腺病毒/HSV_1-tk/GCV、ACV系统对卵巢上皮癌细胞旁观者效应的研究

Study on the bystander effect of the Ad/HSV１-tk/GCV，ACV system on ovarian epithelial cancer cells.

目的：研究腺病毒／ＨＳＶ１－ｔｋ／ＧＣＶ、ＡＣＶ系统对卵巢上皮癌细胞旁观者效应。方法：常规重组腺病毒扩增纯化后，测病毒滴度。Ｘ－ｇａｌ染色法测定转染效率，ＰＣＲ检测ＨＳＶ１－ｔｋ的表达，混合细胞培养实验观察旁观者效应。结果：重组腺病毒扩增纯化后测定滴度分别为Ａｄ／ｔｋ*"６２×１０９ｐｆｕ／ｍｌ，ＡｄＬａｃＺ*"１４×１０９ｐｆｕ／ｍｌ。当感染复数为１００时，近１００％的细胞可被感染。ＰＣＲ结果显示，Ａｄｔｋ转染ＴＹＫ后，可见４０４ｂｐ的特异性条带，在同一ＭＯＩ中，有１０％的Ａｄｔｋ／ＴＹＫ细胞存在，ＧＣＶ就可杀死近５０％左右的混合细胞。ＡＣＶ的作用约为ＧＣＶ的１／１０。结论：重组腺病毒Ａｄ／ｔｋ可有效地感染人卵巢上皮癌细胞，腺病毒／ＨＳＶ１ｔｋ／ＧＣＶ、ＡＣＶ系统存在着旁观者效应。

Objective：To study on the bystander effect of Ad/HSV １tk/ GCV，ACV gene therapy system on ovarian epithelial cancer cells.Methods: Recombinant adenovirus was amplificated and purified using routine metho d,the titer was detected.The efficiency of recombinant Adtk transference were ev aluated by regular Xgal staining.The expression of HSV１tk gene was assaye d using PCR.The bystander effect was observed by mixed cell culture.Results:Recombinant adenovirus was amplificated and purified.The titer of Ad/tk was 6.2×１０９pfu/ml and of AdLacZ was 1.4×１０９pfu/ml respectively.When the MOI was 100,approximately 100% cells were infected.PCR assay showed specific 404 bp band.In the same MOI,GCV could killed almost 50% cells when there were 10% Ad /tk＋.The effect of ACV was about 10 times weaker than that of GCV.Con clusions:Replicated-deficient recombinant adenovirus Adtk can affected h uman ovarian epithelial cancer cells effectively.Bystander effect is found in Ad /HSV１-tk/GCV，ACV gene therapy system.