表达hEGF导入基因的角朊细胞促进体外培养的正常角朊细胞增殖

Enhancement effects of pcDNA_3-hEGF modified human keratinocytes on the proliferation of normal human keratinocytes in vitro

目的 :观察含外源性人表皮细胞生长因子 (hEGF)基因改造的角朊细胞 (hEGF -MHK) ,对正常培养的角朊细胞 (NHK)增殖的影响 ;探讨转hEGF基因HK对创面愈合的生物学效应及作用机理。方法 :收集含真核表达质粒pcDNA3-hEGF的hEGF -MHK细胞培养上清 ,采用放射免疫分析法测定收集的上清液中hEGF含量 ;以 10 %的浓度加入由正常人皮肤组织分离培养的NHK细胞培养基内 ;MTT法测定NHK细胞培养 15d内的增殖速率并绘制细胞生长曲线 ;3H -TdR掺入法测定细胞培养 72h内的DNA合成速率。结果 :转基因HK细胞培养上清内含有高水平的免疫活性hEGF ,培养 7～ 9d的测定值为每天约 ( 37.89±4.13～ 35 .79± 4.33) μg·L 1,而正常HK培养上清、转染空载体的HK培养上清内均低于 5 μg·L 1;加 10 %的转基因HK细胞培养上清的NHK增殖明显加快 ,较对照组提前 4d以上融合达到生长平台期 ;转基因HK细胞培养上清作用 2 4h后可使NHK细胞DNA合成率明显提高。结论 :含hEGF基因真核表达质粒的hEGF -MHK细胞具有表达分泌hEGF的功能 ,分泌的hEGF可促进NHK细胞生长增殖 ,其机理为启动NHK细胞DNA合成 ;说明hEGF -MHK细胞可借助旁分泌途径刺激正常角朊细胞增生 。

Objective: By observation of effects produced by pcDNA 3-hEGF modified human keratinocytes (hEGF-MHK) on the proliferation of normal human keratinocytes (NHK), to investigate the biological effects of hEGF-MHK on wound healing.Methods:The supernatants were collected from normal human keratinocytes, pcDNA 3-neo and pcDNA 3-hEGF modified human keratinocytes cultures and the concentrations of hEGF in the supernatants were measured by radioactive immunoassay. Then 10% of the supernatants were added into the cultivated normal human keratinocytes respectively.Cell proliferation and DNA synthesis rate were determined by MTT assay and 3 H-TdR incorporation.Results: hEGF were about 37.89±4.13 μg·L 1 to 35.79±4.33 μg·L 1 per day in supernatants of hEGF-MHK from day 7 to day 9 after culture. At the same time, less than 5 μg·L -1 per day were detectable in supernatants of NHK and pcDNA 3-neo modified human keratinocytes. NHK proliferation was enhanced observably by the addition of 10% of hEGF-MHK supernatant, which could stimulate the DNA synthesis of NHK.Conclusion:hEGF-MHK could secrete active hEGF to enhance NHK proliferation and DNA synthesis. These results demonstrate that hEGF-MHK might promote the wound healing processin vivo.