How Atypical Penile Curvature Influence Clinical Outcomes in Patients with Peyronie's Disease Receiving Collagenase Clostridium Histolyticum Therapy?

Inhibitory effect of luteolin on the growth, migration and vasculogenic mimicry formation of a melanoma cell line B16 / 中华皮肤科杂志

Objective To evaluate the effect of luteolin on the growth,migration and vasculogenic mimicry formation of a melanoma cell line B16.Methods In vitro cultured B16 melanoma cells were divided into 4 groups:low-,middle-and high-dose luteolin groups treated with 2.5,5,10 μmol/L luteolin respectively,and control group treated with 0.1％ dimethyl sulfoxide (DMSO).Scratch assay,Transwell invasion assay and vascular channel formation assay were performed to assess the migration,invasion of and vascular channel formation by melanoma cells.A model of subcutaneous transplanted B 16 melanoma was established in 12 C57 mice,which were randomly and equally divided into 4 groups:control group gavaged with ultrapure water,low-,middle-and high-dose luteolin groups gavaged with 10,20,40 mg/kg luteolin respectively every day.The above treatment for the tumor-bearing mice lasted till day 28,and then these mice were sacrificed.Meanwhile,the lung and tumor tissues of the mice were excised,and the growth,metastasis and vasculogenic mimicry of transplanted melanoma were observed.Immunofluorescence and immunohistochemical studies were performed to evaluate the effects of luteolin on the expression of vascular endothelial cadherin (VE-cadherin),vascular endothelial growth factor receptor 1 (VEGFR1),VEGFR2,matrix metalloproteinase-2 (MMP-2) and MMP-9 in the transplanted melanoma.Means were compared among several groups by using one-way analysis of variation or rank sum test.Results In vitro study showed that the relative scratch width at 48 hours significantly differed among the control group,low-,middle-and high-dose luteolin groups (0.47 ± 0.04,0.64 ± 0.04,0.73 ± 0.03,0.84 ± 0.04 respectively;F =34.51,P ＜ 0.001),and the migration ability of B16 cells was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.05).At 24 hours,there were significant differences in the number of cells crossing the Transwell membrane among the control group,low-,middle-and high-dose luteolin groups (281.00 ± 8.79,169.00 ± 15.35,92.00 ± 14.79 and 57.00 ± 13.72 respectively;F =275.30,P ＜ 0.001),and the invasive ability was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (P ＜ 0.01).Meanwhile,the number of formed vascular channels also differed among the above 4 groups (20.00 ± 2.77,11.00 ± 1.28,7.00 ± 1.86 and 2.00 ± 1.32 respectively;F =48.61,P ＜ 0.001),and the number of vascular channels was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.01).In vivo study showed that the tumor size significantly differed among the control group,low-,middle-and high-dose luteolin groups (5.10 ± 1.72,4.02 ± 2.13,2.98 ± 0.92,1.49 ± 1.13 cm3 respectively;F =28.76,P ＜ 0.001),and was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (t =3.86,7.11 and 13.06 respectively,all P ＜ 0.01).CD31-PAS double staining showed that the number of vasculogenic mimicry was significantly higher in the control group than in the low-,middle-and high-dose luteolin groups (all P ＜ 0.01).In vivo and in vitro studies both showed that the expression of vasculogenic mimicry-related markers in the cells or mouse tumor tissues was significantly lower in the high-dose luteolin group than in the control group (P ＜ 0.05).Conclusion Luteolin can effectively inhibit the growth,metastasis and vasculogenic mimicry formation of melanoma.

