In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test, which is marked by inhibition of enzyme activity. Four compounds, namely daidzin, genistin, matrine and oxymatrine, were selected as test subjects. The natural antitumor drug camptothecin was used as the control. The inhibitory effect was detected by two experimental methods: direct detection of trypsin activity inhibition and hydrolysis of bovine serum albumin by trypsin. The results showed the inhibitory effects of the four natural compounds on trypsin, and the inhibition rates of the four natural compounds were significantly different. The enzyme activity assay showed that the inhibitory effect of matrine was better than that of oxymatrine, indicating that trypsin had a good screening resolution. The inhibitory effect was significantly increased with the increased ratio of sample to trypsin, suggesting the structure-activity correlation and dose-effect correlation of the screening methods. Altogether, the experimental method of screening antitumor activity of natural compounds by trypsin has good application values. Since porcine trypsin is similar to human trypsin in terms of molecular structure and performance, it is more applicable for screening of antitumor efficacy of natural pharmacodynamic compounds.
Humans ; Trypsin/chemistry* ; Alkaloids/pharmacology*

BACKGROUNDMost epidermal cells used in skin tissue engineering are obtained from the skins of fetuses or prepuces, which can not be widely used in culturing and transplanting autologous epidermis for patients with extensive burn wounds. To solve the problem, in this study, we cultured epidermal cells from different parts of human body in vitro, and detected their growth activity.

METHODSNormal epidermal cells obtained from the prepuce, scalp, and axilla of male patients, were cultured and passaged. Their growth characteristics including adherent rate and growth activity were compared. Data were analyzed by homogeneity test of variance.

RESULTSIn primary culture, the growth of epidermal cells from the prepuce was significantly faster than that of the epidermal cells from the scalp and axilla. In the cells obtained from the prepuce, 80% confluence was achieved on day 12, while on day 16 and day 20 in the cells from the scalp and axilla, respectively. However, no significant difference was detected in their growth and proliferation in the second passage.

CONCLUSIONSAlthough the growth of epidermal cells obtained from the scalp and axilla is slower than that from the prepuce in primary culture, stable cell line can be established and used in preparation of auto-epidermal grafts for patients with extensive burn wounds. Therefore, the scalp and axillary skin should be considered as important sources of epidermal cells other than the prepuce.

Adult ; Cell Proliferation ; Cell Survival ; Epidermis ; cytology ; Humans ; Male ; Trypsin ; pharmacology

The study was purposed to investigate the apoptosis of HL-60 cells induced by recombinant common buckwheat trypsin inhibitor (rBTI) and its mechanism. The inhibition rate of rBTI on HL-60 cells was detected by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide); the morphology of HL-60 nuclei was observed by fluorescence microscopy; the apoptosis cells of HL-60 detected by agarose gel electrophoresis and the changes of apoptosis rate was assayed by flow cytometry (FCM), when the HL-60 cells were treated with different concentration of rBTI for 24 hours. The results showed that the growth of HL-60 cells was inhibited evidently after treatment with rBTI in a dose-dependent manner, but there were minimal effects on normal human peripheral blood mononuclear cells (PBMNCs). The nuclei of HL-60 cells showed the characteristics of apoptosis, the analysis by flow cytometry indicated that the apoptosis rate of HL-60 cells was 52% after treatment with rBTI (100 microg/ml), DNA analyzed by agarose gel electrophoresis showed "ladder" pattern. It is concluded that rBTI obviously inhibits growth of HL-60 and induces its apoptosis which provides a foundation for use of recombinant common buckwheat trypsin inhibitor to cure the acute myeloid leukemia.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Fagopyrum ; chemistry ; HL-60 Cells ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Trypsin Inhibitors ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo observe the effect of isolating the chondrocytes from articular cartilage with the method of three-step enzymatic digestion, and the biological characteristics of the isolated chondrocytes during cultivation in vitro in order to evaluate their biological activity.

METHODSThe method of three-step enzymatic digestion was designed that the articular cartilage was digested one by one with the 1 g/L trypsin and 1 g/L EDTA, 1 g/L hyaluronidase and 2 g/L collagenase I in the culture medium to isolate chondrocytes. The harvesting and viability rate of the primary chondrocytes were detected. During the passage cultivation in vitro, the changes of the chondrocytes shape and growth were observed, the changes of the collagen type I and II and aggrecan in the extracellular matrix were investigated and detected.

