仿生搏动流体应力对小口径组织工程血管的生物学作用及相关机制研究
发布时间：2018-08-18 19:07
【摘要】：背景： 1、制造小口径组织工程血管是当前的研究热点，仿生应力对小口径组织工程血管的生长和成熟非常重要，但目前的很多灌注培养式生物反应器尚不能提供模拟生理血流的动力学环境，而且未提供足够的生物学数据来证明其优越性； 2、小口径组织工程血管的功能和生物力学特性都离不开平滑肌细胞的良好增殖，但平滑肌细胞在接种到支架材料后的增殖情况并不令人满意，从而影响小口径组织工程血管的功能和生物力学强度。目的： 1、构建小口径组织工程血管，体外仿生搏动流体应力条件下进行培养，观察对小口径组织工程血管的生物学作用； 2、构建平滑肌细胞受仿生搏动应力模型，探讨仿生搏动应力促平滑肌细胞增殖的转录后调控机制。 方法： 1、（1）酶消化法分离获得内皮细胞和平滑肌细胞，显微镜观察细胞形态，VIII因子相关抗原免疫组化染色和α肌动蛋白免疫荧光染色分别鉴定内皮细胞和平滑肌细胞的表型； （2）用不使用胰酶的化学方法制备脱细胞兔主动脉支架材料，观察大体形态，行组织学观察、扫描电镜和透射电镜观察，CCK-8法评价生物相容性，计算接种细胞黏附率，评价亲水性，接种内皮细胞和平滑肌细胞后行组织学观察、扫描电镜和透射电镜观察； （3）内皮细胞和平滑肌细胞接种到脱细胞兔主动脉，构建小口径组织工程血管，置入我们自行研制的小口径组织工程血管生物反应器，分别给予仿生搏动流体应力和静态培养后检测：观察大体形态，行HE染色、Masson染色和弹力纤维染色，扫描电镜观察，Real-time PCR检测Collagen I、III、IV、碱性成纤维细胞生长因子和内皮素-1mRNA表达，免疫荧光检测钙调蛋白、α肌动蛋白、VIII因子相关抗原和血管性假性血友病因子，NO和6-keto-PGF1a浓度检测，血小板黏附实验，生物力学检测。 2、（1）分组：①动态培养组；②静态培养组；③动态培养+慢病毒组；④静态培养+慢病毒组；⑤动态培养+IGF-1R siRNA组；⑥静态培养+IGF-1R siRNA组。检测：细胞增殖、细胞周期、细胞凋亡，Real-time PCR检测IGF-1R、骨桥蛋白mRNA，Western Blot检测IGF-1R、骨桥蛋白、pTyr-IGF-1R、p-AKT； （2）分组：①动态培养组；②静态培养组。检测：microRNA芯片检测，microRNA数据库预测，Real-time PCR检测差异表达的microRNA； （3）分组：①动态培养组；②静态培养组；③动态培养+慢病毒组；④静态培养+慢病毒组；⑤动态培养+microRNA-223组；⑥静态培养+microRNA-223组；⑦动态培养+microRNA-153组；⑧静态培养+microRNA-153组。检测：靶基因验证实验、细胞增殖、细胞周期、细胞凋亡，Real-time PCR检测IGF-1R、骨桥蛋白mRNA，WesternBlot检测IGF-1R、骨桥蛋白、pTyr-IGF-1R、p-AKT。 结果： 1、（1）平滑肌细胞成束平行的排列，呈典型的“峰与谷”样生长，α肌动蛋白免疫荧光呈现强阳性；内皮细胞呈典型的“鹅卵石”样生长，排列密集，VIII因子相关抗原免疫组化染色为强阳性； （2）①脱细胞兔主动脉质地与新鲜兔主动脉相比无明显变化；②HE染色：结构疏松，质地均匀，自身细胞基本完全脱除；Masson染色：结构疏松，胶原纤维之间可见较多空隙；弹力纤维染色：结构疏松，弹力纤维之间可见较多空隙；③扫描电镜检测：外表面毛糙，主要由粗大的胶原纤维交织而成，孔隙比内表面多，内表面光滑；④对平滑肌细胞和内皮细胞的细胞毒性评价为0～1级；⑤平滑肌细胞和内皮细胞的黏附率分别为64.32%±2.03%和52.77%±1.19%；⑥种子细胞-脱细胞兔主动脉复合物的检测：HE染色：平滑肌细胞生长良好，并有部分迁移至管壁内部，内皮细胞附着于内表面，生长良好；扫描电镜：外表面和内表面均被平滑肌细胞和内皮细胞附着，并且部分平滑肌细胞向支架表面的孔隙内生长；透射电镜：平滑肌细胞胞