Expression of collagenases (matrix metalloproteinase-1, 8, 13) and tissue inhibitor of metalloproteinase-1 of retrodiscal tissue in temporomandibular joint disorder patients

OBJECTIVES: The aim of this study was to reveal how collagenases (matrix metalloproteinase [MMP]-1, 8, 13) and tissue inhibitor of metalloproteinase 1 (TIMP-1) are expressed in immunohistochemistry of retrodiscal tissue in temporomandibular joint disorder patients. MATERIALS AND METHODS: This study was conducted on 39 patients who underwent discoplasty or discectomy. Immunohistochemical staining was undertaken and expression levels of MMP-1, 8, 13, and TIMP-1 were evaluated. The status of internal derangement of disc, osteoarthritis, and joint effusion were analyzed using magnetic resonance imaging (MRI). Disc status observed during operation was also categorized. RESULTS: The more severe disc derangement was observed on MRI, the more increased expression of MMPs and TIMP-1 appeared. Regarding MMP-13 expression, 86.7% of late-stage disc displacement patients showed grade II or III. Expression level of MMPs or TIMP was not statistically significant associated with joint effusion level. In perforation and/or adhesion groups, all patients showed grade II or III expression of MMP-13. Once perforation occurred, MMP-13 showed increased expression with statistical significance. CONCLUSION: MMP-1 and MMP-13 expression seem to be related to progression of osteoarthritis whereas MMP-8 does not seem to have a specific role with regard to temporomandibular joint disorders. TIMP-1 is considered to be partly related to internal derangement rather than osteoarthritis, but it is not significant.

Comparison between two different experimental models of osteoarthritis in rabbits. Intra-articular collagenase injection and anterior cruciate ligament transection

ABSTRACT PURPOSE: To compare two different experimental models of osteoarthritis in rabbits: intra-articular collagenase injection and anterior cruciate ligament transection. METHODS: Ten adult rabbits were randomly divided in two groups: COLL (collagenase group) and ACLT (anterior cruciate ligament transection). The COLL group was treated with 0.5 ml collagenase solution (2mg collagenase/0.5 ml sterile PBS), and the ACTL group was subjected to anterior cruciate ligament. After six and twelve weeks, respectively, the animals in the COLL and ACTL groups were euthanized. The gross appearance and histological examinations conducted in the cartilage articular surface was blindly scored according to the criteria developed by Yoshimi et al. (1994) and Mankin et al. (1971), respectively. RESULTS: The gross morphologic observation, macroscopic score and histological examinations have demonstrated that the ACTL group presented the highest scores, and lesions more severe than those in the COLL group. CONCLUSIONS: Both methods, anterior cruciate ligament transection and collagenase, applied to the stifle joint of the rabbits have effectively induced degenerative changes in the cartilage tissue, through statistically significant analysis (p≤0.05). The ACTL method has presented more severe lesions.

Histological analysis of chondrons isolated from age-related normal rabbit knee / 中华骨科杂志

Objective To explore the histological properties of isolated chondrons and chondrocytes from rabbit knee cartilage,and to determine if these properties vary with age.Methods Three groups of rabbit knees were evaluated according to different age:(1) young (2 months,n=10);(2) adult (8 months,n=10);and (3) old (31 months,n=10).The cartilage structure,proteoglycan,collagen-2,and collagen-6 content were determined by light microscopic using hematoxylin-eosin (HE),Toluidine Blue,and col-2,6 staining.The chondrons were enzymatically isolated using 0.3 g/L dispase and 0.2 g/L collagenase-2 by shaking for 3 hours.The morphology and composition of isolated chondrons were observed by HE and collagen-6 immunostaining staining after overnight coverslip monolayer culture under a microscopy.Results The chondrocytes became sparser and the total content of proteoglycans and collagen-2 were decreased in the articular cartilage with age.Compared to the chondrocytes,the surrounding rim or capsule was more obvious in the isolated chondrons,and they exhibited obvious differences in shape.The cells within one cluster from different age groups were similar to the morphology observed in cartilage in situ.The adult and old chondrons generally possessed a thicker pericellular matrix with more enclosed cells,and the chondrons contained more cells can reach 47％.Conclusion These findings further suggest that the properties of the chondrons and pericellular matrix have an important influence on the biomechanical microenvironment of the knee joint cartilage degeneration that occurs with age.

