| Objective:To investigate the role of TGF-β and its downstream signal Smad in the development of diabetic retinopathy. Through the research of diabetic retinal tissue of rats with TGF beta, Smad2/3, and the protein alpha Sam, MMP-2, PAI-1, COL-1, VEGF and CTGF expression changes, revealing the mechanism of diabetic retinopathy of TGF beta/Smad signaling pathway, from the molecular level to improve diabetes pathogenesis of retinopathy of feasible gene therapy or drug treatment.Methods:1. In vivo experiment (1) animal model establishment: male SD rats, weighing 200±25g, STZ (the product of sigma company, pH was 4.0, dose of 55mg /kg, dissolved in 0.1 mmol/L sodium citrate and 0.2 mmol/L sodium dihydrogen phosphate) a large one-off intraperitoneal injection, in 48h after collect tail blood to measure glucose late every 2 weeks were measured blood glucose and body weight, blood glucose concentration> 16.7 mmol/L for diabetic rats. Body weight and blood glucose were measured at fourth,8,12 weeks after the experimental animals. (2) RT-PCR, blotting Western was used to determine the, a-SMA, MMP-2, p-Smad2/3, PAI-1, Col-1, VEGF, CTGF, TGF-β and RNA, and the dynamic changes of the retina in the retina.2. in vitro experiment:(1) rat retinal pigment epithelial cells, retinal capillary endothelial cells and Muller cells were cultured in vitro. The third generation cells were identified as the experimental subjects. (2) TGF beta stimulation test:the three kinds of cells to 1 x 104 inoculation in 35 mm Petri dish,12 hours after attaching to the wall, discard supernatant, each dish added serum-free RPMI 1640, were added to the 1,10,50 ng/ml TGF beta 1 were cultured for 24h,48H and 72h. (3) the effect of TGF- beta on the expression of TGF- beta/Smad signaling pathway in rat retinal capillary endothelial cells, retinal pigment epithelial cells and Muller cells. Real-time RNA Quantitative was used to detect the RNA expression level of the related factors, and the protein expression level of the above factors was detected by semi quantitative blotting western.Results:1.the rat model with type 1 diabetes mellitus was successfully prepared and maintained a high blood glucose environment in vivo for several weeks, that is, the model of DR was 84.4% (P<0.05).2. RF/6A cells and ARPE-19 cells were treated with different concentrations of sugar, and most of the factors were expressed at the highest level in 25mM sugar treatment. Muller cells were treated with different concentrations of sugar, and the factors were expressed at higher concentration.3.after 25mM treatment at different time points, RF/6A cells and ARPE-19 cells Q-PCR detection showed that all the factors were in the 25mM sugar treatment 24h, the highest expression level. And the expression levels of Smad3 and TGF-P Q-PCR detection of Muller cells in a longer time to express higher.4.different concentration of TGF-β treatment, RF/6A cell Q-PCR detection can be seen in the treatment of 5ngTGF- beta, the factors have reached the first peak. And at different concentrations of TGF-β treatment, Smad3 and TGF-P expression levels also showed a similar trend. Q-PCR and blot Western were detected in Muller cells treated with different concentrations of TGF-β,5ng TGF were found to be the first peak in the treatment of the expression of VEGF, CTGF, COL-1, TGF-β. And a-SMA, PAI, Smad2, MMP2 are in the 50ng TGF beta treatment to reach the peak.5.1-3 month diabetic rats, the expression changes of various factors in the retina, the expression levels of COL-1, a-SMA and Smad3 were inversely proportional to the time, while the expression levels of TGF-β and Smad2 were proportional to the time.Conclusion:1. According to the dose of 55mg/kg streptozotocin (STZ dissolved in O.1mol/l citrate buffer, pH 4.2) a large dose to rats by intraperitoneal injection, given normal diet can be successfully prepared with type 1 diabetes rat model, and maintain a number of weeks in vivo high blood glucose caused by diabetic retinopathy, namely Dr animal model. The molding rate is 84.4% (P<0.05).2.after treatment with different concentrations of sugar, it was found that in vitro RF/6A cells and ARPE-19 cells were more sensitive to the treatment of 25mM, while Muller cells were less sensitive to high glucose.3. after different time of 25mM treatment, RF/6A cells and ARPE-19 cells had the highest expression level of each factor when 25mM glucose was treated with 24h. And the expression level of Smad3 and TGF-β Muller cells were more tolerant to high glucose, and the factors of 48h and 72h reached the maximum expression.4.after treatment with different concentrations of TGF, and the factors of RF/6A cells were treated with 5ng TGF-β, all the factors reached the first peak. And the expression level of Smad3 and TGF-β beta in different concentration of TGF-βtreatment also showed similar trend. It was further confirmed that the expression levels of Smad3 in RF/6A and Muller cells, which are the downstream signaling factors of TGF- beta, were in a dose dependent manner in TGF-β5. after the establishment of a stable 1-3 month rat model, the expression levels of COL-1, a-SMA and Smad3 were found to be inversely proportional to the time, while the expression level of TGF-β and Smad2 was proportional to the time. This study further demonstrated the role of TGF- beta/Smad pathway in the development of diabetic retinopathy. |
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