CAJ | 학술논문

目的探索小鼠精原干细胞(SSCs)冷冻保存方法以及解冻后体外快速增殖的条件. 方法实验以6d龄雄性昆明小白鼠为材料,两步酶消化法分离小鼠睾丸生殖细胞,Percoll非连续密度梯度离心法富集小鼠精原干细胞,随后加入不同的冷冻液以及采用不同的降温速率冷冻小鼠精原干细胞.以MEMα为基本培养基,加入10%胎牛血清和100μg/L的胶质细胞源性神经营养因子, WST-8比色法分析培养SSCs 复苏后的增殖率,运用碱性磷酸酶细胞化学染色和RT-PCR技术,对培养的SSCs进行鉴定. 结果冷冻液中添加10%二甲基亚砜、10%胎牛血清、0.07mol/L蔗糖时,以1℃/min程控降温方式冷冻小鼠SSCs,细胞解冻后活率最高,达84%以上;采用非程控降温方式冻存SSCs,尽管细胞解冻后活率相对于程控降温方式略低,但具有方法简单、易于操作、无需昂贵仪器的优点,复苏后SSCs贴壁时间为8～12h,24h可见细胞分裂,48h细胞出现迅速增殖,96h可见较多含20～25细胞的细胞团,此时精原干细胞增殖近5倍. 结论本实验所用培养条件,可以使经长期冷冻的SSCs短期快速增殖.
목적 탐색소서정원간세포(SSCs)냉동보존방법 이급해동후체외쾌속증식적조건. 방법 실험이6d령웅성곤명소백서위재료,량보매소화법분리소서고환생식세포,Percoll비련속밀도제도리심법부집소서정원간세포,수후가입불동적냉동액이급채용불동적강온속솔냉동소서정원간세포.이MEMα위기본배양기,가입10%태우혈청화100μg/L적효질세포원성신경영양인자, WST-8비색법분석배양SSCs 복소후적증식솔,운용감성린산매세포화학염색화RT-PCR기술,대배양적SSCs진행감정. 결과 냉동액중첨가10%이갑기아풍、10%태우혈청、0.07mol/L자당시,이1℃/min정공강온방식냉동소서SSCs,세포해동후활솔최고,체84%이상;채용비정공강온방식동존SSCs,진관세포해동후활솔상대우정공강온방식략저,단구유방법 간단、역우조작、무수앙귀의기적우점,복소후SSCs첩벽시간위8～12h,24h가견세포분렬,48h세포출현신속증식,96h가견교다함20～25세포적세포단,차시정원간세포증식근5배. 결론 본실험소용배양조건,가이사경장기냉동적SSCs단기쾌속증식.
Objective To explore the conditions and methods for cryopreservation and proliferation of mouse spermatogonial stem cells (SSCs). Methods SSCs were isolated from six-day-old Kunming mouse using two-step enzymatic digestion and Percoll discontinuous density gradient centrifugation. Cells were frozen with different freezing medium and cooling rate. After thaw, they were cultured in mimimum essential medium alpha (MEMα) supplemented with 10% fetal calf serum (FCS) and 100μg/L glial cell line-derived neurotrophic factor (GDNF). The survived and proliferating SSCs were examined by WST-8 colorimetric assay. Alkaline phosphatase andreverse transcription-polymerase chain reaction (RT-PCR) were performed to confirm if the cultured 96 hours germ cells were still stem cells. Results The best method to cryopreserve SSCs is using cryoprotector containing 10% dimethyl sulfoxide(DMSO), 10% FCS, 0.07mol/L sucrose and 1℃/min cooling rate, and the viability of cells in this method is more than 84%;Although the cell viability in non-programmed freezing method is less than that in the programmed freezing method, it is a simple and effective cropreservation method for mouse SSCs. What is more, the anchoring time of SSCs in this method is 8-12 hours after thaw, SSCs begin to proliferate 24 hours later, and rapid proliferation appears on the 48 hours, colonies are composed by 20-25 cells in 96 hours, when SSCs proliferated nearly 5 times.Conclusion The culture condition we used is suitable for proliferation of frozen-thawed SSCs.