The Effect Of P27kip1 Gene Transfection Mediated By Nanoparticles On Nanoparticles On Neointimal Proliferation And Apoptosis In Autogenous Vein Grafts

The stenosis of coronary artery and peripheral artery is a kind of serious disease caused by atherosclerosis. This disease threatens peoples life, make them amputated and mutilated. Due to improvement of people's living standard and change of life style, the incidence rate of arteriosclerosis has a tendency of growing year by year. Some people died because of myocardial infarction. Some need amputation because of limb necrosis. The effective therapeutic method is to apply all kinds of by - pass operation. But the long — term effect is not good because of restenosis. The research of mechanisms of restenosis and prevention and treatment for it become the key points to improve curative effect. At present , the autogenous vein graft's by - pass operation is still a chief therapeutic method for the stenosis of coronary artery and peripheral artery.After years of decades research we know that the Intima Hyperplasia (IH) and Vascular smooth muscle cell( VSMC) proliferation is a major cause of graft failure. Now we believed that the foundation of pathology of IH is SMCs migration from tunica media to intima. Matrix also takes an important role in this process. As the last gateway, Cell cycle pays an important role in controlling of cell proliferation and differentiation. In each period of cell cycle, many particular regulating factors interact to decide process of cell cycle. These regulating factors are divided into three species; Cyclin, Cyclin depended kinase (CDK) and Cyclin depended kinase inhabitor (CDKI). CDK is a kind of kinase that has phosphorylation function. This phoshorylation is regulated by activation of Cyclin. The two form a compound that has the ability to phosphorylate. EveryCyclin compounds CDK separately. These compounds make pRB phosphorylated and then pRB can release E2F, which combined together with pRB. E2F can integrate with promoter of CDK1 or Cyclin as a transcription factor. So transcription carries out and synthesis of some factors and DNA make cell cycle undertake smoothly. CKIs are negative regulators that affect the process of cell cycle and cells biological behavior. CKIs can restrain the activity of kinases by combined with Cyclin - CDKs. There are two major families of CKIs : InK4 family includes P16/INK4A, P15/INK4B, pl8/INK4C and pl9/INK4D, Cip/kip family, includes p21/cip, p27kipl, p57/kip2.As a member of Cip/kip family, p27kipl can refrain the activity of Cyclin and Cyclin - CDK, which cause inhibiting of the pRB phosphorylation, and then cell proliferation is blocked at G1 period. Meanwhile, p27kipl has another function that can promote the apoptosis of the cell. The transfection of p27kipl gene has been widely used in prevention of tumor cell proliferation recent years. But little is known about it in vein grafs. In our experiment we study the effect of p27kipl on SMC proliferation and apoptosis in vein grafs. Nanoparticle, composed of macromolecule polymer such as polylacticpolyglycolicacidco - polymer ( PLGA) , polylacticacid ( PLA) , is a new drug releasing carrier whose diameter is wthin 10 ~ 1000 nm. The drug could be packed inside the Nanoparticle or adsorbed on the surface or combined with Nanoparticle by chemical bond. Mechanism of drug releasing includes diffusing, unbinding, or degradation. The releasing time from several minutes to several months and the carrying drug include protein, As carrier for gene transfection, Nanoparticle can enhance the stability because of avoiding hydrolysis and send the target gene to the focus and bring it into the cell. The target gene can be expressed in local position. In our experiment , we construct the p27kipl gene mediated by PLGA Nanoparticle and trans-feet the gene to VSMC to observe the expression and biological behavior of p27kip1 gene. Our work includes three parts listed below.Part oneMethodsBiodegradable co - polymer of polylactic - polyglycolic acid ( PLGA) was used as matrix to prepare p27kip1 nanoparticles using an emulsification / solvent evaporation technique. This kind of nanoparticles was characterized. A Submi-cro Laser Defractometer (PCS Brookhaven Co. USA, BI - 9000 AT correlator, BI200SM photometer) was used to assess the size distribution, Package efficiency, release progress invitro. The particle morphology was observed by scanning electron microscopy (SEM). GFP, which packed by the same size of PLGA, is transiect into the autogenous grafted vein to observe the distribution of Nanoparticles in the tissue.ResultsThe size of the Nanoparticle (NP) p27kipl DNA complex is between 243 -343nm. The average size is 288. 