| Introduction:At present, the morbidity of atherosclerotic obliterans is increasing. The main treatment is bypass operation using autogenous vein grafts. But it often results in stenosis and occlusion due to the migration and proliferation of vascular smooth muscle cells (VSMCs) and excessive deposition of extracellular matrix (ECM). Schwartz found that VSMC occupied 11 % of volume in human stenostic vascular tissue, with the left ECM. That means ECM plays a more important role in vascular stenosis.Transforming growth factor - β1 (TGF - β1) as a pleiotropic growth factor is one of the key factors that modulate metabolism of ECM in tissues and organs, TGF - β1 signals combining with its type I receptor (TGFβ - R I ). Our study explores the expression of TGF - β1 and TGFβ - R I using the autogenous vein grafting model in Wistar rats. We also explore the expression of the main degrading enzyme of vascular ECM, matrix metalloproteinase - 1 ( MMP -1) , and its inhibitor, tissue inhibitor of Metalloproteinase - 1 (TIMP - 1) , and their relations with vascular stenosis.Nowadays, there is no effective pharmaceutical treatment to stenosis of vein grafts. Gene therapy offers a new road. The key points of gene therapy are the selects of specific gene and carriers. Our study transfected antisense gene of TGF - β1 carried by silicon nanoparticles to the wall of grafted veins locally and watched their roles in vascular stenosis in order to explore a new way to prevent and treat vascular stenosis.Materials1. Main reagents: pMAMneo -Anti TGF - β1 plasmids, silicon nanoparti-cles, monoclone antibodies of TGF - β1. TGFβ - R I , MMP - 1 and TIMP - 1, SABC immunohistochemical kits, RT - PCR reacting kit.2. Main Instruments: SXB - 1 operating microscope, microsurgical instruments, computer image analysis system, PTC - 100TM type DNA expander, JSM -T300 scan electron microscope, et al.Methods1. Construction of animal model: Male Wistar rats weighing 250 ~ 300g were anesthetized. Under the operating microscope, the right internal jugular vein,4 ~5mm long, was transplanted to the ipsilateral carotid artery with end -to - end anastomosis. The method of local transfection is that the distal end of the artery was anastomosed to the vein, and a gene catheter was induced to the proximal end of the vein. Then the same volume of gene carried by nanoparti-cles, or only the nanoparticles, or natrichloride solution was irrigated to the vein with the ligation at both ends. Keep the pressure for 30 minutes, suck out the fluid, and spread the fluid to the external membrane of the grafted vein. At last the proximal end of vein was anastomosed to the artery, and the incision was closed.2. The construction of antisense TGF — β1 carried by nanoparticles: Cyclo-hexane, deionized water, and ethyl - silicon were mixed, and aqueous ammonia was added. After the mixture was reacted for 24 hours at room temperature, it was rinsed with acetone and dried in vacuum. Nanoparticles weighing 0. 09g was dissolved in 6 ml deionized water, sonicated and dispersed for 2 hours, then autoclaved. 50 ml nanoparticles mixed with 4 mg antisense TGF - β1 gene and 1 mol/l NaCl solution were reacted for 15 minutes at room temperature for future use.Indexes examined: ①The thickness of intima and vascular wall was meas-ured using HE and Verhoffe staining and computer analysis system. ②The expression of TGF - β1 and TGFβ - R I was examined by immunohistochemical method. ③The protein expression of TGF - β1, MMP - 1, and TIMP - 1 was examined by Western Blotting. ④Their mRNA expression was examined by RT - PCR. ⑤The morphology and size of nanoparticles were measured by scanning electron microscope.Results1. Intimal thickness was not increased remarkably on Day 3 after transplantation. But on Day 7, intima thickened significantly , containing abundant VSMCs embedded in ECM, compared with controls, t = 10.218, P<0.05. Intimal thickness reached a peak on Day 14 with elastic fibers ruptured at the site of intimal hyperplasia. Two weeks after gene transfection , intima of vein grafts thickened significantly, with the results of(47. 3 ±6. 4) μm and(44. 2 ±8. 1) μm. The intimal hyperplasia of nanoparticles - DNA group was inhibited , with the result of(20. 5 ±4.7) μm,P <0.01 compared with controls.2. Immunohistochemistry analysis showed TGF — β1 protein expression of grafted veins on Day 3,7, and 14 were significantly higher than controls (P < 0. 05). The positive expression of TGFp - R I was remarkably increased on Day 7 after operation, peaked on Day 14, and decreased afterwards.3. Western blotting showed that the expression of TGF - β1 increased significantly on Day 3, peaked on Day 7, and returned to the baseline on Day 28. There were no significant differences in MMP - 1 expression among Day 3 group, Day 7 group and controls. But the protein expression on Day 14 and 28 were significantly decreased. The protein expression of TIMP - 1 was significantly increased on Day 14 and 28. TGF - β1 protein expression of gene transfection group was significantly lower than controls.4. By means of RT - PCR, the mRNA expression of MMP - 1 ,TIMP - 1, and TGF - β1 has the similar results with Western blotting. The expression of TGF - β1 mRNA of gene transfection group was significantly lower than controls.5. Watched by scanning electron microscope, the diameter of nanoparticles is about 50 ~ 100nrn, which was uniformed and spherical.DiscussionAutogenous saphenous vein is often used in lower extremity circulation bypass grafting operations, but stenosis, even occlusion of the grafted veins often occurs after vein transplantation. In human restenosis specimens, VSMCs accounted for only about 11 % of neointimal volume, with the remaining volume of ECM. So to say that ECM plays a more important role in the vascular stenosis.TGF - β1 as a pleiotropic growth factor, is a principal determinant in regulating ECM metabolism. Our research demonstrated that TGF - β1 increased on Day 3 after operation, and peaked on Day 7. As shown above, intimal hyper-plasia occurred on Day 7 and Day 14. It means that TGF - β1 may have relations with intimal hyperplasia.TGF - β1 combining with its type I and type Ⅱ receptor acted on Smad protein to transduce the extracellar signals to nuclear. Our research found that the expression of TGFβ - R I increased significantly on Day 7, and peaked on Day 14. It' s later than TGF - β1. That probably means that the overexpression of TGF - β1 may induce the expression of its type I receptor.TGF - β1 plays an important role in many fibrotic diseases such as nephritic and hepatic fibrosis through regulating the balance between MMPs and TIMPs. Our study in the field of vascular grafting demonstrated that MMP - 1 expression decreased significantly on Day 14 and Day 28, but TIMP -1 expression increased in the same period, so the balance between them was broken. The degradation of ECM was more than synthesis that incurred excessive ECM accumulation and intimal hyperplasia. As shown above, TGF - β1 overexpression occurred earlier than MMP - 1 expression shortage and TIMP - 1 overexpression, that means TGF - β1 may result in ECM deposition through destroying the balance between MMP - 1 and TIMP - 1.With the advancement of nanotechnology, nanoparticle, as a novel gene carrier, has been used widely, because it is able to pass through the tissues eas-...
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