目的 對比Ki67法與羧基熒光素乙酰乙酸（carboxyfluoreseein di-acetate succinimidyl ester，CFSE）法檢測間充質(zhì)干細胞（mesenchymal stem cell，MSCs）對淋巴細胞的增殖抑制能力，開發(fā)MSCs免疫調(diào)控功能評價的新方法。方法 選取5批臍帶來源的MSCs，流式細胞術(shù)進行表面標志物（陽性CD73/CD90/CD105/CD44、陰性CD34/CD45/CD14/CD19）檢測；試劑盒誘導(dǎo)后染色進行成脂、成骨、成軟骨三系分化能力檢測；流式細胞術(shù)進行增殖指數(shù)檢測；CCK8法進行群體倍增時間檢測；生物學有效性檢測合格后與相同供者來源的外周血單個核細胞（peripheral blood mononuclear cell，PBMC）進行共培養(yǎng)，分別用CFSE法與Ki67法檢測MSCs對PBMC的增殖抑制率。結(jié)果 制備的MSCs生長狀態(tài)良好，CD73/CD90/CD105/CD44表達率均≥98%，CD34/CD45/CD14/CD19表達率均≤3%，成脂、成骨、成軟骨三系分化能力良好，增殖指數(shù)為35.8±9.9，倍增時間為（19.1±1.2） h，增殖情況良好。CFSE法檢測5批MSCs對PBMC的增殖抑制率為（91.20±0.70）%，Ki67法檢測5批MSCs對PBMC的增殖抑制率為（89.99±1.45）%，2種方法比較，差異無統(tǒng)計學意義。結(jié)論 Ki67法可作為評價MSCs免疫調(diào)控功能的新方法，有效避免傳統(tǒng)方法在檢測過程中的局限性。

Objective Ki67 method was compared with carboxyfluoreseein di-acetate succinimidyl ester (CFSE) method was used to detect the inhibition of mesenchymal stem cells (MSCs) on lymphocyte proliferation, to proposed a new idea for the evaluation of the immunoregulatory function of MSCs. Methods Five batches of MSCs from umbilical cord were selected. Surface markers (positive CD73/CD90/CD105/CD44, negative CD34/CD45/CD14/CD19) were detected by flow cytometry. After induction of the kit, staining was performed to detect the differentiation ability of lipid, osteogenic and chondrogenic lines. Proliferation index was detected by flow cytometry. CCK8 method was used to detect population doubling time. After qualified biological validity test, the Peripheral blood mononuclear cells (PBMC) from the same donor were co-cultured with MSCs,and then, CFSE and Ki67 methods were used to detect the inhibition rate of MSCs on lymphocyte proliferation, respectively. Results The expression rates of CD73/ CD90/CD105/CD44 were ≥ 98%, and CD34/CD45/CD14/CD19 were ≤ 2%. The proliferation index of MSCs was 35.8±9.9, and the multiplication time was (19.1±1.2) h. The proliferation was good. The proliferation inhibition rate of five batches of MSCs on PBMC was (91.20±0.70) % by CFSE method, and (89.99±1.45)% by Ki67 method. There was no statistical difference between the two methods. Conclusion Ki67 method can be used as a new method to evaluate the immunoregulatory function of MSCs, so as to avoid the limitations of traditional methods in the detection process.

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