Effect Of Adenovirus-mediated Transfer Of HSV-tk/GCV Controlled By Human Midkine Promoter On Human Hepatoma In Vitro

Background:New development in molecular biology leads to a possible approach to cancer therapy by using suicide gene.In this strategy,restriction and controllability of the transgene expression in tumor cells is most crucial issues.Heretofore,exciting achievement has been made on the specific expression of suicide genes in hepatocellular carcinoma (HCC) utilized by specific transcriptional regulatory unit corresponding to tumor-specific or organ-specific genes,such as alpha-fetoprotein(AFP).The previous studies suggested the feasibility of the AFP promoter-mediated gene therapy for HCC.However,relatively low frequency of AFP expression in human HCC may hamper its clinical application, although serums AFP were elevated in 70%of HCC patients.Being a heprin-binding growth factor,midkine(MK) possesses a variety of activities relating to tumrigenesis which include those of mitogenicity,angiogenicity and antiapoptosis,suggesting a potential role of MK for oncogenesis.Moreover,the high level of MK expression has been reported for a number of tumors,such as bladder cancer, colorectal cancer,as well as HCC.Although the mechanisms underlying tumor-specific expression of MK gene need to be clarified,several reports on MK gene promoter-drived suicide genes expression in tumor cells have been published.Especially,the transcriptional activity of MK gene promoter was as strong as that of the enhancer-linked AFP promoter in human HCC specimens.The MK gene promoter could activate the HSV-TK gene in HCC cells to a similar extent as the AFP promoter.Moreover,this study suggested that the MK gene the promoter is applicable to AFP-low/nonproducing.In this study,the human MK promoter was obtained by PCR.And then,the recombinant adenovirus containing thymidine kinase gene of Herpes simplex virus drived by human midkine gene promoter was constructed.Both AFP positive and negative human HCC cell lines were used to explore the killing effect of AdMKTK/GCV,including morphological change and bystander effect.Part One: Cloning of human midkine promoter gene fragmentObjective:To clone the 2335bp fragment(-2285～+50) of human midkine promoter gene.Methods:As a template,the human genome DNA was extracted from healthy human blood firstly.And then,the polymerase chain reaction(PCR) was applied to clone the 2335bp fragment of human midkine promoter gene.After primary identification with agarose gel electrophoresis,the corresponding DNA fragment was purified and cloned into p GEM T vector.Finally,the recombinant plasmid was comfirmed with DNA sequencing.Result:Besides of a fragment about 1400bp,another fragment about 2300bp was obtained by PCR.This DNA fragment was in accordance with the human midkine promoter dominantly with 8 mutant base pairs.Conclusions:We have got the 2335bp fragment of human midkine promoter successfully.Part Two: Construction of recombinant adenovirus with human midkine promoter and thymidine kinase gene of Herpes simplex virusObjective:To construct the replication-deficient recombinant adenovirus containing human mindkine gene promoter and thymidine kinase gene of Herpes simplex virus(HSV-TK).Methods:Based on Adeno-X^TM expression system,CMV promoter of pShuttle was replaced by MK promoter.The SHV TK gene,which was obtained from plasmid pHSV-106, was subcloned into pShuttle vector under MK promoter.The proper p Shuttle-MK-TK was identified by special restriction endonuclease analysis.Both of the plasmid pShuttle-MK-TK and Adeno-X DNA were digested with restriction endonuclease PI-SceI and I-CeuI,the target fragment(MK+TK) was subcloned into the Adeno-X DNA with ligation in vitro and the recombinant Adeno-MK-TK was obtained.After linearized by digesting with restriction endonucleases PacI,the plasmid Adeno-MK-TK was transfected into HEK 293 to produce recombinant virus stocks.Using PCR method,the virus stocks were identified whether it contained MK and TK gene.Results:The restriction endonuclease analysis showed that the MK promoter and TK gene were inserted into the pShuttle successfully.And,the TK gene was located on the downstream of the MK promoter.PCR products were in coincidence with anticipation.Conclusion:By ligation in vitro,the recombinant HSV-TK gene under MK promoter mediated by adenovirus vector was constructed successfully.Part Three: Effect of adenovirus-mediated transfer of HSV-Tk/GCV drive by human midkine promoter on human hepatoma in vitroObjective:To observe and examine the killing effects of the herpes simplex virus thymidine kinase/gancclovir(HSV-TK/GCV) suicide gene system transferred by adenovirus on human HCC in vitro.Methods:The AFP-positive human HCC cell line BEL-7402 and AFP-negative human HCC cell line SMMC-7721 were infected with the recombinant adenoviral vector containing HSV-tk gene derived by human midkine promoter.The intracellular transcription of this recombinant was detected by RT-PCR,and the effects of ganciclovir(GCV) on killing the human HCC cells were observed by using microscope and examined by MTT.Results:In vitro,GCV exerts remarkable killing effects and the "bystander effect" on AFP-positive BEL-7402 as well as AFP-negative cells SMMC-7721.The killing effect was enhanced following to the GCV concentration preconditioned with same recombinant adenovirus.Conclusion:Both of AFP-positive and AFP-negative human HCC cells with HSV-TK gene expressing can be killed by suicide gene HSV-TK which is controlled by human midkine promoter.It hoped that the recombinant adenovirus(AdrMKTK)could be used in specific gene therapy on the HCC,especially to AFP-negative ones.