胎盘间充质干细胞传代后的增殖能力

Proliferative abilities of placenta-derived mesenchymal stem cells at different passages

背景:胎盘间充质干细胞的增殖能力比骨髓间充质干细胞增殖能力强,但是否每个代次胎盘间充质干细胞的增殖能力都相同尚无定论。目的:分析不同代次胎盘间充质干细胞的增殖能力。方法:采用酶消化法分离人胎盘间充质干细胞,并利用流式细胞仪检测干细胞表面标志物,并进行成脂成骨诱导分化。采用细胞计数和BrdU方法检测细胞的增殖能力,根据倍增时间公式计算倍增时间,并进行长期传代。结果与结论:胎盘间充质干细胞阳性表达CD44、CD90和CD105,阴性表达CD14、CD34和CD45;成脂诱导21 d后油红O染色镜下可见红色脂肪滴,成骨诱导35 d茜素红染色镜下可见钙盐结节。细胞从第1代开始计数,直到第15代共获得3.5×1011个细胞。P5代比P10和P15代胎盘间充质干细胞的增殖能力强,倍增时间短。长期传代,胎盘间充质干细胞传代到第25代时,其中3例标本细胞形态完全发生改变,呈铺路石样;另外2例标本几乎无贴壁细胞。提示胎盘间充质干细胞可以长期传代到第25代,不同代次的胎盘间充质干细胞增殖能力不同,P5代增殖能力明显高于P10和P15代,临床应用应以P5代之前为宜。

BACKGROUND：The mesenchymal stem cells which are from the placenta have better proliferative potential than the bone marrow mesenchymal stem cells. It is inconclusive, however, whether placenta-derived mesenchymal stem cells at different passages have the same proliferative ability.METHODS： Placenta-derived human mesenchymal stem ceils were isolated with enzyme digestion, and flow cytometry was used to detect the cell surface markers. After osteogenic and adipogenic induction, cell proliferation detection was carried out using cell counting and BrdU analysis. Cell doubling time was calculated based on formula. The maximum cell passage was determined by long term cell culture until the cell died or differentiated. RESULTS AND CONCLUSION： CD44, CD90 and CD105 were highly expressed in placenta-derived mesenchymal stem cells, while CD14, CD34 and CD45 were negatively expressed. After 21 days of adipogenic induction, oil red O staining showed red fat droplets; after 35 days of osteogenic induction, alizarin red staining showed calcium nodules. Cell counting started from passage 1 to 25, and cell number reached to 3.5×10^11 at passage 15. Cell proliferation at passage 5 was stronger with shorter doubling time than that at passages 10 and 15. When cells reached to passage 25, three specimens completely altered their cell morphology with cobblestone structure, and another two specimens lost adherent features. These findings indicate that