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AIM: To explore the effect of Snail1 siRNA on high-glucose induced tubular epithelial-to-mesenchymal transition (TEMT). METHODS: Subconfluent renal tubular epithelial cells were incubated in serum-free DMEM for 24 h to arrest and synchronize the cell growth. Then cells were treated with normal glucose (5.5 mmol/L D-glucose) or high glucose (25 mmol/L D-glucose) for 72 h. Meanwhile 19.5 mmol/L D-manntiol was used as high osmotic control. Snail1 siRNA was transfected into tubular epithelial cells. In parallel, cells were transfected with non-specific siRNA which served as the control data sets. Cells were then treated with 25 mmol/L D-glucose for 72 h. RNA and cell lysates were collected to determine the protein and mRNA levels of Snail1, TGF-β_1, α-SMA, vimentin and E-cadherin. RESULTS: Transfection caused the decreases in Snail1 at mRNA and protein levels by 62% and 68% respectively as compared to those in untransfected cells cultured in high glucose medium. Western blotting exhibited that Snail1 siRNA transfection restored E-cadherin protein expression by 61% compared to that in high-glucose-treatment cells, whereas it inhibited high-glucose-induced induction of α-SMA protein by 58%. Similarly, RT-PCR revealed that Snail1 siRNA transfection dramatically suppressed the high-glucose-induced mRNA expressions of α-SMA and vimentin by 72% and 61%, respectively, while E-cadherin mRNA increased by 53%. CONCLUSION: Our study provides direct evidence that Snail1 is able to control TEMT.
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AIM: To observe the changes in infiltrating macrophages (M?)and extracellular matrix (ECM) in the kidney in the progressive course of nephrotoxic nephritis (NTN). METHODS: NTN model was established with rabbit-anti-rat nephrotoxic serum. On day 3, 7, 15, 30 and 90, renal biopsies were performed. Renal histology was checked under light microscopy with HE and Masson's trichrome staining sections. M?, fibronectin (FN), type Ⅲ and Ⅳ collagen were examined with immunohistochemistry ABC method. RESULTS: Infiltration of M? appeared on day 3 of NTN and preceded changes of FN and collagen. On day 15 of NTN, all nephritic animals had significant proteinuria, increased serum creatinine, infiltration of M? and deposit of entracellular matrix. On day 90 of NTN, seven nephritic animals improved significantly, while other five developed renal scarring with diffuse infiltration of M? which positively paralleled to renal function and deposit of FN, type Ⅲ and Ⅳ collagen. CONCLUSION: M? infiltrating into renal tissue enhances deposit of ECM and therefore plays important roles in progression or improvement of NTN.
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Objective:To investigate the purified methods of human anti-D antibody from IgG contained anti-D. Methods:The IgG was separated by the column ion-exchange chromatography(CIEC) from the plasma in which the content of anti-D was 0.814 ?g/ml. Then the IgG preparation contained anti-D was purified by the affinity chromatography(AC) with the O group, RhD positive red blood cell (genotype CCDee). Results:The content of non anti-D IgG were reduced about 90% by the method of AC and the proportion of anti-D could be significantly increased in the final preperation. The quality of final preparation attained reqirements of national standard of biologics. Conclusion:This method is able to purify anti-D from IgG contained anti-D and offer a reference for plasma products.
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AIM: To observe the expression of transforming growth factor ? 1 (TGF-? 1), MAPK 1/3 and fibronectin (FN) in the development of renal tubulointerstitial disease. METHODS: Wistar male rats were randomly divided into normal control group, diabetic group of 1week, 2 weeks, 4 weeks and 8 weeks. Diabetic model was induced by peritoneal injection of streptozotocin. Immunohistochemistry was employed to detect the expression of TGF-? 1, MAPK 1/3 and FN in the kidney. TGF-? 1 protein in the renal cortex was checked by Western blot. BG, Scr and UP were analysed by biochemical methods, and the morphological changes in renal tubulointerstitium were also examined under microscopy on sections stained with HE and PAS. RESULTS: The expression of MAPK 1/3 and FN was observed, but not the expression of TGF-? 1 in normal renal tissue. Positive staining of TGF-? 1 was observed in the renal tubulo-interstitium in 1-week diabetic group and thereafter it increased in the course of diabetes. A continuous increase in the expression of MAPK 1/3 and FN was also observed in two - week diabetic rats. Chronologically the expression of TGF-? 1,MAPK 1/3 and FN and the ratio of KW/BW were positively correlative with each other in diabetic animals except one -week diabetic rats. There was also a positive correlation between MAPK 1/3 and FN in l -week diabetic rats. CONCLUSION: Our data suggest that TGF-? 1 appears in the renal tubulointerstitium in early period of diabetes and then its signal is mediated by MAPK 1/3 cascades to accelerate production of FN ,and in turn leads to renal hypertrophy and tubulointerstitial fibrosis. [
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AIM: To observe the dynamic changes of expression of PKC? , TGF-? 1 and ?-SMA in glomeruli of diabetic rats induced by the alloxon and to invesitigate their roles in the diabetic nephropathy(DN). METHODS: Rats were randomly divided into four groups: normal control group (group A), diabetic group of one week (group B), diabetic group of one month (group C), diabetic group of two months (group D). Immunohistochemistry and Western blotting were used to detect the expression of PKC?, TGF-? 1 and ?-SMA in renal tissue of all groups. Blood glucose, triglycerides, cholesterol, creatinine and urine protein were analysed by chemical methods. The morphological changes of renal tissue were checked through microscopy. RESULTS: The expression of PKC? and TGF-? 1 in renal tissue of diabetic groups were increased comparing with those of nomal control group( P