【作者】 陈海金；

【导师】 黄宗海；

【作者基本信息】 第一军医大学 ， 普外科， 2007， 博士

【摘要】 研究背景我国大肠癌发病率从20年前的十万分之十上升到了今天的十万分之三十,在大城市恶性肿瘤的排名中大肠癌从第六位上升至第二位。大肠癌发病率正以4%的年增长率迅速上升,但治愈率却没有得到根本改变。传统的治疗方法有其自身的局限性,因此寻找新的疗法迫在眉睫。肿瘤自杀基因疗法是近年来肿瘤基因治疗中一个重要的研究策略,尤其是自杀基因的选择与重组,载体系统的优化,基因的靶向性转移表达,以及自杀基因的联合治疗等均有助于提高自杀基因特异性表达,增强杀伤肿瘤细胞的作用。肿瘤自杀基因治疗是将自杀基因导入肿瘤靶细胞并使之表达,基因编码的酶将无毒的药物前体转化为具有细胞毒性的代谢物,从而诱导靶细胞产生自杀作用,杀伤肿瘤细胞,同时还通过旁观者作用杀伤邻近未转染的肿瘤细胞。目前研究最多的且被美国FDA批准应用于临床试验的自杀基因系统主要有单纯疱疹病毒Ⅰ型胸苷激酶/丙氧鸟苷系统（HSV-TK/GCV）和胞嘧啶脱氨酶/5-氟胞嘧啶系统（CD/5-FC）。此两系统的抗肿瘤效能已在多种肿瘤的体内外实验中得到证实。日本、荷兰、墨西哥等地的学者已开始进行HSV-TK基因治疗前列腺癌的临床试验。目前的临床试验认为,针对人前列腺癌的原位基因治疗安全而有效,具有良好的抗癌生物学活性。鉴于不同类型肿瘤细胞对自杀基因系统的敏感性不同、单一自杀基因系统治疗时部分肿瘤细胞易产生耐药以及CD/5-FC和HSV-TK/GCV系统的作用机理具有很强的互补性,联合应用此两系统不仅可以提高肿瘤细胞杀伤作用,改善治疗效果,而且可以扩大肿瘤治疗谱,减少耐药现象发生。众所周知,肿瘤的自杀基因治疗虽已成为基因治疗中研究热点之一,但目的基因的靶向性及基因转移的低效率是这一疗法广泛开展的主要障碍。实验研究中大多使用逆转录病毒载体和腺病毒载体进行基因转染治疗,与其它几种常用的病毒载体相比较,逆转录病毒载体病毒滴度低,只能感染分裂期细胞,容纳外源基因的DNA片段长度不超过8 kb。而腺病毒载体感染细胞时,病毒DNA游离在细胞核内,并不整合到染色体上,在体内不能实现稳定的长期表达,且反复应用容易引起免疫反应,因而在应用上这两种病毒载体都受到一定的限制。慢病毒载体最大的特点是可以感染分裂期及非分裂期细胞,容纳外源性目的基因的片段大,可以在体内长期地表达,免疫反应小,安全性较好,可在更多范围的宿主细胞内生成高滴度的病毒,已成为当前基因治疗中载体研究的热点。同时,如能使自杀基因选择性地作用于目的肿瘤细胞而不在正常细胞中表达,将能使该治疗策略更加完善。既往研究利用肿瘤特异性基因调控元件调控自杀基因表达,如以erbB-2、α-人乳白蛋白（hALA）启动子驱动自杀基因进行乳腺癌基因治疗,甲胎蛋白（AFP）基因启动子用于肝癌自杀基因疗法,癌胚抗原（CEA）基因启动子用于肠道肿瘤自杀基因疗法等。上述治疗方案中,每一特异启动子仅能进行针对某一类型肿瘤细胞靶向基因治疗,而绝大多数恶性实体瘤的生物学特性为生长迅速,致使其滋养血管不能及时供应所有肿瘤细胞,该特点使前体药物难以接近所有肿瘤细胞。选择一合适启动子既靶向肿瘤血管内皮细胞,又靶向肿瘤细胞,如此既可杀伤肿瘤细胞,又可靶向破坏肿瘤滋养血管使得其灌流区大量肿瘤细胞缺血性坏死,这样即高效放大了自杀基因的疗效。大量研究证明,KDR在正常人体组织中呈低水平表达,而在绝大多数肿瘤中呈现高表达,且KDR不仅表达于血管内皮细胞,还表达于肿瘤细胞,其表达水平与血管内皮细胞更新速度及肿瘤恶性程度呈正相关。它不仅促进血管内皮细胞分裂、增殖,且诱导肿瘤血管增生,并促使肿瘤细胞生长转移。在肿瘤组织内KDR表达明显高于正常内皮细胞,血管的发生对于肿瘤的生长和转移起着关键的作用。因此,将KDR作为靶点,可为治疗和诊断肿瘤提供了新的理论依据。