目的 研究含胎牛血清（FBS）培養(yǎng)基、無血清培養(yǎng)基（SFM）對不同擴增代次間充質(zhì)干細(xì)胞（MSCs）生物特征的影響。方法 制備人臍帶間充質(zhì)干細(xì)胞（hUC-MSCs）并進行表面標(biāo)志物及三系分化鑒定；用含10% FBS的DMEM/F-12完全培養(yǎng)基及SFM（1～4）連續(xù)培養(yǎng)制備的MSCs，取P3/P5/P10/P15代次，顯微鏡下觀察形態(tài)，用細(xì)胞計數(shù)儀進行細(xì)胞直徑分析；計數(shù)并繪制生長曲線；實時熒光定量PCR（qRT-PCR）檢測端粒酶逆轉(zhuǎn)錄酶（TERT）、p53、p21、p16、53上調(diào)凋亡調(diào)控因子（PUMA） mRNA表達(dá)；進行軟瓊脂成克隆能力分析、核型分析。結(jié)果 制備的MSCs經(jīng)鑒定均符合要求；在含血清培養(yǎng)條件下，MSCs生長狀態(tài)、大小和增殖能力相對穩(wěn)定； SFM培養(yǎng)下，MSCs生長狀態(tài)、大小和增殖能力差別較大，前期增殖速度快，細(xì)胞形態(tài)更小更細(xì)長，后期增殖速度明顯降低；端粒酶檢測結(jié)果顯示，在含血清培養(yǎng)下MSCs中TERT的mRNA表達(dá)相對穩(wěn)定，與同代次FBS培養(yǎng)條件比較，P3、P5代中各SFM培養(yǎng)條件下TERT的表達(dá)量均顯著升高（P＜0.01、0.001）； P10代中SFM-3、SFM-4，P15代中SFM-4培養(yǎng)條件下TERT的表達(dá)量均顯著升高（P＜0.05、0.01）；軟瓊脂克隆檢測結(jié)果顯示，血清/SFM-2/3/4培養(yǎng)的不同代次MSCs均無陽性克隆形成，SFM-1培養(yǎng)的P5代MSCs在每孔300、600個鋪板條件下，有陽性克隆形成；與含血清培養(yǎng)條件相比，無血清培養(yǎng)下，p53、p21、p16、PUMA在P10代均發(fā)生顯著性增高（P＜0.001）；但各條件MSCs的核型中均未發(fā)現(xiàn)染色體異?；蚧儯砻骱诵途欠€(wěn)定的。結(jié)論 傳統(tǒng)含血清培養(yǎng)的MSCs生長更穩(wěn)定，安全性高，提示細(xì)胞研發(fā)機構(gòu)的MSCs如果涉及無血清培養(yǎng)，要做好工藝穩(wěn)定性、安全性及有效性驗證，以保證細(xì)胞質(zhì)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

天津市科技計劃項目細(xì)胞制品的成藥性及轉(zhuǎn)化研究資助項目（23ZGCXQY00050）； 天津市科技計劃項目細(xì)胞和基因治療產(chǎn)品概念驗證平臺建設(shè)資助項目（24ZYCGCG00600）