靶向示踪SPIO标记的OECs移植治疗大鼠脊髓损伤的研究

发布时间：2018-05-18 00:28

  本文选题：嗅鞘细胞 + 脊髓损伤　； 参考：《宁夏医科大学》2016年硕士论文

【摘要】：目的1.从SD新生鼠嗅黏膜取材原代培养提纯鉴定嗅鞘细胞。2.探讨嗅鞘细胞在体外模拟的脊髓损伤缺氧内环境中是否诱导自噬,并检测自噬对嗅鞘细胞增殖能力的影响。3.验证嗅鞘细胞移植对大鼠半横断损伤的治疗作用。4.探讨SPIO体外标记嗅鞘细胞的合适方案,以及体外成像特点。方法1.用酶消化法和改良的Nash差速贴壁法分离提纯嗅鞘细胞,采用CCK-8法绘制生长曲线,选出增殖较好的细胞代次;利用免疫荧光法鉴定细胞属性和纯度。2.采用氧浓度为2%作为低氧条件(低氧处理组),培养SD新生鼠嗅黏膜来源的OECs,以正常条件下培养的OECs作为对照组,采用自噬抑制剂3-MA处理后的OECs继续在低氧条件下培养作为3-MA低氧处理组,在相差显微镜下观察细胞形态;对照组、4 h低氧处理组、8 h低氧处理组,Western blotting法检测各组OECs中HIF-1α(Hypoxia inducible factor-1,低氧诱导因子-1α)、自噬标志蛋白LC3Ⅰ/Ⅱ和自噬标志基因Beclin-1的蛋白表达水平;对照组、自噬抑制剂3-MA处理的OECs然后在正常条件下培养作为3-MA处理组、低氧组、3-MA低氧处理组,采用CCK-8法检测各组OECs的增殖能力。3.建立SD大鼠脊髓左侧半横断损伤模型,实验组伤后立即注射嗅鞘细胞,对照组只注射完全培养基,单纯损伤组不注射。于1、2、4、6、8周对各组实验鼠进行大鼠后肢运动功能(BBB)评分。明确嗅鞘细胞移植对脊髓半横断损伤大鼠的治疗效果。4.用多聚左旋赖氨酸包被过的SPIO纳米粒子标记OECs,普鲁士蓝染色和透射电镜下观察标记结果,对标记后的嗅鞘细胞进行细胞活性和增殖能力的检测并实现体外MRI成像。结果1.经原代分离、提纯培养的SD新生鼠嗅黏膜来源嗅鞘细胞大多数呈梭形,典型的双极、三极细胞形态。激光共聚焦显微镜下GFAP和P75双染阳性的为嗅鞘细胞。根据计算公式其纯度可达87%。2.与对照组比较,低氧组嗅鞘细胞中HIF-1α表达水平增高(P0.05),LC3Ⅰ向LC3Ⅱ的转化明显,Beclin-1表达水平增高(P0.05);自噬抑制组中嗅鞘细胞增殖能力降低(P0.05)。3.实验组与对照组和单纯损伤组比较,P0.05,具有统计学差异;对照组与单纯损伤组在各个时间点比较P0.05,无统计学差异;随着时间的推移各组BBB评分也在升高。4.成功应用SPIO标记嗅鞘细胞且不影响其生物学特性,MRI体外成像显示1×106数量级细胞在轴位和矢状位的T2WI和SWI序列上信号可较好显示。结论1.低氧诱导的自噬对大鼠嗅鞘细胞的增殖能力具有促进作用。2.嗅鞘细胞移植对脊髓半横断损伤有治疗作用。3.成功应用SPIO标记嗅鞘细胞且不影响细胞基本生物学特性和增殖能力,并实现MRI体外成像,为以后嗅鞘细胞移植入脊髓的示踪问题奠定了基础。
[Abstract]:Objective 1. Olfactory ensheathing cells. 2. 2 were isolated from olfactory mucosa of newborn SD rats. To investigate whether olfactory ensheathing cells induce autophagy in anoxic environment of spinal cord injury in vitro and to detect the effect of autophagy on the proliferation of olfactory ensheathing cells. To verify the therapeutic effect of olfactory ensheathing cell transplantation on hemitranverse injury in rats. 4. 4. Objective: to investigate the appropriate scheme of SPIO labeling olfactory ensheathing cells in vitro and the imaging characteristics in vitro. Method 1. Olfactory ensheathing cells were isolated and purified by enzyme digestion method and modified Nash differential adherence method. The growth curve was drawn by CCK-8 method and the cells with better proliferation were selected. The cell properties and purity were identified by immunofluorescence method. OECs derived from olfactory mucosa of newborn SD rats were cultured under hypoxic conditions (oxygen concentration of 2%) and OECs cultured in normal condition as control group. OECs treated with autophagy inhibitor 3-MA continued to be cultured in hypoxic condition as 3-MA hypoxia group, and the morphology of cells was observed under phase contrast microscope. Western blotting assay was used to detect the expression of HIF-1 伪 -Hypoxia inducible factor-1, hypoxia inducible factor-1 伪, autophagy marker LC3 鈪，

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