体外动态灌注OECs-DBM预血管化与受区血管融合机制研究

发布时间：2018-05-16 07:53

本文选题：动态灌注培养 + 晚期内皮祖细胞　；参考：《昆明医科大学》2016年博士论文

【摘要】：[目的]探讨在体外通过动态灌注系统培养晚期内皮祖细胞-脱钙骨基质复合体,对晚期内皮祖细胞在脱钙骨基质上的铺展粘附、增殖、分化和基质孔隙中成管能力的影响及其可能的机制。探讨动态灌注预血管化的晚期内皮祖细胞-脱钙骨基质复合体的血管在体内与受区血管可能的融合机制。[方法]1.密度梯度离心法分离培养人外周血晚期内皮祖细胞,并通过细胞形态观察,流式细胞检测,结合凝集素及摄取乙酰化低密度脂蛋白实验和Matrigel基质胶体外成管实验对细胞进行综合鉴定。分别用10/30/50/70/100 MOI值的慢病毒介导eGFP试转染OECs,计算细胞转染效率,MTT比色法绘制不同MOI值下晚期内皮祖细胞的生长曲线,并与对照组细胞进行比较,优选出最优的MOI值,挑选耐嘌呤霉素单细胞克隆并扩增培养建立稳定细胞株,并判断其对晚期内皮祖细胞的增殖、细胞表面标记及体外成管特性有无影响。2.构建体外动态灌注培养系统,将eGFP慢病毒稳定转染晚期内皮祖细胞株接种于脱钙骨基质上,分别进行静态接种培养、动态灌注培养和抑制剂实验,通过荧光显微镜实时动态观察eGFP标记的晚期内皮祖细胞在脱钙骨基质上的铺展粘附和增殖情况,于培养第6天扫描电子显微镜下观察细胞在脱钙骨基质孔隙中的体外成管情况,RT-PCR及Western blot检测分析不同培养条件下的细胞CD34、VEGFR-1、VEGFR-2、VE-cadherin、Tie2和VEGF的mRNA及蛋白表达水平。3.建立裸鼠背部血管化动态观察窗模型,结合尾静脉注射德克萨斯红标记的右旋糖酐标记血浆,光镜及荧光显微镜下观察微血管,比较单纯观察窗模型及其结合德克萨斯红标记的右旋糖酐在微血管观察中的优劣势,并记录微血管开始显影时间,最佳观测时间及显影持续时间。往裸鼠背部血管化动态观察窗中植入静态培养和动态灌注培养的OECs-DBM支架,分别在植入后第1、3、5、7、9、12天荧光显微镜下动态观察并计数支架边缘长入血管数量,计算支架边缘及中心区荧光累积光密度值来反映支架获得的血液灌注量。在植入后第12天切取支架及其周围组织进行硬组织切片HE染色,在光镜及荧光显微镜下观察支架上荧光细胞的分布及其与受体血管融合情况。[结果]1.晚期血管内皮祖细胞表现为典型的鹅卵石样形态,能较为特异的结合凝集素及摄取乙酰化低密度脂蛋白,在Matrigel基质胶上可形成血管管腔样结构,流式细胞仪检测显示其表达细胞表面标记CD34和VEGFR-2,基本不表达CD133。当MOI=50时,慢病毒介导的eGFP转染晚期内皮祖细胞转染效率较高且对细胞生长增殖无影响,筛选出的eGFP-OECs稳定转染细胞株在荧光显微镜下发出明亮稳定的绿色荧光,细胞的生物学形状及功能保持稳定。2.与静态培养组相比较,动态灌注组脱钙骨基质上的细胞密度和细胞投射面积更大,扫描电镜观察显示动态灌注培养组在脱钙骨基质孔隙中形成三维血管腔样结构的数量多于静态培养组。RT-PCR及Western blot检测显示,与静态培养组相比,动态灌注培养组细胞CD34 mRNA表达及蛋白合成下调,内皮细胞标记VEGFR-1、VEGFR-2、VE-cadherin和Tie2mRNA表达及蛋白合成上调。抑制剂实验显示PI3K和mTOR抑制剂能够抑制流体剪切应力促进细胞铺展粘附、增殖、分化和体外成管的作用。3.成功构建裸鼠背部血管化观察窗模型。与单纯观察窗模型相比,其结合德克萨斯红标记的右旋糖酐在微血管观察中更为精确。OECs-DBM支架边缘新长入血管计数结果显示两组支架植入后周围都有微血管长入,随着培养时间的延长,长入的微血管数量增多。在支架植入后第3、5、7天,动态灌注组的新长入血管数目较静态培养组多。动态灌注组的长入血管数量上升速率较静态灌注组快。动态灌注组支架边缘区域的荧光累积光密度值在所有观察时间点均高于静态培养组支架,而两组的支架中心区荧光累积光密度值差异更为显著。硬组织切片HE染色显示两组支架外均未见绿色荧光细胞。[结论]通过动态灌注系统给予脱钙骨基质上晚期内皮祖细胞持续不断的流体剪切应力,能够经由PI3K/Akt/mTOR信号传导通路促进晚期内皮祖细胞在脱钙骨基质上的铺展粘附、增殖、向成熟内皮细胞分化和基质孔隙中三维成管的能力,促进脱钙骨基质体外预血管化。与静态培养组相比,体外动态灌注培养预血管化的OECs-DBM支架能以长入式融合的方式与受区血管融合,更快更充分的获得受区的血液灌注,研究成果可能用于促进组织工程产品的临床应用。
[Abstract]:[Objective] to explore the effect and possible mechanism of advanced endothelial progenitor cells (endothelium progenitor cells, decalcified bone matrix complex) in vitro by dynamic perfusion system on the ability of spreading and adhesion, proliferation, differentiation and matrix pore formation on decalcified bone matrix. A possible fusion mechanism between the blood vessels of the matrix complex in the body and the recipient vessels. [method]1. density gradient centrifugation was used to isolate and culture the advanced endothelial progenitor cells of human peripheral blood, and through cell morphology observation, flow cytometry, binding of lectin and uptake of acetylated LDL and Matrigel matrix colloidal outer tube test. The 10/30/50/70/100 MOI value of eGFP was used to transfect OECs, and the transfection efficiency was calculated. MTT colorimetric method was used to plot the growth curve of late endothelial progenitor cells under different MOI values. Compared with the control group, the optimal MOI value was optimized, and the single cell clone and amplification of purinomycin were selected and amplified and established. To stabilize the cell line and determine its proliferation to advanced endothelial progenitor cells, the cell surface markers and the characteristics of the in vitro culture have no effect on the.2. construction in vitro, and the eGFP lentivirus stable transfection of late endothelial progenitor cells is inoculated on the decalcified bone matrix for static inoculation, dynamic perfusion culture and inhibitor. The spread and adhesion of eGFP labeled advanced endothelial progenitor cells on decalcified bone matrix were observed by fluorescence microscopy in real time. The cell formation in vitro of decalcified bone matrix was observed under sixth days of scanning electron microscope, and RT-PCR and Western blot were used to detect CD34, V of cells under different culture conditions. EGFR-1, VEGFR-2, VE-cadherin, Tie2 and VEGF mRNA and protein expression level.3. established the dynamic observation window model of the nude mouse back vascularization, combined with the tail vein injection of Texas red labeled dextran plasma, light microscope and fluorescence microscope to observe the microvascular, compared the simple observation window model and the right of Texas red label right The advantages and disadvantages of maleic anhydride in microvascular observation, and the recording time of the microvasculature, the best observation time and the duration of development were