骨髓間充質(zhì)干細(xì)胞的內(nèi)皮分化及材料對(duì)分化影響的研究
發(fā)布時(shí)間：2018-12-12 18:26
【摘要】：心血管疾病大部分需要血管移植治療,目前小直徑(6mm)血管材料都面臨著易形成血栓、內(nèi)膜增生的困擾。解決這些問(wèn)題關(guān)鍵在于促進(jìn)材料表面形成功能化的內(nèi)皮層,有效抑制血栓形成和內(nèi)膜增生,維持長(zhǎng)期通暢。天然內(nèi)皮細(xì)胞(endothelial cells, ECs)來(lái)源和增殖能力有限,骨髓間充質(zhì)干細(xì)胞(bone mesenchymal stem cells, BMSCs)分離方法簡(jiǎn)單、具有自我更新和多向分化能力,因而研究者希望誘導(dǎo)BMSCs形成IECs(induced endothelial cells)。另外,材料的功能修飾也是促材料內(nèi)皮化的有效途徑。本課題組前期利用RGD蛛絲蛋白pNSR32、聚己內(nèi)酯(polycaprolacton, PCL)和殼聚糖(chitosan,CS)靜電紡絲制備pNSR32/PCL/CS三維納米纖維支架,其力學(xué)性能良好,細(xì)胞和血液相容性良好,在大鼠體內(nèi)能至少維持8w結(jié)構(gòu)完整和血管通暢。 為解決天然ECs來(lái)源有限的問(wèn)題,本研究以分離獲得的大鼠BMSCs(rBMSCs)為基礎(chǔ),體外誘導(dǎo)其內(nèi)皮分化,考察IECs的生理功能,并研究pNSR32/PCL/CS支架對(duì)rBMSCs內(nèi)皮分化及IECs生物學(xué)功能的影響,主要研究結(jié)果如下： 1.全骨髓貼壁培養(yǎng)法分離純化的rBMSCs呈長(zhǎng)梭形、形態(tài)均一,具有向成骨細(xì)胞、脂肪細(xì)胞分化的潛能,表達(dá)特定的表面標(biāo)志物；rBMSCs增殖曲線呈S形,細(xì)胞周期檢測(cè)其增殖指數(shù)為21.71%；rBMSCs骨架蛋白與細(xì)胞長(zhǎng)軸平行,發(fā)育良好。證實(shí)了分離培養(yǎng)的細(xì)胞為高純度的rBMSCs,具有強(qiáng)大的遷移、增殖和分化能力。 2.利用血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor, VEGF)、成纖維細(xì)胞生長(zhǎng)因子(fibroblast growth factor, bFGF)誘導(dǎo)rBMSCs內(nèi)皮分化,獲得的IECs呈典型的“鋪路石”形態(tài),能表達(dá)vWF因子；IECs的NO合成能力與天然ECs無(wú)顯著性差異；IECs在Matrigel基質(zhì)上能形成網(wǎng)狀毛細(xì)血管樣結(jié)構(gòu),具有成血管能力。 3.添力pNSR32的pNSR32/PCL/CS材料較PCL/CS更利于rBMSCs內(nèi)皮分化,PECAM-1表達(dá)水平較高,達(dá)天然ECs水平；細(xì)胞粘附率及骨架蛋白表達(dá)結(jié)果表明在pNSR32/PCL/CS和PCL/CS上IECs均能很好地粘附、鋪展；pNSR32/PCL/CS促進(jìn)IECs分泌NO,成血管基因Angl表達(dá)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.
【學(xué)位授予單位】：福建師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R318.08
本文編號(hào)：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.
【學(xué)位授予單位】：福建師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R318.08
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 王露露;胡亮;劉超;周露;李慶平;;大鼠骨髓間充質(zhì)干細(xì)胞體外培養(yǎng)可自發(fā)分化為內(nèi)皮細(xì)胞[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2013年08期
2 陸樹洋;孫曉寧;王春生;;小口徑人工血管及其內(nèi)皮化策略的研究進(jìn)展[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版);2013年01期
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