不同力学刺激对人BMSCs向软骨细胞分化的实验研究

发布时间：2018-05-19 03:33

本文选题：人类 + 骨髓间充质干细胞　；参考：《福建医科大学》2009年硕士论文

【摘要】： 目的:软骨缺损或损伤是常见的疑难病症之一。软骨细胞几乎没有迁徙能力,增生能力差,再生能力低,因此软骨缺损或损伤后难以自体修复。而且自体或异体移植成形的软骨其生物性能、耐磨性、韧性均欠佳,易蜕变。随着材料学、生命科学的发展,人们提出了一种新的方法—组织工程。骨髓间充质干细胞(BMSCs)具有分化为骨、软骨、肌腱、脂肪等组织的多向分化潜能,而且骨髓间充质干细胞取材方便,易于培养,自体移植无明显免疫排斥反应,因此可作为组织工程理想的种子细胞。本实验旨在通过BMSCs体外分离、培养、纯化、增殖条件下,将第3代BMSCs粘附到支架材料上,研究不同力学刺激对BMSCs向软骨细胞分化的影响,为今后的软骨损伤提供新的治疗方法。方法:应用密度梯度离心加贴壁培养筛选法分离纯化并增殖BMSCs,通过流式细胞仪检测BMSCs表面的CD44、CD45抗原标记,再结合BMSCs的生物学特征及定向分化潜能,以鉴定BMSCs。将第3代BMSCs制成2X107/ml细胞浓度,接种致孔径为200微米,孔隙率为85%的聚乳酸-聚羟基乙酸共聚物(PLGA)上,分别施以不同的离心力即100g、200g、300g。经过4周的受力后,通过HE染色、Ⅱ型胶原免疫组化、蛋白聚糖定量测定等方法来检测软骨细胞分泌的特异性标志物,以表明BMSCs向软骨细胞分化时的适宜力学刺激参数。结果:使用密度梯度离心加贴壁培养筛选法能够成功从人全骨髓中分离纯化BMSCs,获得的BMSCs能够自我更新增殖,但传至第8代时BMSCs增殖速度减慢,且胞体逐渐变得宽大、扁平,折光性能降低,出现老化现象。BMSCs能够很好得粘附在PLGA上生长,并分泌细胞基质,逐渐填满支架孔隙。在本实验的条件下,100g组、200g组、300g组所分泌的软骨细胞特异性基质均明显多于静止组,P0.01,差异具有统计学意义。而在受力组中,200g组所分泌的软骨细胞特异性基质均多于100g组、300g组,P0.01,差异具有统计学意义。100g组与300g组所分泌的软骨细胞特异性基质差异无统计学意义,P0.05。结论:密度梯度离心加贴壁培养筛选法能够便捷得从人全骨髓中分离纯化出BMSCs,BMSCs具有自我更新的能力,但不具有保留原有细胞特征无限增殖的能力。孔径为200微米,孔隙率为85%的PLGA支架,能够很好得为BMSCs提供粘附场所,从而能够更多的保留细胞所分泌的基质,更贴近细胞所生存的环境,有利于细胞生长发育。同时PLGA具有一定的强度,便于进行组织移植,为组织损伤进行移植治疗时提供前提。在本实验条件下,力学刺激有利于BMSCs向软骨细胞分化,而200g组更有利于BMSCs向软骨细胞分化。
[Abstract]:Objective: cartilage defect or injury is one of the most common diseases. Cartilage cells have almost no migratory ability, poor proliferative ability and low regeneration ability. Therefore, the cartilage defect or injury is difficult to repair after injury. Moreover, the biological properties, wear resistance and toughness of the cartilage formed by autologous or allograft are poor and easy to change. With material science and Life Science A new method, tissue engineering, is proposed. Bone marrow mesenchymal stem cells (BMSCs) have the multidirectional differentiation potential that differentiate into bone, cartilage, tendon and fat, and bone marrow mesenchymal stem cells are easily obtained, easy to be cultured and autologous transplantation without obvious immune rejection, so it can be used as the ideal seed of tissue engineering. This experiment aims to attach the third generation of BMSCs to the scaffold material under the conditions of isolation, culture, purification and proliferation of BMSCs in vitro, to study the effect of different mechanical stimuli on the differentiation of BMSCs to cartilage cells, and to provide a new method for the treatment of cartilage injury in the future.
Methods: the density gradient centrifugation and adherent culture screening method were used to isolate and proliferate BMSCs. The CD44 and CD45 antigens on the surface of BMSCs were detected by flow cytometry, then the biological characteristics of BMSCs and the potential of directional differentiation were combined to identify the BMSCs. concentration of 2X107/ml cells in third generation BMSCs, the inoculated pore diameter was 200 microns, and the porosity was 85%. On the poly (lactic acid polyglycolic acid) copolymer (PLGA), different centrifugal forces, namely, 100g, 200g, and 300g., were subjected to 4 weeks of force, and the specific markers of cartilage cell secretion were detected by HE staining, immunohistochemistry of type II collagen and quantitative determination of proteoglycan, so as to demonstrate the appropriate mechanical stimulation of BMSCs to chondrocyte differentiation. Parameters.
Results: the density gradient centrifugation and adherent culture screening method can successfully separate and purify BMSCs from the whole bone marrow, and the obtained BMSCs can self renew and proliferate, but the proliferation rate of BMSCs is slow at the time of eighth generation, and the cell body gradually becomes larger, flat, the refractive performance is reduced, and the aging phenomenon.BMSCs can be good to adhere to the PLGA. In this experiment, the specific matrix of chondrocytes secreted in group 100g, group 200g and 300g was significantly more than that in static group, P0.01, and the difference was statistically significant. In the group of stress group, the specific matrix of the soft bone cell secreted by 200g group was more than that of the 100g group, 300g group and P0.01. Statistically significant difference in chondrocyte specific matrix secretion between group.100g and group 300g, P0.05.
Conclusion: the density gradient centrifugation and adherent culture screening method can be convenient to separate and purify BMSCs from the whole bone marrow, and BMSCs has the ability of self renewal, but it does not have the ability to retain the unlimited proliferation of the original cell characteristics. The PLGA scaffold with a pore size of 200 microns and a porosity of 85% can provide a place of adhesion for BMSCs, so that it can be more effective. The matrix secreted by many retained cells is more close to the living environment of the cells and is beneficial to the growth and development of cells. At the same time, PLGA has a certain strength to facilitate tissue transplantation and provides a prerequisite for transplantation treatment for tissue damage. In this experimental condition, the mechanical stimulation is beneficial to the differentiation of BMSCs into chondrocytes, and the 200g group is more beneficial to BM. SCs differentiating into chondrocytes.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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