浆内富含肌丝，纵向平行排列，胞内有密斑和密体，内皮细胞间存在细胞连接，胞浆内可见W-P小体；⑦亲水性：浸泡15分钟后吸水率227±21.2%，浸泡12小时后饱和(519%±23%)，而后维持在这一水平； （3）①仿生搏动流体应力培养的小口径组织工程血管管腔通畅，色泽与天然血管接近；②HE染色：仿生搏动流体应力培养的血管中平滑肌细胞和内皮细胞增殖的数量较静态培养的数量更多；③扫描电镜：仿生搏动流体应力培养的血管平滑肌细胞向邻近的细胞伸出很多突起相连，静态培养平滑肌细胞沿着支架形成纺锤形状，细胞数量也非常少；仿生搏动流体应力培养的血管管腔内面形成更完整的单层内皮细胞层，并更广泛的分布；④弹力纤维染色：天然血管和仿生搏动流体应力培养的血管中染色较为丰富；Masson染色：胶原表达没有显著差异；Real-time PCR检测：仿生搏动流体应力培养的平滑肌细胞Collagen I、III和IV基因表达水平明显升高；⑤免疫荧光检测：仿生搏动流体应力培养的血管中，平滑肌细胞沿环形方向伸展排列，Calponin和a-actin免疫荧光强度更强，数量更多；仿生搏动流体应力培养的血管中，内皮细胞几乎铺满管腔内壁，VIII因子相关抗原和血管性假性血友病因子免疫荧光强度更强，内皮细胞数量更多；⑥Real-time PCR检测：仿生搏动流体应力培养的血管中碱性成纤维细胞生长因子和内皮素-1mRNA的表达较静态培养明显升高；⑦NO和6-keto-PGF1a检测：仿生搏动流体应力培养的血管的内皮细胞能够产生大量NO和6-keto-PGF1a，数量比静态培养更接近天然血管；⑧血小板黏附实验：在仿生搏动流体应力培养的血管的管腔内面粘附的血小板很少；⑨力学特性：仿生搏动流体应力培养的血管的拉伸弹性恢复率、断裂伸长率、抗拉强度明显高于静态培养的血管。 2、（1）①动态培养静脉平滑肌细胞，与静态培养静脉平滑肌细胞相比较，细胞增殖明显增强，凋亡明显减弱；动态培养干扰IGF-1R mRNA翻译后的静脉平滑肌细胞，与动态培养的静脉平滑肌细胞相比较，细胞增殖明显减弱，凋亡明显增强；②Real-timePCR检测：动态培养静脉平滑肌细胞，与静态培养静脉平滑肌细胞相比较，IGF-1R mRNA的表达明显上调；动态培养干扰IGF-1R mRNA翻译后的静脉平滑肌细胞，与动态培养的静脉平滑肌细胞相比较，IGF-1R mRNA的表达明显下调；③Western Blot检测：动态培养静脉平滑肌细胞，与静态培养静脉平滑肌细胞相比较，，IGF-1R、pTyr-IGF-1R、p-AKT的表达明显上调；动态培养干扰IGF-1R mRNA翻译后的静脉平滑肌细胞，与动态培养的静脉平滑肌细胞相比较，IGF-1R、pTyr-IGF-1R、p-AKT的表达明显下调； （2）①动态培养的静脉平滑肌细胞组中筛选出2个表达上调2倍以上的microRNA，6个表达下调2倍以上的microRNA；②Real-time PCR对芯片结果进行验证,microRNA-153的表达在动态培养静脉平滑肌细胞中约为静态培养静脉平滑肌细胞中的1/2，microRNA-223的表达在动态培养静脉平滑肌细胞中约为静态培养静脉平滑肌细胞中的1/4；③Real-time PCR检测发现随着时间的延长，microRNA-153和microRNA-223的表达呈下降趋势，并均在4小时达到最低，此后microRNA-153和microRNA-223的表达一直维持在最低水平； （3）①EGFP/RFP报告系统结果显示：IGF-1R是microRNA-153和microRNA-223作用的靶基因，并且microRNA-223对靶基因IGF-1R的调控作用强于microRNA-153；②Real-time