HyTK基因转移联合ganciclovir对瘢痕成纤维细胞的体外杀伤作用

增生性瘢痕和瘢痕疙瘩的过度增生主要是由于高度增生活性的成纤维细胞的数量异常增多及细胞外基质合成增加所致．用逆转录病毒载体介导单纯疱疹病毒胸苷激酶基因（ＨＳＶｔｋ）的转移,随后应用药物９－（１,３－二羟基－丙氧基－甲基）鸟嘌呤（ｇａｎｃｉｃｌｏｖｉｒ,ＧＣＶ）可选择性地杀死增生细胞．采用组织块贴壁法在体外原代培养成功增生性瘢痕病人的成纤维细胞（ＦＢ）．重组逆转录病毒ＧＴＫ转染ＦＢ细胞后,用ｈｙｇｒｏｍｙｃｉｎＢ筛选出阳性细胞克隆（ＦＢ／ＧＴＫ）,经ＰＣＲ方法检测证明ＨｙＴＫ基因已成功地导入ＦＢ中,但不含可复制的辅助病毒．分别用不同浓度的ＧＣＶ作用于ＦＢ／ＧＴＫ及ＦＢ,光镜下观察不同时间后细胞形态变化及ＭＴＴ法检测细胞活性．结果表明,ＧＣＶ浓度大于０．１μｍｏｌ／Ｌ时即对ＦＢ／ＧＴＫ有显著的杀伤作用,且具有强有力的“旁观者效应”

Cytotoxic Effect of Ganciclovir on Scar Derived Fibroblasts in vitro Transfected with HyTK Gene

Hypertrophic scar and keloid are due to abnormal increase of proliferating fibroblasts and overproduction of extracellular matrix components.It has been shown that the transfer of the herpers simplex virus thymidine kinase(HSV tk)gene into proliferatiog cells could confer their susceptibility to the antiviral drug ganciclovir(GCV),and thus selectively produce"killing"effect on the proliferationg cells exposed to GCV.Scar derived fibroblasts were transfected with recombinant retroviral vector GTK,and by selection with hygromycin B and the positive cells clones were obtained.PCR results showed that the HyTK gene was transferred successfully into fibroblasts and replication competent virus was absent.FB/GTK cells exposed to GCV for different hours were observed under light microscope and the cell was determined by MTT colorimetric cell proliferation assay.These results suggest that FB/GTK cells exposed to GCV(>0.1 μmol/L)can be killed remarkablely with a powerful bystander effect.