CAJ | 학술논문

目的探讨不同培养条件对孕中期羊水干细胞分离及扩增能力的影响.方法收集2007年9月-2008年6月在解放军总医院妇产科因医疗指征引产的15例孕16～24周产妇.经腹部穿刺抽取羊水10～20 ml,收集细胞并根据培养条件不同分为4组:(1)低糖Dulbecco改良Eagle培养基(LD培养基)+10%胎牛血清(10%血清组);(2)LD培养基+20%胎牛血清(20%血清组);(3)LD培养基+15%胎牛血清+碱性成纤维生长因子(bFGF,bFGF组);(4)LD培养基+10%胎牛血清+0.1%明胶铺板(明胶组).比较各组培养的原代细胞集落数和成纤维样细胞集落数、细胞形态、可传代次数等;采用流式细胞仪、RT-PCR技术对各组干细胞特异性标志物进行鉴定;诱导各组向脂肪细胞分化;并行染色体核型分析.结果 (1)10%血清组、20%血清组、bFGF组及明胶组的羊水干细胞扩增培养的成功率分别为60%、73%、73%及60%,10%血清组及明胶组分别与20%血清组及bFGF组比较,差异均无统计学意义(P>0.05);各组细胞集落数分别为(0.9±0.5)、(2.6±1.5)、(2.9±1.5)、(1.1±0.8)个,10%血清组及明胶组分别与20%血清组及bFGF组比较,差异均有统计学意义(P<0.01);各组成纤维样细胞集落数比例分别为46%、49%、64%及44%,各组分别比较,差异均无统计学意义(P>0.05).(2)各组第2代细胞经诱导均可分化为脂肪样细胞.(3)bFGF组有5例持续传代5次以上,细胞形态稳定,细胞数多达1 ×107个以上,与其他各组比较,差异均有统计学意义(P<0.01).(4)染色体核型分析各组均正常.(5)流式细胞仪及RT-PCR技术检测显示,bFGF组传代的细胞均能表达干细胞的标志物阶段特异性胚胎抗原4(SSEA-4)、Oct-4及Nanog基因,而其他各组在传代中细胞出现分化或停止生长.结论孕中期羊水中可成功分离获得具有干细胞特性的细胞群,适当的血清浓度及添加bFGF能增加羊水干细胞扩增培养的效率.
목적 탐토불동배양조건대잉중기양수간세포분리급확증능력적영향.방법 수집2007년9월-2008년6월재해방군총의원부산과인의료지정인산적15례잉16～24주산부.경복부천자추취양수10～20 ml,수집세포병근거배양조건불동분위4조:(1)저당Dulbecco개량Eagle배양기(LD배양기)+10%태우혈청(10%혈청조);(2)LD배양기+20%태우혈청(20%혈청조);(3)LD배양기+15%태우혈청+감성성섬유생장인자(bFGF,bFGF조);(4)LD배양기+10%태우혈청+0.1%명효포판(명효조).비교각조배양적원대세포집락수화성섬유양세포집락수、세포형태、가전대차수등;채용류식세포의、RT-PCR기술대각조간세포특이성표지물진행감정;유도각조향지방세포분화;병행염색체핵형분석.결과 (1)10%혈청조、20%혈청조、bFGF조급명효조적양수간세포확증배양적성공솔분별위60%、73%、73%급60%,10%혈청조급명효조분별여20%혈청조급bFGF조비교,차이균무통계학의의(P>0.05);각조세포집락수분별위(0.9±0.5)、(2.6±1.5)、(2.9±1.5)、(1.1±0.8)개,10%혈청조급명효조분별여20%혈청조급bFGF조비교,차이균유통계학의의(P<0.01);각조성섬유양세포집락수비례분별위46%、49%、64%급44%,각조분별비교,차이균무통계학의의(P>0.05).(2)각조제2대세포경유도균가분화위지방양세포.(3)bFGF조유5례지속전대5차이상,세포형태은정,세포수다체1 ×107개이상,여기타각조비교,차이균유통계학의의(P<0.01).(4)염색체핵형분석각조균정상.(5)류식세포의급RT-PCR기술검측현시,bFGF조전대적세포균능표체간세포적표지물계단특이성배태항원4(SSEA-4)、Oct-4급Nanog기인,이기타각조재전대중세포출현분화혹정지생장.결론 잉중기양수중가성공분리획득구유간세포특성적세포군,괄당적혈청농도급첨가bFGF능증가양수간세포확증배양적효솔.
Objective To investigate the effects of different culture conditions on the isolation and expansion of stem cells from second-trimester amniotic fluids.Methods Amniotic fluids were obtained from 15 pregnant women undergone amniocenteses for medical indications between 16-24 gestation weeks by transabdominal amniocenteses from September 2007 to June 2008.Amniotic fluids(10-20 ml)samples were collected and each WaS cultured under different conditions or groups.(1)Low-glucose DMEM(LD) medium supplemented with 10%of fetal bovine serum(group of 10% FBS);(2)LD medium with 20%of FBS(group of 20%FBS);(3)LD medium with 15%of FBS and 4 ng/ml of basic fibroblast growth factor (group of bFGF);(4)LD medium with 10%of FBS as well ag the culture plate coated with gelatin(group of gelatin).The effects of different conditions were evaluated by comparing the number of primary colonies,the cell morphology and the ability of expansion.The isolated stem cells were identified by flow cytometry,RT-PCR and differentiation ability to edipocyte.Resuits (1)The success rates of primary culture of the group of 10%FBS,20%FBS,bFGF and gelatin were 60%,73%,73%and 60% respectively(P>0.05).The numbers of colonies were 0.9±0.5,2.6±1.5,2.9±1.5,1.1±0.8(P<0.01 when group of 10%FBS and gelatin compared with group of 20%FBS and bFGF);among the primary colonies,fibroblast-like colonies accounted for 46%,49%,64%.44%respectively(P>0.05).(2)The second passage cells obtained from all of these four groups could difierentiate into adipocyte after induction.(3)In the group of bFGF,stem cells were isolated from 5 samples and expanded to nearly 107 cells after 5 passages(P<0.01 compared with other groups).(4)Karyotype were normal in all samples.(5)Stem cells from bFGF group showed positive expression of SSEA-4.Oct-4 and Nanog gene detected by flow cytometry and RT-PCR.Conclusion Stem cells can be isolated from second-trimester amniotic fluids;moderate serum concentration and supplementation of bFGF can improve the efficiency of isolation and expansion of amniotic fluid of stem cells.