PCR检测：静态培养microRNA-153和microRNA-223分别转染后的静脉平滑肌细胞，与静态培养的静脉平滑肌细胞相比较，microRNA-153和microRNA-223的表达明显上调，上调约4倍左右；动态培养microRNA-153和microRNA-223分别转染后的静脉平滑肌细胞，与静态培养microRNA-153和microRNA-223分别转染后的静脉平滑肌细胞相比较，microRNA-153和microRNA-223的表达明显下调；③动态培养microRNA-153和microRNA-223分别转染后的静脉平滑肌细胞，与动态培养静脉平滑肌细胞相比较，细胞增殖明显减弱，凋亡明显增强；动态培养microRNA-223转染后的静脉平滑肌细胞，与动态培养microRNA-153转染后的静脉平滑肌细胞相比较，细胞增殖减弱更加明显，凋亡增强更加明显；④Real-time PCR检测：动态培养microRNA-153和microRNA-223分别转染后的静脉平滑肌细胞，与动态培养静脉平滑肌细胞相比较，IGF-1R mRNA的表达无明显差异；动态培养microRNA-223转染后的静脉平滑肌细胞，与动态培养microRNA-153转染后的静脉平滑肌细胞相比较，IGF-1R mRNA的表达无明显差异；⑤Western Blot检测：动态培养microRNA-153和microRNA-223分别转染后的静脉平滑肌细胞，与动态培养静脉平滑肌细胞相比较，IGF-1R的表达明显下调；动态培养microRNA-223转染后的静脉平滑肌细胞，与动态培养microRNA-153转染后的静脉平滑肌细胞相比较，IGF-1R、pTyr-IGF-1R、p-AKT的表达下调更加明显。结论： 1、酶消化法分离内皮细胞和平滑肌细胞方法简单可行，细胞获取率高，获得细胞的细胞形态和免疫表型符合内皮细胞和平滑肌细胞的特征； 2、用不使用胰酶的化学方法制备脱细胞兔主动脉支架材料，方法简便可行，制备的脱细胞兔主动脉支架材料的亲水性、细胞黏附性、细胞相容性和组织学等特性均适合作为小口径组织工程血管的支架材料； 3、仿生搏动流体应力可以有效促进平滑肌细胞和内皮细胞增殖并引导平滑肌细胞的同向排列和内皮细胞的均匀分布，可能会引导小口径组织工程血管形成接近于天然血管的组织结构；仿生搏动流体应力可以有效促进胶原和弹力纤维的分泌，可能会引导小口径组织工程血管具备接近于天然血管的生物力学性能；仿生搏动流体应力可以有效促进平滑肌细胞和内皮细胞成熟表型的表达和细胞因子的分泌，可能会引导小口径组织工程血管向天然血管的方向成熟分化；仿生搏动流体应力可以有效促进内皮细胞舒张血管和抗血栓形成功能的发挥，可能会引导小口径组织工程血管拥有接近于天然血管的舒张血管和抗血栓形成能力； 4、IGF-1R的表达上调、活性及其下游信号通路激活在仿生搏动应力促进静脉平滑肌细胞的增殖和减少其凋亡中起重要作用； 5、仿生搏动应力可以促进静脉平滑肌细胞IGF-1R相关的microRNA-223和microRNA-153表达下调；仿生搏动应力作用4小时，microRNA-223和microRNA-153下调幅度最大，此后维持在这一表达水平； 6、microRNA-223和microRNA-153的表达下调在仿生搏动应力促进静脉平滑肌细胞的增殖和减少其凋亡中，通过上调IGF-1R的表达、激活其活性及其下游信号通路起重要作用。
[Abstract]:Background:
1. Fabrication of small-caliber tissue-engineered blood vessels is a hot research topic at present. Bionic stress is very important to the growth and maturation of small-caliber tissue-engineered blood vessels. However, many perfusion-culture bioreactors can not provide a dynamic environment to simulate physiological blood flow, and do not provide enough biological data to prove their superiority.
