Research Of Biological Effects And Related Mechanism Of Bionic Pulsating Fluid Stress To Small-diameter Tissue-engineering Vascular

Background:1. In present, manufacture of small-diameter tissue engineering vascular is a research hotspot. The bionic stress is very important for the growth and maturation of the small-diametertissue engineering vascular. But many perfusing culture bioreactors can not yet mimicphysiological blood flow dynamics environment now and does not provide sufficientbiological data to prove its superiority.2. Function and biomechanical properties of the small-diameter vascular tissueengineering are inseparable from good proliferation of smooth muscle cells.The proliferationof smooth muscle cell inoculated into the scaffold is not satisfactory, and thereby affect thefunction of the small-diameter vascular tissue engineering and biomechanical strength.Objective:1. Build small-diameter tissue engineering vascular. After cultured under conditions ofbionic pulsating fluid stress in vitro, observe biological effects to small-diameter tissue-engineering vascular.2. Build a model of venous smooth muscle cells stimulated by bionic pulsating fluidstress. Study the post-transcriptional regulation mechanism of promoting proliferation ofsmooth muscle cell.Methods:1.(1) Isolate and obtain endothelial cells and smooth muscle cells by digestion withenzyme. Observe cell morphology with microscope. Identify phenotype of endothelial cellsand smooth muscle cells by immunohistochemical staining of factor VIII related antigen andimmunofluorescence staining of a-actin.(2) Scaffolds of acellular rabbit aorta are prepared by chemical methods without trypsin. Observe morphology and histological strcture of scaffold and observe scaffold by scanningelectron microscope and transmission electron microscope. CCK-8method evaluatebiocompatibility of scaffold. Calculate the adhesion rate of seeding cells. Evaluate hydrophilicof scaffold. After endothelial cells and smooth muscle cells are seeded, observe histologicalstrcture of seeding cells and observe seeding cells by scanning electron microscope andtransmission electron microscope.(3) Build small-diameter tissue engineering vascular with arterial smooth muscle cellsand endothelial cells seeded onto acellular rabbit aorta scaffolds. They are placed into ourself-developed small-diameter vascular tissue engineering bioreactor and are cultured withbionic pulsatile fluid stress and static culture respctively. Observe morphology andhistological strcture of vascular and observe vascular by Masson staining, elastic fibersstaining and scanning electron microscope. Real-time PCR detects of Collagen I, III, IV, basicfibroblast growth factor and endothelin-1mRNA expression. Immunofluorescence detectscalponin, a-actin, factor VIII related antigen and von Willebrand factor. Detect concentrationof NO and6-keto-PGF1a. Carry on platelet adhesion assay and biomechanical test.2.(1) Groups:①Dynamic culture group.②Static culture group.③Dynamic culture+lentivirus Group.④Static culture+lentivirus group.⑤Dynamic culture+IGF-1R siRNAgroup.⑥Static culture+IGF-1R siRNA group. Detection: cell proliferation, cell cycle,apoptosis, Real-time PCR detection of IGF-1R, osteopontin mRNA, Western Blot detection ofIGF-1R, osteopontin, pTyr-IGF-1R, p-AKT.(2) Groups:①Dynamic culture group.②Static culture group. Detection: microRNAmicroarray, microRNA database prediction, Real-time PCR detection of differences ofmicroRNAs expression.(3) Groups:①Dynamic culture group.②Static culture group.③Dynamic culture+lentivirus Group.④Static culture+lentivirus group.⑤Dynamic culture+microRNA-223group.⑥Static culture+microRNA-223group.⑦Dynamic culture+microRNA-153group.⑧Static culture+microRNA-153group. Detection: target gene validation experiment, cellproliferation, cell cycle, apoptosis, Real-time PCR detection of IGF-1R, osteopontin mRNA,Western Blot detection of IGF-1R, osteopontin, pTyr-IGF-1R, p-AKT.Results:1.(1) Smooth muscle cells parallel to arrange in bundles and form typical "peak and valley" like morphology. Immunofluorescence staining of a-actin shows strong positive.Endothelial cells show characteristic "cobblestone" like morphology, arrange in a dense.Factor VIII related antigen immunohistochemical staining shows strong positive.(2)①Texture is no significant change compared with fresh rabbit aorta.②HE staining:structure is loose, texture is uniform. Its own cells essentially are completely removal. Massonstaining: structure is loose. Many gaps are visible between collagen fibers. Elastic fibersstaining: structure is loose. Many gaps are visible between elastic fibers.③Scanning electronmicroscopy: outer surface is rough, is mainly composed of interwovening coarse collagenfibers and has more pores than inner surface. Inner surface is smooth.④Cytotoxicity tosmooth muscle cells and endothelial cells is0to1.⑤Adhesion rates of smooth muscle cellsand endothelial cells respectively are64.32%±2.03%and52.77%±1.19%.