摘要
目的探讨小鼠角膜上皮细胞永生化细胞系的建立方法,并鉴定其表型的稳定性。方法提取小鼠角膜上皮原代细胞,以猴空泡病毒40大T抗原(SV40-LT)慢病毒感染小鼠角膜上皮原代细胞建立永生化细胞系。通过HE染色与免疫荧光染色对角膜上皮组织表型和标记物(K12、Pax6、Ki-67)进行鉴定。提取原代角膜上皮细胞并对其角膜上皮细胞分层蛋白K14进行鉴定,观察细胞明场视野评估细胞形态学特征。对转染后的P1和P8代细胞的部分标志物(K12、Pax6、Ki-67、ABCG2、K14)进行鉴定。采用结晶紫染色法观察转染后的P1和P8代细胞克隆的形成能力。结果小鼠角膜上皮组织中K12、Pax6、Ki-67染色阳性;P1、P8代小鼠角膜上皮细胞中K12、Pax6、Ki-67、K14和ABCG2染色阳性,上述标志物阳性率比较,差异均无显著性(P>0.05),P1与P8代小鼠角膜上皮细胞克隆能力无明显差异(P>0.05)。结论使用SV40-LT慢病毒转染小鼠角膜上皮,成功建立永生化小鼠角膜上皮细胞系,表型稳定且稳定增殖。
Objective To investigate the method for establishing an immortalized mouse corneal epithelial cell line,and to identify the stability of its phenotype.Methods Primary mouse corneal epithelial cells were extracted and transfected with Simian Vacuolating Virus 40 Large T Antigen(SV40-LT)lentivirus to establish an immortalized cell line.HE staining and immunofluorescent staining were used to identify the phenotypes and markers(K12,Pax6,Ki-67)of corneal epithelium.Primary cor-neal epithelial cells were extracted to identify K14 protein in corneal epithelial cell layer,and bright field observation was performed to evaluate cell morphological characteristics.Several markers(K12,Pax6,Ki-67,ABCG2,K14)were identified for P1 and P8 cells after transfection.Crystal violet staining was used to observe the colony-formation ability of P1 and P8 cells after transfection.Results Mouse corneal epithelium was stained positive for K12,Pax6 and Ki-67,while the P1 and P8 generations of mouse cor-neal epithelial cells were stained positive for K12,Pax6,Ki-67,K14,and ABCG2;there were no significant differences in the positive rates of the above markers(P>0.05).There was no significant difference in colony-formation ability between P1 and P8 cells(P>0.05).Conclusion The immortalized mouse corneal epithelial cell line is successfully established by transfection with SV40-LT lentivirus,which has stable phenotype and proliferation.
作者
白莹
王一卉
葛欢欢
卞雯晗
陈鹏
BAI Ying;WANG Yihui;GE Huanhuan;BIAN Wenhan;CHEN Peng(Department of Human Anatomy,Histology and Embryology,School of Basic Medicine,Qingdao University,Qingdao 266071,China)
出处
《精准医学杂志》
2024年第2期134-138,143,共6页
Journal of Precision Medicine
基金
国家自然科学基金资助项目(81970782)。