2. The function and biomechanical properties of small-caliber tissue-engineered blood vessels can not be separated from the good proliferation of smooth muscle cells. However, the proliferation of smooth muscle cells after implantation of scaffolds is not satisfactory, which affects the function and biomechanical strength of small-caliber tissue-engineered blood vessels.
1. To construct small-diameter tissue-engineered blood vessels and culture them in vitro under the condition of bionic pulsating fluid stress.
2. To construct a bionic pulsatile stress model of smooth muscle cells and explore the post-transcriptional regulation mechanism of the proliferation of smooth muscle cells induced by bionic pulsatile stress.
Method:
1. (1) Endothelial cells and smooth muscle cells were isolated by enzymatic digestion. Cell morphology was observed under microscope. The phenotypes of endothelial cells and smooth muscle cells were identified by factor VIII associated antigen immunohistochemical staining and alpha actin immunofluorescence staining respectively.
(2) The acellular rabbit aorta scaffolds were prepared by chemical method without trypsin. The morphology of the scaffolds was observed by histological observation, scanning electron microscopy and transmission electron microscopy. The biocompatibility was evaluated by CCK-8 method. The adhesion rate of the inoculated cells was calculated and the hydrophilicity was evaluated. Observation by electron microscope.
(3) Endothelial cells and smooth muscle cells were inoculated into the aorta of acellular rabbits to construct small-caliber tissue-engineered blood vessels, and then implanted into a small-caliber tissue-engineered blood vessel bioreactor developed by ourselves. Bionic pulsatile fluid stress and static culture were performed respectively. The gross morphology was observed, HE staining, Masson staining and elastic fiber staining were performed. The expression of Collagen I, III, IV, basic fibroblast growth factor and endothelin-1 mRNA, calmodulin, alpha actin, factor VIII related antigen and von Willebrand factor, NO and 6-keto-PGF1a, platelet adhesion test and biomechanical test were detected by Real-time PCR and immunofluorescence.
2. (1) Grouping: dynamic culture group; static culture group; dynamic culture + lentivirus group; static culture + lentivirus group; _dynamic culture + IGF-1R siRNA group; _static culture + IGF-1R siRNA group. Detection: cell proliferation, cell cycle, apoptosis, Real-time PCR detection of IGF-1R, osteopontin mRNA, Western Blot detection of IGF-1R Osteopontin, pTyr-IGF-1R, p-AKT;
(2) Grouping: dynamic culture group; static culture group; detection: microRNA chip detection, microRNA database prediction, Real-time PCR detection of differentially expressed microRNA;
(3) Grouping: dynamic culture group; static culture group; dynamic culture + lentiviral group; static culture + lentiviral group; static culture + microRNA-223 group; static culture + microRNA-223 group; dynamic culture + microRNA-153 group; static culture + microRNA-153 group; detection: target gene validation experiment, cell proliferation, fine Cell cycle, apoptosis, Real-time PCR for IGF-1R, osteopontin mRNA, Western Blot for IGF-1R, osteopontin, pTyr-IGF-1R, p-AKT.
Result:
1. (1) Smooth muscle cells were arranged in bundles parallel to each other, showing a typical "peak and valley" like growth, and alpha actin immunofluorescence showed a strong positive; endothelial cells showed a typical "cobblestone" like growth, dense arrangement, and factor VIII related antigen immunohistochemical staining showed a strong positive;
(2) The texture of acellular rabbit aorta had no obvious change compared with fresh rabbit aorta; HE staining: the structure was loose, the texture was uniform, and the cells were completely removed; Masson staining: the structure was loose, and there were more gaps between collagen fibers; elastic fiber staining: the structure was loose, there were more gaps between elastic fibers; Electron microscopic examination showed that the outer surface was rough, mainly composed of thick collagen fibers, with more pores than the inner surface and smooth inner surface. The cytotoxicity of smooth muscle cells and endothelial cells was evaluated as 0-1 grade. _The adhesion rates of smooth muscle cells and endothelial cells were 64.32% + 2.03% and 52.77% + 1.19% respectively. HE staining: smooth muscle cells grew well, and some of them migrated to the inner wall, endothelial cells adhered to the inner surface and grew well; scanning electron microscopy: smooth muscle cells and endothelial cells adhered to the outer surface and inner surface, and some of the smooth muscle cells grew into the pores of the scaffold surface; The cytoplasm of smooth muscle cells was rich in myofilaments, arranged in parallel and longitudinal directions, and there were spots and dense bodies in the cells. There were junctions between endothelial cells and W-P bodies in the cytoplasm.