9nm. Package efficiency is 60%. The content of DNA measured by photometer is 3% . Release progress invitro can last for more than 18d. GFP is observed in the intimal layer.ConclusionPLGA nanoparticle can packet the p27kip1 gene as a carrier. PLGA (300nm diameter) can diffuse into intimal layer of autogenous grafted veinPart TwoMethodsAutogenous vein graft model was established in 120 rats by transplanting in-ternal branch of jugular vein to carotid artery by end - to - end anastomosis. After anesthetizing with 10%chloral hydrate (2ml/kg) , Incision in cervical part, freeing jugular vein and common carotid artery, cutting jugular vein 0. 5cm, washing with heparin saline, using 11 - 0 no - injure suturing, end - to - end anastomosis jugular vein into common carotid artery. Three groups were studied: (1) p27 group; p27kipl gene mediated by NP were transfected into the veins before anastomosis; the veins were immerged in the nanoparticle suspension and incubated for 30 min at 37°C , (2) Control group: the vein were transfected by simple NP; (3) Grafting group; The veins were grafted with no transfection. The grafted veins were harvested at 3 d, 7d, 14d and 28d respect(?)ely after the operation. The exogenous p27kipl mRNA and protein expression in veins were determined by RT - PCR and Western blot. The β actin was used as a control in RT - PCR. Data are presented as mean SEM, The statistical significance of differences between the normal and treated groups was determined by T test.Result1. RT - PCR Result; The RT - PCR products detected in p27kipl group corresponding to an expected 273 bp product. The signal for TheBactin was also observed at 587 bp for positive control. There is no product occurs in both control group and simple grafting group. The expression of p27Kip1 mRNA was found to be equal at each time point.2. Western Blot Result;The expression of p27kip1 protein in p27kip1 group was detected at 3d strongly and last to 28d. For both simple grafting group and control group p27kipl protein occurs at 7d and reaches the peak at 14d and then decreased gradually.ConclusionP27kipl gene transfection mediated by NP complex enabled to produce the mRNA and protein expression of p27kipl gene. NP is an effective gene trans-fecting carrier.The regulation of p27kip1 protein expression maybe at the transcription level.Part ThreeMethodSmooth muscle cell of rats thoracic aorta is cultured were invitro. After i-dentified by α- actin Immunonistochemistry, the cells are transfected by p27kip1 — NP and blank - NP respectively. The no transfected are treated as Control. 48hs later, the proliferation of the SMC1 is determined by MTT method and the apoptosis is detected by flowcytometry. Autogenous vein graft model was established in 96 rats. The groups, the process of transplanting and transfection-an and time point of harvesting are the same as part two. Intimal Hyperplasia (IH) was observed by pathology. The expression of PCNA,E2F were detected by Imrnunohistochemistry and the presence of apoptotic VSMC was demonstrated by TUNEL method. Analyzed by computer digitizing system.ResultThe result of MTT shows that the proliferation of SMC after transfecton by p27kipl - NP was inhibited by 18.6% compared with Control group. 68.4% of the SMC is restricted in G, and the ratio of apoptosis up to 15. 38%. While they are 1. 09% ,1.79% respectively for Control group and blank NP group.IH of the grafted vein respectively for 7d ~28d in P27 group was inhibited significantly (p <0.01). Immunohistochemical analysis of PCNA indicated decreased positive cell in the p27 group compared with the control group at 7d ~ 28d (p <0.01). The expression of E2F was decreased in p27 group at 7d -14d (p <0. 01). There is no significant difference between Control group and Grafting group in expression of E2F and PCNA. Apoptosis of VSMC in the p27...