: The study population consisted of 116 Korean subjects (65 males, 51 females; mean age, 30.4 yr, range, 1-88 yr) diagnosed with idiopathic chronic pancreatitis (ICP), idiopathic recurrent acute pancreatitis (IRAP), or idiopathic acute pancreatitis (IAP). We analyzed sequences of targeted regions in the PRSS1, SPINK1, CFTR, and CTRC genes, copy numbers of PRSS1 and SPINK1, and clinical data from medical records. RESULTS: We identified three types of pathogenic PRSS1 variants in 11 patients, including p.N29I (n=1), p.R122H (n=1), and p.G208A (n=9). Sixteen patients exhibited heterozygous pathogenic variants of SPINK1, including c.194+2T>C (n=12), p.N34S (n=3), and a novel pathogenic splicing variation c.194+1G>A. A heterozygous CFTR p.Q1352H pathogenic variant was detected in eight patients. One patient carried a heterozygous CTRC p.P249L pathogenic variant, which is a known high-risk variant for pancreatitis. All patients had normal PRSS1 and SPINK1 gene copy numbers. Weight loss occurred more frequently in patients carrying the p.G208A pathogenic variant, while pancreatic duct stones occurred more frequently in patients with the c.194+2T>C pathogenic variant. CONCLUSIONS: Pathogenic variants of PRSS1, SPINK1, and CFTR were associated with idiopathic pancreatitis, while pathogenic variants of CTRC were not. Copy number variations of PRSS1 and SPINK1 were not detected.


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Asian Continental Ancestry Group/genetics , Carrier Proteins/genetics , Child , Child, Preschool , Chymotrypsin/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA Copy Number Variations , Female , Heterozygote , Humans , Infant , Male , Middle Aged , Pancreatitis, Chronic/genetics , Polymorphism, Genetic , Republic of Korea , Trypsin/genetics , Young Adult
18.
Acta Physiologica Sinica ; (6): 103-109, 2015.
Article in Chinese | WPRIM | ID: wpr-255966

ABSTRACT

The aim of the present study was to observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn Sprague Dawley (SD) rats was isolated and digested with 0.25% trypsin for 20, 30, or 40 min. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each of 20 and 30 min groups was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed, respectively. Immunofluorescence for glial fibrillary acidic protein (GFAP) was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptotic rates of purified astrocytes. The results showed that, the cells being digested for 20 min usually reached confluence at 9 d after seeding. When the digestion time was extended to 30 min, the cells grew faster and reached confluence at 7 d after seeding, meanwhile the morphology of astrocytes was normal, GFAP positive rate (70.2 ± 4.0)% being much higher than that of the 20 min group (P < 0.05). Compared with 20 min group, 40 min group showed higher GFAP positive rate, whereas the cell proliferation was slower, and cell injury was more obvious. After shaking at constant temperature, two times of trypsin digestion could decrease the number of contaminated cells after passage. The GFAP positive rates of two-time-digestion groups in passage 1 (P1) were higher than those of corresponding control groups, and the GFAP positive rate of 30 min + two-time-digestion group in P1 reached (98.1 ± 1.7)%, which was equivalent to that of the 20 min + control group in P3. However, the apoptotic rate showed no significant difference between these two groups. Based on above mentioned results, we conclude that 30 min + two-time of trypsin digestion effectively improves the purity of astrocytes and shortens the time of primary culture and purification, suggesting that it is a rapid and effective method to obtain astrocytes with high purity in vitro.


Subject(s)
Animals , Astrocytes , Cell Biology , Cell Culture Techniques , Cell Proliferation , Cell Separation , Methods , Glial Fibrillary Acidic Protein , Metabolism , Rats , Rats, Sprague-Dawley , Trypsin
19.
Article in English | WPRIM | ID: wpr-257661

ABSTRACT

<p><b>OBJECTIVE</b>To observe the apoptosis of neural stem cells (NSCs) at differential time points after the dissociation of neurospheres by Accutase or trypsin.</p><p><b>METHODS</b>The NSCs were isolated from striatum of human fetals that suffered abortion at 12-16 weeks of pregnancy. The 3(rd)-5(th) passages of NSCs were digested by Accutase or trypsin. Only vortexing was applied, and the triturating by Pasteur pipette was avoided to attenuate the injury to the cells during the dissociation. The single cells were then stained by Annexin V/propidium iodide and Hoechst 33342. The apoptosis rates 2 and 24 hours after passaging were evaluated.</p><p><b>RESULTS</b>The trypan blue staining confirmed that immediately after the dissociation,the viability of cells digested by trypsin was (83.10 ± 6.76)%, which was significantly lower than that digested by Accutase,which was (91.65 ± 4.43)% (P<0.05). The apoptosis of the NSCs digested by Accutase was higher than that digested by trypsin at both 2 and 24 hours after passaging (P<0.01). Four days after the passaging, both the new clone formation rate and diameter of new spheres after trypsin digestion were significantly higher than those after Accutase digestion (P<0.01).</p><p><b>CONCLUSIONS</b>Although the viability of NSCs immediately after the disassociation by trypsin is lower than that digested by Accutase, the apoptosis of NSCs subsequently caused by trypsin is lower than that caused by Accutase. Trypan blue test immediately after the disassociation can not be used as an indicator in estimating the apoptosis of NSCs during the expanding.</p>


Subject(s)
Apoptosis , Collagenases , Female , Humans , Neostriatum , Neural Stem Cells , Peptide Hydrolases , Pregnancy , Trypsin
20.
Article in English | WPRIM | ID: wpr-29877

ABSTRACT

BACKGROUND AND OBJECTIVES: One of the most cellular source used for cartilage tissue engineering are mesenchymal stem cells (MSCs). In present study, human MSCs were used as cellular source. Since scaffold plays an important role in tissue engineering the aim of this study is to assess fibrin scaffold ability in chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs). METHODS: ADMSCs were isolated and cultured in DMEM medium supplemented with 10% FBS. Also ADMSCs expanded and characterised by flow cytometry. ADMSCs expressed CD44, CD90, CD105 but not CD34. After trypsinization, cells were entered within the fibrin scaffold. Then, chondrogenic medium was added to the scaffold. Seven days after cell culture, cell viability and proliferation were assessed by MTT test. Finally, 14 days after the ending of chondrogenic differentiation, analysis of chondrogenic genes expression was evaluated by RT-PCR and Real time PCR. Also, formation and development of chondrocyte cells was analysed by histological and immunohistochemistry evaluations. RESULTS: Viability and proliferation as well as chondrogenic genes expression within fibrin scaffold increased significantly compared with control group (cells free scaffold). Also, histological and immunohistochemistry evaluation showed that chondrocyte cells and collagen type II are formed on fibrin scaffold. CONCLUSIONS: Fibrin is a suitable scaffold for chondrogenic differentiation of ADMSCs.


Subject(s)
Cartilage , Cell Culture Techniques , Cell Survival , Chondrocytes , Collagen Type II , Fibrin , Flow Cytometry , Humans , Immunohistochemistry , Mesenchymal Stem Cells , Real-Time Polymerase Chain Reaction , Tissue Engineering , Transforming Growth Factor beta3 , Trypsin
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