Spring annotations requirelogin12/8/2023 ![]() Reverse zymography was similarly performed with the exception that conditioned medium from baby hamster kidney (BHK) cells, which express gelatinase A, was included in the gel mix with the gelatin (1 ml of conditioned medium was added to 15 ml of the gel mix). The gel was then stained for 4 hours in Coomassie Brilliant Blue. The gel was then rinsed three times in water, followed by a 24-hour incubation at 37☌ in incubation buffer (50 mmol/L Tris/HCl, pH 7.5, and 5 mmol/L CaCl 2). After electrophoresis, the gel was washed once for 15 minutes and then overnight in wash buffer (2.5% Triton X-100, 50 mmol/L Tris/HCl, pH 7.5, and 5 mmol/L CaCl 2) to remove the sodium dodecyl sulfate (SDS). Gelatin was included to a final concentration of 1 mg/ml. Thus, excessive TIMP production is not a sufficient explanation for the observed extracellular matrix accumulation, but complex changes in the local MMP/TIMP balance may underlie the pathomechanisms of fibrosis. These findings indicate a likely surplus in the BDL model of fibrosis of free gelatinases as compared with the TIMPs. This appears to result from the formation of TIMP/MMP complexes. No corresponding increase in TIMP protein activity was detected by reverse zymography. TIMP-2 and TIMP-3 transcripts become detectable on day 10 and remained stable afterwards. TIMP-1 transcripts appeared at day 2, increased until day 10, and remained elevated throughout the study period. The increase in gelatinase activities was accompanied by an increase in the TIMP mRNA transcripts. We found that the proteolytic activities of MMP-2 and MMP-9 increased by 2 days after ligation, reached maximal levels at day 10, and remained high through the study period, whereas the gelatinolytic activities in plasma were unchanged. In addition, we analyzed free gelatinase and TIMP activities by zymography and reverse zymography, respectively. Expression of four members of the matrix metalloproteinase (MMP) family (MMP-2/gelatinase A, MMP-3, MMP-9/gelatinase B, and MMP-13) and three tissue inhibitors of metalloproteinases-1, -2, and -3 (TIMP-1, TIMP-2, and TIMP-3) were evaluated by Northern blot analysis of RNA from liver tissue isolated at 0, 2, 5, 10, 20, and 30 days after either a BDL or sham operation. ![]() ![]() ![]() A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the expression and activities of collagen-degrading proteinases and their inhibitors during the progression of fibrosis. ![]()
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