直流电场对人脂肪间充质干细胞增殖和干细胞生物学特性的影响

Effects of direct current electric field on proliferation and biological characteristics of human adipose derived mesenchymal stem cells

目的探讨直流电场对体外培养的人脂肪间充质干细胞(human adipose-derived stem cells,hASCs)增殖的影响。方法将手术中获得的脂肪组织采用酶原消化法制备hASCs,待细胞培养传代至第三代接种于6孔板内,24 h后进行以下处理:(1)分别给予0(未刺激组)、50、100、150、200 mV/mm直流电场刺激,刺激频率为1 h/d,连续刺激3 d。在倒置显微镜下观察细胞的形态及生长特点。收集各实验组细胞进行细胞计数、CCK-8实验检测细胞的增殖情况。(2)通过流式细胞仪分析直流电场刺激对hASCs干细胞表型标志CD105、CD73、CD90、CD14、CD34、CD31、CD45表达的影响。(3)诱导分化检测直流电场刺激对hASCs分化能力的影响。(4)蛋白免疫印迹检测刺激组和未刺激组(0 mV/mm)细胞增殖相关通路蛋白的表达。结果50、100、150 mV/mm细胞的增殖率和光密度值明显高于未刺激组(P均<0.01)。当直流电场的刺激强度增加到200 mV/mm时细胞的增殖率明显低于未刺激组(P<0.001),而细胞的光密度值高于未刺激组(P<0.05)。直流电场刺激前后hASCs均高表达CD105、CD73、CD90,低表达CD14、CD31、CD34、CD45。且直流电场刺激前后hASCs均能分化为骨细胞和脂肪细胞,但刺激组的分化速度较未刺激组快。刺激组细胞增殖相关蛋白p-ERK1/2表达高于未刺激组(P<0.01)。结论直流电场可能通过调控ERK的磷酸化促进人脂肪间充质干细胞的增殖,并且不改变其干细胞生物学特性。

Objective To investigate the effect of direct current(DC) electric field on the proliferation of human adipose-derived stem cells(hASCs) in vitro. Methods The fat tissue obtained in operation was digested with zymogen to prepare hASCs. After the cell culture was passaged to the third generation, the hASCs were inoculated in a 6-well plate. After 24 h, the cells were cultured under a stimulation of 0, 50, 100, 150 and 200 mV/mm DC electric field, at a frequency of 1 h/d for 3 continuous days. Cell morphology and growth were observed under an inverted microscope. Cell counting and CCK8 assay were carried out to measure cell proliferation. Flow cytometry was performed to detect the expression of stem cell phenotype markers, CD105, CD73, CD90, CD14, CD34, CD31 and CD45. The effect of DC electric field stimulation was observed after differentiation induction. Western blotting was used to detect the expression of proteins related to cell proliferation in stimulated and unstimulated cells. Results The proliferation rate and absorbance value were significantly higher in the cells after 50, 100 and 150 mV/mm stimulation than those without(all P<0.01). When the stimulation intensity of the DC electric field was increased to 200 mV/mm, the proliferation rate was significantly lower(P<0.001), but the absorbance value was statistically higher than those of the 0 mV/mm stimulation(P<0.05). The stimulation also resulted in remarkably higher expression of CD105, CD73 and CD90, and lower expression of CD14, CD31, CD34 and CD45. Moreover, the hASCs could differentiate into bone cells and adipocytes with and without DC electric field stimulation, but the stimulated cells had faster differentiation than those without. Compared with the unstimulated cells, the expression of cell proliferation related protein p-ERK1/2 was significantly increased in the stimulated cells(P<0.01). Conclusion The DC electric field promotes the proliferation of hASCs probably by regulating the phosphorylation of ERK, but has no effect on the biological characteristics of the stem cells.