动物实验,糖尿病,PTA z-bo 2020-09-03 410

　　Background: Percutaneous transluminal angioplasty (PTA) is widely used for diabetic peripheral vascular disease (PVD) due to its high initial success rate. However, as many as 70% of patients develop restenosis within a year, which limits this clinical application. Attempts to prevent or reduce restenosis through antiplatelet drugs, glucocorticoids and calcium channel blockers can all lead to adverse drug reactions. The lack of a true restenosis model limits the possibility of investigating potential therapies. The installation of angioplasty stenosis animal models has been used to study pharmacological and mechanical methods. In most studies, the abuse of conventional arterial angioplasty balloons to replicate the vascular response of PTA people (single injury model) is to establish an atherosclerosis model rather than a restenosis model. Therefore, it is very important to establish a reliable animal model to study potential treatments to prevent PTA restenosis in patients with PVD. Statins are hydroxymethylglutaryl-coenzyme A (HMG-CoA)-(CoA) reductase inhibitors with anti-inflammatory and anti-proliferative effects. Previous studies have shown that statins can reduce inflammation and intimal hyperplasia in animal models of balloon injury. For these reasons, it is clinically used as a routine treatment for PTA patients to control the development of restenosis. However, in our daily work, we found that the clinical effect of statins in inhibiting restenosis is secondary. In this experiment, a double injury restenosis model of New Zealand white rabbits was established to simulate the proliferation process of restenosis after PTA hyperglycemia. Atorvastatin is the most widely used statin. Therefore, in this study, atorvastatin was used to test the inhibitory effect of statins on restenosis. This animal model is used to study the potential treatment of restenosis after PVD treatment in diabetic patients or to understand the mechanism of restenosis itself.

　　Method: Animal: New Zealand male white rabbit (1-7.7 kg). The rabbits were individually housed in standard rabbit cages and stored in a 12:12 light:dark cycle. One week before the start of the experiment, an atherogenic diet (cholesterol 1%) was given, and the animals were sacrificed after feeding. During the study, all rabbits can eat freely. Test protocol: Induction of experimental diabetes: After fasting overnight, a freshly prepared 80 mg/kg alloxan solution was injected through the rabbit's ear vein. After the injection of alloxan, animals have free access to food and water. After confirming hypoglycemia, take a 20% glucose solution orally to prevent hypoglycemic shock. The blood glucose level was measured one week after the alloxan injection, and animals with blood glucose levels higher than 300 mg/dl were considered as diabetic and used for testing. Double injury surgery: (1) Balloon-induced endothelial injury surgery causes atherosclerotic plaque formation. The rabbit was anesthetized with phenobarbital for the first week after the induction of diabetes. Use standard surgical procedures to properly dissect the great saphenous artery, and insert a 2.5mm wire guiding balloon catheter to damage the artery as described above. Ultrasound confirmed that about 4 weeks after the first surgery, severe atherosclerosis damaged the artery. (2) Next, PTA causes restenosis plaques. After dissecting the distal end of the right femoral artery, insert a balloon catheter into the damaged internal artery, and perform PTA at the initial damaged site. After the above processing, color Doppler shows shown arteriosclerosis. The restenosis model caused by successful double injury surgery was randomly divided into 4 groups: 7-day restenosis group (1 group, n = 6) 14-day restenosis group (2 groups, = 6); 28-day restenosis group (3 Group, n=6) and 28-day atorvastatin treatment group (group A, n=6). The dose of atorvastatin (2.5 mg/kg/d). Oral gavage of atorvastatin was started on the day of double injury surgery. Except that the balloon was not inserted, six sham-operated diabetic rabbits underwent the same operation. Obtain histological and morphological analysis: animals (A and C animals on day 28) were sacrificed at each time point (7, 14, 28 days after double injury surgery). Collect ear blood samples to detect serum total cholesterol, triglycerides, low-density lipoprotein and high-density lipoprotein cholesterol levels. The damaged arterial segment was obtained, fixed with 4% formaldehyde, embedded in paraffin, and cut into 5 micron sections. The gross morphology was observed by HE staining, and Masson's trichrome staining was performed for collagen and smooth muscle cells (SMC). Image analysis software is used for morphological analysis of intimal growth. Intimal growth is estimated by the ratio of the area of the intima to the inner elastic layer (the cross-sectional area of the lumen becomes narrower) and the ratio of the area of the intima to the interior.

