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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。
Objective To explore a new method for the isolation and culture of human umbilical cord Wharton’s jelly mesenchymal stem cells (MSC). Methods The umbilical cord tissue was removed and the artery and vein were removed and cut into 2 ~ 5 mm3. The tissue fragments were immersed in a mixture of 4 g / L type I collagenase and 1 g / L hyaluronidase for 1 h at 37 ° C. After digestion with 0.25% trypsin for 30 min under the same conditions, the resulting digestion solution was filtered through a 70 μm cell strainer to prepare a single cell suspension, which was cultured and passaged. The P1, P3, and P7 umbilical cord Wharton’s jelly MSCs were drawn and the cell growth curve was drawn. The P3 generation cells were taken and the cell surface marker molecules were detected by flow cytometry. Osteoblasts and adipogenic medium Adipogenic differentiation, alizarin red staining and oil red O staining results. Results P1 and P3 on the umbilical cord Wharton’s jelly MSCs proliferation, and P1 generation of cell proliferation stronger than the P3 generation, P7 generation of cell proliferation than P3 cells weakened. P3 High expression of CD90 (99.8%), CD105 (100%) and CD166 (100%), low expression of CD45 (0.3%), CD14 (0.1%), CD34 (0.2%) and CD79a 0.3%), did not express HLA-DR. P3 generation Wharton’s jellyMSC after osteogenic induction, alizarin red staining visible red nodules; After adipogenic induction, oil red O staining shows lipid deposition. Conclusion Wharton’s jelly MSC obtained by this method has good activity and proliferation ability, which provides ideal seed cells for subsequent experimental research and clinical application.