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目的:观察原浆型星形胶质细胞作为饲养层细胞对神经干细胞(NSC)分化行为的影响。方法:通过无血清培养的方法,将原代培养所获得的NSC克隆经纯化和标记后,以原浆型星形胶质细胞作为饲养层细胞,10d后进行NF-200免疫荧光染色,在显微镜下随机选取20个视野,记数神经元和标记NSC的比例。通过CX32免疫组化染色观察分化细胞间的信号联系;利用Fura-3探针标记和药物刺激检测分化神经元的电活动变化。结果:①在原浆型星形胶质细胞培养条件下,NSC分化为神经元的比例高达75%;②部分分化细胞CX32染色为阳性;③分化神经元在药物刺激下,可发生Ca2+活动的变化。结论:原浆型星形胶质细胞具有较好地促进NSC向神经元分化,且分化的神经细胞具有生物学活性,可作为神经组织工程研究中一种新的种子细胞。
Objective: To observe the effect of primary astrocyte as feeder cells on the differentiation of neural stem cells (NSCs). Methods: NSC clones obtained by primary culture were purified and labeled by serum-free culture method. Primary myeloid astrocytes were used as feeder cells. After 10 days, NF-200 immunofluorescence staining was performed. 20 fields were randomly selected to record the proportion of neurons and labeled NSC. CX32 immunohistochemical staining was used to observe the signal transduction between differentiated cells. Fura-3 probe labeling and drug stimulation were used to detect the changes of electrical activity of differentiated neurons. RESULTS: ① In primary astrocyte culture, the percentage of NSCs differentiated into neurons was as high as 75%. ② The differentiated cells were positive for CX32 staining. ③ The differentiated neurons could change in activity of Ca2 + under drug stimulation . CONCLUSION: Primary astrocyte can promote the differentiation of NSC into neurons, and the differentiated neural cells have biological activity, which can be used as a new kind of seed cells in neuro-tissue engineering.