recorded. The static and dynamic perfusion OECs-DBM scaffolds were implanted into the dynamic observation window of the back vascularization of nude mice, and the dynamic observation and count branches were observed on the 1,3,5,7,9,12 day fluorescence microscope after the implantation. The number of blood vessels on the edge of the frame and the value of fluorescence accumulated light density of the stent were calculated to reflect the amount of blood perfusion obtained by the scaffold. The stent and its surrounding tissue were cut by HE staining at twelfth days after implantation. The distribution of fluorescein on the scaffold and the fusion relationship with the receptor vessel were observed under the light and fluorescence microscope. [results] [results]1. advanced vascular endothelial progenitor cells showed a typical cobblestone like morphology, which could be more specific binding lectin and acetylated LDL, and could form vascular lumen like structure on Matrigel matrix gel. Flow cytometry showed that the expression of cell surface markers CD34 and VEGFR-2, basically not CD133. when MOI= was expressed. At 50, the transfection efficiency of lentivirus mediated eGFP transfected advanced endothelial progenitor cells was higher and no effect on cell growth and proliferation. The selected eGFP-OECs stable transfected cell lines emitted bright and stable green fluorescence under the fluorescence microscope, and the biological shape and function of the cells remained stable and.2. was compared with the static culture group, and the dynamic perfusion group was removed. The cell density and the cell projection area on the calcium bone matrix were larger. The scanning electron microscope showed that the number of three-dimensional vascular cavities formed in the pore of decalcified bone matrix was more than that in the static culture group.RT-PCR and Western blot. Compared with the static culture group, the expression of CD34 mRNA and the egg in the dynamic perfusion culture group were compared with that of the static culture group. White synthesis down-regulation, endothelial cell markers VEGFR-1, VEGFR-2, VE-cadherin and Tie2mRNA expression and up regulation of protein synthesis. Inhibitor experiments showed that PI3K and mTOR inhibitors could inhibit fluid shear stress and promote cell spreading and adhesion, proliferation, differentiation and in vitro formation of tubes..3. successfully constructed the nude mouse back vascularization observation window model. Compared with the window model, it combined with the Texas red labeled dextran in the microvascular observation more accurate.OECs-DBM stent edge new long entry blood vessel count results showed that two groups of stent implantation around the microvascular length, with the extension of culture time, the increase of the number of microvasculature increased. After the stent implantation at the 3,5,7 day, dynamic irrigation. The number of new long entry vessels in the injection group was more than that in the static culture group. The increase rate of the amount of blood vessels in the dynamic perfusion group was faster than that in the static perfusion group. The fluorescence cumulative light density value of the stent edge area in the dynamic perfusion group was higher than that in the static culture group at all observation time points, but the difference of the fluorescence cumulative light density value of the two groups was more different. HE staining of hard tissue sections showed that no green fluorescent cells were found outside the two groups. [Conclusion] the continuous fluid shear stress was given to late endothelial progenitor cells on the decalcified bone matrix through the dynamic perfusion system, and could promote the spread and adhesion of late inner progenitor cells to the decalcified bone matrix via the PI3K/Akt/mTOR signal transduction pathway. Proliferation, the ability to differentiate into mature endothelial cells and the ability of three-dimensional tube formation in the matrix pores to promote the prevascularization of the decalcified bone matrix in vitro. Compared with the static culture group, the prevascularized OECs-DBM scaffold in vitro can fuse with the recipient blood vessels in a long entry mode, and study the blood perfusion faster and more fully. The results may be used to promote the clinical application of tissue engineering products.

【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R68;R318.08

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