(3) Bionic pulsatile fluid stress culture of small-caliber tissue engineering vascular lumen unobstructed, color and color close to natural vessels; HE staining: bionic pulsatile fluid stress culture of vascular smooth muscle cells and endothelial cells proliferation more than static culture; Scanning electron microscopy: bionic pulsatile fluid stress culture of blood The smooth muscle cells of the tubules extend many processes to adjacent cells and connect with each other. Static cultured smooth muscle cells form a spindle shape along the scaffold, and the number of cells is very small. A more complete monolayer of endothelial cells is formed in the lumen of the vascular lumen cultured by bionic pulsing fluid stress, and more widely distributed. 4 Elastic fiber staining: natural blood vessels. Masson staining showed no significant difference in collagen expression; Real-time PCR showed that the expression levels of Collagen I, III and IV genes in cultured smooth muscle cells were significantly increased; _Immunofluorescence detection: blood vessels cultured under bionic pulsatile fluid stress. In the bionic pulsatile fluid stress cultured vessels, endothelial cells almost covered the lumen wall, factor VIII associated antigen and von Willebrand factor immunofluorescence intensity was stronger, the number of endothelial cells was more; The expression of basic fibroblast growth factor (bfgf) and endothelin-1 (et-1) mRNA in blood vessels cultured under pulsatile fluid stress was significantly higher than that in static culture. _NO and 6-keto-PGF1a detection: endothelial cells cultured under pulsatile fluid stress could produce large amounts of NO and 6-keto-PGF1a, which were closer to those cultured in static culture. Natural blood vessels; _Platelet adhesion test: There were few platelets adhered to the lumen of blood vessels cultured by bionic pulsating fluid stress; _Mechanical properties: The elastic recovery rate, breaking elongation rate and tensile strength of blood vessels cultured by bionic pulsing fluid stress were significantly higher than those cultured by static fluid stress.
2. (1) The proliferation and apoptosis of VSMCs cultured dynamically were significantly enhanced and weakened compared with those cultured statically. The proliferation and apoptosis of VSMCs cultured dynamically interfered with the translation of IGF-1R mRNA were significantly weakened compared with those cultured dynamically. The expression of IGF-1R mRNA was up-regulated in the cultured VSMCs compared with the static VSMCs, while the expression of IGF-1R mRNA was down-regulated in the VSMCs after the translation of IGF-1R mRNA. Measurement: The expression of IGF-1R, pTyr-IGF-1R and p-AKT was significantly up-regulated in cultured VSMCs, and the expression of IGF-1R, pTyr-IGF-1R and p-AKT was significantly down-regulated in cultured VSMCs after interfering with the translation of IGF-1R mRNA.
(2) Two microRNAs with up-regulation of more than two times and six microRNAs with down-regulation of more than two times were screened out in the dynamic cultured VSMC group; and (2) Real-time PCR was used to verify the results of the microRNA-153 chip. The expression of microRNA-153 in the dynamic cultured VSMC was about 1/2 of that in the static cultured VSMC and microRNA-22. The expression of microRNA-153 and microRNA-223 in VSMCs was about 1/4 of that in VSMCs cultured in static state. Flat;
(3) The results of EGFP/RFP reporting system showed that IGF-1R was the target gene of microRNA-153 and microRNA-223, and the regulatory effect of microRNA-223 on target gene IGF-1R was stronger than that of microRNA-153. Real-time PCR detection: static culture of microRNA-153 and microRNA-223 were transfected into venous smooth muscle cells, which were flat with static culture of venous smooth muscle cells. The expression of microRNA-153 and microRNA-223 was up-regulated by about 4 times in SMCs, and the expression of microRNA-153 and microRNA-223 in VSMCs transfected with microRNA-153 and microRNA-223 was up-regulated by microRNA-153 and microRNA-223 respectively. (3) The proliferation and apoptosis of VSMCs transfected with microRNA-153 and microRNA-223 were significantly decreased compared with those of VSMCs transfected with microRNA-153, and the VSMCs transfected with microRNA-223 were cultured dynamically to smooth veins after microRNA-153 transfection. The expression of IGF-1R mRNA in the vein smooth muscle cells transfected with microRNA-153 and microRNA-223 was not significantly different from that in the vein smooth muscle cells transfected with microRNA-223. The expression of IGF-1R mRNA in VSMCs transfected with microRNA-153 was not significantly different from that in VSMCs transfected with microRNA-153. _Western Blot assay: The expression of IGF-1R in VSMCs transfected with microRNA-153 and microRNA-223 was detected by dynamic culture. The expression of IGF-1R, pTyr-IGF-1R and p-AKT in the vein smooth muscle cells transfected with microRNA-223 was significantly down-regulated compared with the vein smooth muscle cells transfected with microRNA-153.