Optimization the methodology of isolating human ubilical cord mesenchymal stromal cells from Wharton's jelly and examination of their passage effect on immune phenotype using flow cytometry / 天津医药

Objective To observe the effects of different collagenase digestions on isolating human umbilical cord mesenchymal stromal cells (MSC) from Wharton’s jelly, to exam their differentiation ability and to investigate their passage effect on the immune phenotype. Methods Human umbilical cord samples were digested by collagenaseⅠorⅡorⅣfor 4-18 hours then were passed through sieves . Cells were collected by centrifugation then inoculated in DMEM/F12 medium at concentration within range of 4.8×103-1×104/cm2 to compare the effect of different digestions on MSC. Von kossa staining and tetracycline fluorescence was used to label the osteogenic differentiation capacity of MSC. Also RT-PCR was employed to identify the differentiate capacity of MSC into myocardial-like cells. The immunophenotype of MSCs were detected by flow cytometry after subculture. Results Using collagenaseⅠdigestion, the number of MSCs isolated from human umbilical cord in Wharton’s jelly and their vitality were much higher while the period to show cell extension and primary culture time were shorter than those using collagenaseⅡorⅣdigestions. The analysis of surface marker revealed that the expression of positive markers include CD29, CD44, CD73, CD90 and CD105 did not change with passages while the negative markers such as CD31, CD34 and HLA-DR increased significantly with passages;Differential experiments induced in vitro show that human umbilical cord MSC in wharton’s jelly had the ability to differentiate into osteoblasts and myocardial-like cells. Con?clusion The human umbilical cord MSC in Wharton’s jelly was successfully isolated by collagenaseⅠdigestion. This meth?od was simple with a high success rate while cell loss and damage were minimum. This makes large-scale cultivation possi? ble. Negative markers increased with cell passages. This phenomenon revealed that MSC showed directional differentiation.

The changes and significance of the extracellular matrix in larynx of old guinea pig / 中华老年医学杂志

Objective To study the changes and significance of the hyaluronic acid (HA) and collagen in the larynx of old guinea pig.Methods Six young guinea pigs (4 months old) and 6 old guinea pigs (2 years old) were selected as control group and aged group for this study (n=6 for each)The expressions of HA and collagen in the larynx of guinea pigs were detected using Alcian blue and Masson trichrome methods,respectively.The images were managed using Image-Pro Plus 6.0 For Window.Results The optical density (expression level) of HA in the larynx was lower in aged group than in control group (0.69 vs.0.80,F=13.45,P＜0.05).The optical density(expression level) of collagen in the larynx was higher in aged group than in control group (0.71 vs.0.63,F=9.56,P＜0.05).Conclusions The content of HA in larynx is decreased and collagen in larynx is increased in old guinea pig which may result in presbyphonia change through higher viscoelastic properties of vocal cords.

Effects of different collagenase digestion on yield rate of purified islets in rats / 中华器官移植杂志

ObjectiveTo compare the yield rate of rats islets between different collagenase digestion groups.MethodsThe SD rats were randomly divided into two groups as following by using random digits table:collagenase P group (pancreas digested by 1 mg/ml collagenase P) and type Ⅴ collagenase group (pancreas digested by 1 mg/ml type Ⅴ collagenase).After pancreas digestion,rat islet cells in two groups were culture,purified and stained with DTZ.The mean islet number and islet equivalent (IEQ) before and after purification were measured under an inverted microscope.The viability of purified islets was assessed by fluorescence staining of aridine orange (AO) and propidium iodide (PI) under the fluorescence microscopy.After purification and culture for two days,islets function was evaluated by insulin releasing tests in the two groups.ResultsBefore purification,there was no significant difference in the islets number obtained from the pancreas between two groups (P＞0.05),but there was significant difference in the IEQ (P＜0.05).After purification,the islets number in type Ⅴcollagenase group and collagenase P group was (485 ± 113)/pancrease and (643 ± 82)/pancrease,and IEQ was (674 ± 157)/pancreas and (989 ± 126)/pancreas,respectively (P＜0.05).Islet viability in type Ⅴcollagenase group and collagenase P group was (96.13 ±1.13) ％ and (96.38 ± 0.92) ％ respectively (P＞0.05).The results of insulin releasing tests revealed there was no significant difference in islet function stimulated by hypoglycemia and hyperglycemia between two groups (P＞0.05).ConclusionTwo types of collagenase are suitable for the islets digestion in rats.The stability of digestion and yield rate of purified islets in collagenase P group are higher than in type Ⅴ collagenase group.

