大鼠骨髓间充质干细胞来源的血管内皮细胞贴附ePTFE的实验研究

发布时间：2019-03-18 17:48

【摘要】： 目的: 应用大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)来源的血管内皮细胞贴附ePTFE血管壁,并加入肝素共同培养,观察其对细胞贴附效率的影响,探讨提高细胞贴附ePTFE的方法。方法: 1.MSCs分离和诱导分化培养:在无菌条件下,取SD大鼠长骨骨髓,经密度梯度离心法获取形态均一的梭形细胞;加入10ng/ml内皮生长因子(vascularendothelial growth factor,VEGF)和2ng/ml碱性成纤维细胞生长因子(basicfibroblast growth facotr,bFGF)共同培养。 2.诱导细胞的鉴定:取传代后第二代实验组和对照组的两组细胞,进行免疫组织荧光染色,观察Ⅷ因子相关抗原(von Willebrand factor,vWF)的表达情况,及应用透射电镜观察细胞分化后细胞浆W-P小体的超微结构。 3.细胞贴壁和贴壁率的测定:将诱导的血管内皮细胞分成4组,种植于膨体聚四氟乙烯(ePTFE)人工血管上并分别加入浓度为0、10ug/ml、20ug/ml、30ug/ml的肝素培养,每4h检测内皮细胞贴附率,绘制细胞贴壁曲线。 4.统计学分析:实验数据以(?)±s表示,采用SPSS13.0软件方差齐性检验和单因素方差分析法进行统计学检测。结果: 1.经密度梯度离心法获取的MSCs漩涡状生长,形态呈梭形;诱导后细胞的vWF免疫组织荧光染色呈阳性表达,胞浆呈黄绿色,细胞轮廓清晰;电镜下可见血管内皮细胞特征性的W-P小体。 2.诱导的血管内皮细胞在ePTFE的贴附率逐渐升高,于20h时间点达到最高,4组贴比率分别是88.34%、96.48%、91.98%、85.69%。 3.肝素能显著提高血管内皮细胞在ePTFE贴壁率,浓度为20ug/ml肝素的效果最高。结论: 1.大鼠MSCs具有向血管内皮细胞分化的能力,能够为血管内皮化及组织工程提供理想的种子细胞。 2.肝素对MSCs来源的血管内皮细胞有重要的影响,具有能够有效地提高血管内皮化效率的作用,为人工血管的进一步发展提供了新的思路。
[Abstract]:Purpose: The vascular endothelial cells derived from the rat bone marrow mesenchymal stem cells (MSCs) were applied to the ePTFE blood vessel wall, and the heparin co-cultured to observe the effect of the heparin on the cell attachment efficiency and to explore the improvement of the cell adhesion ePTFE. Method. Methods:1. MSCs were isolated and induced to differentiate and cultured: in the condition of aseptic, the bone marrow of SD rats was taken, and the fusiform cells with uniform morphology were obtained by density gradient centrifugation;10 ng/ ml of endothelial growth factor (VEGF) and 2 ng/ ml of basic fibroblast growth factor (basic fibroblast growth factor) were added. th facotr,b 2. The expression of von Willebrand factor (vWF) and the cell differentiation were observed by transmission electron microscope. 3. The ultrastructure of the W-P body of the pulp.3. The measurement of the adherence and the attachment rate of the cells: the induced vascular endothelial cells were divided into 4 groups, the artificial blood vessels of the expanded polytetrafluoroethylene (ePTFE) were grown and the concentration of 0,10 ug/ ml,20 ug/ ml and 30 ug/ ml of the heparin culture were respectively added, and the endothelial cells were detected every 4 hours. The cell attachment rate and the cell adherence curve were plotted. 4. Statistical analysis: The experimental data is expressed as (?)% s, and the SPSS13.0 software is used for the performance inspection. Inspection and inspection list The results were as follows:1. The proliferation and morphology of the MSCs obtained by density gradient centrifugation were in the form of the shuttle. The fluorescent staining of the vWF-induced cells in the post-induced cells showed a positive expression, and the cytoplasm was in the form of yellow-green and cell-shaped. 2. The adhesion of the induced vascular endothelial cells to ePTFE gradually increased, the highest in the time of 20 h, and the ratio of the 4 groups was 8. 8.34%, 96.48%, 91.98%, 85.69%. The cells are The effect of ePTFE adherence was the highest in 20 ug/ ml of heparin. The ability to differentiate into the vascular endothelial cells can provide the ideal seed cell for the vascular endothelialization and the tissue engineering.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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