微电流刺激表皮角朊细胞体外增殖的培养模型

A culture model of epidermal keratinocyte proliferation in vitro stimulated by microcurrent

目的:建立微电流作用于角朊细胞的体外模型,观察微电流对角朊细胞增殖等细胞行为的影响。方法:实验于2003-08/2004-08在第三军医大学烧伤研究所实验室完成。所用细胞来自西南医院泌尿外科3例行包皮环切术患者的手术切除包皮标本,患者均知情同意,每个标本均独立进行实验,分为对照组和电刺激组。模型建立:在细胞培养皿中部组建3.0cm×1.0cm×1.0cm大小的培养小室,底部分别用镍铬合金丝或纯铂丝作为电极,同时自行制作输出电流范围为4～1800μA的微电流发生器。电刺激后观察两种电极周围是否发生影响细胞生长的副反应,选择未发生副反应的电极应用于模型中。把手术切除的包皮皮片用中性蛋白酶和胰蛋白酶消化以获取角朊细胞,精制无血清培养基培养。电刺激组给予一定强度频率和时间脉冲交流电刺激后,用碘化丙啶染色,选流式细胞术分析两组角朊细胞周期变化。结果:①镍铬电极的培养模型,制作比较复杂,电极周围易发生通电引起的副反应,电刺激1d后,电极附近生出淡绿色电解反应产物;纯铂电极的培养模型,制作过程相对简单,持续7d电刺激后,电极周围无化学反应,该模型选用了纯铂电极。②自制的微电流发生器输出功率稳定,电流强度可在4.0～1800μΑ之间调节。③角朊细胞DNA合成前期对照组比电刺激组所占百分率明显降低[(52.76±3.57)%,(46.63±0.38)%,t=2.953,P<0.05];合成期、合成后期和分裂期电刺激组所占百分率稍高于对照组[(29.54±16.27)%,(27.02±18.27)%;(23.84±16.48)%,(20.21±17.90)%],说明该组角朊细胞DNA合成活跃,细胞分裂数目增多。④在纯铂电极的培养模型中,电刺激4d后,角朊细胞呈多角形,核分裂相多见,细胞生长旺盛,在小室中的融合现象更加明显。结论:微电流刺激下,角朊细胞DNA的合成活跃,细胞分裂数目增多。自制纯铂电极培养模型输出功率稳定,电流强度可控,能满足细胞长期电刺激培养的需要,为进一步研究电流对细胞增殖的影响提供了技术平台。

AIM:To establish a culture model of keratinocytes(KCs) stimulated by microcur rent and observe the effect of microcurrent on behaviors such as KC proliferatio n in vitro. METHODS:The experiment was finished in the Research Institute of Burns,Southw est Hospital,Third Military Medical University from August 2003 to August 2004.A ll the cells were from the excised foreskin specimens of three patients treated with circumcision in the Department of Urinary Surgery,and according to patients ' consent of experiment protocol,each specimen was experimented separately to di vide them into experiment group and control group.Cell culture dish was modified to form a culture chamber at a size of 3.0 cm× 1.0 cm× 1.0 cm.The two electro des at the bottom of the chamber were made of either thread of nickel- chromium alloy or pure platinum thread,and connected to the self- prepared microcurrent power supply with an output of 4 to 1800 μ A.After electronic stimulation,the surrounding two electrodes were observed to determine whether adverse reaction h appened during cell growth,and the ones without adverse reaction were used in th e model.The human KCs suspension was prepared from the circumcised foreskin flap s by dispase and trypsin digestion.The KCs were cultured in this model with defi ned serum- free keratinocyte culture medium.The experiment group was stimulated by alternating current with appropriate intensity and time.The cell cycle alter ation of KCs in the two groups was evaluated by propidium iodide(PI) staining an d flow cytometry. RESULTS:① The culture model was prepared complicatedly by electrodes made of nickel- chromium alloy,and adverse reaction happened easily surrounding electr odes.After 1- day current stimulation,slightly green products were found near t o electrodes.Oppositely,the culture model was prepared easily by electrodes made of pure platinum thread.On the electrodes which had been stimulated by microcur rent for 7 days,no visual chemical reaction were found.② In the self- made mic rocurrent power supply,the output power was stable,and current strength could be regulated from 4.0 to 1 800 μ Α .③ The percentage of KCs in the G0/G1 period in the control group was significantly lower than that in the experiment group[ (52.76± 3.57)% vs (46.63± 0.38)% ,t=2.953,P< 0.05],and that in the G1 perio d and the G1/G2 and division period was significantly higher in the experiment g roup than in the control group[(29.54± 16.27)% ,(27.02± 18.27)% vs(23.84± 1 6.48)% ,(20.21± 17.90)% ].So it was shown that DNA synthesis of KCs in the ex periment group was more active,and the division number was more.④ After the cul ture model prepared by pure platinum thread stimulated for 4 days,the KCs were p olygonal,karyokinesis phases were more,and KCs grew more vigorously,and cell fus ion was found more in the chamber as compared with those prepared by nickel- ch romium alloy. CONCLUSION:Stimulated by microcurrent,DNA synthesis of KCs is active,and numb er of divided cells is increased.The culture model prepared by the electrodes ma de of self- made pure platinum thread is in stable output and with controllable current strength,which is sufficient to culture KCs for a long term.It provides a new technical platform to research cell proliferation under stimulation of mi crocurrent.