Efficacy of plasma radiofrequency ablation at low temperature in disc combined with collagenase injection out of disc in patients with cervical intervertebral disc herniation / 中华麻醉学杂志

Objective To evaluate the efficacy of plasma radiofrequency ablation at low temperature in disc combined with collagenase injection out of disc in patients with cervical intervertebral disc herniation.Methods Fifty-six patients suffering from cervical intervertebral disc herniation with headache,dizziness,and pain in the neck and in the shoulder were randomly divided into 2 groups ( n =28 each):collagenase injection out of disc group (group C) and plasma radiofrequency ablation at low temperature in disc combined with collagenase injection out of disc group (group R).All operations were carried out under CT guidance.Results At the sixth month of follow up after treatment,the remission rates of headache,dizziness and pain in the neck and in the shoulder were 86％,79％,and 93％ in group C and 96％,93％,and 100％ in group R,.respectively,with significant difference between the two groups ( P ＜ 0.05 ) Conclusion The efficacy of plasma radiofrequency ablation at low temperature in disc combined with collagenase injection out of disc is much better than collagenase single in patients with cervical intervertebral disc herniation.

Induction of metalloproteinases expression by TLR ligands in human fibroblast like synoviocytes from juvenile idiopathic arthritis patients.

Background & objective: Juvenile idiopathic arthritis (JIA) is characterized by chronic synovitis, cartilage damage and bone erosion. Both genetic and environmental factors and microbes probably play a role in pathogenesis. Microbes are recognized by Toll like receptors (TLRs) and activate innate immune response. We studied the ability of bacterial and viral products to produce matrix metalloproteinases (MMPs) and cytokines by fibroblast like synoviocytes (FLS) from patients with JIA. Methods: FLS were cultured from synovial fluid (SF) of patients with JIA and subsequently stimulated for 48 h by different TLR ligands [peptidoglycan (PG) for TLR2, poly(I-C) for TLR3, lipopolysaccharide (LPS) for TLR4, flagellin for TLR5, imiquimod for TLR7 and CpG DNA for TLR9]. Later the production of IL6, IL8, MMP-1, MMP-3, tissue inhibitors of metalloproteinase (TIMP1) was measured in the culture supernatants by ELISA. Expression of TLR2, TLR4, TLR7 and TLR9 was studied in FLS derived from JIA patients by RT-PCR. Results: IL6, IL8, MMP3 and MMP1 production was induced on stimulation of FLS with TLR2 ligand, TLR3 ligand, TLR4 ligand, TLR5 ligand but not with TLR7 ligand and TLR9 ligand. There was no effect of these ligands on the production of TIMP thus the balance was tilted in favour of MMPs after TLR ligation. TLR2, TLR4 and low expression of TLR9 was found but, no expression of TLR7 was found in FLS from JIA patients. Interpretation & conclusion: TLR pathway stimulation by microbial products or endogenous ligands could be involved in the production of MMPs in JIA and may contribute to disease pathology. Thus it may be beneficial to inhibit TLR pathway to reduce cartilage destruction.

Efficacy of percutaneous laser disc decompression combined with injection of collagenase through a target location for treatment of lumbar intervertebral disc protrusion / 中华麻醉学杂志

