脂质体介导的单纯疱疹胸苷激酶基因在人前列腺癌细胞中的表达

目的：提取并鉴定已构建的EB病毒表达载体pDR2-TK，利用脂质体介导的基因转染技术将pDR2-TK转染人前列腺癌细胞并对单纯疱疹胸苷激酶（HSV－TK）表达状况进行检测。方法：采用DNA大量制备及纯化系统提取pDR2-TK，酶切和DNA测序进行鉴定，采用阳离子脂质体法将pDR2-TK导入激素非依赖性人前列腺癌细胞系PC－3m，逆转录PCR（RT－PCR）法和SABC免疫组化法检测TK mRNA和蛋白的表达。结果：扩增提取的质粒经PstⅠ和EcoR Ⅴ酶切后各获得4个及2个片段，与原基因酶切图谱一致；所提取质粒PCR产物经DNA测序，与NCBI公布的HSV－TK基因序列对照，证实所提取质粒含目的基因序,旨质体法转染PC－3m细胞后，mRNA和蛋白均有HSV－TK的表达，其蛋白表达率约为22％。结论：pDR2-TK质粒含有目的的基因HSV－TK，阳离子脂质体法可将pDR2-TK导入人前列腺癌细胞并获得较高效率的表达。

Expression of HSV-TK gene mediated by liposome in human prostatic cancer cells

Purpose:To extract and identify of pDR2-TK, an EBV-based expression vector, and to transfer the vector into human prostatic cancer cells by using liposome-mediated gene delivery technique, Herpes Simplex virus-thymidine kinase (HSV-TK) expression in the cancer cells were evaluated as well. Method:pDR2-TK was extracted by a preparation and purification system and was evaluated by restrctional enzymatic- digestion and DNA sequencing analysis. The plasmid vector was introduced into the targeted PC-3m cells utilizing the cationic liposome-mediated gene delivery technique. In addition, mRNA and protein expression of HSV-TK were investigated by RT-PCR and SABC immunohistochemical stain respectively. Result:The amplified plasmid as digested into 4 and 2 fragments after enzymes Pst I and EcoR V incubation , and coincidence with the objective gene map. DNA sequence analysis also indicated that the vector contain the destination gene. After having been transfecting by liposome, the target PC-3m cells were demonstrated to obtain mRNA and protein expression of HSV-TK, and the positive ratio of protein stain was approximate 22%. Conclusion:The vector pDR2-TK carried the objective HSV-TK gene and could be efficiently transferred by liposome into human prostatic cancer cell.