CAJ | 학술논문

背景:到目前为止,国际上通用的动物胚胎干细胞细胞系大多是从129品系和C57BL/6系小鼠中获得的,少部分来自BALB/C系,来自于中国昆明鼠则鲜有报道.目的:分离培养昆明系小鼠胚胎干细胞,并对其生物学特性进行初步鉴定.设计、时间及地点:以细胞为对象的观察性实验,于2006-05/2007-06在南昌大学第一附属医院泌尿外科研究所完成.材料:昆明小鼠3.5 d胚龄的囊胚.方法:自昆明系小鼠3.5 d胚龄的囊胚分离内细胞团细胞,接种于小鼠原代胚胎成纤维细胞饲养层上,对分离的内细胞团细胞进行扩增、传代培养.主要观察指标:观察胚胎干细胞集落的生长情况,对其碱性磷酸酶表达进行染色分析,采用免疫荧光法检测其阶段特异性胚胎抗原1表达,以反转录-聚合酶链反应检测其特异性表达因子c-Myc,Nanog,Oct3/4和Sox2的基因表达情况,并对其体内外分化能力对进行鉴定分析.结果:分离、培养自内细胞团的细胞呈典型的胚胎干细胞集落样生长,碱性磷酸酶染色及阶段特异性胚胎抗原1荧光染色阳性,细胞表达c-Myc,Nanog,Oct3/4和Sox2等基因,细胞在体内外均能分化成来源于不同胚层的多种细胞类型.结论:成功从昆明小鼠囊胚中分离并建立一株胚胎干细胞系,其生物学特性与其他胚胎干细胞株相符.
배경:도목전위지,국제상통용적동물배태간세포세포계대다시종129품계화C57BL/6계소서중획득적,소부분래자BALB/C계,래자우중국곤명서칙선유보도.목적:분리배양곤명계소서배태간세포,병대기생물학특성진행초보감정.설계、시간급지점:이세포위대상적관찰성실험,우2006-05/2007-06재남창대학제일부속의원비뇨외과연구소완성.재료:곤명소서3.5 d배령적낭배.방법:자곤명계소서3.5 d배령적낭배분리내세포단세포,접충우소서원대배태성섬유세포사양층상,대분리적내세포단세포진행확증、전대배양.주요관찰지표:관찰배태간세포집락적생장정황,대기감성린산매표체진행염색분석,채용면역형광법검측기계단특이성배태항원1표체,이반전록-취합매련반응검측기특이성표체인자c-Myc,Nanog,Oct3/4화Sox2적기인표체정황,병대기체내외분화능력대진행감정분석.결과:분리、배양자내세포단적세포정전형적배태간세포집락양생장,감성린산매염색급계단특이성배태항원1형광염색양성,세포표체c-Myc,Nanog,Oct3/4화Sox2등기인,세포재체내외균능분화성래원우불동배층적다충세포류형.결론:성공종곤명소서낭배중분리병건립일주배태간세포계,기생물학특성여기타배태간세포주상부.
BACKGROUND:Up to date,inbred embryonic stem cells(ESCs) line mainly derived from 129 mouse strain and C57BL/6 strain,occasionally from BALB/c mouse strain,however,few reports concerning ESCs lines derived from Ch inese Kunmingmouse strain.OBJECTIVE:To isolate and culture mouse ESCs of kunming species,additionally,to identify its biological properties.DESIGN,TIME AND SETTING:The experiment with cells as observed subjects was conducted in the institute of Urology,First Affiliated Hospital of Nanchang University from May 2006 to June 2007.MATERIALS:Blastocysts of Kunmin mice with 3.5 days of embryonic age.METHODS:Inner cell mass from 3.5 days embryonic age were isolated from Kunming species mice,cultured on feeder layers of primary mice embryonic fibroblasts,and then the cells were isolated and subsequently cultured.MAIN OUTCOME MEASURES:Colony growth was observed and determined by alkaline phosphatase (AKP) staining;The expression of stage-specific embryonic antigen (SSEA)-1,and cell specific genes of c-Myc,Nanog,Oct3/4 and Sox2 were measured by immunofluorescence and RT-PCR;finally,the ability of differentiation in vitro and in vivo was identified.RESULTS:The ESCs-like colonies presented typical morphological characteristics of ESCs,which was positive to AKP and SSEA-1,and could express several cell specific genes,such as c-Myc,Nanog,Oct3/4 and Sox2,and differentiate into various cell types in vitro and in vivo.CONCLUSION:An ESCs line is successfully dedved from Kunming mice,which has typical biological characteristics of ESCs.