Objective To investigate the efficacy of percutaneous laser disc decompression(PLDD)combined with injection of collagenase through a target location for treatment of lumbar intervertebral disc protrusion.Methods Ninety patients with lumbar intervertebral disc protrusion scheduled for discolysis,aged 31-52 yr,weighing 58-70 kg,were randomly divided into 3 groups: PLDD group(group P,n = 29),collagenase injection group(group C,n = 31),PLDD combined with injection of collagenase through a target location group(group PC,n = 30).The puncture was performed under the guidance of CT.Group P was treated using PLDD.Group C was treated with collagenase injection.Group PC was treated with injection of collagenase after PLDD was completed.The therapeutic effect was assessed before operation and on day 7,30,60 and 90 after operation using M-JOA score.Results M-JOA grade was significantly higher at the each time point after operation in group P and PC,and on day 30,60 and 90 after operation in group C than that before operation(P ＜ 0.05).M-JOA grade was significantly lower on day 30 after operation in group P,while higher on day 30,60 and 90 after operation in group C and PC than that on day 7 after operation(P ＜ 0.05).M-JOA grade was significantly lower at the each time point after operation in group P and C than in group PC.Conclusion The therapeutic effect of PLDD combined with collagenase injection through a target location is stable for treatment of lumbar intervertebral disc herniation and better than that of PLDD or collagenase injection alone.

Development of a chromogenic assay for blood-plasma collagenase Ⅳ and its initial application / 中华检验医学杂志

Objective To establish a chromogenic assay for blood-plasma collagenase Ⅳ in order to evaluation the reference range of collagenase Ⅳ in the plasma of healthy individuals.Methods The assay was based on measurement of terminal amino group with succinylated gelatin as substrates and TNBS as chromogenic reagent.The optical density of each reaction was determined at 405 nm using a Sunrise microplate reader.Chromatographic and detection conditions were optimized and performance of the methed was evaluated by recovery experiments and precision experiments.It was compared with ELISA.The levels of collagenase Ⅳ in 112 health persons'plasma were determined and the data were analyzed by SPSS statistical software.Results The whole determing time was within 1.5 h,the linear range of this method was 1.5-10.0 mg/L,and the minimum detection limit was 0.965 mg/L.They were well correlated with ELISA results (R~2=0.999 7,P＜0.01).The within-run CV was less than 3.16%and between-run CV was less than 9.81%.The 95%confidence interval of collagenase Ⅳ in healthy plasma was 33.38-49.80 mg/L.Conclusion This chromogenic assay for blood-plasma collagenase Ⅳ can be used for measurement of collagenase Ⅳ in blood-plasma and the reference range of collagenase Ⅳ in healthy plasma was established.

Additive Effects of Intra-articular Injection of Growth Hormone and Hyaluronic Acid in Rabbit Model of Collagenase-induced Osteoarthritis

In a rabbit model of collagenase-induced osteoarthritis, the additive effects of intra-articular recombinant human growth hormone (GH) administration to hyaluronic acid (HA) were evaluated. After intra-articular collagenase injection, mature New Zealand white rabbits (n=30) were divided into 3 groups. Group 1 (control rabbits) received once weekly intra-articular saline injections for 4 weeks. Group 2 rabbits received 6 mg HA injections, and group 3 rabbits were injected with 6 mg HA and 3 mg recombinant human GH. These injections were initiated 4 weeks after collagenase injections. Lameness was observed for 9 weeks after collagenase injections. Macroscopic and histopathological knee joint findings were also evaluated at the end of 9 weeks after collagenase injections. Although all animals had lameness after collagenase injections, the duration and severity of lameness were significantly shorter and less severe in group 3 than group 1 and 2 (P<0.01). Macroscopic scores showed that femoral condyles of group 3 rabbits received significantly less cartilage damage than those of groups 1 and 2 rabbits (P<0.01). Histopathological score was also the lowest in group 3 (P<0.01). These results suggest that co-injection of intra-articular HA and recombinant human GH is more effective than HA injections alone in an osteoarthritis model.