　　Immunohistochemical analysis: paraffin tissue sections, deparaffinized, gradient ethanol hydration. EDTA buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween20, pH 9.0) in the microwave was used for 20 minutes of heat-induced antigen recovery. After cooling, the sections were incubated with 3% hydrogen peroxide. After blocking with PBS containing 5% goat serum, the sections were incubated with primary antibody overnight at 4°C. After washing with PBS, the sections were incubated with the secondary antibody at 37°C for 30 minutes. Through the peroxidase substrate solution, PBS containing 0.02% (w/v) H2O2 and 0.1% (w/v) 3,3-diaminobenzidine hydrochloride can see a positive reaction. Hematoxylin counterstaining. For negative control, use mouse IgG antibody instead of primary antibody. Cell proliferation was measured using anti-proliferating cell nuclear antigen (PCNA) antibody (1:400 dilution). Count PCNA positive cells. The PCNA index is calculated according to the following formula: PCNA positive cells/total cells x 100%.

　　Immunofluorescence staining: immunofluorescence staining. In short, 3% hydrogen peroxide incubation will inhibit endogenous peroxidase activity, and block with 5% (v/v) goat serum for 1 hour. After washing with PBS, the sections were incubated with FITC-labeled smooth muscle actin antibody (αSMA) (1:100) for 4 hours. After the second wash, prepare slices with dapi-fluormount-g. Examine the specimen with a fluorescence microscope. Results: Animals: A total of 45 New Zealand white rabbits were used at the beginning of the experiment. After the injection of alloxan, blood sugar increased by 30 due to insufficient blood sugar, and fatal deaths in 5 and 10 were excluded. Of the 30 diabetic rabbits, 24 underwent double injury surgery and all achieved restenosis. Measure the weight and blood sugar level of diabetic rabbits. Throughout the study, blood glucose levels were maintained above 300 mg/dl. On the 28th day, the total serum cholesterol of group C was 18.75±0.44 mmol/L, group 3 was 18.47±0.54 mmol/L, and group A was 9.44±0.27 mmol/L. There was no significant difference in serum total cholesterol between groups c and 3, while the difference between groups a and 3 was statistically significant. Serum low-density lipoprotein cholesterol levels were also similar (10.2±0.35 mmol/L in group C, 10±0.34 mmol/L in group 3, and 4.60±0.18 mmol/L in group A). Serum triglyceride levels (group C 2.01±0.04 mmol/L, group 3 2.15±0.05 mmol/L, group A 1.36±0.05 mmol/L). There was no significant difference in serum HDL cholesterol levels between the two groups. Histopathological study and time course of dual injury model: Intimal thickening after primary injury involves SMC embedded in stromal cells after PTA as a matrix. After double injury, there are obvious signs of trauma, including extensive fracture of the internal elastic layer and dissection of the inner membrane into the matrix. In the restenosis schedule, 18 rabbits were used to observe the restenosis process. The results showed that compared with the rabbit arteries in group C, the c intima gradually increased during double injury surgery. The stenosis rates on the 7, 14 and 28 days were 21±5.85%, 60.93±12.46% and 90.02±3.11%, respectively. Similarly, the membrane/medium ratios on the 7, 14 and 28 days were 0.35±0.05, 1.63±0.29 and 1.86±0.14, respectively. The maximum expression of PCNA appeared on the 7th day and then gradually decreased. Immunohistochemistry of α-actin (labeled as smooth muscle cells) showed that at each time point, the inner membrane was almost completely surrounded by smooth muscle cells. The effect of atorvastatin on stenosis rate and intimal/medial area in the dual injury model: 28 days of atorvastatin treatment did not reduce the stenosis rate of bilateral ilia arteries in rabbits. It matches the stenosis rate. In the three medial groups, the intima/media ratio increased significantly, and atorvastatin treatment did not reduce the increase in the intima/media ratio in group A. The effect of atorvastatin on cell proliferation and SMC migration: PCNA and Masson trichrome staining and αSMA immunofluorescence were performed for cell proliferation to further clarify the effect of atorvastatin on restenosis in the dual injury model. It is possible to evaluate the migration of neointimal smooth muscle cells. The PCNA index increased significantly after arterial injury in the three groups. The PCNA index did not change after atorvastatin treatment, nor did it change compared with group a. The results of α-actin immunohistochemistry showed that there were a large number of smooth muscle cells in the inner membrane of the atorvastatin treatment group (group A) and the non-statin treatment group (group 3), and there was no significant difference between the two groups. Being displayed. Conclusion: In conclusion, it is possible to repeatedly develop animal peripheral arterial restenosis models that mimic human restenosis after PTA. This model will help to study the mechanism of restenosis and treatment intervention plans. It was found that atorvastatin has no therapeutic effect in suppressing restenosis. Further research is needed to find more effective treatments.