锌对大鼠骨髓间充质干细胞DNA和蛋白质含量及增殖周期的影响

Effects of zinc on DNA and protein content as well as reproductive cycle in rat bone marrow mesenchymal stem cells

背景：锌通过直接和间接作用对DNA聚合酶和RNA聚合酶产生影响,在细胞的DNA复制、RNA转录、增殖、分化等活动中起重要作用。目的：探讨锌对大鼠骨髓间充质干细胞DNA和蛋白质含量及增殖周期的影响。设计、时间及地点：细胞学体外对照观察,于2008-09/12在辽宁医学院完成。材料：清洁级1月龄SD大鼠7只,由辽宁医学院动物中心提供。方法：全骨髓法体外分离培养大鼠骨髓间充质干细胞,传至第3代时,对照组向细胞中加入含体积分数为15%胎牛血清的DMEM培养液,锌处理组向上述培养液中加入硫酸锌,使锌终浓度分别达到0.1,0.2,0.5,1.0,2.0,4.0,6.0mg/L。胰酶消化后按5×10^7L^-1密度接种,培养21d。主要观察指标：细胞表面抗原的表达,MTT法检测细胞活性,流式细胞仪测定细胞DNA含量、蛋白质含量和增殖周期。结果：第3代细胞表面抗原CD106呈阳性表达。锌对骨髓间充质干细胞活性的促进作用呈量效关系,0.2-4.0mg/L锌处理组细胞活性显著高于对照组（P〈0.01）,尤以质量浓度为2.0mg/L时最为明显;当锌的质量浓度达6.0mg/L时,细胞活性明显降低（P〈0.01）。培养7,14,21d与对照组比较,2.0mg/L锌处理组骨髓间充质干细胞DNA含量、蛋白质含量均显著升高（P〈0.01）;细胞增殖指数、S期和G2^＋M期细胞百分率均明显升高,G0/G1期细胞百分率则显著下降（P〈0.01）。结论：0.2-4.0mg/L锌能促进大鼠骨髓间充质干细胞的增殖、DNA复制和蛋白质合成,且2.0mg/L为最佳质量浓度。

BACKGROUND： Zinc has effects on DNA polymerase and RNA polymerase by direct and indirect action, and plays important roles in DNA duplication, RNA transcription, proliferation and differentiation of cells. OBJECTIVE： To explore effects of zinc on rat bone marrow mesenchymal stem cells （BMSCs） DNA and protein content as well as reproductive cycle. DESIGN, TIME AND SETTING： The cytology in vitro controlled observation study was performed at the Liaoning Medical University from September to December 2008. MATERIALS： A total of 7 clean Sprague Dawley rats aged 1 months were supplied by Animal Center, Liaoning Medical University. METHODS： Rat BMSCs were isolated and cultured in vitro by the whole bone marrow method. At the third passage, BMSCs in the control group were incubated with DMEM containing 15% fetal bovine serum. BMSCs in the zinc group were incubated in above-mentioned DMEM, supplemented by with zinc sulfate. The final concentration of zinc was 0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 6.0 mg/L. After digested by trypsin, BMSCs were incubated at 5×10^7/L for 21 days. MAIN OUTCOME MEASURES： Cell surface antigen expression. MTT assay was used to detect cell activity. Flow cytometry was utilized to measure DNA content, protein content and reproductive cycle. RESULTS： At passage 3, cells were positive for CD106. Zinc had dose-effect relationship on promotion of BMSC activity. Cell activity was significantly higher in the 0.2 4.0 mg/L zinc group compared with the control group （P〈 0.01）, especially at 2.0 mg/L. Cell activity significantly decreased at 6.0 mg/L （P 〈 0.01）. At 7, 14, 21 days, BMSC DNA content and protein content significantly increased in the 2.0 mg/L zinc group compared with the control group （P 〈 0.01）. Cell percentage in the S and G2＋M phases and cell proliferation index significantly increased, but cell percentage in the Go/G1 phases （P〈 0.01）. CONCLUSION： Zinc （0.2 4.0 mg/L） has the effect of promoting the rat BMSC proliferation, DNA reproduction and protein synthesis, and the 2.0 mg/L is the optimal concentration.