| ObjectiveIntimal hyperplasia (IH) , an event that produces a decrease in the area of the vessel lumen of autologous vein grafts, is a major obstacle to long - term patency of many types of vascular reconstruction. IH, characterized by exuberant smooth muscle cell (SMC) proliferation and both extracellular matrix synthesis and accumulation, is the most common pathological process leading to graft failure. Overall, 20 -40% of grafts by 1 -2 years have occluded, and up to 40 -60% of vein grafts become stenotic by 10 years.Researchers and doctors have seeked the treatment of intimal hyperplasia in the past years, however, the precise mechanism of the disease is still unclear. There is a hopeful way with the development of molecular biology, gene therapy. With the method of molecular biology, some special genes can be transferred into target tissue or organ, repair the gene which lost normal function or depress the over express of some genes.Cytosine deaminase ( CD) is one of the promising suicide gene therapeutic strategies. CD is an enzyme found in bacteria and fungi but not in mammalian cells. Insertion and expression of the CD gene in target cells by means of an efficient gene transferring system, can help to catalyze the non - toxic prodrug , 5 - fluorocytosine (5 - FC) , a widely used fungicide , into the highly toxic agent 5 -fluorouracil (5 -FU) , which has the ability to block DNA synthesis.This method has been studied to therapy tumor or restenosis of injuryed artery. The purpose of this study was to investigate the anti - metastatic effect and whether it could repress the intimal hyperplasia of vein graft with in situ trans-duction of adenovirus encoding cytosine deaminase ( AdCD) followed by the systemic use of 5 - FC in a rat model.MethodsExperiment I : This study was firstly to investigate the efficiency and time curve of expression of marker gene ( LacZ) in a rat vein graft model in vivo. Adenovirus vector carried LacZ gene dwelled in vein graft for 30min, For improving the transfer gene efficiency, physiological pressure (100mm Hg) were used in situ before the vein graft was transplanted into carotid artery. On the 3 day, 1 week, 2 week and 4 week after vein transplant, the samples were collected and X - gal stain on the grafts, positive of X - gal cells were counted with microscope. Some organs, heart, brain, liver, lung, spleen, kidney et al were stained with X - gal and were observed.Experiment II : In situ transduction of the CD gene, followed by systemic use of 5 - FC at a daily dosage of 500 mg/ kg for 10 days, was performed in the rat model. Samples were collected at the different time point same as Experiment . Half of the vein grafts were analyzed with HE, IHC and in situ hybridization; other half were analyzed with RT - PCR or FCM.Experiment HI : Digestion with protein enzyme, collect the cells and analyzed with FCM to investigate the role of apoptosis. Tissue slides were stain with TUNEL to research the apoptosis cell in situ.ResultsOn the normal vein and control vein grafts, negative X - gal was detected. On 3 day post - operation, both low and high PFU groups were stained with X -gal, and no significant difference between the two groups (34.6% ±5. 6% VS. 37. 2% ± 5. 8% , P > 0. 05 ). From 1 week to 2 weeks, the X - gal positive cells decreases gradually. There is no X - gal stain on the 4 weeks in low PFU group, but positive in high group. More import, X - gal was detected at kidney in high PFU group. |
PDF Full Text Request