1. Enzymatic digestion is a simple and feasible method for isolation of endothelial cells and smooth muscle cells. The cell morphology and immunophenotype of the obtained cells conform to the characteristics of endothelial cells and smooth muscle cells.
2. Acellular rabbit aorta scaffolds were prepared by chemical method without trypsin. The method was simple and feasible. The acellular rabbit aorta scaffolds were suitable for scaffolds with small diameter for tissue engineering.
3. Bionic pulsatile fluid stress can effectively promote the proliferation of smooth muscle cells and endothelial cells and induce the co-arrangement of smooth muscle cells and the uniform distribution of endothelial cells, which may lead to the formation of small-diameter tissue-engineered blood vessels close to the structure of natural blood vessels; bionic pulsatile fluid stress can effectively promote collagen and elastic fibers. Biomimetic pulsatile fluid stress can effectively promote the expression of mature phenotype of smooth muscle cells and endothelial cells.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.11
本文编号：2190398
[Abstract]:Background:
1. Fabrication of small-caliber tissue-engineered blood vessels is a hot research topic at present. Bionic stress is very important to the growth and maturation of small-caliber tissue-engineered blood vessels. However, many perfusion-culture bioreactors can not provide a dynamic environment to simulate physiological blood flow, and do not provide enough biological data to prove their superiority.
2. The function and biomechanical properties of small-caliber tissue-engineered blood vessels can not be separated from the good proliferation of smooth muscle cells. However, the proliferation of smooth muscle cells after implantation of scaffolds is not satisfactory, which affects the function and biomechanical strength of small-caliber tissue-engineered blood vessels.
1. To construct small-diameter tissue-engineered blood vessels and culture them in vitro under the condition of bionic pulsating fluid stress.
2. To construct a bionic pulsatile stress model of smooth muscle cells and explore the post-transcriptional regulation mechanism of the proliferation of smooth muscle cells induced by bionic pulsatile stress.
Method:
1. (1) Endothelial cells and smooth muscle cells were isolated by enzymatic digestion. Cell morphology was observed under microscope. The phenotypes of endothelial cells and smooth muscle cells were identified by factor VIII associated antigen immunohistochemical staining and alpha actin immunofluorescence staining respectively.
(2) The acellular rabbit aorta scaffolds were prepared by chemical method without trypsin. The morphology of the scaffolds was observed by histological observation, scanning electron microscopy and transmission electron microscopy. The biocompatibility was evaluated by CCK-8 method. The adhesion rate of the inoculated cells was calculated and the hydrophilicity was evaluated. Observation by electron microscope.
(3) Endothelial cells and smooth muscle cells were inoculated into the aorta of acellular rabbits to construct small-caliber tissue-engineered blood vessels, and then implanted into a small-caliber tissue-engineered blood vessel bioreactor developed by ourselves. Bionic pulsatile fluid stress and static culture were performed respectively. The gross morphology was observed, HE staining, Masson staining and elastic fiber staining were performed. The expression of Collagen I, III, IV, basic fibroblast growth factor and endothelin-1 mRNA, calmodulin, alpha actin, factor VIII related antigen and von Willebrand factor, NO and 6-keto-PGF1a, platelet adhesion test and biomechanical test were detected by Real-time PCR and immunofluorescence.
