不同培养代次对经血源子宫内膜干细胞生物学活性的影响

该研究明确了不同培养代次经血源子宫内膜干细胞(menstrual blood derived endometrial stem cells,MenSCs)的生物学活性差异,为深入研究MenSCs生物学特性及其潜在临床应用提供理论支持。该研究使用钙黄绿素AM(Calcein-AM)染色检测体外培养至第三代(passage 3,P3)、P9和P15代MenSCs的形态;β-半乳糖苷酶染色检测不同培养代次MenSCs的衰老程度;活性氧试剂盒检测不同培养代次MenSCs中活性氧的变化;随后利用MTT、流式细胞术及细胞活死染色在接触式共培养条件下检测P3、P9和P15代MenSCs对小鼠脾淋巴细胞活性、细胞周期、死亡情况以及脾脏淋巴细胞中CD3^+和CD19^+淋巴细胞比例的影响。结果表明,随着培养代次的增加MenSCs细胞面积显著增大,在培养至P15代时MenSCs开始出现大量丝状伪足。衰老程度及活性氧的含量也随培养代次的增加而显著升高;随后与MenSCs接触式共培养体显著增加了小鼠脾淋巴细胞的活性,降低淋巴细胞死亡率,且随培养代次的增加,MenSCs对促进淋巴细胞存活、降低死亡的能力显著降低;进一步细胞周期检测发现,MenSCs无刺激淋巴细胞增殖分裂活性,但可显著降低淋巴细胞死亡及碎片化,且培养代次的增加可显著降低MenSCs维持淋巴细胞存活的能力;此外,不同培养代次MenSCs在体外均对小鼠脾淋巴细胞中CD3^+和CD19^+细胞亚群百分比无显著性影响。综上,MenSCs随着体外培养时间和代次的增加,出现明显的生物学活性降低等特征,且对淋巴细胞活性的调节能力显著降低,上述结果为临床应用中保障MenSCs质量、平衡细胞培养代次和细胞数量及保证稳定的MenSCs临床治疗效果提供理论支持。

The Effect of Subculture Passage on the Biological Characteristics of Menstrual Blood Derived Endometrial Stem Cells

The biological characteristics of MenSCs(menstrual blood derived endometrial stem cells)cultured in vitro at different passages(passage 3,9 and 15)were examined,in order to provide support for further studies on the potential clinical applications of MenSCs.Therefore,in this study,the morphology of MenSCs was observed after Calcein-AM staining.The senescence of MenSCs was detected byβ-galactosidase staining.The production of ROS(reactive oxygen species)in MenSCs was detected by ROS probe staining.The activity of mouse derived spleen lymphocytes co-cultured with MenSCs was determined by conventional MTT assay and Live/Dead staining,respectively.Additionally,both the cell cycle of mouse derived spleen lymphocytes co-cultured with MenSCs and the activation of CD3^+ and CD19^+ lymphocytes in mouse derived spleen lymphocytes co-cultured with MenSCs were analyzed by flow cytometry.Consequently,our results showed that no matter the cell size of MenSCs,or the degree of senescence and ROS production in MenSCs had significant increases as the subculture passage increased in vitro,also a large number of filamentous pseudopodia was observed in P15 MenSCs.Subsequently,the mouse derived spleen lymphocytes co-cultured with MenSCs exhibited a superior metabolic activity than the lymphocytes cultured alone,the following Live/Dead staining results also confirmed a lower death rate in the mouse derived spleen lymphocytes co-cultured with MenSCs,and P3 MenSCs exhibited the optimal viabilitykeeping capacity.Additionally,the further cell cycle examination showed no influence on the proliferation of mouse derived spleen lymphocytes,no matter co-cultured with or without MenSCs,but the percentage of debris in lymphocytes co-cultured with MenSCs had a significant decrease,which suggested MenSCs were capable of decreasing the death rate of co-cultured mouse derived spleen lymphocytes.Finally,there was no significant change in the percentage of CD3^+ and CD19^+ lymphocytes in mouse derived spleen lymphocytes,no matter co-cultured with or without MenSCs,which suggested the low immunogenicity of MenSCs.In summary,with the increased subculture passages in vitro,MenSCs not only exhibited a significant decrease in their viability,but also the MenSCs derived viability-keeping capacity for lymphocytes was significantly down-regulated.These results will contribute to balance the quality and quantity of MenSCs used in clinic,and then guarantees the therapeutic effect of MenSCs based therapies.