中华鳖胚胎肝成纤维细胞的原代培养及其聚肌苷酸胞苷酸(Poly I:C)刺激模型构建

为建立中华鳖胚胎肝成纤维细胞的体外分离培养体系,研究聚肌苷酸胞苷酸(Poly I:C)刺激对细胞干扰素生成通路相关因子的影响,从而最终构建基于该成纤维细胞的干扰素生成通路激活模型,以发育至23期的中华鳖胚胎为实验材料,采用胰蛋白酶消化法得到肝成纤维细胞,结合细胞形态学、生长曲线和PCR法进行鉴定,并分析其生物学特性.之...

Primary culture of fibroblast from embryonic liver of Chinese soft-shelled turtle (Pelodiscus sinensis) and establishment of polyinosinic acid cytidylic acid (Poly I:C) stimulation cell model

The aims of the present study were to establish an in vitro isolation and culture system for the embryonic liver fibroblast of Chinese soft-shelled turtle(Pelodiscus sinensis), and to study the effect of polyinosinic acid cytidylic acid (Poly I:C) stimulation on the key factors of the cellular interferon production pathway, finally, to establish the activation model of interferon production pathway based on the fibroblast. The embryo developed to stage 23 was used to obtain the liver fibroblast by trypsin digesting. After that, the cell was identified by PCR method. Its biological characteristics including cell morphology and growth pattern were analyzed. Besides, the gene and protein expression levels of the factors related to the interferon production signal pathway were detected at 24 h after cell transfection with Poly I:C. The results showed that the obtained embryonic liver fibroblasts were in good growth condition and had been successfully passed to the 20^th generation. The growth curve of the cell was S-shaped, which conformed to the cell proliferation law. Fibroblast marker genes VIM and ACTA2 were confirmed. Before and after cryopreservation, the cell survival rates were 90.51% and 82.95%, respectively. After Poly I:C stimulation, the protein expressions of RIG-Ⅰ and MAVS were up-regulated, the gene expression of RIG-Ⅰ was up-regulated. Whereas, the gene expressions of m⁶A and YTHDF2 were down-regulated. In addition, the change of cell morphology was recorded. In conclusion, the study successfully established an in vitro isolation and culture system for embryonic liver fibroblasts and constructed an activation model of interferon production pathway based on embryonic liver fibroblasts of Chinese soft-shelled turtle.