骨髓间充质干细胞的内皮分化及材料对分化影响的研究
发布时间：2018-12-12 18:26
【摘要】：心血管疾病大部分需要血管移植治疗,目前小直径(6mm)血管材料都面临着易形成血栓、内膜增生的困扰。解决这些问题关键在于促进材料表面形成功能化的内皮层,有效抑制血栓形成和内膜增生,维持长期通畅。天然内皮细胞(endothelial cells, ECs)来源和增殖能力有限,骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs)分离方法简单、具有自我更新和多向分化能力,因而研究者希望诱导BMSCs形成IECs(induced endothelial cells)。另外,材料的功能修饰也是促材料内皮化的有效途径。本课题组前期利用RGD蛛丝蛋白pNSR32、聚己内酯(polycaprolacton, PCL)和壳聚糖(chitosan,CS)静电纺丝制备pNSR32/PCL/CS三维纳米纤维支架,其力学性能良好,细胞和血液相容性良好,在大鼠体内能至少维持8w结构完整和血管通畅。 为解决天然ECs来源有限的问题,本研究以分离获得的大鼠BMSCs(rBMSCs)为基础,体外诱导其内皮分化,考察IECs的生理功能,并研究pNSR32/PCL/CS支架对rBMSCs内皮分化及IECs生物学功能的影响,主要研究结果如下： 1.全骨髓贴壁培养法分离纯化的rBMSCs呈长梭形、形态均一,具有向成骨细胞、脂肪细胞分化的潜能,表达特定的表面标志物；rBMSCs增殖曲线呈S形,细胞周期检测其增殖指数为21.71%；rBMSCs骨架蛋白与细胞长轴平行,发育良好。证实了分离培养的细胞为高纯度的rBMSCs,具有强大的迁移、增殖和分化能力。 2.利用血管内皮生长因子(vascular endothelial growth factor, VEGF)、成纤维细胞生长因子(fibroblast growth factor, bFGF)诱导rBMSCs内皮分化,获得的IECs呈典型的“铺路石”形态,能表达vWF因子；IECs的NO合成能力与天然ECs无显著性差异；IECs在Matrigel基质上能形成网状毛细血管样结构,具有成血管能力。 3.添力pNSR32的pNSR32/PCL/CS材料较PCL/CS更利于rBMSCs内皮分化,PECAM-1表达水平较高,达天然ECs水平；细胞粘附率及骨架蛋白表达结果表明在pNSR32/PCL/CS和PCL/CS上IECs均能很好地粘附、铺展；pNSR32/PCL/CS促进IECs分泌NO,成血管基因Angl表达量提高一倍,利于血管新生和成熟。
[Abstract]:Most cardiovascular diseases require vascular transplantation. At present, small diameter (6mm) vascular materials are prone to thrombosis and intimal hyperplasia. The key to solve these problems is to promote the formation of functional endodermis on the surface of materials, effectively inhibit thrombosis and intimal hyperplasia, and maintain long-term patency. The (endothelial cells, ECs) source and proliferation ability of natural endothelial cells is limited. The method of isolation of (bone mesenchymal stem cells, BMSCs) from bone marrow mesenchymal stem cells is simple and has the ability of self-renewal and multi-differentiation. So the researchers want to induce BMSCs to form IECs (induced endothelial cells). In addition, functional modification of materials is also an effective way to promote material endothelialization. In the early stage, the scaffolds of pNSR32/PCL/CS nanofibers were prepared by electrostatic spinning of RGD arachnoprotein pNSR32, polycaprolactone (polycaprolacton, PCL) and chitosan (chitosan,CS). The scaffolds have good mechanical properties and good cell and blood compatibility. At least 8 weeks of intact structure and patency of blood vessels could be maintained in rats. In order to solve the problem of limited sources of natural ECs, the endothelial differentiation of rat BMSCs (rBMSCs) was induced in vitro and the physiological function of IECs was investigated based on the isolated rat BMSCs (rBMSCs). The effects of pNSR32/PCL/CS scaffold on endothelial differentiation of rBMSCs and biological function of IECs were studied. The main results are as follows: 1. The rBMSCs isolated by whole bone marrow adherent culture was fusiform, homogeneous in morphology, able to differentiate into osteoblasts and adipocytes, and expressed specific surface markers. The proliferation curve of rBMSCs was S-shaped. The proliferation index of rBMSCs was 21.71%. The cytoskeleton protein of rBMSCs was parallel to the long axis of the cells and developed well. It was confirmed that the cells isolated and cultured were high purity rBMSCs, with strong ability of migration, proliferation and differentiation. 2. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF),) fibroblast growth factor (fibroblast growth factor, bFGF) was used to induce endothelial differentiation of rBMSCs. The obtained IECs showed typical "paving stone" morphology and expressed vWF factor. The NO synthesis ability of IECs was not significantly different from that of natural ECs, and IECs could form reticular capillary structure on Matrigel matrix and had vascular forming ability. 3. The pNSR32/PCL/CS material of pNSR32 was more favourable to rBMSCs endothelial differentiation than that of PCL/CS, and the expression of PECAM-1 was higher than that of PCL/CS, and reached the level of natural ECs. The results of cell adhesion rate and cytoskeleton protein expression showed that IECs could be well adhered to and spread on both pNSR32/PCL/CS and PCL/CS, and pNSR32/PCL/CS enhanced the expression of NO, angiogenic gene Angl in IECs, which was beneficial to angiogenesis and maturation.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R318.08
