| OBJECTIVE To investigate the therapeutic effects of herpes simplex virus â…¡ thymidine kinase (HSV-II-tk) and angiostatin fusion gene which were regulated by human AFP promoter on human primary hepatocellular carcinoma in vitro. METHOD From clinical PHC sample cloning human AFP promoter by PCRXonstructed eukaryon expression vector pcDNA3/AFP/tk/Angio by genetic engineering techniques.The pcDNA3/AFP/tk/Angio was transfected into HPHC SMMC-7721 and Bel 7402 by cation liposome Lipofectamine.RT-PCR,MTT,flow cytometry (FCM) and transmission electron microscope were used to detect whether HSV-â…¡-tk and angiostatin gene was expressed and whether the products of HSV-â…¡-tk may inhibit the growth of experimental cells about SMMC-7721 and Bel 7402.RESULTS 1.The sequencing date shows that coding region of human AFP promoter is 300bp.2.The purpose fragment which inserted pcDNA3/tk/Angio was confirmed by sequencing.3.A 1100bp specific fragment which is the same the HSV-â…¡-tk was obtained by RT-PCR,and it showed that the HSV-II-tk gene was expressed in the Bel 7402 that is expressed AFP.The result of MTT showed that can inhibit the growth of Bel 7402 and cannot inhibit the growth of SMMC-7721,the different of which was statistically significant(P<0.05).The apoptosis peak can be seen in the pcDNA3/AFP/tk/Angio group of Bel 7402 in FCM measuring, whereas we cannot see it in the contrast group of Bel 7402 and in the all group of SMMC-7721.The apoptotic body can be seen in the pcDNA3/AFP/tk/Angio group of Bel 7402 in FCM measuring, whereas we cannot see it in the contrast group of Bel 7402 and in the all group of SMMC-7721 by the transmission electron microscope. CONCLUSIONS Human AFP promoter was cloned. The eukaryon expression vector pcDNA3/AFP/tk/Angio was constructed.After being transfected by Lipofectamine, HSV-II-tk gene can be expressed in Bel 7402 cells.The HSV-â…¡-tk/GCV system can efficiently inhibit the growth of Bel 7402 cells that is expressed AFP.
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