The Primary Study Of Promoting Effect Of Atorvastatin On Vascularization In TypeⅠ Collagen Protein Scaffold Of Rat

ObjectiveIn Recent years,tissue engineering study based on growth factors,seed cells and biomaterials has been greatly developed.But the clinical application is not very satisfied.The main reason is the deficiency of vascular net on the graft which caused insufficient nutrition supply to the cells.Some reserches have proved that seed cells can only survive in the extent 150 to 200μm around blood diffusion.If the problem of vascularization can not be solved the application of large volum tissue engineering transplatations can not be succeeded in the clinical.Recently there are many reports about tissue engineering transplatations vascularization by various vascular growth factors and transgenic technology.Because the vascular growth factors have proper limitations,and many probloms have not been solved in transgenic therapy.the application in clinical is limited.So it is important to look for a convenient,effective and useful drug which can induce angiogenesis to replace vascular growth factors,and it is of significant in clinic.Atorvastatin is an inhibitor of HMG-CoA reducing enzyme,and commonly used to decrease the blood fat in clinical.The co-culture research about vessel neogenesis in vitro have manifested that Atorvastatin can promote the vessel strcture formation。If Atorvastatin can be used to promote vascularization in tissue engineering transplatation,it will bring new hope to the vascularization in tissue engineering.The experiment typeⅠcollagen protein scaffold which was embedded into subcutaneously of rats.And the rats were intragastriced with Atorvastatin every day,By observing VEGF,CD34 cells,vessel structure and the expression of VEGFmRNA,we can observe wheather Atorvastatin could promote vascularization or not in tissue engineering tissue. Methods1,The morphology observation of typeⅠcollagen protein scaffold(1) Eosin staining:take moiety typeⅠcollagen protein scaffold,put in freezing embedding medium,after quick freezing using liquid nitrogen,making frozen sections, and the thickness is 8μm.the sections were fixation with 4%paraformaldehyde in 30 min.after water douche,the sections were staining with eosin.take pictures in the light microscope.(2) Scanning electron microscope:moiety typeⅠcollagen protein scaffold was take,using 2.5%Glutaral to fixation the materialising Alcohol dehydration,drying in critical point,conspergere gold,use Scanning electron microscope scaning and take pictures.2,Embeding typeⅠcollagen protein scaffold in vivo and groupingForty Wistar Rats weighting 90-100g were embedded typeⅠcollagen protein scaffold volumed 125 mm~3 in subcutaneously at middle point in the back of thigh.Then the rats were randomly divided into control group(n=20)and experimental group(n=20).3,Using Atorvastatin intragastriclyAfter one day,the rats of experimental group were intragastriced with atrorvastatin (1mg/KG/d),while the rats of control group were intragastricted with the same dose saline.After 14 days,all the rats were sacrificed,and the typeⅠcollagen protein scaffold was dislodged.Some typeⅠcollagen protein scaffold was embedded in freezing embedding medium,and after qickly freezing by liquid nitrogen,the typeⅠcollagen protein scaffold was freezed in -70°C refrigerator for immunohistochemistry analysis,and another typeⅠcollagen protein scaffold was putted in Trizol and freezed in -70°C refrigerater for RT-PCR detection.4,Immunohistochemistry stainTypeⅠcollagen protein scaffold witch was embedded in freezing embedding medium was cuted into 8μm frozen section.The sections were stained with VEGF and CD34 antibody.then observed by light microscope. 5,RT-PCRThe expression of VEGFmRNA in typeⅠcollagen protein scaffold of two groups was analysed by RT-PCR and the product of RT-PCR was indentified by ionophoresis using 1.5%sepharose with 150 voltage in 30 miniute.The images were scanned with Tanon gel electrophoresis image analysis system.6,Statistical treatmentAll the data were analyzed by spss 13.0 statistics software.Results1,The morphology of typeⅠcollagen protein scaffoldTypeⅠcollagen protein scaffold was red,and the structure was rarefaction,the typical porous structure were seen and the aperture size was 150-200μm.By the scanning electron microscope,the scaffold surface was smooth and glossy and a few reductus were formed.2,General state of ratsAfter 1 hour postoperation,all rats awake.The incisal opening was dry and neat.The activity and foodintake were normal.then the weight of rats increased gradually.After 14 days,the incision healed perfectly in all rats without infection.3,Result of immunohistochemistryCD34 cells were located in marginal part of the frame,and buffy drops scattered in cytoplasm uniformly.More CD34 cells formed blood vessel structure whose diameters were between 6 to 10μm.The number of CD34 cells and blood vessel structures in experimental group were more than that of the control group.VEGF were located in vascular endothelial cell and extracellular matrix around the vascular endothelial cells.The buffy drops scattered in cytoplasm and extracellular matrix uniformly.Blood vessel structures did not formed in positive position.Some akaryocytes scatterd around positive position.The area of positive position and akaryocytes in experimental group were more than those in control group.4,Result of RT-PCRThe expression of VEGFmRNA in experimental group was stronger than that of the control group. 5,Result of statistical analysisCD34,VEGF and VEGFmRNA in the experimental group were stronger than that in the control group.P<0.05,the difference had statistical significance.ConclusionAtorvastatin can up-regulate the expression of VEGFmRNA,stimulate the secretion of VEGF,increase the number of CD34 cells and promote the formation of blood vessels constructed by CD34 cells in typeⅠcollagen protein scaffold.