⑥Detection ofseeding cells-acellular rabbit aorta complexes: HE staining: smooth muscle cells grow welland some migrate into the internal wall. Endothelial cells attached to inner surface grow well.Scanning electron microscopy: smooth muscle cells and endothelial cells respectively attachto outer surface and inner surface. And some of smooth muscle cells migrate into the porosityof the surface of scaffold. Transmission electron microscope: Cytoplasm of smooth musclecells is rich in muscle wire. Smooth muscle cells arrange in parallel to the longitudinal. Thereare dense plaques and dense bodies in smooth muscle cells. Cell connection exists betweenthe endothelial cells. There are W-P bodies in cytoplasm of endothelial cells.⑦Hydrophilic:water absorption is227±21.2%after soaked for15minutes, up to saturation value519%±23%after12hours immersion, and then maintains at this level.(3)①Lumen of small-diameter tissue engineering vascular cultured with bionicpulsating fluid stress is patent. Color is close to natural blood vessels.②HE staining:proliferation quantity of smooth muscle cells and endothelial cells of vascular cultured withbionic pulsating fluid stress is more than those with static culture.③Scanning electronmicroscopy: smooth muscle cells cultured with bionic pulsating fluid stress stretch out a largenumber of projections extending to adjacent cells and connect with each other. Smoothmuscle cells with static culture form spindle-shaped along the scaffold. And the number ofcells is also very small. There are more complete endothelial cell layer on inner surface ofvascular lumen cultured with bionic pulsating fluid stress and more widely distribute.④Elastic fibers staining: staining is more abundant in natural blood vessels and vascular cultured with bionic pulsating fluid stress. Masson staining: there are no obvious difference inthe expression of collagen. Real-time PCR detection: collagen I, III and IV gene expressionlevels were significantly higher in smooth muscle cells with bionic pulsating fluid stressculture.⑤Immunofluorescence: smooth muscle cells stretch and arrange along thecircumferential direction in vacular with bionic pulsating fluid stress culture. Calponin anda-actin immunefluorescence intensity are stronger and the number of smooth muscle cells ismore. Endothelial cells almost cover the inner surface of vascular lumen with bionic pulsatingfluid stress culture. Factor VIII related antigen and Von Willebrand factor immunef-luorescence intensity are stronger and the number of endothelial cells is more.⑥Real-timePCR detection: expression of basic fibroblast growth factor and endothelin-1mRNA ofvascular with bionic pulsating fluid stress cultur are significantly higher than those with staticculture.⑦NO and6-keto-PGF1a detection: endothelial cells of vascular with bionicpulsating fluid stress cultur are capable of producing large amounts of NO and6-keto-PGF1a.Amount closer to natural blood vessels than those with static culture.⑧Platelet adhesionassay: little adhesion of platelets is on the inner surface of vascular lumen with bionicpulsating fluid stress culture.⑨Mechanical properties: elastic recovery rate, elongation atbreak, tensile strength of vascular with bionic pulsating fluid stress culture are significantlyhigher than those with static culture.2.(1)①Cell proliferation is obviously enhanced and apoptosis is obviously weakenedin venous smooth muscle cells with dynamic culture, compared to venous smooth musclecells with static culture. Cell proliferation is obviously weakened and apoptosis is obviouslyenhanced in venous smooth muscle cells in which IGF-1R mRNA translation is interferedwith dynamic culture, compared to venous smooth muscle cells with dynamic culture.②Real-time PCR detection: IGF-1R mRNA expression is obviously up-regulated in venoussmooth muscle cells with dynamic culture, compared to venous smooth muscle cells withstatic culture. IGF-1R mRNA expression is obviously down-regulated in venous smoothmuscle cells in which IGF-1R mRNA translation is interfered with dynamic culture,compared to venous smooth muscle cells with dynamic culture.③Western Blot detection:IGF-1R、pTyr-IGF-1R、p-AKT expression is obviously up-regulated in venous smooth musclecells with dynamic culture, compared to venous smooth muscle cells with static culture.IGF-1R、pTyr-IGF-1R、p-AKT expression is obviously down-regulated in venous smooth muscle cells in which IGF-1R mRNA translation is interfered with dynamic culture,compared to venous smooth muscle cells with dynamic culture.(2)①Two microRNAs which are up-regulated more than2times and six microRNAswhich are down-regulated more than2times are filtered out in venous smooth muscle cellswith dynamic culture.②Microarray results are verified by Real-time PCR, expression ofmicroRNA-153in venous smooth muscle cells with dynamic culture is approximate1/2ofexpression of microRNA-153in venous smooth muscle cells with static culture, expression ofmicroRNA-223in venous smooth muscle cells with dynamic culture is approximate1/4ofexpression of microRNA-223in venous smooth muscle cells with static culture.