一种分离培养人脐带Wharton's jelly间充质干细胞的新方法

目的探讨一种分离培养人脐带Wharton′s jelly间充质干细胞(mesenchymal stem cell,MSC)的新方法。方法取人脐带组织,除去动静脉后剪碎成2～5 mm3,将组织碎片浸入4 g/LⅠ型胶原酶和1 g/L透明质酸酶的混合液中,于37℃处理1 h,再用0.25%胰蛋白酶在相同条件下消化30 min,得到的消化液经70μm细胞滤网过滤后,制备单个细胞悬液,培养并传代。取P1、P3、P7代脐带Wharton′s jelly MSC,绘制细胞生长曲线;取P3代细胞,采用流式细胞术检测细胞表面标记分子,并分别采用成骨和成脂诱导培养基进行成骨及成脂诱导分化,茜素红染色和油红O染色观察结果。结果 P1、P3代脐带Wharton′s jelly MSC的增殖能力强,且P1代细胞的增殖能力强于P3代,P7代细胞的增殖能力较P3代细胞有所减弱。P3代脐带Wharton′s jelly MSC高表达CD90(99.8%)、CD105(100%)和CD166(100%),低表达CD45(0.3%)、CD14(0.1%)、CD34(0.2%)和CD79a(0.3%),不表达HLA-DR。P3代Wharton′s jellyMSC经成骨诱导后,茜素红染色可见红色结节;经成脂诱导后,油红O染色可见脂质沉积。结论本方法获得的Wharton′s jelly MSC活性好,增殖能力强,为后续实验研究及临床应用提供了理想的种子细胞。

A novel method for isolation and culture of mesenchymal stem cells from Wharton's jelly of human umbilical cord

Objective To develop a novel method for isolation and culture of mesenchymal stem cells(MSCs) from Wharton′s jelly of human umbilical cord.Methods Human umbilical cord tissues were collected,from which arteries and veins were removed,and minced into pieces each at a sized of 2 - 5 mm3.The pieces were treated with the mixture of 4 g/L collagenase Ⅰ and 1 g/L hyaluronidase at 37℃ for 1 h,then digested with 0.25% trypsin under the same condition for 30 min.The digested suspension was filtered with a 70 μm nylon mesh and prepared into a single-cell suspension,then cultured and subcultured.The growth curve of MSCs of passages 1,3 and 7(P1,P3 and P7)was plotted,while those of P3 were determined for surface marker by flow cytometry then subjected to osteogenic and adipogenic differentiations,and the results were observed by Alizarin red and oil red O staining.Results MSCs of P1 and P3 showed strong proliferation ability,while the proliferation ability of P1 was stronger than that of P3.However,the proliferation ability of P7 was weakened as compared with that of P3.The expression rates CD90(99.8%),CD105(100%)and CD166(100%)were high in MSCs of P3,while those of CD45(0.3%),CD14(0.1%),CD34(0.2%) and CD79a(0.3%)were low,and no HLA-DR was expressed.Red calcium node was observed by Alizarin red staining in MSCs of P3 after osteogenic differentiation.However,after adipogenic differentiation,liquid vacuoles were observed by oil red O staining.Conclusion The MSCs obtained from Wharton′s jelly of human umbilical cord showed high activity and strong proliferation ability,which provided an ideal cell seeds for further laboratory study and clinical application.