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

Effects of different culture conditions on isolation and expansion of stem cells from second-trimesteramniotic fluids

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10-20ml，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1×10^7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。

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 as 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 adipoeyte. Results （ 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 differentiate 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 ceils 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 ceils.