目的:观察脉冲Nd:YAG激光照射体外单层培养KB细胞后的形态改变及损伤后HSP70, c-Fos的表达情况,初步探讨较强脉冲激光对细胞的损伤效应及损伤修复机制.方法:建立单层培养细胞的脉冲Nd:YAG激光损伤模型,每个脉冲能量密度为160 J/cm2～186 J/cm2或220 J/cm2～257 J/cm2,分别于照后即刻、2 h和6 h,用台盼蓝染色、TUNEL检测分析该激光对KB细胞的损伤特点,免疫组化法检测HSP70, c-Fos的表达水平.结果:当照射剂量为220 J/cm2～257J/cm2时,照后即刻,光斑中央细胞形态严重破坏,直接坏死;周围细胞形态未发生明显改变.2 h后周围细胞TUNEL着色也增强,呈强阳性.照后6 h光斑中央及周围细胞着色均减弱.TUNEL着色区直径随时间先扩大后缩小.当照射剂量为160 J/cm2～186 J/cm2时,细胞内HSP70、c-Fos表达随时间先显著增强,而后减弱至正常.结论:脉冲Nd:YAG激光在所选剂量下,可以引起单层KB细胞的损伤,包括即刻坏死、延迟性死亡及可逆性损伤.HSP70、c-Fos的高表达说明它们在保护受损细胞、修复激光所致损伤中发挥重要作用.

Objective: To investigate the character of morphological change after Nd:YAG laser irradiation on KB cells, and determine if two protective proteins(HSP70, c-Fos) are involved in the cellular stress response and repair processes. Methods: Monolayer KB cell cultures on glass-slides were irradiated with Nd:YAG laser. The pulse energy density was controlled within the range of 160 J/cm~2～186 J/cm~2 or 220 J/cm~2～257 J/cm~2 in single pulse mode. The cultures were then analyzed by means of trypan blue staining and TUNEL examination. HSP70 and c-Fos were examined with immunocytochemical staining. Results:KB cells at the center of laser beam were damaged instantly, the results of trypan blue staining and TUNEL were strong positive, while cells near the boundary of laser beam had hardly morphological change and were stained weakly. At 2h after irradiation, the TUNEL staining inside and near the laser beam was both strong positive. But at 6h, the staining weakened. The diameter of staining area changed with the time after irradiation (increased at first and then decreased) . The expression of HSP70 and c2Fos firstly increased obviously and then decreased to normal level gradually. The expression of above two proteins is the highest at 2h after irradiation.Conclusions :Pulse Nd :YAGlaser can damage monolayer KB cells , including immediate necrosis , delayed death and reversible damage. High expression of HSP270 and c2Fos after irradiation shows that the two proteins may play an important role in protecting cells and repairing damage induced by Nd :YAGlaser.