Research Of Reparation And Mechanism Of Lentivirus Gene Carrying BFGF For Rabbit Internal Carotid Artery Aneurysm Wall

Background Intracranial aneurysm is the main reason of spontaneous subarachnoid hemorrhage, accounting for 25% of intracranial hemorrhage, which causes high mortality and morbidity. Surgical clipping of aneurysms is the main method of treating the clinical aneurysms. Interventional treatment of intracranial aneurysms has also developed rapidly in the past ten years, but some complex aneurysms cannot be treated by surgery or surgical clipping. So the research for a conservative treatment to prevent or delay the rupture has practical significance.Reseach about changes on the vessel wall and inflammatory cells, extracellular matrix enzyme involved in the process of aneurysm formation gets deeper in recent years. Aneurysm formation may be precisely because of arterial blood flow shear stress increasing in the the bifurcation or the bending part ,which leads to the corresponding changes of endothelial cells in the structure and function, with a number of local factors synthesized and released.This leads to the biological changes in the arterial wall, including endothelial degeneration and lack of cells, smooth muscle cell contraction or apoptosis, which leads to the formation of aneurysms.Although aneurysm formation, growth and rupture is a continuous process, but it may be divided into two stages: the formative stages and the growth-rupture stage. The former factor is the blood flow shear stress. The reseach core about vascular biology mechanism is endothelial cells and smooth muscle cells, focusing on what mechanism about endothelial cells affecting smooth muscle cells and extracellular matrix, especially the basement membrane and internal elastic layer, because only their damage can lead to the formation of aneurysms. The latter factor is mechanical stress related to blood pressure.The reseach core of vascular biology mechanism is about changes and causes of smooth muscle cells and the extracellular matrix mostly constituted of ollagenШwhen vascular endothelial cells are damaged.Fibroblast growth factor is a class of peptide olecules which plays a role in regulating cell growth by specific receptor binding with the cell membrane. FGFs family are biological factors with a wide range of biological effects , promoting embryo development, variation of cell growth, playing an important role in tissue formation and repair, inflammation, thrombosis, tumorigenesis and metastasis and other physiological and pathological processes .It can promote proliferation and diviation of fibroblasts, epithelial cells and endothelial cells. FGFs can promote angiogenesis and promote fibroblast proliferation. Hoshina et al who adopted the role of bFGF gene in experimental abdominal aortic aneurysms in 2004 found bFGF can promote smooth muscle cell proliferation and repair the aneurysm wall; in 2007 Shiraya et al found that atorvastatin can inhibit the expansion of experimental abdominal aortic aneurysms . In this article we intend to carry bFGF gene with the lentiviral vector to interfere aneurysms, observing if it has effect on repair of aneurysms, which provides a theoretical basis to prevent or delay the rupture of the aneurysm as conservative treatment .The first part: induction of aneurysm in carotid arteries of rabbits to observe the phenotype of VSMC from aneurysm wallPartⅠObjective To identify developmental changes in vascular smooth muscle cells (VSMC) during aneurysm formation using an elastase-induced rabbit carotid artery aneurysm model.Methods Aneurysm was induced by exposure to elastase. Tissues from developing aneurysms were isolated at various time points for histological, immunohistochemical and RT-PCR analysis.Results During the aneurysm growth, the vascular media became thinner and elastic fibres became fragmented. The expression of smooth muscle marker SM22αand cytoskeleton proteins (SMα-actin,β-Tubulin and Desmin) expression was decreased while platelet-derived growth factor (PDGF) was increased, which correlated with a remodeling of VSMC from contractile to synthetic functions.Conclusion During the development of aneurysm, VSMC is remodeled from the contractile phenotype to a synthetic phenotype as a potential repair mechanism. Further studies may reveal the causative effects between these events.The second part: ovservation of the effects of fibroblast growth factor on the proliferation VSMC cultured in vitoPartⅡObjective To research the effects of fibroblast growth factor on the proliferation vascular smooth muscle cells cultured in vito and its mechanism.Methods Add bFGF andb FGF + R1-respectively into vascular smooth muscle cells in vitro.5-BrDU labeled with the proliferation of VSMC and others was used to detect the expression of VSMC Phenotypic SM22αand HRG-1mRNA of proliferation negative regulation by RT-PCR.Results After bFGF intervened in vascular smooth muscle cells cultured in serum,the expression of SM22αand HRG-1 was downregulated and marker-positive proliferated cells by 5-BrDU increased.While SM22αand HRG-1 was significantly upregulated when bFGF and R1- affected them. The results showed that bFGF had effect on vascular smooth muscle proliferation and phenotype of transformation.Conclusions bFGF had effect on vascular smooth muscle proliferation and transformation of phenotype.The mechanism is achieved through R1 receptor.The third part: build and discrimination of the lentivirus vector with stable expression of bFGFPartⅢObjective Applicate molecular biology technology to build lentivirus vector with stable expression of bFGF.Methods Design PCR primers of bFGF gene ,and use Taq enzyme to amplificate bFGF With pCI-bFGF plasmid as a template. Digest the pGC-FU lentiviral vector for formation of linear vector to amplificate gene fragments with bFGF exchange reaction .Use 293T packaging cells to package and build viral vectors .The titer of lentivirus is determined by fluorescence and Real-time Q-PCR.Results Discrimination of recombinant lentiviral plasmids is completely correct.Drops of purified virus after amplified is 1.0×108TU/ml.Conclusions The virus could stably express bFGF.The results show that the packaging of lentivirus carrys bFGF successfully. It may become a new means of repairing aneurysm wall later.The fourth part :the research into lentivirus vector carrying bFGF gene to repair rabbit aneurysm wallPartⅣObjective Observe the effct of bFGF on the repair of aneurysm wall by injecting recombinant lentivirus with bFGF to the aneurysm model. Methods Injected lentivirus drops carrying bFGF daily from the rabbit ear after models were founded .The experiment is divided into two parts. Every other week a group of rabbits were killed to collect samples. The expression of smooth muscle SM22αm RNA and HRG-1mRNA is detected by Real-time PCR . The PDGF expression of aneurysm wall was observed by immunohistochemical label.Results The aneurysms intervented by lentiviral drops had smaller size than the control group, with lighter degree of elastic fiber fracture and a larger number of smooth muscle cells.The expression of smooth muscle SM22αm RNA and HRG-1mRNA was decreased.Conclusions Recombinant lentivirus carrying bFGF can repair damaged aneurysms and inhibit the aneurysm dynamic growth. It provides an experimental basis for conservative treatment of intracranial aneurysms in the future.