【摘要】 目的:以昆明白小鼠胎鼠成纤维细胞作为饲养细胞,观察其不同接种密度对人胚胎干细胞的克隆生长情况及分化率的影响。方法:实验于2003-07/2005-03华中科技大学同济医学院附属同济医院完成。首先进行小鼠胚胎成纤维细胞的分离培养。选取2～5代的小鼠胚胎成纤维细胞,待其至对数生长期倒掉或吸掉培养基,加入含10mg/L丝裂霉素C的细胞全培养液5mL,置37℃,体积分数0.05的CO2饱和湿度温箱中培养二三小时,在6孔板中加入0.1%明胶包被,放置30min以上,倒掉或吸掉含丝裂霉素C的培养基,磷酸盐缓冲液(-)清洗5遍,尽量除去丝裂霉素C,加入2mL0.05%的胰蛋白酶消化细胞,镜下观察,当细胞间出现裂隙,细胞变圆时(约2～4min),立即加入等量的全培养液终止消化,并用吸管反复吹打,使之成为单细胞悬液,在细胞计数板中央放置专用的盖玻片,用玻璃虹吸管吸取细胞,让虹吸管在盖玻片上或下侧的计数板凹槽处流出悬液,至盖玻片下被液体充满为止。用细胞计数板计算活细胞数,并分别按3×108、5×108、1×109L-1密度接种,机械法平均分人胚胎干细胞克隆,5个克隆/孔接种于不同密度的小鼠胚胎成纤维细胞上。观察克隆的生长情况及分化率,分化率=(完全分化克隆数+部分分化克隆数)/(完全分化克隆数+部分分化克隆数+未分化的克隆数)。结果:胚胎干细胞克隆在3×108L-1密度的小鼠胚胎成纤维细胞上生长极易分化,贴壁48h后无生长,72～96h分化率(98.0±2.0)%;接种于5×108L-1的小鼠胚胎成纤维细胞上后,胚胎干细胞生长与分化共存,贴壁后48h部分克隆生长,下次传代前分化率分化效率为(30.0±5.0)%;在1×109L-1小鼠胚胎成纤维细胞上,胚胎干细胞克隆贴壁后48h即开始生长,分化率为(1.0±0.2)%。3组之间分化率差异有显著性意义(P<0.05)。结论:以昆明白小鼠胎鼠成纤维细胞作为饲养细胞,1×109L-1可以起到促进胚胎干细胞的增殖,并抑制其分化的作用。更多还原

【Abstract】 AIM: To investigate the influences of different density of mouse embryonic fibroblast (MEF),from Kunming white foetus mice as feeder cell,on the growth and differentiation rate of human embryonic stem cell (hESC). METHODS: The experiment was conducted at Tongji Hospital,Tongji Medicine College,Huazhong University of Science and Technology between July 2003 and March 2005. Firstly,MEF was isolated and cultured. Generation 2-5 MEF were selected. When reached logarithm growth phase,it was poured or the medium was removed. 5 mL medium containing 10 mg/L mitocin-C was added ,kept at 37 ℃,and then cultured in saturated humidity warm box with the CO2 of 0.05 fraction volume for 2 or 3 hours. It was coated with 0.1% gelatin in 6-hole plate,laid for over 30 minutes,and then threw away or the medium with mitocin-C was removed,washed with phosptat buffer(-) for 5 times so as to remove the mitocin-C. 2 mL 0.05% trypsin digestive cells were added,and it was observed under microscope. When crevice appeared,cells became round (about 2-4 minutes),digestion was stopped by adding medium of the same volume,and blew up with straws repetitively to make it into monoplast suspension. Special-used cover glass was put in the center of cell counting chamber. Cells were sucked in by glass siphon,and cell suspension flew out at bucket of up or down-sides of counting chamber,until the cover glass was filled with fluid. Living cell were inoculated at concentrations of 3×108,5×108,1×109 L-1 after their number counted. Colonies were divided evenly by mechanical method,about 5 colonies were planted on medium with different MEF density and the growth and differentiation of the colonies were observed. The differentiation rate was equal to colonies fully and partial differentiated /total number of the colonies. RESULTS: Colonies on MEF with a density of 3×108 L-1 were most likely to undergo differentiation. Colonies would not grow until 48 hours after adhering with a differentiation rate reached up to (98.0±2.0)% at 72-96 hours after adhering. Growth and adhering coexisted when colonies were inoculated on 5×108 L-1 MEF. A part of the colonies begun to grow at hour 48 after adhering and the differentiated rate was (30.0±5.0)% before next passage. When planted on 1×109 L-1 MEF,colonies began to grow at hour 48 after adhering and the differentiated rate was (1.0±0.2)%. The differentiation rates were significantly different among these three groups (P < 0.05). CONCLUSION: Kunming white foetus mice as feeder cell of MEF at 1×109 L-1 can induce the proliferation of ESC,and inhibit its differentiation.更多还原