本文编号：2375082
[Abstract]:Most cardiovascular diseases require vascular transplantation. At present, small diameter (6mm) vascular materials are prone to thrombosis and intimal hyperplasia. The key to solve these problems is to promote the formation of functional endodermis on the surface of materials, effectively inhibit thrombosis and intimal hyperplasia, and maintain long-term patency. The (endothelial cells, ECs) source and proliferation ability of natural endothelial cells is limited. The method of isolation of (bone mesenchymal stem cells, BMSCs) from bone marrow mesenchymal stem cells is simple and has the ability of self-renewal and multi-differentiation. So the researchers want to induce BMSCs to form IECs (induced endothelial cells). In addition, functional modification of materials is also an effective way to promote material endothelialization. In the early stage, the scaffolds of pNSR32/PCL/CS nanofibers were prepared by electrostatic spinning of RGD arachnoprotein pNSR32, polycaprolactone (polycaprolacton, PCL) and chitosan (chitosan,CS). The scaffolds have good mechanical properties and good cell and blood compatibility. At least 8 weeks of intact structure and patency of blood vessels could be maintained in rats. In order to solve the problem of limited sources of natural ECs, the endothelial differentiation of rat BMSCs (rBMSCs) was induced in vitro and the physiological function of IECs was investigated based on the isolated rat BMSCs (rBMSCs). The effects of pNSR32/PCL/CS scaffold on endothelial differentiation of rBMSCs and biological function of IECs were studied. The main results are as follows: 1. The rBMSCs isolated by whole bone marrow adherent culture was fusiform, homogeneous in morphology, able to differentiate into osteoblasts and adipocytes, and expressed specific surface markers. The proliferation curve of rBMSCs was S-shaped. The proliferation index of rBMSCs was 21.71%. The cytoskeleton protein of rBMSCs was parallel to the long axis of the cells and developed well. It was confirmed that the cells isolated and cultured were high purity rBMSCs, with strong ability of migration, proliferation and differentiation. 2. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF),) fibroblast growth factor (fibroblast growth factor, bFGF) was used to induce endothelial differentiation of rBMSCs. The obtained IECs showed typical "paving stone" morphology and expressed vWF factor. The NO synthesis ability of IECs was not significantly different from that of natural ECs, and IECs could form reticular capillary structure on Matrigel matrix and had vascular forming ability. 3. The pNSR32/PCL/CS material of pNSR32 was more favourable to rBMSCs endothelial differentiation than that of PCL/CS, and the expression of PECAM-1 was higher than that of PCL/CS, and reached the level of natural ECs. The results of cell adhesion rate and cytoskeleton protein expression showed that IECs could be well adhered to and spread on both pNSR32/PCL/CS and PCL/CS, and pNSR32/PCL/CS enhanced the expression of NO, angiogenic gene Angl in IECs, which was beneficial to angiogenesis and maturation.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R318.08
【参考文献】
相关期刊论文 前2条
1 王露露;胡亮;刘超;周露;李庆平;;大鼠骨髓间充质干细胞体外培养可自发分化为内皮细胞[J];南京医科大学学报(自然科学版);2013年08期
2 陆树洋;孙晓宁;王春生;;小口径人工血管及其内皮化策略的研究进展[J];复旦学报(医学版);2013年01期
，本文编号：2375082
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