脐带间充质干细胞长期体外培养的生物学特性及遗传稳定性

Biological characters and genetic stability of umbilical cord mesenchymal stem cells after long-term culture in vitro

目的观察脐带间充质干细胞(umbilical cord mesenchymal stem cells,UC-MSCs)体外长期培养的生物学特性及遗传稳定性。方法从剖宫产足月健康新生儿脐带中分离培养UC-MSCs,并连续传代,显微镜下观察细胞形态。分别检测P3和P12代UC-MSCs的增殖能力、免疫表型、向成脂肪细胞和成骨细胞的分化诱导率、衰老情况、成纤维细胞集落形成单位(CFU-F)及染色体核型。结果 P3代UC-MSCs为形态相对均一的梭形贴壁细胞,呈平行排列生长或旋涡状生长;P12代UC-MSCs宽大扁平,贴壁性减退,漂浮细胞增多,胞浆内出现黑色颗粒和空泡;P3和P12代UCMSCs均表达CD73、CD90、CD105,不表达CD34、CD45、HLA-DR;P3较P12代UC-MSCs的细胞增殖能力及向成脂肪细胞分化诱导率均明显升高(P均<0.05);向成骨细胞分化诱导率及细胞衰老率均明显降低(P均<0.05);成纤维细胞集落数及染色体中期分裂相明显增多(P<0.05)。结论长期体外培养的UC-MSCs生物学特性发生改变,遗传学特性稳定,但染色体中期分裂相减少。

Objective To observe the biological characters and genetic stability of umbilical cord mesenchymal stem cells （UC-MSCs） after long-term culture in vitro. Methods Primary culture of UC-MSCs were isolated from umbilical cords of matare healthy newborns delivery by caesarean section, then subcultured and observed for morphology by microscopy. The UC-MSCs of passages 3 and 12 were determined for proliferation ability, immune phenotype, induced adipogenic and osteogenie differentiation rates, senescence, colony forming unit of fibroblasts （CFU-F） and caryotype. Results UC-MSCs of passage 3 were in relatively even fusiform and adherent, which were in parallel or circinate arrangement. However, the UC-MSCs of passage 12 were wide, flat and low adherent, of which the floating ones increased, and black particles and vacuoles appeared in cytoplasm. CD73, CD70, CD105 were expressed, while CD34, CD45 and HLA-DR were unexpressed in both UC-MSCs of passages 3 and 12. As compared with those of passage 12, the proliferation ability and induced adipogenic differentiation rate increased, significantly （each P 〈 0. 05）, while the induced osteogenic differentiation rate and senescence rate decreased significantly （each P 〈 0. 05 ）, and the fibroblast colony count and metaphase chromosome increased （P 〈 O. 05）. Conclusion The biological characters of UC-MSCs after long-term culture in vitro changed, while the genetics was stable. However, the metaphase chromosome decreased.