RESULTS(1) The extracellular matrix of articular cartilage was completely dissolved by the three-step enzymatic digestion, and the chondrocytes were completely isolated from the solid matrix. The number of the harvested chondrocytes from every gram of wet cartilage was 50.3 x 10(6) on average, and their viability rate was 98.8% on average. (2) The primary and first passage chondrocytes had triangle or multi-angle shape, and became elliptic shape at the growing confluence with the positive immunohistochemical stain of collagen type II and the strong heterochromia to toluidine blue. The content of sulfate glycosaminoglycans (GAG) in the extracellular matrix of the primary passage cells was (92 +/- 10) microg/cm(2). The chondrocytes after the third passaging gradually became spindle shape with the negative stain of collagen type II and the weak heterochromia to toluidine blue. The content of sulfate GAG of the fourth passage cells was (48 +/- 12) microg/cm(2).

CONCLUSION(1) The method of three-step enzymatic digestion can make the extracellular matrix of articular cartilage completely degraded, and has advantages in the high efficiency of harvesting primary chondrocytes, high cellular viability rate and simple manipulation. (2) The primary and first passage chondrocytes have fine biological activity, and the chondrocytes after the third passaging have lost their special biological activity.

Animals ; Cartilage, Articular ; cytology ; drug effects ; Cell Culture Techniques ; Cell Separation ; methods ; Cells, Cultured ; Chondrocytes ; cytology ; Collagenases ; pharmacology ; Female ; Hyaluronoglucosaminidase ; pharmacology ; Male ; Rabbits ; Trypsin ; pharmacology

Animals ; Aquaporins/metabolism* ; Disease Models, Animal ; Glycoproteins/pharmacology* ; Humans ; Lung/metabolism* ; Protective Agents/pharmacology* ; Respiratory Distress Syndrome/physiopathology* ; Sus scrofa ; Trypsin Inhibitors/pharmacology* ; Up-Regulation

OBJECTIVETo study the effects of ulinastatin on coagulation in children who underwent open-heart surgery with cardiopulmonary bypass (CPB).

METHODSFifty children who underwent open-heart surgery for ventricular septal defect were randomly divided into two groups: ulinastatin treatment and control. Before CPB, ulinastatin (1.0×10(4) U/kg) was added to CPB priming fluid only in the ulinastatin treatment group. Activated partial thromboplasin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen and international normalized ratio (INR) were measured both before and at 1 hr, 6 hrs and 24 hrs after CPB.

RESULTSThe PT in the ulinastatin group was more prolonged than in the control group at 1 hr after CPB (18.7 ± 0.7 s vs 15.5 ± 0.5 s) and 6 hrs after CPB (17.5 ± 0.6 s vs 15.0 ± 0.6 s). The APTT in the ulinatatin group was also significantly more prolonged than in the control group at 6 hrs after CPB (38.7 ± 3.1 s vs 35.3 ± 3.1 s) and 24 hrs after CPB (34.2 ± 3.0 s vs 31.1 ± 2.6 s).

CONCLUSIONSUlinastatin may prolong PT and APTT after CPB, and thus affects coagulation in children.

Blood Coagulation ; drug effects ; Cardiac Surgical Procedures ; Cardiopulmonary Bypass ; Female ; Glycoproteins ; pharmacology ; Humans ; Infant ; Male ; Partial Thromboplastin Time ; Prothrombin Time ; Trypsin Inhibitors ; pharmacology

