本文選題：人類 + 骨髓間充質(zhì)干細胞　； 參考：《福建醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的:軟骨缺損或損傷是常見的疑難病癥之一。軟骨細胞幾乎沒有遷徙能力,增生能力差,再生能力低,因此軟骨缺損或損傷后難以自體修復(fù)。而且自體或異體移植成形的軟骨其生物性能、耐磨性、韌性均欠佳,易蛻變。隨著材料學(xué)、生命科學(xué)的發(fā)展,人們提出了一種新的方法—組織工程。骨髓間充質(zhì)干細胞(BMSCs)具有分化為骨、軟骨、肌腱、脂肪等組織的多向分化潛能,而且骨髓間充質(zhì)干細胞取材方便,易于培養(yǎng),自體移植無明顯免疫排斥反應(yīng),因此可作為組織工程理想的種子細胞。本實驗旨在通過BMSCs體外分離、培養(yǎng)、純化、增殖條件下,將第3代BMSCs粘附到支架材料上,研究不同力學(xué)刺激對BMSCs向軟骨細胞分化的影響,為今后的軟骨損傷提供新的治療方法。 方法:應(yīng)用密度梯度離心加貼壁培養(yǎng)篩選法分離純化并增殖BMSCs,通過流式細胞儀檢測BMSCs表面的CD44、CD45抗原標記,再結(jié)合BMSCs的生物學(xué)特征及定向分化潛能,以鑒定BMSCs。將第3代BMSCs制成2X107/ml細胞濃度,接種致孔徑為200微米,孔隙率為85%的聚乳酸-聚羥基乙酸共聚物(PLGA)上,分別施以不同的離心力即100g、200g、300g。經(jīng)過4周的受力后,通過HE染色、Ⅱ型膠原免疫組化、蛋白聚糖定量測定等方法來檢測軟骨細胞分泌的特異性標志物,以表明BMSCs向軟骨細胞分化時的適宜力學(xué)刺激參數(shù)。 結(jié)果:使用密度梯度離心加貼壁培養(yǎng)篩選法能夠成功從人全骨髓中分離純化BMSCs,獲得的BMSCs能夠自我更新增殖,但傳至第8代時BMSCs增殖速度減慢,且胞體逐漸變得寬大、扁平,折光性能降低,出現(xiàn)老化現(xiàn)象。BMSCs能夠很好得粘附在PLGA上生長,并分泌細胞基質(zhì),逐漸填滿支架孔隙。在本實驗的條件下,100g組、200g組、300g組所分泌的軟骨細胞特異性基質(zhì)均明顯多于靜止組,P0.01,差異具有統(tǒng)計學(xué)意義。而在受力組中,200g組所分泌的軟骨細胞特異性基質(zhì)均多于100g組、300g組,P0.01,差異具有統(tǒng)計學(xué)意義。100g組與300g組所分泌的軟骨細胞特異性基質(zhì)差異無統(tǒng)計學(xué)意義,P0.05。 結(jié)論:密度梯度離心加貼壁培養(yǎng)篩選法能夠便捷得從人全骨髓中分離純化出BMSCs,BMSCs具有自我更新的能力,但不具有保留原有細胞特征無限增殖的能力�？讖綖�200微米,孔隙率為85%的PLGA支架,能夠很好得為BMSCs提供粘附場所,從而能夠更多的保留細胞所分泌的基質(zhì),更貼近細胞所生存的環(huán)境,有利于細胞生長發(fā)育。同時PLGA具有一定的強度,便于進行組織移植,為組織損傷進行移植治療時提供前提。在本實驗條件下,力學(xué)刺激有利于BMSCs向軟骨細胞分化,而200g組更有利于BMSCs向軟骨細胞分化。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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