CAJ | 학술논문

目的建立无血清悬浮培养分离人胰腺癌细胞系PANC1干细胞球的方法.方法采用无血清悬浮法培养PANC1细胞,显微镜下观察干细胞球形成率;流式细胞仪检测细胞CD133表达和细胞周期;以含10%FBS培养基诱导干细胞球细胞分化,荧光显微镜下观察细胞形态及CK18的表达;干细胞球细胞接种NOD/SCID小鼠皮下,观察其成瘤能力.结果在无血清悬浮培养条件下存活的PANC1细胞形成干细胞球,体外连续传代培养20代始终保持4%0～5%0的干细胞球形成率.干细胞球细胞CD133表达率(5.91±0.7)%,G0/G1期细胞占(80.99±2.60)%,与原代PANC1细胞的(1.44±0.52)%和(69.01±5.03)%相差显著(P＜0.05).将于细胞球细胞置含血清培养基中培养.细胞逐渐恢复原代细胞形态,并表达上皮标志蛋白CK18,2×103的干细胞即在NOD/SCID小鼠皮下成瘤.结论无血清悬浮法培养PANC1细胞可分离出肿瘤干细胞,其具有自我更新、多向分化和成瘤能力.
목적 건립무혈청현부배양분리인이선암세포계PANC1간세포구적방법.방법 채용무혈청현부법배양PANC1세포,현미경하관찰간세포구형성솔;류식세포의검측세포CD133표체화세포주기;이함10%FBS배양기유도간세포구세포분화,형광현미경하관찰세포형태급CK18적표체;간세포구세포접충NOD/SCID소서피하,관찰기성류능력.결과 재무혈청현부배양조건하존활적PANC1세포형성간세포구,체외련속전대배양20대시종보지4%0～5%0적간세포구형성솔.간세포구세포CD133표체솔(5.91±0.7)%,G0/G1기세포점(80.99±2.60)%,여원대PANC1세포적(1.44±0.52)%화(69.01±5.03)%상차현저(P＜0.05).장우세포구세포치함혈청배양기중배양.세포축점회복원대세포형태,병표체상피표지단백CK18,2×103적간세포즉재NOD/SCID소서피하성류.결론 무혈청현부법배양PANC1세포가분리출종류간세포,기구유자아경신、다향분화화성류능력.
Objective To establish the method of suspension culture for stem cells from human pancreatic cancer cell line PANC1.Methods PANC1 cells were cultured in serum-free medium under floating-culture system.Tumor cell spheres were observed by optical microscope.Expression of CD133 and cell cycle were detected by flow cytometry.Cancer stem cells were induced to differentiate with 10%FBS,and expression of CK18,was evaluated with immunofluorescence microscope.Spheres cells were injected into the subcutaneous space of NOD/SCID rat and tumor formation was monitored weekly.Results PANC1 cells could form the stem cells spheres,and the rate of sphere formation was stable between 4%0 and 5%0 after 20 passages in vitro.The expression of CD133(5.91±0.7%)and proportion of G0/G1 phase cell(80.99±2.60%)was significantly increased in spheres cells compared with parental PANC1 cells(1.44±0.52%and 69.01±5.03%),and the difference was statistically significant(P＜0.05).When these spheres cells were cultured in media with serum,these cells gradually returned to the status of parental cells and expressed CK18,2×103 sphere cells injection could initiate tumor fornmtion in NOD/SCID rat.Conclusions Tumor spheres stem cellscould be generated under serum-free floating-culture system.The sphere cells possessed the capacities of self renew,difierentiation,and tumorigenic potential.