| Diabetic retinopathy (DR) is the most common and serious microvascular complications in diabetes, and is one of the four main causes of blindness. So clarifying the pathogenesis mechanisms of DR and questing for valid prevention and cure method are very important to people. The microvascular are mainly composed of endothelial cells and pericytes. The selective loss of retinal microvascular pericytes is the earliest histopathological hallmark in DR. Advanced glycation end products (AGE) induced by the long-term chronic and high glucose, is the main causes of the loss of pericyte in DR. Transforming growth factor-β (TGF- β) and oxidative stress participate in the pathogenesis of AGE. Moreover antioxidant (Ginkgo biloba L.) has certain prevention and therapeutical effects on the diabetic microvascular complications. Therefore, investigating the pathogenesis of AGE on pericytes and studying the protection of Ginkgo biloba L. on DR are very important. This experiment we adopted the method of filtering through two sieves and removing contamination to establish primary culture of bovine retinal microvascular pericytes. Then we applied the method of MTT, flow eytophotometer and immunofluorescence stain techniques to investigate the effects of AGE and Ginkgo biloba L. joined with AGE on proliferation, cell cycle, apoptotic cell numbers and TGF-β expression in cultured pericytes. In vivo animal experiment, we adopted STZ at small amount for many times to establish 1 type diabetes rat model, then we used retinal stretched preparation techniques to investigate the loss and TGF- β expression in pericytes of normal, DM and Ginkgo biloba L. group. Our results are as follows: 1. According to the results of phase contrast microscope, immunocytochemical stain and transmission electron microscopy techniques, the purity of pericytes were more than 95%. Pericytes had the characteristic of pleomorphism, growth slowing, cell process, overlapping row for non-contact inhibition, α-SMA antibody staining positive, and Ⅷ factor correlated antigen antibody staining negative. Its nucleus was big, and had several nucleoli by electron microscope. Besides pericytes had microvillus, rough endoplasmic reticulum, mitochondrion, Golgi complex, free ribosome, microfilament, and without Ⅷ factor corpuscles. 2. When pericytes were cultured with AGE for 24 h, pericytes growth was significantly retarded in a dose-dependent manner (P<0.01). And pericytes cultured with AGE , which S period cell numbers(38.49±1.65%)increased, G2-M period(0.90±0.44%)decreased and apoptotic cell numbers(13.73±1.80%)increased(P<0.01).Moreover TGF-β expression was strong positive in 100ug/mlAGE group by immunofluorescence stain techniques. 3. Ginkgo biloba L. promoted pericytes proliferation in a dose-dependent manner cultured with 100μg/ml AGE for 24h. Ginkgo biloba L. retarded the effects of AGE on cell cycle, reduced apoptotic cell numbers (P<0.01), and decreased TGF-β expression. 4. The blood sugar and serum AGE level of DM rat were significant higher than control (P<0.01). Observed from retinal stretched preparation, the capillary was distorted, irregular, caliber discrepancy, and capillary had emphraxis formation, furthermore pericytes quantity reduced obviously (P<0.01).TGF-β was overexpression in diabetic pericytes(P<0.01), and serum AGE level was associated with pericytes loss(P<0.01).5,After treatment of Ginkgo biloba L., capillary was even, had no significant distort and expansion, and caliber was well-distributed, moreover capillary emphraxis was not observed. The pericytes loss was lower than DM , but had no obvious statistical significance(P>0.05).Ginkgo biloba L. decreased the TGF- β expression of pericytes in DM (P<0.01). These results suggested that AGE inhibit proliferation, block cell cycle at S period, and promote TGF- β expression of pericytes in vitro. However Ginkgo biloba L. inhibited the above-mentioned effects. The loss of retinal pericytes was not only associated with AGE, but also possibl... |
PDF Full Text Request