Análise do padrão de expressão de MMP-2, -9 e -8 em tecido humano pulpar normal e inflamado / Analysis of the expression perfile of MMP-2, -9 and -8 in normal and inflamed human pulp tissue

As metaloproteinases da matriz (MMPs) foram relacionadas a diversas doenças inflamatórias como artrite e também ao câncer. O presente trabalho tem por objetivo estabelecer o papel da MMP-2, MMP-9 e MMP-8 no processo de inflamação pulpar. Foram adotadas as seguintes hipóteses nulas: (1) o padrão de expressão das MMP-2, MMP-9 e MMP-8 não sofre alteração nos diferentes estágios da polpa humana: normal, reversível, transição, irreversível ou necrose; (2) não há diferença de expressão das MMP-2, -9 e MMP-8, considerando-se um mesmo estágio de inflamação tecidual pulpar. Os métodos utilizados foram: (I) Obtenção dos espécimes, que foram divididos em grupos de acordo com critérios adotados de semiologia subjetiva e objetiva. Obtiveram-se os seguintes grupos: GI (Controle) dentes hígidos (n=7); GII (Pulpite Reversível n=4); GIII (Pulpite Transição n=4); GIV (Pulpite Irreversível/Necrose n=8). Logo após exodontia, os dentes obtidos foram cortados ligeiramente abaixo da junção amelodentinária e fixados em formol a 10% por 48h. Foram lavados em água corrente (24h) para então serem processados histologicamente. Foram obtidas secções de 4m, aderidas em lâminas silanizadas e submetidas à imunomarcação (Técnica da Peroxidase), utilizando os anticorpos anti MMP-2, MMP-9 e MMP-8 humanos. A presença de imunomarcação foi realizada através da análise semi-quantitativa por escores, sendo que a quantificação de marcação por corte seguiu o seguinte escore: 0= ausente; 1= leve; 2= moderada; 3= intensa. Realizou-se teste estatístico não paramétrico Kruskal-Wallis, p<0,05. As comparações intergrupos revelaram, para CO: (1)MMP-2 - GI=GII=GIII, GIII=GIV, GI>GIV (p<0,01) e GII>GIV (p<0,05); (2)MMP-9 GI=GII=GIV, GII=GIII e GIII>GI (p<0,01); (3)MMP-8 GI=GII=GIII=GIV. Na região central da polpa, obteve-se: (1)MMP-2 GI=GII=GIII, GIII=GIV, GI>GIV (p<0,001) e GII>GIV (p<0,01); (2)MMP-9 GI=GII=GIII, GIII=GIV, GIV>GI (p<0,001) e GIV>GII (p<0,01); (3)MMP-8 GI=GII, GIII=GIV, GIII>GI (p<0,05),...

The matrix metalloproteinases (MMPs) have been related to various inflammatory diseases, such as arthritis, as well as to cancer. The aim of the present study was to establish the role of MMP-2, MMP-9 and MMP-8 in the process of dental pulp inflammation. The following null hypotheses were adopted: (1) the pattern of MMP-2, MMP-9 and MMP-8 expression does not undergo alteration in the following different stages of human pulp: normal, reversible, transition, irreversible or necrosis; (2) there is no difference in the expression of MMP-2, -9 and MMP-8, when considering the same stage of pulp tissue inflammation. The methods used were: (I) Obtainment of specimens, which were divided into groups according to the subjective and objective criteria of semiology adopted. The following groups were obtained: GI (Control) healthy teeth (n=7); GII (Reversible Pulpitis n=4); GIII (Transition Pulpitis n=4); GIV (Irreversible Pulpitis/Necrosis n=8). Soon after extraction the teeth obtained were cut slightly below the amelodentinal junction and fixed in 10% formol for 48h. They were washed under running water (24h) and were histologically processed afterwards. Sections of 4m were obtained, adhered to silanized slides, and submitted to immunomarking (Peroxidase Technique), using human anti MMP-2, MMP-9 and MMP- 8 antibodies. The presence of immunomarking was determined through semi-quantitative analysis by scores, and marking by cut was quantified using the following score: 0= absent; 1= slight; 2= moderate; 3= intense. The Kruskal-Wallis non-parametric statistical test was performed, p<0.05. Intergroup comparisons revealed the following: for CO: (1)MMP-2 - GI=GII=GIII, GIII=GIV, GI>GIV (p<0.01) and GII>GIV (p<0.05); (2)MMP-9 GI=GII=GIV, GII=GIII and GIII>GI (p<0,01); (3)MMP-8 GI=GII=GIII=GIV. In the central region of the pulp, the following results were obtained: (1)MMP-2 GI=GII=GIII, GIII=GIV, GI>GIV (p<0.001) and GII>GIV (p<0.01); (2)MMP-9 GI=GII=GIII, GIII=GIV, GIV>GI...