③Real-timePCR results demonstrate that expression of microRNA-153and microRNA-223is adownward trend with time, and is the lowest at4hours. After that microRNA-153andmicroRNA-223’s expression keep at the minimum level.(3)①Results of EGFP/RFP reporting system show that IGF-1R is the target gene ofmicroRNA-153and microRNA-223. And microRNA-223is stronger in the regulation oftarget gene IGF-1R than microRNA-153.②Real-time PCR detection: microRNA-153andmicroRNA-223expression is obviously up-regulated in venous smooth muscle cells in whichmicroRNA-153and microRNA-223are respectively transfected with static culture, comparedto venous smooth muscle cells with static culture. The fold is about4times. MicroRNA-153and microRNA-223expression is obviously down-regulated in venous smooth muscle cells inwhich microRNA-153and microRNA-223are respectively transfected with dynamic culture,compared to venous smooth muscle cells in which microRNA-153and microRNA-223arerespectively transfected with static culture.③Cell proliferation is obviously weakened andapoptosis is obviously enhanced in venous smooth muscle cells in which microRNA-153andmicroRNA-223are respectively transfected with dynamic culture, compared to venoussmooth muscle cells with dynamic culture. Cell proliferation is more obviously weakened andapoptosis is more obviously enhanced in venous smooth muscle cells in which microRNA-223is transfected with dynamic culture, compared to venous smooth muscle cells in whichmicroRNA-153is transfected with dynamic culture.④Real-time PCR detection: IGF-1RmRNA expression has no significant difference in venous smooth muscle cells in whichmicroRNA-153and microRNA-223are respectively transfected with dynamic culture,compared to venous smooth muscle cells with dynamic culture. IGF-1R mRNA expression has no significant difference in venous smooth muscle cells in which microRNA-223istransfected with dynamic culture, compared to venous smooth muscle cells in whichmicroRNA-153is transfected with dynamic culture.⑤Western Blot detection: IGF-1R,pTyr-IGF-1R, p-AKT expression is obviously down-regulated in venous smooth muscle cellsin which microRNA-153and microRNA-223are respectively transfected with dynamicculture, compared to venous smooth muscle cells with dynamic culture. IGF-1R, pTyr-IGF-1R, p-AKT expression is more obviously down-regulated in venous smooth muscle cells inwhich microRNA-223is transfected with dynamic culture, compared to venous smoothmuscle cells in which microRNA-153is transfected with dynamic culture.Conclusions:1. Method of enzyme digestion to separate endothelial cells and smooth muscle cells issimple and feasible. Rate to obtain cells is high. Cell morphology and immunity phenotype ofobtained cells correspond with the characteristics of endothelial cells and smooth musclecells.2. The use of chemical acellular method of preparation of acellular rabbit aorta scaffoldis simple and feasible. Hydrophilic, cell adhesion, cell compatibility and histologicalcharacteristics of prepared acellular rabbit aorta scaffold are suitable for use as small diametertissue engineering vascular scaffold.3. Bionic pulsating fluid stress can effectively stimulate the proliferation of smoothmuscle cells and induce the same direction arranging of smooth muscle cells and the uniformdistribution of endothelial cells. So it maybe can induce the small-diameter tissue engineeringvascular to form the tissue and structure similar to that of natural blood vessels. Bionicpulsatile fluid stress can effectively promote the secretion of collagen and elastic fibers. So itmaybe can induce small-diameter tissue engineering vascular to have the biomechanicalproperties similar to that of natural blood vessels. Bionic pulsatile fluid stress can effectivelypromote the expression of mature phenotype and cytokine secretion of smooth muscle cellsand endothelial cells. So it maybe can induce the small-diameter tissue engineering vascularto maturation of the direction of natural blood vessels. Bionic pulsatile fluid stress caneffectively promote functions of relaxation of vascular and anti-thrombotic formation ofendothelial cells. So it maybe can induce small-diameter tissue engineering vascular to havethe capacities of vasodilatation and anti-thrombotic similar to that of natural blood vessels. 4. Up-regulation of IGF-1R expression and further activating of the activity of IGF-1Rand its downstream signaling pathways are very important for bionic pulsatile stress topromote venous smooth muscle cell proliferation and reduce apoptosis.5. Bionic pulsatile stress can promote downregulation of microRNA-223andmicroRNA-153which are related with IGF-1R in venous smooth muscle cells. After4hoursof bionic pulsatile stress, microRNA-223and microRNA-153reduce to maximum, andthereafter maintain at this level of expression.6. Down-regulation of microRNA-223and microRNA-153expression is very importantfor bionic pulsatile stress to promote venous smooth muscle cell proliferation and reduceapoptosis by up-regulating IGF-1R expression and further activate the activity of IGF-1R andits downstream signaling pathways.