2. (1) Grouping: dynamic culture group; static culture group; dynamic culture + lentivirus group; static culture + lentivirus group; _dynamic culture + IGF-1R siRNA group; _static culture + IGF-1R siRNA group. Detection: cell proliferation, cell cycle, apoptosis, Real-time PCR detection of IGF-1R, osteopontin mRNA, Western Blot detection of IGF-1R Osteopontin, pTyr-IGF-1R, p-AKT;
(2) Grouping: dynamic culture group; static culture group; detection: microRNA chip detection, microRNA database prediction, Real-time PCR detection of differentially expressed microRNA;
(3) Grouping: dynamic culture group; static culture group; dynamic culture + lentiviral group; static culture + lentiviral group; static culture + microRNA-223 group; static culture + microRNA-223 group; dynamic culture + microRNA-153 group; static culture + microRNA-153 group; detection: target gene validation experiment, cell proliferation, fine Cell cycle, apoptosis, Real-time PCR for IGF-1R, osteopontin mRNA, Western Blot for IGF-1R, osteopontin, pTyr-IGF-1R, p-AKT.
Result:
1. (1) Smooth muscle cells were arranged in bundles parallel to each other, showing a typical "peak and valley" like growth, and alpha actin immunofluorescence showed a strong positive; endothelial cells showed a typical "cobblestone" like growth, dense arrangement, and factor VIII related antigen immunohistochemical staining showed a strong positive;
(2) The texture of acellular rabbit aorta had no obvious change compared with fresh rabbit aorta; HE staining: the structure was loose, the texture was uniform, and the cells were completely removed; Masson staining: the structure was loose, and there were more gaps between collagen fibers; elastic fiber staining: the structure was loose, there were more gaps between elastic fibers; Electron microscopic examination showed that the outer surface was rough, mainly composed of thick collagen fibers, with more pores than the inner surface and smooth inner surface. The cytotoxicity of smooth muscle cells and endothelial cells was evaluated as 0-1 grade. _The adhesion rates of smooth muscle cells and endothelial cells were 64.32% + 2.03% and 52.77% + 1.19% respectively. HE staining: smooth muscle cells grew well, and some of them migrated to the inner wall, endothelial cells adhered to the inner surface and grew well; scanning electron microscopy: smooth muscle cells and endothelial cells adhered to the outer surface and inner surface, and some of the smooth muscle cells grew into the pores of the scaffold surface; The cytoplasm of smooth muscle cells was rich in myofilaments, arranged in parallel and longitudinal directions, and there were spots and dense bodies in the cells. There were junctions between endothelial cells and W-P bodies in the cytoplasm.
(3) Bionic pulsatile fluid stress culture of small-caliber tissue engineering vascular lumen unobstructed, color and color close to natural vessels; HE staining: bionic pulsatile fluid stress culture of vascular smooth muscle cells and endothelial cells proliferation more than static culture; Scanning electron microscopy: bionic pulsatile fluid stress culture of blood The smooth muscle cells of the tubules extend many processes to adjacent cells and connect with each other. Static cultured smooth muscle cells form a spindle shape along the scaffold, and the number of cells is very small. A more complete monolayer of endothelial cells is formed in the lumen of the vascular lumen cultured by bionic pulsing fluid stress, and more widely distributed. 4 Elastic fiber staining: natural blood vessels. Masson staining showed no significant difference in collagen expression; Real-time PCR showed that the expression levels of Collagen I, III and IV genes in cultured smooth muscle cells were significantly increased; _Immunofluorescence detection: blood vessels cultured under bionic pulsatile fluid stress. In the bionic pulsatile fluid stress cultured vessels, endothelial cells almost covered the lumen wall, factor VIII associated antigen and von Willebrand factor immunofluorescence intensity was stronger, the number of endothelial cells was more; The expression of basic fibroblast growth factor (bfgf) and endothelin-1 (et-1) mRNA in blood vessels cultured under pulsatile fluid stress was significantly higher than that in static culture. _NO and 6-keto-PGF1a detection: endothelial cells cultured under pulsatile fluid stress could produce large amounts of NO and 6-keto-PGF1a, which were closer to those cultured in static culture. Natural blood vessels; _Platelet adhesion test: There were few platelets adhered to the lumen of blood vessels cultured by bionic pulsating fluid stress; _Mechanical properties: The elastic recovery rate, breaking elongation rate and tensile strength of blood vessels cultured by bionic pulsing fluid stress were significantly higher than those cultured by static fluid stress.
