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目的探讨锌对人脐带血间充质干细胞(human umbilical cord blood-drived mesenchymal stem cells,hUCBMSCs)细胞周期蛋白D2(cyclin D2)、细胞周期蛋白依赖性激酶4(cyclin-dependent kinase4,CDK4)、DNA和总蛋白含量的影响。方法采用密度梯度离心法分离培养hUCBMSCs。传代培养时将实验分为7组,对照组采用含15%FBS的DMEM培养基行传代培养,6个锌处理组于上述培养基中加入ZnSO4?7H2O,使锌终浓度分别为0.5、1.5、2.5、3.5、4.5、5.5mg/L。用流式细胞仪检测第3代hUCBMSCs表面抗原CD29、CD34、CD44和CD45;MTT法检测细胞活性,筛选最佳锌浓度组定为实验组。取实验组和对照组第3代hUCBMSCs绘制细胞生长曲线,流式细胞仪检测细胞DNA含量和增殖周期,Western blot检测总蛋白及cyclin D2和CDK4含量。结果 hUCBMSCs表面抗原CD29和CD44阳性表达率均>70%。MTT检测示,2.5mg/L浓度组细胞活性最高,定为实验组。培养7、14、28d,实验组hUCBMSCs的总蛋白含量、DNA含量、cyclin D2和CDK4含量均明显高于对照组(P<0.01);细胞增殖指数、S期细胞百分比均较对照组明显升高(P<0.01)。结论 0.5～4.5mg/L浓度的锌能增强hUCBMSCs活性,其中2.5mg/L为最佳浓度,可促进hUCBMSCs增殖和DNA复制,增加细胞cyclin D2、CDK4和总蛋白含量。
Objective To investigate the effect of zinc on the expression of cyclin D2, cyclin-dependent kinase 4 (CDK4), human umbilical cord blood-drived mesenchymal stem cells (hUCBMSCs) And total protein content. Methods The hUCBMSCs were isolated and cultured by density gradient centrifugation. In the subculture, the experiment was divided into 7 groups, the control group was subcultured with DMEM medium containing 15% FBS, and the 6 zinc treatment groups were added ZnSO4? 7H2O to the above medium, the final zinc concentrations were 0.5, 1.5, 2.5, 3.5, 4.5 and 5.5 mg / L. The third generation of hUCBMSCs surface antigen CD29, CD34, CD44 and CD45 were detected by flow cytometry. Cell viability was detected by MTT assay and the best zinc concentration group was selected as the experimental group. The 3rd generation of hUCBMSCs in the experimental group and the control group were drawn cell growth curve, DNA content and proliferation cycle were detected by flow cytometry, Western blot detection of total protein and cyclin D2 and CDK4. Results The positive rates of CD29 and CD44 on hUCBMSCs were both> 70%. MTT test showed that 2.5mg / L concentration of cells in the highest group of activity, as the experimental group. The total protein content, DNA content, the content of cyclin D2 and CDK4 in hUCBMSCs in experimental group were significantly higher than those in control group (P <0.01) at 7, 14 and 28 days. The cell proliferation index and the percentage of S phase cells were significantly higher than those in control group (P <0.01). Conclusions Zinc 0.5 ~ 4.5mg / L can enhance the activity of hUCBMSCs, of which 2.5mg / L is the best concentration, which can promote the proliferation and DNA replication of hUCBMSCs and increase the content of cyclin D2, CDK4 and total protein.