CAJ | 학술논문

目的探讨通过Nunc Upcell温度敏感型培养皿获得尿道下裂患儿尿源性细胞膜片的可行性.方法排尿时,对尿道下裂患儿尿源性细胞进行收集和原代培养,传代后计数细胞并绘制生长曲线,观察细胞形态学特征,RT PCR、细胞免疫荧光染色鉴定尿源性细胞特异mRNA及蛋白表达情况；将尿道下裂患儿尿源性细胞转移入Thermo公司的Nunc Upcell中,根据温度的变化获得该细胞的细胞膜片.结果镜下观察和生长曲线图显示:尿道下裂患儿来源的尿源性细胞具有与正常人的尿源性细胞类似的体外增殖能力,培养至第十代生长仍很旺盛,可以达到109个细胞,能够满足组织工程所需要的细胞数量；2组细胞具有类似的群体倍增时间；RT-PCR和细胞免疫荧光染色显示这2组尿源性细胞均表达尿道上皮细胞和平滑肌细胞表面标志物；利用Nunc Upcell温度敏感型培养皿可以转移得到完整的细胞膜片,且转移后的细胞仍保持旺盛的增殖能力.结论尿道下裂患儿来源的尿源性细胞可在体外大量增殖,可作为一种新型的种子细胞用于泌尿系统组织工程;结合使用新型的Nunc Upcell温度敏感型细胞培养皿,可获得尿道下裂患儿尿源性来源细胞的细胞膜片,为将来进行尿道缺损修复的动物及临床试验奠定了良好基础.
목적 탐토통과Nunc Upcell온도민감형배양명획득뇨도하렬환인뇨원성세포막편적가행성.방법 배뇨시,대뇨도하렬환인뇨원성세포진행수집화원대배양,전대후계수세포병회제생장곡선,관찰세포형태학특정,RT PCR、세포면역형광염색감정뇨원성세포특이mRNA급단백표체정황；장뇨도하렬환인뇨원성세포전이입Thermo공사적Nunc Upcell중,근거온도적변화획득해세포적세포막편.결과 경하관찰화생장곡선도현시:뇨도하렬환인래원적뇨원성세포구유여정상인적뇨원성세포유사적체외증식능력,배양지제십대생장잉흔왕성,가이체도109개세포,능구만족조직공정소수요적세포수량；2조세포구유유사적군체배증시간；RT-PCR화세포면역형광염색현시저2조뇨원성세포균표체뇨도상피세포화평활기세포표면표지물；이용Nunc Upcell온도민감형배양명가이전이득도완정적세포막편,차전이후적세포잉보지왕성적증식능력.결론 뇨도하렬환인래원적뇨원성세포가재체외대량증식,가작위일충신형적충자세포용우비뇨계통조직공정;결합사용신형적Nunc Upcell온도민감형세포배양명,가획득뇨도하렬환인뇨원성래원세포적세포막편,위장래진행뇨도결손수복적동물급림상시험전정료량호기출.
Objective To investigate the feasibility of acquiring cell sheet using temperature-responsive culture dishes (Nunc Upcell) in children with hypospadias.Methods The clean urine were collected from children with hypospadias.The urine-derived cells were cultured and characterized by cell growth curve,RT-PCR and immunofluorescene staining was employed to identify the mRNA and protein expression in them.Afterwards,cells were then seeded on Nunc Upcell dishes,and acquired the cells sheet according to differential temperature.Results The phase contrast microscopic observation and cell growth curve revealed that the expansion capacity of urine-derived cells from children with hypospadias were identical to those of normal ones.Cells could actively grow to passage 10 and number is up to 109 cells,which completely satisfy the application of tissue engineering.Two groups of cells had the similar population doubling time and expressed urothelial and smooth muscle cell surface markers could be noted in RT PCR and immune-staining.Furthermore,the intact cell sheets could be obtained from the temperature-responsive culture dishes and cells were able to maintain ideal proliferation capacity after transferring.Conclusions The urine-derived cells from children with hypospadias have in vitro expand extensively and can be served as an alternative cell source for urinary tract tissue engineering.The intact cell sheets can also be obtained via temperature-responsive culture dishes,which provide a better platform for the following study of urethral reconstruction or other urological tissue repair.