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目的建立体外稳定获取大数量及高纯度脊髓神经元的培养方案,比较当前国内外最常用的无血清培养基(Neurobasal+B27,NCM)与传统含血清培养基(DMEM/F12+10%胎牛血清+5%马血清,SCM)对体外培养脊髓神经元生长状态的影响。方法取E14-15d Wistar大鼠的胚胎脊髓组织行胰酶消化获取脊髓神经元的单细胞悬液,分别用NCM与SCM进行培养,差速贴壁法及Ara-C干预纯化培养的神经元。于接种后第1、2、3、4、5、6、7d倒置相差显微镜下观察细胞生长状态的变化,并用免疫荧光细胞化学法比较两种培养基培养下获取的神经元纯度。结果采用NCM培养的脊髓神经元生长良好,对环境变化如换液及加入Ara-C更为耐受;NCM培养的神经元的纯度为(87.70±8.70)%,SCM培养的神经元纯度为(78.61±7.00)%,前者纯度更高(P=0.019)。结论NCM较之SCM更加适合脊髓神经元的体外培养。
OBJECTIVE: To establish a culture protocol for obtaining large numbers and high-purity spinal cord neurons stably in vitro. Compared with the most commonly used serum-free medium (Neurobasal + B27, NCM) at home and abroad, the traditional culture medium containing DMEM / F12 + 10% Effects of Serum + 5% Horse Serum and SCM on the Growth Status of Spinal Cord Neurons in. Methods The embryonic spinal cord tissues from E14-15d Wistar rats were harvested and trypsinized to obtain single cell suspension of spinal cord neurons. Cultured in NCM and SCM respectively, the neurons were purified by differential adherence and Ara-C treatment. The changes of cell growth state were observed under inverted phase contrast microscopy on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th day after inoculation, and the purity of neurons obtained from the two culture media was compared by immunofluorescence cytochemistry. Results The spinal cord neurons cultured with NCM grew well and were more tolerant to environmental changes such as fluid exchange and addition of Ara-C. The purity of neurons cultured in NCM was (87.70 ± 8.70)% and the purity of neurons cultured in SCM was ( 78.61 ± 7.00)%, the former purity higher (P = 0.019). Conclusion NCM is more suitable than SCM for the culture of spinal neurons in vitro.