壳聚糖对视网膜神经细胞生长的促进作用及其作为基因载体的初步研究

发布时间：2018-04-03 16:27

  本文选题：视网膜神经节细胞　切入点：壳聚糖　出处：《华中科技大学》2012年硕士论文

【摘要】：目的：研究壳聚糖对体外分层及纯化培养的鼠视网膜神经节细胞生长的促进作用；制备载基因壳聚糖纳米粒，研究其特性及其对质粒gp130-pDNA3.1的保护能力。 方法：取SD乳鼠视网膜，在解剖显微镜下将视网膜分成内、外两层，分别将内外层及全层视网膜制成细胞悬液，各自进行培养。实验组加入含10%壳聚糖的完全培养基，对照组不加壳聚糖。每天观察细胞生长状态，MTT法检测细胞存活率。各层细胞分别用Thy1.1抗体鉴定神经节细胞，计算各层细胞的节细胞率。两步法提纯视网膜神经节细胞。计算提纯率。提纯后分别加入含10%血清、10%壳聚糖及鼠神经生长因子（NGF）的培养基，观察细胞生长状态。 复凝集法制备壳聚糖纳米粒，测定其粒径大小及zata电位；计算包封率，凝胶阻滞实验观察壳聚糖对质粒gp130-pDNA3.1的包裹能力,DNAaseⅠ消化实验观察壳聚糖对质粒的保护作用。 结果：分层培养细胞的存活率：各层细胞第1、2d存活率最高，MTT示：OD外层1.189±0.158，OD内层1.084±0.150，OD全层1.381±0.089；3d后随着培养时间的延长，，细胞存活率逐渐降低（P＜0.05）：外层OD3d0.835±0.120，OD4d0.805±0.116，OD5d0.782±0.107；内层：OD3d0.388±0.095，OD4d0.371±0.084，OD5d0.347±0.052；全层：OD3d0.523±0.047，OD4d0.340±0.084，OD5d0.227±0.057；相同时间点实验组（壳聚糖组）比对照组（不含壳聚糖）的存活率高（P＜0.05）。经免疫组化鉴定，内层细胞节细胞率最高(19.248±0.999)%，其次为全层(4.986±0.635)%，外层节细胞率最低（0.748±0.177）%。两步法提纯神经节细胞：提纯率可达95%以上。纯化的神经节细胞培养48h后测量最大轴突长度，壳聚糖组：62.5μm；NGF组：37.5μm；空白对照组：12.5μm。 复凝集法制备的载基因壳聚糖纳米粒分布均匀，平均直径约719nm，zata电位约17.2±0.8mV；包封率可达100%，琼脂糖凝胶电泳显示壳聚糖与gp130-pDNA3.1能有效结合；被壳聚糖包裹的gp130-pDNA3.1不会被DNAaseⅠ降解。结论：混合培养的各层细胞中，内层细胞的节细胞率最高。在混合培养的各层细胞中加入壳聚糖，能促进其生长并提高存活率。加入壳聚糖或NGF后，能提高纯化的神经节细胞的存活率。 复凝集法制备的载基因壳聚糖纳米粒分布均匀，带正电荷，能够有效包裹并保护质粒gp130-pDNA3.1。
[Abstract]:Aim: to study the effects of chitosan on the growth of cultured rat retinal ganglion cells in vitro and to study the properties of chitosan nanoparticles and their protective effect on plasmid gp130-pDNA3.1.Methods: the retina of newborn SD rats was divided into inner and outer layers under anatomic microscope. The inner and outer layers of retina and the whole layer of retina were made into cell suspensions and cultured separately.In the experimental group, 10% chitosan was added to the complete medium, while in the control group, the chitosan was not added.Cell survival rate was determined by MTT assay.The ganglion cells were identified by Thy1.1 antibody and the ganglion cell rate was calculated.Retinal ganglion cells were purified by two-step method.Calculate the purification rate.After purification, the cell growth state was observed by adding 10% serum 10% chitosan and rat nerve growth factor (NGF) medium respectively.Chitosan nanoparticles were prepared by complex agglutination method, their particle size and zata potential were measured, the encapsulation efficiency was calculated, and the encapsulation ability of chitosan to plasmid gp130-pDNA3.1 was observed by gel block assay. The protective effect of chitosan on plasmid was observed.Results: the survival rate of the cells in stratified culture: the highest survival rate was observed on the 1st day of each layer. The outer layer of OD was 1.189 卤0.158 and the inner layer of OD was 1.084 卤0.150. The whole layer of OD was 1.381 卤0.089 after 3 days, and with the extension of culture time, the survival rate of the cells in each layer was higher than that in the control group.缁嗚優瀛樻椿鐜囬

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