嗅鞘细胞原代培养及其与脊髓损伤修复材料的作用研究

发布时间：2018-01-30 04:57

  本文关键词： 嗅鞘细胞 PRGD 蛋白吸附 亲水性 细胞亲和性　出处：《武汉理工大学》2007年硕士论文　论文类型：学位论文

【摘要】： 近年来，利用嗅鞘细胞移植治疗脊髓神经损伤已成为最为引人注目的方法，但是单纯用细胞移植来修复脊髓损伤，缺乏理想的载体，细胞存活时间短，且不能很好地迁移，从而影响了神经修复和功能恢复。因此，为嗅鞘细胞选择合适的支架材料是亟待解决的问题。这些合成材料需要具有生物相容性好、对嗅鞘细胞的黏附性强的特点，，同时细胞黏附在其表面以后能够维持很好的活性并持续生长。 本研究比较了几种分离与纯化嗅鞘细胞的方法，优选了改良的NASH法作为实验方法，从新生大鼠的嗅球提取嗅鞘细胞，并对其进行了纯化，采用GFAP、P~(75)以及S-100免疫组化染色法鉴定其特性及纯度，结果显示采用改良的NASH法提取和分离的嗅鞘细胞的数量最多，且纯度最高，达80％以上，是一种经济而有效的方法，为本课题的研究提供了嗅鞘细胞。 本研究合成了聚乳酸(PLA)和聚(羟基乙酸-L-赖氨酸-乳酸)，简称PLGK，以及将PLGK接枝了RGD，合成了PRGD材料，选用1mg／mL的血清白蛋白(BSA)与三种材料作用，通过Bradford法，分别在不同的作用时间，用酶标仪测定并计算三种材料表面的蛋白吸附量，结果显示PLA在最初的2h，蛋白吸附量较PLGK和PRGD多，随：着时间的延长，PRGD吸附蛋白的量有所增加，4小时后，高于其他两者；将三种材料PLA、PLGK和PRGD分别与5％的BSA37℃震荡孵育12h，取出干燥后，采用FT-IR测试三种材料表面，通过比较三种材料与BSA作用前后的光谱图，分析材料对BSA的吸附情况，这一结果与蛋白吸附结果一致。 分别在三种材料PLA、PLGK和PRGD表面滴加一滴去离子水，在常温下采用ZAP高温显微镜观测，并摄像，图像处理系统测试水接触角，比较三种材料的亲／疏水性。通过测试水接触角，三种材料的亲水性由强到弱依次为PRGD＞PLGK＞PLA。 将纯化后的嗅鞘细胞分别接种于PLA、PLGK和PRGD材料表面，共培养3天后，通过MTT检测OD值来评价材料表面细胞活性；细胞计数法计算细胞粘附率；环境扫描电镜以及S-100染色后荧光显微镜观察细胞在材料表面的生长状态，结果显示，材料PRGD表面细胞数目多，活性好，细胞粘附率高，细胞生长良好。 综上所述，材料PRGD的亲水性强，蛋白吸附性能好，嗅鞘细胞在其表面黏附性高，细胞生长活性强，生长状态良好；材料PRGD是一种细胞亲和性强、生物相容性好的材料。
[Abstract]:In recent years, olfactory ensheathing cell transplantation has become the most attractive method for the treatment of spinal cord nerve injury, but only with cell transplantation to repair spinal cord injury, lack of ideal carrier, cell survival time is short. Therefore, the selection of appropriate scaffold materials for olfactory ensheathing cells is an urgent problem. These synthetic materials need to have good biocompatibility. The cell adhesion to olfactory ensheathing cells is strong, and cell adhesion on its surface can maintain good activity and continue to grow. In this study, several methods of separation and purification of olfactory ensheathing cells were compared. The improved NASH method was selected as the experimental method to extract and purify olfactory ensheathing cells from olfactory bulb of newborn rats. The results showed that the number of olfactory ensheathing cells extracted and isolated by modified NASH method was the highest. The highest purity of above 80% is an economical and effective method, which provides olfactory ensheathing cells for this study. Polylactic acid (PLA) and poly (glycolic acid-L-lysine-lactic acid) (PLGK) were synthesized, and PLGK was grafted onto PRGD. One mg / mL of serum albumin (BSA) was used to react with three kinds of materials. By Bradford method, the reaction time was different. The amount of protein adsorbed on the surface of the three materials was measured and calculated by enzyme labeling instrument. The results showed that the amount of protein adsorbed on the surface of PLA was more than that of PLGK and PRGD at the first 2 h, and with the prolongation of the time. The amount of PRGD adsorbed protein increased after 4 hours, and was higher than that of the other two. The three materials PLAGK and PRGD were incubated at 5% BSA37 鈩

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