CAJ | 학술논문

目的：探讨源于小鼠的诱导多能干细胞（ induced pluripotent stem cells ， iPSCs）体外定向分化为神经元的方法，为大量稳定地获得神经元及提供种子细胞治疗脊髓损伤奠定体外实验基础。方法：源于小鼠的iPSCs在不同的诱导培养基中经悬浮、贴壁、再悬浮和再贴壁培养。免疫荧光检测iPSCs多能性标志物、神经干细胞及其分化细胞标志物的表达。 RT-PCR检测神经元及胶质细胞基因表达并行神经元基因测序鉴定，流式细胞术检测iP-SCs定向分化为神经干细胞及进一步分化为神经元的比例。结果：源于小鼠的iPSCs表达干细胞多能性标志物Sox2、Oct4和SSEA1。悬浮培养可以形成类球形拟胚体；经诱导及机械分离后的细胞可形成神经球，神经球经诱导后可分化为神经元。免疫荧光显示iPSCs可诱导出表达nestin的神经球，贴壁培养的神经球可分化为分别表达微管相关蛋白2（MAP-2）、胶质细胞原纤维酸性蛋白（GFAP）及髓磷脂碱性蛋白（MBP）的神经元、星形胶质细胞和少突胶质细胞。 RT-PCR及基因鉴定结果显示形成小鼠神经元；流式细胞术检测结果显示分化细胞中神经干细胞和神经元的比例分别为63．93％±1．47％和21．40％±1．70％。结论：源于小鼠的诱导多能干细胞能够稳定地在体外定向分化为神经元，为脊髓损伤的治疗提供了一个可靠的细胞来源。
목적：탐토원우소서적유도다능간세포（ induced pluripotent stem cells ， iPSCs）체외정향분화위신경원적방법，위대량은정지획득신경원급제공충자세포치료척수손상전정체외실험기출。방법：원우소서적iPSCs재불동적유도배양기중경현부、첩벽、재현부화재첩벽배양。면역형광검측iPSCs다능성표지물、신경간세포급기분화세포표지물적표체。 RT-PCR검측신경원급효질세포기인표체병행신경원기인측서감정，류식세포술검측iP-SCs정향분화위신경간세포급진일보분화위신경원적비례。결과：원우소서적iPSCs표체간세포다능성표지물Sox2、Oct4화SSEA1。현부배양가이형성류구형의배체；경유도급궤계분리후적세포가형성신경구，신경구경유도후가분화위신경원。면역형광현시iPSCs가유도출표체nestin적신경구，첩벽배양적신경구가분화위분별표체미관상관단백2（MAP-2）、효질세포원섬유산성단백（GFAP）급수린지감성단백（MBP）적신경원、성형효질세포화소돌효질세포。 RT-PCR급기인감정결과현시형성소서신경원；류식세포술검측결과현시분화세포중신경간세포화신경원적비례분별위63．93％±1．47％화21．40％±1．70％。결론：원우소서적유도다능간세포능구은정지재체외정향분화위신경원，위척수손상적치료제공료일개가고적세포래원。
AIM:To study the induction method of mouse induced pluripotent stem cells (iPSCs) that differ-entiate into neurons in vitro.METHODS:Mouse iPSCs were cultured in non-adherent culture dishes for 2 d to form em-bryoid bodies (EBs).The EBs were cultured for consecutive 2 d in the presence of retinoic acid (RA), and then were plated in the serum-free medium for adherent culture .Seven days later , Pasteur pipette was used to detach the differentia-ted cells around adherent EBs into “fragment” cell colonies with the help of dissecting microscopes , and these“fragments”were transferred to culture dishes with neural stem cell medium .Another 7 days later, the cells were plated onto the culture dishes using differentiation medium containing fetal bovine serum ( FBS) and RA.The morphological changes of the cells were observed under inverted microscope .The iPSCs markers Oct4, Sox2 and SSEA1, the neural stem cell (NSC) marker nestin, the neuronal marker microtubule-associated protein 2 (MAP-2), the astrocyte marker glial fibrillary acidic protein ( GFAP) and oligodendrocyte marker myelin basic protein ( MBP) were detected by immunofluorescence method .The mR-NA expression of GFAP, nestin,β3-tubulin, MAP-2 and MBP was detected by RT-RCR.MAP-2 gene sequence was iden-tified.The proportions of NSCs differentiated from iPSCs and neurons from NSCs were detected by flow cytometry .RE-SULTS:Mouse iPSCs strongly expressed Oct4, Sox2 and SSEA1, and formed spherical EBs by suspended culture .The EBs were induced by RA and serum-free medium in adherent culture for 2 d, and rosette structure was observed under the microscope .“Fragments” separated by Pasteur pipette from the rosette structure formed neurosphere-like colonies .After the colonies were cultured in adherent condition for 5 d to 7 d in the presence of RA and FBS , the typical neurite was ob-served under the microscope .The neurospheres expressed nestin and their differentiated derivatives expressed MAP -2, GFAP and MBP, respectively.RT-PCR analysis and gene sequencing showed that the neurons were induced successfully . The results of flow cytometry demonstrated that 63.93%±1.47%of iPSCs differentiated into NSCs and 21.4%±1.70%of NSCs differentiated into neurons .CONCLUSION: Mouse iPSCs proliferate stably and differentiate into neurons in vitro, which provide a reliable source for the treatment of spinal cord injury .