利用月经血建立的经血源性基质干细胞系

Derivation of mesenchymal stem cell lines from menstrual blood

背景:经血源性基质干细胞被证实是一种新型的成体干细胞,目前国内关于其系统的建系方法报道极少。目的:建立经血源性基质干细胞系,并鉴定其表型和多能性。方法:收集健康成年女性的月经血,通过密度梯度离心法分离出经血源性基质干细胞进行培养,观察细胞的形态及增殖特性;采用CCK-8法检测各株细胞的增殖能力,定期收集细胞进行核型分析,采用流式细胞术检测细胞表面抗原CD34,CD38,CD44,CD45,CD73,CD90,CD105,Oct-4的表达,对细胞进行体外成脂和成骨诱导分化并鉴定。结果与结论:成功从6名健康志愿者的经血中分离获得4株经血源性基质干细胞系。细胞呈现典型的梭状结构,具有46,XX核型,能在体外稳定迅速增殖,传代后大约48 h进入对数生长期,表达CD44,CD73,CD90,CD105及Oct-4,不表达CD38,CD34,CD45;成脂诱导22 d后油红O染色阳性,成骨诱导2周后茜素红染色阳性。实验表明经血源性基质干细胞具备扩增迅速、核型稳定、多潜能性等优点,通过建立经血源性基质干细胞系,为进一步的临床治疗应用以及科学研究奠定了基础。

BACKGROUND： Menstrual blood-derived mesenchymal stem cells are proved to be a new type of adult stem cells. To date, little is reported on the methods of establishing this kind of cell lines systematically. OBJECTIVE： To isolate, culture, identify and finally establish menstrual blood-derived mesenchymal stem cell lines. METHODS： Menstrual blood-derived mesenchymal stem cells were isolated from the menstrual blood of healthy female donors and cultured accordingly using density gradient centrifugation method. Cell morphology and proliferation characteristics were observed, and proliferative ability was detected by cell counting kit-8 assay. Cells were collected periodically for karyotype analysis. Expressions of cell surface antigens such as CD34, CD38, CD44, CD45, CD73, CD90, CD105 and Oct-4 were analyzed using flow cytometry. Cell pluripotency were identified by inducing adipogenesis and osteogenesis in vitro. RESULTS AND CONCLUSION： We successfully established four menstrual blood-derived mesenchymal stem cell lines from the menstrual blood of six healthy volunteers. The cultured cell lines had a typical spindle shape and kept normal 46, XX karyotype. The cells grew rapidly and entered into the logarithmic growth phase at 48 hours after passaging. The results of flow cytometry showed that menstrual blood-derived mesenchymal stem cells were positive for CD44, CD73, CD90, CD105 and Oct-4, while negative for CD38, CD34 and CD45. Menstrual blood-derived mesenchymal stem cells were positive for oil red O staining at 22 days after adipogenic induction and also positive for alizarin red staining at 2 weeks after osteogenetic induction. In conclusion, our study suggests that menstrual blood-derived mesenchymal stem cells with stable karyotype and pluripotency can proliferate quickly, and we can provide a new resource for stem cell therapy and regenerative medicine through establishing the menstrual blood-derived mesenchymal stem cell lines.