【作者】 陆春明；

【导师】 顾红光；

【作者基本信息】 第三军医大学 ， 外科学， 2006， 硕士

【摘要】 研究背景:随着研究的不断深入,使得借助自杀基因治疗恶性肿瘤成为可能。在癌症的自杀基因治疗中,实现目的基因在靶细胞的定位和可控性表达是自杀基因治疗的关键。目前,利用肝癌特异性转录调控元件（transcriptional regulatory elements,TREs）,如肝癌细胞特异性合成的甲胎蛋白（alpha-fetoprotein,AFP）基因的增强子/启动子以调控自杀基因于肝癌细胞的特异性表达,从而实现对肝癌组织的靶向性研究取得了长足的进步。不容忽视的是,近30%的肝癌细胞并不表现为AFP阳性,凸现出上述靶向性治疗策略的局限性。作为一类新的肝素结合生长/分化因子,Midkine基因与肿瘤的发生存在某种联系,其在肝细胞癌中呈现高表达。此前,已有研究者将Midkine启动子运用于其他类型恶性肿瘤的自杀基因治疗。新近的文献证实,运用Midkine基因启动子（promoter）2335bp（-2285～+50）片段驱动单纯疱疹病毒胸苷激酶基因（Thymidine kinasegene of Herpes simplex virus,HSV-TK）作用于AFP阳性与AFP阴性肝细胞癌,其效果远优于Midkine基因609bp片段。更为重要的是,AFP阴性肝细胞癌在上述情况下同样显示对丙氧鸟苷（GCV）的易感性（susceptibility）,从而提示在肝细胞癌自杀基因的靶向性治疗方面,以midkine驱动较AFP增强子驱动具有一定的优越性。以上述研究为基础,本研究拟采用PCR法获取人midkine基因启动子片段,将HSV-TK基因置于其调控之下,并构建重组腺病毒AdMKTK。观察AdMKTK/GCV对体外培养的AFP阳性和AFP阴性人肝癌细胞株的杀伤作用,以进一步明确将其运用于肝癌自杀基因治疗的可行性。第一部分人Midkine基因启动子片段的克隆目的:克隆人midkine启动子基因2335bp片段。方法:健康人血中提取人类基因组DNA,以此作为模板,聚合酶链式反应技术克隆人midkine启动子基因2335bp片段。琼脂糖凝胶电泳初步鉴定PCR产物,切取目的条带纯化并将其克隆入p GEM T载体中。选取阳性克隆进行DNA测序。结果:电泳结果显示,PCR产物包含有一大小约为2300bp条带和一大小约为1400bp的条带。较大片段的测序结果显示,其与Genbank中的人midkine启动子片段吻合。结论:成功克隆人midkine启动子基因2335bp片段。第二部分人MidkiBe启动子调控HSV-TK基因重组腺病毒的构建目的:构建含以人midkine（MK）启动子调控的TK基因的重组复制缺陷型腺病毒。方法:以Adeno-X^TM表达试剂盒为基础,应用分子克隆技术,将穿梭质粒pShuttle的CMV启动子替换为MK启动子,并将由p HSV-106获取HSV-TK基因的编码序列亚克隆至其下游,酶切鉴定阳性重组穿梭质粒pShuttle-MK-TK。再通过I-CeuI和PI-SceI两个稀有酶切位点,将目的基因与腺病毒质粒DNA（p Adeno-X）进行体外连接,获得含目的基因的重组腺病毒质粒DNA,后者经限制性内切酶Pac I切割后,两端露出反向末端重复序列（ITR）,利用脂质体转染293细胞,获得含有目的基因重组腺病毒上清,PCR检测。结果:酶切结果显示,MK启动子与TK基因均正向插入pShuttle中,TK位于MK启动子的下游。PCR检测显示重组腺病毒中含有MK启动子及TK基因片段。结论:利用体外连接法成功构建含以人MK启动子调控的TK基因的重组腺病毒。第三部分人Midkine启动子驱动HSV-TK自杀基因体外抗肝癌作用目的:观察以人midkine（MK）启动子调控单纯疱疹病毒胸苷激酶基因（HSV-TK）/丙氧鸟苷（GCV）自杀基因系统在体外对人肝癌细胞的杀伤效应。方法:以含有人midkine（MK）启动子调控的HSV-TK基因的重组腺病毒分别感染体外培养的甲胎蛋白阳性人肝癌细胞BEL-7402和甲胎蛋白阴性人肝癌细胞SMMC-7721,逆转录聚合酶链式反应（RT-PCR）检测HSV-TK基因在上述两种细胞中的转录表达,观察GCV对人肝癌细胞的杀伤作用。结果:GCV在体外对重组腺病毒感染的甲胎蛋白阳性人肝癌细胞BEL-7402及甲胎蛋白阴性人肝癌细胞SMMC-7721均有明显的杀伤作用,又以前者为著。相同的病毒滴度,其杀伤作用随着GCV浓度的增高而增强。均具有旁观者效应。结论:在体外,表现HSV-TK基因的甲胎蛋白阳性及阴性人肝癌细胞均可被人midkine启动子调控的自杀基因HSV-TK杀伤。重组腺病毒AdrMKTK有望用于肝癌的尤其是甲胎蛋白阴性肝癌的特异性基因治疗。更多还原

【Abstract】 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.更多还原