2. (1) The proliferation and apoptosis of VSMCs cultured dynamically were significantly enhanced and weakened compared with those cultured statically. The proliferation and apoptosis of VSMCs cultured dynamically interfered with the translation of IGF-1R mRNA were significantly weakened compared with those cultured dynamically. The expression of IGF-1R mRNA was up-regulated in the cultured VSMCs compared with the static VSMCs, while the expression of IGF-1R mRNA was down-regulated in the VSMCs after the translation of IGF-1R mRNA. Measurement: The expression of IGF-1R, pTyr-IGF-1R and p-AKT was significantly up-regulated in cultured VSMCs, and the expression of IGF-1R, pTyr-IGF-1R and p-AKT was significantly down-regulated in cultured VSMCs after interfering with the translation of IGF-1R mRNA.
(2) Two microRNAs with up-regulation of more than two times and six microRNAs with down-regulation of more than two times were screened out in the dynamic cultured VSMC group; and (2) Real-time PCR was used to verify the results of the microRNA-153 chip. The expression of microRNA-153 in the dynamic cultured VSMC was about 1/2 of that in the static cultured VSMC and microRNA-22. The expression of microRNA-153 and microRNA-223 in VSMCs was about 1/4 of that in VSMCs cultured in static state. Flat;
(3) The results of EGFP/RFP reporting system showed that IGF-1R was the target gene of microRNA-153 and microRNA-223, and the regulatory effect of microRNA-223 on target gene IGF-1R was stronger than that of microRNA-153. Real-time PCR detection: static culture of microRNA-153 and microRNA-223 were transfected into venous smooth muscle cells, which were flat with static culture of venous smooth muscle cells. The expression of microRNA-153 and microRNA-223 was up-regulated by about 4 times in SMCs, and the expression of microRNA-153 and microRNA-223 in VSMCs transfected with microRNA-153 and microRNA-223 was up-regulated by microRNA-153 and microRNA-223 respectively. (3) The proliferation and apoptosis of VSMCs transfected with microRNA-153 and microRNA-223 were significantly decreased compared with those of VSMCs transfected with microRNA-153, and the VSMCs transfected with microRNA-223 were cultured dynamically to smooth veins after microRNA-153 transfection. The expression of IGF-1R mRNA in the vein smooth muscle cells transfected with microRNA-153 and microRNA-223 was not significantly different from that in the vein smooth muscle cells transfected with microRNA-223. The expression of IGF-1R mRNA in VSMCs transfected with microRNA-153 was not significantly different from that in VSMCs transfected with microRNA-153. _Western Blot assay: The expression of IGF-1R in VSMCs transfected with microRNA-153 and microRNA-223 was detected by dynamic culture. The expression of IGF-1R, pTyr-IGF-1R and p-AKT in the vein smooth muscle cells transfected with microRNA-223 was significantly down-regulated compared with the vein smooth muscle cells transfected with microRNA-153.
1. Enzymatic digestion is a simple and feasible method for isolation of endothelial cells and smooth muscle cells. The cell morphology and immunophenotype of the obtained cells conform to the characteristics of endothelial cells and smooth muscle cells.
2. Acellular rabbit aorta scaffolds were prepared by chemical method without trypsin. The method was simple and feasible. The acellular rabbit aorta scaffolds were suitable for scaffolds with small diameter for tissue engineering.
3. Bionic pulsatile fluid stress can effectively promote the proliferation of smooth muscle cells and endothelial cells and induce the co-arrangement of smooth muscle cells and the uniform distribution of endothelial cells, which may lead to the formation of small-diameter tissue-engineered blood vessels close to the structure of natural blood vessels; bionic pulsatile fluid stress can effectively promote collagen and elastic fibers. Biomimetic pulsatile fluid stress can effectively promote the expression of mature phenotype of smooth muscle cells and endothelial cells.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.11
本文编号：2190398
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