Imersão do pâncreas por 24 horas em solução de colagenase a 4ºC no isolamento de ilhotas de Langerhans / Immersion of pancreas for 24 hours in collagenase solution at 4ºC in Langerhans islets isolation

Objetivos: testar se a imersão do pâncreas em solução de 1 ou 2 mg/ml de colagenase em 4ºC por 24 horas, no isolamento experimental de ilhotas de Langerhans, aumenta o rendimento de ilhotas por grama de tecido pancreático. Métodos: estudo experimental com camundongos, realizado no Laboratório de Nefrologia do Instituto de Pesquisas Biológicas do Hospital São Lucas da PUCRS, Porto Alegre, RS. Após sacrifício dos animais sob anestesia, os pâncreas foram retirados e triturados, sendo divididos em quatro grupos, conforme a técnica utilizada para isolar as ilhotas. A) colagenase 1mg/mL, a 4ºC por 24 horas e posterior aquecimento a 39ºC por 15 minutos. B) colagenase 2mg/mL com as mesmas etapas anteriores. C) colagenase 1mg/mL com aquecimento da solução no mesmo dia da retirada, a 39ºC por 15 minutos. D) colagenase 2mg/mL com aquecimento da solução no mesmo dia da retirada, a 39ºC por 15 minutos. Verificamos a viabilidade das ilhotas através do teste do azul tripano. Resultados: as medianas da quantidade de ilhotas isoladas nos grupos A, B, C e D foram 9.142, 8.285, 2.813 e 3.199 respectivamente. O teste de Kruskal- Wallis demonstrou diferença significativa, com valor de H = 17,44 com a = 0,01 e, na comparação dos grupos entre si, demonstrou que não há diferença entre as soluções de colagenase com concentrações de 1 e 2 mg/dL. Os grupos com a imersão do tecido pancreático em solução de colagenase por 24 horas obtiveram três vezes mais ilhotas, quando comparados aos submetidos à digestão imediata, conforme teste de Dunn com a<0,05. O teste do azul tripano demonstrou uma vitalidade maior que 95% em todos os grupos. Conclusões: sugere-se que a imersão em colagenase por tempo mais prolongado melhora o processo de digestão do tecido pancreático, aumentando o rendimento de ilhotas isoladas por grama de tecido pancreático. A diferença de concentração entre as soluções de colagenase não afetou o resultado.

Aims: To test whether the immersion of the pancreas in solutions of 1 or 2 mg/mL of collagenase in 4°C for 24 hours, for the isolation of Langerhans islets, rises the yield of islets/ grams of pancreatic tissue. Methods: Experimental study with mouses, performed in the Laboratory of Nephrology of the Instituto de Pesquisas Biológicas do Hospital São Lucas da PUCRS, Porto Alegre, RS. After the animals have been sacrified under anesthesia, the pancreas were removed and divided in four groups, according the technique used for isolating the islets. A) collagenase 1mg/mL, in 4ºC for 24 hours and heating for 39ºC for 15 minutes. B) collagenase 2mg/mL with the same previous described steps. C) collagenase 1mg/ mL and heating of the solution in the same day, in 39ºC for 15 minutes. D) collagenase 2mg/mL and heating of the solution in the same day, in 39ºC for 15 minutes. We verified the viability of the islet through the trypan blue test. Results: The median numbers of isolated islets in the groups A, B, C and D were 9142, 8285, 2813 e 3199, respectively. Kruskal-Wallis test showed significant difference, with the value of H = 17,44 with a = 0,01, and in the comparison between the groups, there was o difference in the solutions of collagenase with concentrations of 1 and 2 mg/dL. Groups with immersion of pancreatic tissue in collagenase solution for 24 hours had three times more islets when compared to groups submitted to immediate digestion, according to Dunn test with a <0,05. The trypan blue test showed viability higher than 95% in all groups. Conclusions: We suggest that the immersion in solution of collagenase for longer time improves the process of digestion of pancreatic tissue, rising the yield of islets/ grams of pancreatic tissue. Different concentration between the collagenase solutions did not affect the final result.

