自杀基因表达产生旁观者效应的初步研究

Primary study on mechanisms of bystander effect caused by expression of suicide gene

目的：构建单纯疱疹病毒胸苷激酶基因（ＨＳＶ－ｔｋ）的重组逆转录病毒载体，初步探讨转染有ＨＳＶ－ｔｋ基因的人肝癌细胞产生旁观者效应的作用机制。方法：ＥｃｏＲⅠ和ＰｖｕⅡ酶切质粒ＰＨＳＶ－１０６，回收１．９６ｋｂＨＳＶ－ｔｋ基因片段，将其定向克隆人逆转录病毒载体ＬＸＳＮ的ＥｃｏＲⅠ／ＨｐａⅠ酶切位点。经ｌｉｐｏｆｅｃｔｉｎ将ＨＳＶ－ｔＫ基因转染人肝癌细胞系，测定转染细胞对ＧＣＶ或ＡＣＶ的敏感性。

effect mechanisms of bystander effect caused by humen hepatocellular carcinoma cells expressing HSV-tk gene.Method: Recombinant retroviral vector containing herpes simplex thymidine kinase(HSV-tk) gene was constructed and introduced into human hepatocellular carcinoma cell lines (SMMC-7721) with Lipofectin as a delivery system, HSV-tk gene transduced HCC cells were cocultured with corresponding parental cell in different proporations in the presence of Ganciclovir (GCV 100μg/ml) . Under the conditions of different cell density or special media, the results were measured by means of MIT colrimetric assay,3H-TdR assay,flow cytometric analysis. Result:The HSV-tk-expressing cells exhibited higher sensitivity to ganciclovir (GCV) and acyclovir (ACV) . In addition, parental HCC cells were markedly reduced when they were cocultured with their HSV-tk gene transduced cells at a high population at which most cells were in contact with one another. However, when cocultured at low population at which no cells were in contact,a weak but statisti-caly significant bystander effect was observed. Media conditioned by transduced cells with GCV resulted in a weak cytotoxic effect of untransduced cells.Conclution: These results indicate cationic Liposome is a efficient gene delivery system,HSV-tk-expressing cells in vitro exhibited higher sensitivity to GCV or ACV mediated by Lipofectin.The bystander effect of suicide gene is very apparent in vitro, whose mechanism may be a combination of several factors. Metabolic coorperation mediated by 'Gap Junction' plays a major role in cell-cell contact and cell-cell contact plays a major causative role in the bystander ef-fect.Minor contributor to this phenomenon are some cytotoxic substance to parental cells released from transduced dead cells.