不同电刺激波形对脂肪间充质干细胞定向排列的影响

发布时间：2018-08-20 19:21

【摘要】：目的探讨不同电刺激波形对脂肪间充质干细胞(adipose derived mesenchymal stem cells,ADSCs)定向排列和细胞存活率的影响。方法取5周龄SD大鼠(体质量100~150 g)体外分离培养ADSCs。用第3~5代细胞制备细胞爬片,放入自制电场生物反应器中,分别施以电场强度为1、2、3、4、5、6 V/cm的直流电刺激(分别设为A1、A2、A3、A4、A5、A6组);电场强度6 V/cm、占空比50%、频率1 Hz和2 Hz的方波电刺激(分别为B1、B2组);电场强度6 V/cm、脉宽2 ms、频率1 Hz和2 Hz的双相脉冲波电刺激(分别为C1、C2组)。对照组(D组)不施加电刺激。采用倒置显微镜定时拍照法观察ADSCs在电场作用过程中的形态学变化,计算细胞排列的平均对齐指数(orientation factor,OF);采用罗丹明-鬼笔环肽荧光染色分析电场刺激后细胞的骨架排列;活死染色法分析细胞存活率;钙离子荧光染色标识电刺激中细胞内钙离子分布,分析细胞钙离子活动功能。结果 ADSCs对直流电刺激的响应与电场强度相关,电场强度越高,形成垂直排列的细胞越多;各时间点B1、B2组及C1、C2组均未发现明显的细胞定向排列现象。除A5、A6组平均OF显著高于D组(P0.05)外,其余各组平均OF与D组比较差异均无统计学意义(P0.05)。细胞骨架染色分析表明,A5、A6组细胞形成紧密的束状细胞骨架结构,且趋向于垂直电场矢量方向排列。细胞活死染色显示,除A4、A5、A6组细胞存活率显著低于D组(P0.05)外,其余各组细胞存活率与D组比较差异均无统计学意义(P0.05)。钙离子荧光染色显示,A4、A5、A6组细胞内的钙离子荧光强度稍高于D组,其余各组细胞内的钙离子荧光强度与D组比较无显著差别。结论直流电场强度5 V/cm和6 V/cm刺激可引起ADSCs定向排列,直流电场强度低于5 V/cm以及方波和双相脉冲波均未引起细胞定向排列;电刺激后细胞存活率与电场强度成负相关。
[Abstract]:Objective to investigate the effects of different electrical stimulation waveforms on the orientation and survival rate of adipose mesenchymal stem cells (adipose derived mesenchymal stem cells). Methods ADSCs were isolated from 5 week old SD rats (body weight 100 g) in vitro. Cell climbing tablets were prepared from the 3rd generation cells and put into a self-made electric field bioreactor. The electric field was stimulated with electric field intensity of 1 ~ 2 ~ 3 ~ 3 ~ 4 ~ 4 ~ 5 ~ 5 V/cm (A _ (1) A _ (2) A _ (3) A _ (3) A _ (4) A _ (4) A _ (5) A _ (6) group, electric field intensity 6 V / cm, duty cycle 50, square wave stimulation at 1 Hz and 2 Hz (B _ 1B _ 2 group, respectively), electric field intensity 6 V / cm, pulse width 2 Ms, frequency 1 Hz and frequency 1 Hz, respectively. The electric field intensity was 6 V / cm, pulse width was 2 Ms, frequency 1 Hz and frequency 1 Hz. 2 Hz biphasic pulse wave electrical stimulation (C _ 1 C _ 2 group). The control group (group D) did not apply electrical stimulation. The morphologic changes of ADSCs in the process of electric field action were observed by using inverted microscope, and the orientation factor of cell alignment was calculated, and the cytoskeleton arrangement of the cells stimulated by electric field was analyzed by Rhodamine and Gypanopeptide fluorescence staining. The survival rate of cells was analyzed by live staining, and the intracellular calcium distribution was identified by calcium fluorescence staining, and the function of calcium ion activity was analyzed. Results the response of ADSCs to DC stimulation was related to the electric field intensity. The higher the electric field intensity was, the more cells formed vertical alignment, but no obvious cell alignment was found in B1B 2 group and C 1 C 2 group at each time point. Except that the mean of in A5 A6 group was significantly higher than that in D group (P0.05), there was no significant difference between the other groups (P0.05). Cytoskeleton staining showed that the cells in group A _ (5) A _ (6) formed a compact bundle cytoskeleton structure and tended to perpendicular to the direction of electric field vector. Cell death staining showed that the cell survival rate of A4A5A6 group was significantly lower than that of D group (P0.05), and there was no significant difference between the other groups and D group (P0.05). Calcium fluorescence staining showed that the intracellular calcium fluorescence intensity of group A _ (4) A _ (5) A _ (6) was slightly higher than that of group D, while that of other groups had no significant difference from that of group D. Conclusion the direct current electric field intensity of 5 V/cm and 6 V/cm can induce the directional arrangement of ADSCs, but the DC electric field intensity is lower than 5 V/cm and neither square wave nor biphasic pulse wave can induce the directional arrangement of cells, and the cell survival rate is negatively correlated with the electric field intensity.
【作者单位】：成都航空职业技术学院工程实训中心;四川大学电气信息学院医学信息工程系;重庆医科大学附属永川医院骨科;四川大学华西医院心脏大血管外科;
【基金】：国家自然科学基金资助项目(81500213) 四川省教育厅科技项目(17ZA0019)~~
【分类号】：R329.2

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