Study on the effct of MMP-13 and its tissue inhibitors,metalloproteinase-1 on the rat of COPD model / 重庆医科大学学报

Objective:To investigate the expression of the collagenases MMP-13 and its inhibitor, tissue inhibitor metalloproteinase-1 (TIMP-1) in the rat lung of the experimental models of COPD.Methods:Male Wistar rats (10 weeks of age) were divided into two groups—model and control groups.The rat of model group were given intratracheal lipopolysaccharide (LPS),and then animals were exposed to cigarette smoke for 32 days.The lung function was measured,and pathological changes were also observed.Transcriptional levels of MMP-13 and TIMP-1 mRNA extracted from the lungs were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).Results:(1)In COPD model group,the pathological changes of bronchi and lung tissue,the changes of lung function were similar to those of the COPD patients.(2)The mRNA expression of MMP-13 and TIMP-1 in COPD model group were significantly increased compared with those in control group( P

Preventive and Therapeutic Effects of Xianling Gubao Capsules for Postmenopausal Osteoporosis / 广州中医药大学学报

0.05); while in the 12th month, BMD markedly increased in group A and decreased in group B (P 0.05) but ?-CTX level increased after treatment (P

Influence of collagenase with different degrees of activity on the isolation of rats' myocardiocytes / 解放军医学杂志

Objective To investigate the influences of 4 kinds of collagenase with different degrees of activity on the survival rate,contraction and relaxation functions of isolated rats' myocardiocytes.Methods Rats' myocardiocytes were isolated by enzymolysis with 4 kinds of collagenase with different degrees of activity.The survival rate of myocardiocytes was observed immediately after isolation(D),one hour after loading with calcium(E time point) and 10 minutes after electric stimulation(F time point).The contraction and relaxation functions of myocardiocytes,including contraction amplitude(ph),the proportionality of ph/single cardiocyte length(bl),maximal velocity of contraction(+dL/dt) and maximal velocity of relaxation(-dL/dt),were measured with IonOptix video edge tracker.Results From 203U/mg to 299U/mg,with lowering of the activity of collagenase,the isolation time became longer,the survival rate of myocardiocytes declined 1 hour after loading calcium and 10 minutes after electric stimulation(P

Research on abnormal collagen metabolism of colonic wall in patients with anastomotic leakage / 中国普通外科杂志

ObjectiveTo explore the relationship between anastomotic leakage of patients undergoing colonic surgery and the collagen metabolism of colonic wall.Method We measured the overall collagen content of colonic tissue by biochemistry and detected the collagen I, III, MMP-1,MMP-13 by immunohistochemistry in 16 patients with anastomotic leakage compared with 16 control cases. Resultthe overall collagen content and collagen I,III of colonic wall in the leakage group were lower than those in the control group (t=3.417,t=2.841, t=2.261,P

Effect of EGCG on invasion of breast cancer cells and its possible mechanism / 中国病理生理杂志

AIM: To study the effects of (-)-epigallocatechin-3-gallate (EGCG), a tea extract, on the invasion and metastasis of breast cancer cell line MDA-MB-231 and the possible mechanisms in vitro. METHODS: The expression of MUC1 in breast cancer cells treated with or without EGCG was detected by immunohistochemistry. The effect of EGCG on invasion of MDA-MB-231 cells was evaluated using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane (Matrigel). Gelatin zymography was performed to detect the secretion of collagenase-Ⅳ. RESULTS: EGCG reduced the expression of MUC1, significantly suppressed the invasion of tumor cells to basement membrane and reduced the secretion of collagenase-Ⅳ. CONCLUSION: In vitro, EGCG may suppress invasion, metastasis, and collagenase-Ⅳ secretion in MDA-MB-231 cells by inhibiting the production of MUC1.