Huwentoxin-XI (HWTX-XI) is a protein isolated from the crude venom of spider Ornithoctonus huwena. It has 55 amino acid residues containing 6 cysteine residues forming 3 disulfide bonds. It shows potent inhibitory effect on trypsin and voltage-gated potassium channels in rat dorsal root ganglion cells. According to the structure-function relationship of HWTX-XI, we designed two mutants through mutation of potassium channel inhibition related amino acid residues (R5I, R10T,R25A and R5I,R25A) and then expressed them with high purity by using the vector pVT102U on Saccharamyces cerevisiae strain S78; The two mutants had the same trypsin inhibition activity as HWTX-XI, whereas their potassium channel inhibition activity and animal toxicity were much lower than those of HWTX-XI. This study is helpful for designing drugs of trypsin related diseases based on HWTX-XI.
Amino Acid Sequence ; Animals ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutant Proteins ; biosynthesis ; genetics ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Saccharomyces cerevisiae ; genetics ; metabolism ; Spider Venoms ; biosynthesis ; genetics ; pharmacology ; Spiders ; Trypsin Inhibitors ; pharmacology

To investigate the feasibility of making up bovine pericardium into scaffolds for tissue engineering valve, it was decellularized by 0.5% trypsin incubation for three hours. The rats were divided into fresh bovine group, acellular pericardium group and control group, and OD value of rat anti-bovine IgG was measured. Serum levels of IgG showed no difference among the three groups on the 7th day postoperatively (P > 0.05). The OD values of IgG at other time-points displayed distinct difference among the three groups (P < 0.05). Fresh bovine pericardium group produced more IgG than acellular bovine pericardium group did at the same time-point. The IgG level of fresh bovine pericardium group reached the peak postoperatively, but in acellular bovine pericardium group its peak value appeared on the 30th day postoperatively. Histological examination demonstrated that the bovine pericardium was thoroughly decellularized, the fibrous structure was slightly loose, but remained essentially intact. These results indicate that the acellularization procedure can result in a complete removal of cells and decrease of immunogenicity.
Animals ; Antibodies, Anti-Idiotypic ; immunology ; Bioprosthesis ; Cattle ; Cell Separation ; methods ; Immunoglobulin G ; immunology ; Implants, Experimental ; Pericardium ; cytology ; immunology ; Rats ; Rats, Sprague-Dawley ; Tissue Engineering ; methods ; Trypsin ; pharmacology

The effect of trypsin on the separation an subculture of the keratinocytes was investigated in this work. It was found that when 0.25% trypsin was employed for 5 minutes to separate keratinocytes, the number of active keratinocytes and the cells capable of forming colony were higher than those of other experimental conditions. The maximum attached ratio of primary keratinocytes was obtained when skin tissues were treated at 0.05% concentration of trypsin. With the increase of the trypsin concentrations, the attached ratio, attachment rate constant, and colony forming efficiency were all increased. Thus, 0.25% concentration of trypsin was recommended for separating and subculturing the keratinocytes.
Animals ; Cell Count ; methods ; Cell Culture Techniques ; methods ; Cell Separation ; methods ; Cell Survival ; drug effects ; Keratinocytes ; cytology ; drug effects ; Mice ; Stem Cells ; drug effects ; Trypsin ; pharmacology

OBJECTIVETo study the changes of tissue composition and immunogenicity of porcine and human aortic valves after decellularization.

METHODSThree cryopreserved human aortic valves and 4 porcine valves were decellularized with trypsin, and the leaflet tissue was homogenized for SDS-PAGE protein electrophoresis and U-937 migration assay.

RESULTSTrypsin effectively removed the cells from the valve. SDS-PAGE demonstrated an obvious difference in the tissue composition between porcine and human valves. Although decellularization significantly diminished the differences between the valves, decellularized procine aortic valve stilled contained more protein components (between 26 000 and 43 000) than human valve. U-937 migration assay showed an obvious decrease of cell migration in the valves by decellularization (from 832.7×10(3) to 152.4∓31.1×10(3) for porcine valves, P<0.01, and from 644.9×10(3) to 91.2×10(3) for the human valves, P<0.01). Decellularized porcine valves induced a significantly greater cell migration than decellularized human valves (P<0.05).

CONCLUSIONDecellularization with trypsin can effectively decrease the immunogenicity of human or porcine heart valve, but can not completely eliminate the antigen, and decellularized porcine valve still retain strong immunogenicity.

Animals ; Antigens ; isolation & purification ; Aortic Valve ; cytology ; immunology ; Bioprosthesis ; Heart Valve Prosthesis ; Humans ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds ; Trypsin ; pharmacology