Yue Hai- ling ,Peng Dai- zhi,Dong Zheng- xue,Lin Hen g,Li F ang,Zhou Xin,Liu Jing,Research Institute of Burns,Southwest Hospital,Third Milit ary Medical University of Chinese PLA, Chongqing 400038,China
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 number of divided cells is increased.The culture model prepared by the electrodes ma de of self- made pure platinum thread is instable 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.
Xanya Sofra Weiss
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