目的 研究含胎牛血清（FBS）培养基、无血清培养基（SFM）对不同扩增代次间充质干细胞（MSCs）生物特征的影响。方法 制备人脐带间充质干细胞（hUC-MSCs）并进行表面标志物及三系分化鉴定；用含10% FBS的DMEM/F-12完全培养基及SFM（1～4）连续培养制备的MSCs，取P3/P5/P10/P15代次，显微镜下观察形态，用细胞计数仪进行细胞直径分析；计数并绘制生长曲线；实时荧光定量PCR（qRT-PCR）检测端粒酶逆转录酶（TERT）、p53、p21、p16、53上调凋亡调控因子（PUMA） mRNA表达；进行软琼脂成克隆能力分析、核型分析。结果 制备的MSCs经鉴定均符合要求；在含血清培养条件下，MSCs生长状态、大小和增殖能力相对稳定； SFM培养下，MSCs生长状态、大小和增殖能力差别较大，前期增殖速度快，细胞形态更小更细长，后期增殖速度明显降低；端粒酶检测结果显示，在含血清培养下MSCs中TERT的mRNA表达相对稳定，与同代次FBS培养条件比较，P3、P5代中各SFM培养条件下TERT的表达量均显著升高（P＜0.01、0.001）； P10代中SFM-3、SFM-4，P15代中SFM-4培养条件下TERT的表达量均显著升高（P＜0.05、0.01）；软琼脂克隆检测结果显示，血清/SFM-2/3/4培养的不同代次MSCs均无阳性克隆形成，SFM-1培养的P5代MSCs在每孔300、600个铺板条件下，有阳性克隆形成；与含血清培养条件相比，无血清培养下，p53、p21、p16、PUMA在P10代均发生显著性增高（P＜0.001）；但各条件MSCs的核型中均未发现染色体异常或畸变，表明核型均是稳定的。结论 传统含血清培养的MSCs生长更稳定，安全性高，提示细胞研发机构的MSCs如果涉及无血清培养，要做好工艺稳定性、安全性及有效性验证，以保证细胞质量。

Objective The effects of fetal bovine serum (FBS)-containing medium and serum-free medium (SFM) on the biological characteristics of mesenchymal stem cells (MSCs) at different passages were investigated. Methods Human umbilical cord mesenchymal stem cells (hUC-MSCs) were prepared and identified for surface markers and tri-lineage differentiation. MSCs were continuously cultured in DMEM/F-12 complete medium containing 10% FBS and SFM (1-4). Cells at passages P3/P5/P10/P15 were taken for microscopic observation of morphology, cell diameter analysis with a cell counter, cell counting and growth curve drawing. The mRNA expression of telomerase reverse transcriptase (TERT), p53, p21, p16, and p53 upregulated modulator of apoptosis (PUMA) was detected by real-time fluorescence quantitative PCR (qRT-PCR). Soft agar colony formation ability and karyotype analysis were performed. Results The prepared MSCs met the requirements after identification. Under serum-containing culture conditions, the growth state, size and proliferation ability of MSCs were relatively stable. Under SFM culture conditions, the growth state, size and proliferation ability of MSCs varied greatly. The proliferation rate was faster in the early stage, and the cell morphology was smaller and more slender. The proliferation rate decreased significantly in the later stage. Telomerase detection results showed that the mRNA expression of TERT in MSCs under serum-containing culture conditions was relatively stable. Compared with the FBS culture conditions of the same passage, the expression of TERT in SFM-1, SFM-2, SFM-3, and SFM-4 culture conditions at P3 and P5 passages was significantly increased (P <0.01, 0.001). At P10 passage, the expression of TERT in SFM-3 and SFM-4 culture conditions was significantly increased (P <0.05, 0.01). At P15 passage, the expression of TERT in SFM-4 culture conditions was significantly increased (P <0.01). Soft agar colony formation detection results showed that no positive colonies were formed in different passages of MSCs cultured in serum/SFM-2/3/4. In P5 passage MSCs cultured in SFM-1, positive colonies were formed when 300 and 600 cells were seeded per well. Compared with serum-containing culture conditions, the expression of p53, p21, p16, and PUMA in P10 passage MSCs under serum-free culture conditions was significantly increased (P <0.001). However, no chromosomal abnormalities or aberrations were found in the karyotypes of MSCs under all conditions, indicating that the karyotypes were stable. Conclusions Traditional serum-containing culture of MSCs has more stable growth and higher safety. It is suggested that if MSCs from cell research institutions involve serum-free culture, the stability, safety and effectiveness of the process should be verified to ensure cell quality.

R965

天津市科技计划项目细胞制品的成药性及转化研究资助项目（23ZGCXQY00050）； 天津市科技计划项目细胞和基因治疗产品概念验证平台建设资助项目（24ZYCGCG00600）