Construction and identification of recombinant adenovirus expressing rat proteoglycan II / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct recombinant adenovirus expressing rat proteoglycan II (Ad-DCN) and to study its biological features.</p><p><b>METHODS</b>Rat DCN gene was inserted into an E1 and E3-substituted adenovirus shuttle plasmid. And this shuttle plasmid including DCN was recombined with AdEasy-1 in BJ5183-AD-1 electroporation competent cells to form recombinant adenovirus-DCN plasmid, which was further transfected into Ad293 cells to passage adenovirus. The control recombinant adenovirus, Ad-LacZ, was also constructed in the same way. After plaque forming trail and adjusted with PBS, Ad-DCN and Ad-LacZ were obtained with titer 1x10(9) pfu x ml(-1). PCR, Western blot and MTT analysis were used to detect the expression of DCN or the bioactivity of expressed DCN.</p><p><b>RESULT</b>DCN was detected in Ad-DCN infected CHO cells by PCR and Western blot, but not in Ad-LacZ infected CHO cells. MTT analysis results showed that the supernatant from the culture of Ad-DCN infected CHO cells could abrogate the inhibitive effect of TGFbeta1 on proliferation of CCL-64 cells. The proliferation rate of TGFbeta1 + Ad-DCN treated cells was significantly higher than that of TGFbeta1 + Ad-lacz or TGFbeta1 treated cells [(0.5252 +/-0.04 compared with 0.2826 +/-0.02 or 0.2918 +/-0.02) OD, P <0.05] and lower than that of control cells [(0.9332 +/-0.08) OD, P <0.05].</p><p><b>CONCLUSION</b>The constructed recombinant adenovirus can express biologically active decorin.</p>

Expression of matrix metalloproteinase-2 and-9 in kidney of diabetic rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9(MMP-9), transforming growth factor beta(1)(TGF-beta(1)) and IV-collagen (C-IV) in kidneys of diabetic rats.</p><p><b>METHODS</b>Rat diabetic model was induced by streptozotocin (70 mg/kg), and kidneys were examined pathologically and the expressions of MMP- 2, MMP-9, TGF-beta(1) and C-IV were studied by immunohistochemistry. The results were analyzed by imaging quantitative analysis technique.</p><p><b>RESULT</b>Immunoreactive MMP-2 and MMP-9 were mainly expressed in the mesangial cells, endothelial cells, parietal layer of Bowman's capsule and tubular cells. The expression of MMP-2 was significantly weaker in the glomeluri of diabetic rats than that of the control animals (P<0.05), while the expression of TGF-beta(1) and C-IV in the glomeluri of diabetic rats was significantly stronger than that of the controls (P%lt;0.05). The expression of MMP-9 didn't show significant different in glomeluri of the two groups (P>0.05).</p><p><b>CONCLUSION</b>Expression of MMP-2 in glomeluri is decreased in diabetic rats, which may be related to the increased TGF-beta(1) and in turns promote the accumulation of C-IV.</p>

Effects of pioglitazone on expressions of matrix metalloproteinases 2 and 9 in kidneys of diabetic rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The changes in matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions were examined in the kidneys of diabetic rats to investigate the degradative pathway of collagen type IV (C-IV) and the protective effects of pioglitazone on an experimental model of diabetic nephropathy.</p><p><b>METHODS</b>In 54 SD rats used in our study, 18 served as normal controls. Diabetes mellitus was induced in 36 age- and weight-matched rats by intraperitoneal injection of streptozotocin (70 mg/kg); 18 of the diabetic rats were allocated at random to receive pioglitazone [20 mg.kg(-1).d(-1)] in their drinking water and 18 served as diabetic controls. Rats were killed after 2, 4, or 8 weeks of treatment. Kidneys were examined pathomorphologically and the expressions of MMP-2, MMP-9, and C-IV were analyzed by immunohistochemistry, and the results were quantified by image analysis techniques.</p><p><b>RESULTS</b>Diabetes mellitus was associated with a decrease in the expression of MMP-2 in the glomeruli (P < 0.05, vs control). By contrast, MMP-2 expression in the interstitium increased, but not significantly (P > 0.05, vs control). The expression of MMP-9 did not show any change when comparing the three groups (P > 0.05, vs control). STZ-diabetic rats were also associated with an increase in the expression of C-IV in the glomeruli and the interstitium (P < 0.05, vs control). All diabetes-associated changes in MMP-2 expression were attenuated by pioglitazone treatment in association with reduced C-IV accumulation.</p><p><b>CONCLUSIONS</b>These results indicate that a decrease in MMP-2 expression in the glomeruli of diabetic rats may lead to impairment of C-IV degradation and contribute to the matrix accumulation in diabetic nephropathy. Pioglitazone treatment, which can attenuate the decrease of glomerular MMP-2 and the increase of C-IV degradation, has curative effects on diabetic nephropathy.</p>