CAJ | 학술논문

目的了解外源性NO对HaCaT细胞迁移功能的影响及其机制.方法以硝普钠作为NO的供体,在HaCaT细胞培养液中加入0.1、1.0、10.0、100.0、1000.0 μmol/L硝普钠,分别于划痕0 h(划痕即时)及划痕后6、12、24、48 h观察并计算细胞迁移率.根据该结果筛选出硝普钠最佳作用浓度及作用时间,以此为实验刺激条件,应用激光共聚焦显微镜观察细胞骨架的改变,采用蛋白质印迹法、PCR等方法检测实验组(加入10.0 μmol/L硝普钠培养24 h)及阴性对照组整合素β_1、RhoA、cdc42、Rac1蛋白及RhoA、cdc42、Rac1 mRNA表达情况.对各实验结果行单因素方差分析和重复测量的方差分析.结果加入硝普钠处理后各浓度组细胞迁移率随划痕时间的延长而逐渐增加.加入10.0 μmoL/L的硝普钠划痕后6～48 h与划痕0 h比较,差异均有统计学意义(F=31.002,P值均小于0.05).激光共聚焦显微镜显示,阴性对照组细胞纤毛稀少,胞内应力纤维束纤细,而实验组纤毛明显增多,胞内应力性纤维束增粗.实验组整合素β_1蛋白表达水平明显低于阴性对照组(F=8.25,P=0.015);而RhoA的蛋白表达水平为0.92±0.04,高于阴性对照组的0.64±0.04(F=7.25,P<0.05),cdc42、Rac1蛋白表达也高于阴性对照组(F值分别为14.10、6.50,P值均小于0.05).实验组RhoA、cdc42、Rac1的mRNA水平均高于阴性对照组(F值分别为23.67、10.39、9.52,P值均小于0.05).结论适宜浓度的外源性NO可促进HaCaT的增殖及迁移,提示其在皮肤创面修复中起重要作用.其机制可能与整合素β_1表达下降、细胞骨架的改变有关.
목적 료해외원성NO대HaCaT세포천이공능적영향급기궤제.방법 이초보납작위NO적공체,재HaCaT세포배양액중가입0.1、1.0、10.0、100.0、1000.0 μmol/L초보납,분별우화흔0 h(화흔즉시)급화흔후6、12、24、48 h관찰병계산세포천이솔.근거해결과사선출초보납최가작용농도급작용시간,이차위실험자격조건,응용격광공취초현미경관찰세포골가적개변,채용단백질인적법、PCR등방법검측실험조(가입10.0 μmol/L초보납배양24 h)급음성대조조정합소β_1、RhoA、cdc42、Rac1단백급RhoA、cdc42、Rac1 mRNA표체정황.대각실험결과행단인소방차분석화중복측량적방차분석.결과 가입초보납처리후각농도조세포천이솔수화흔시간적연장이축점증가.가입10.0 μmoL/L적초보납화흔후6～48 h여화흔0 h비교,차이균유통계학의의(F=31.002,P치균소우0.05).격광공취초현미경현시,음성대조조세포섬모희소,포내응력섬유속섬세,이실험조섬모명현증다,포내응력성섬유속증조.실험조정합소β_1단백표체수평명현저우음성대조조(F=8.25,P=0.015);이RhoA적단백표체수평위0.92±0.04,고우음성대조조적0.64±0.04(F=7.25,P<0.05),cdc42、Rac1단백표체야고우음성대조조(F치분별위14.10、6.50,P치균소우0.05).실험조RhoA、cdc42、Rac1적mRNA수평균고우음성대조조(F치분별위23.67、10.39、9.52,P치균소우0.05).결론 괄의농도적외원성NO가촉진HaCaT적증식급천이,제시기재피부창면수복중기중요작용.기궤제가능여정합소β_1표체하강、세포골가적개변유관.
Objective To investigate the effect of exogenous nitric oxide ( NO) on the migration of HaCaT cell and its possible mechanism. Methods Sodium nitroprussiate (SNP) was used as the donor of NO. Different concentrations of SNP (0. 1, 1.0, 10.0, 100.0, 1000.0 μmol/L ) were added into nutrient culture medium of HaCaT cells. Cell migration rate was observed and calculated at post scratching hour (PSH) 0 (immediately after scratching) , 6, 12, 24, 48. The most suitable concentration of SNP and culture duration were selected as stimulation condition. Cytoskeletons of HaCaT cells were observed under con-focal laser scanning microscope. The expressions of integrin beta 1, RhoA, Racl and Cdc42 of cells in experiment group (cultured with 10.0 μmol/L SNP for 24 hours) and negative control group were determined at mRNA and protein levels with RT-PCR and Western blot respectively. Data were processed with one-way analysis of variance (ANOVA) and repeated measure ANOVA. Results Migration rate of HaCaT cells in each group increased gradually as time after scratching went on. There were significant differences between PSH 6-48 and PSH 0 in cells cultured with 10. 0 μmol/L SNP ( F = 31.002, P values all below 0. 05 ). Pili were rarely observed in negative control group with slender stress fibers in cells. In comparison, the amount of pili amount increased obviously in experiment group with thickened stress fibers. Compared with those of cells in control group ( RhoA protein expression =0.64 ±0.04) , integrin beta 1 expression decreased obviously ( F =8.25, P =0.015), RhoA (0.92 ±0.04), Cdc42 and Racl were up-regulated at both protein ( with F value respectively 7. 25 , 14. 10, 6. 50, P values all below 0.05) and mRNA levels ( with F value respectively 23. 67 , 10. 39, 9. 52, P values all below 0. 05 ). Conclusions Exogenous NO in suitable concentration can promote the proliferation and migration of HaCaT cell, suggesting it exerts significant effect in wound repair. The changed cytoskeletons and the down-regulated integrin beta 1 expression may be involved in this process.