国内外众多作者报道,KDR在在胃癌、结肠、直肠、卵巢、乳腺癌等多数肿瘤组织中的表达明显增高,并与其生物学行为相关,并伴随肿瘤分级的上升而表达增强。基于上述研究,为弥补单自杀基因的不足、克服以往启动子仅适用于某一肿瘤细胞的缺点、并利用慢病毒载体的优势,本课题设计思路如下:应用慢病毒载体构建KDR启动子驱动的CD/TK融合基因的重组慢病毒,感染血管内皮细胞ECV304及大肠癌细胞LOVO,观察该治疗系统的体外杀伤作用,为重组慢病毒介导KDR启动子驱动的CD/TK融合基因靶向治疗大肠癌的进一步研究提供依据。注:本课题来源于:广东省自然科学基金重点项目（013072）。目的研究慢病毒介导的KDR启动子驱动的CD/TK双自杀基因系统对大肠肿瘤细胞选择性杀伤作用,并为进一步研究奠定基础。首先利用FUGW慢病毒载体构建携带KDR启动子调控的双自杀基因重组慢病毒,将所构建的重组慢病毒感染LOVO细胞和ECV304细胞,测定感染效率,检测转基因的表达,对转基因细胞施以前药（5-FC和/或GCV）,观察转基因细胞对前药的敏感性。最后,以LOVO细胞株建立裸鼠皮下移植瘤动物模型,观察该治疗体系的体内抑瘤效应。方法一、重组慢病毒的构建将FUGW慢病毒载体酶切去掉Ubiqutin启动子,应用PCR扩增出KDR启动子、CD基因、TK基因序列,亚克隆入中间载体pcDNA3,构建pcDNA3-KDRP-CD/TK,然后酶切KDRP-CD/TK片断,插入到FGW中,构建KDR启动子调控下的CD/TK融合基因慢病毒重组质粒FGW-KDRP-CD/TK。重组质粒在2937细胞中包装成病毒,并进一步扩增、纯化,通过荧光显微镜下293T细胞中绿色荧光蛋白（GFP）的表达观察重组病毒的包装与复制,并以PCR方法鉴定重组慢病毒。二、慢病毒介导的FGW-KDRP-CD/TK融合基因系统对LOVO细胞及ECV304细胞的体外杀伤作用用重组慢病毒FGW-KDRP-CD/TK感染LOVO细胞、ECV304细胞及LS174T细胞（对照）,检测目的基因的表达。在前药5-氟胞嘧啶（5-FC）和/或更昔洛韦（GCV）作用下,测定肿瘤细胞的存活率,观察旁观者效应,并分别通过流式细胞仪、透射电镜观察细胞凋亡与坏死。三、慢病毒介导FGW-KDRP-CD/TK融合基因系统对大肠癌LOVO细胞裸鼠模型的体内抑瘤效应采用BALB/C雌性裸鼠,4～6周龄,对数生长期的LOVO细胞接种于裸鼠左侧腋窝皮下,建立大肠癌动物模型,当肿瘤直径达0.5cm时,将荷瘤裸鼠随机分为Ⅰ组:注射重组慢病毒FGW-KDRP-CD/TK与前药5-FC与GCV;Ⅱ组:仅注射前药5-FC与GCV;Ⅲ组:仅注射重组慢病毒FGW-KDRP-CD/TK;Ⅳ组:空白对照,不施加任何处理。每5d测量肿瘤生长状况,估算肿瘤体积,绘制肿瘤生长曲线,25天治疗结束后处死裸鼠,称瘤重,计算肿瘤生长抑制率并观察该治疗体系的体内抑瘤效应,行组织内CD/TK基因表达的检测、肿瘤组织常规病理检查,并观察重要脏器心、肝、脾、肺、肾有无病理变化。结果一、重组慢病毒质粒的鉴定1.重组慢病毒质粒FGW-KDRP-CD/TK的鉴定在重组慢病毒质粒FGW-KDRP-CD/TK的构建过程中,主要采用酶切和测序的方法进行鉴定。将PCR所得的CD片段、TK片段及KDR分别连于T载体（pMD18-T）送上海申友生物技术有限公司测序,结果正确。FGW-KDRP-CD/TK以HindⅢ和BamHI酶切后电泳出现大小约2.4kb（CDglyTK）片段,结果正确。2.重组慢病毒质粒在293T细胞的包装及滴度测定将10μg的转移载体,7.5μg的CMV△R8.2包装质粒,5μg的VSV-G包膜质粒,加入130μL的2mol/L CaCL₂中,磷酸钙法三质粒瞬时共转染293T细胞,24h后通过荧光显微镜即可见GFP荧光表达,72h后收集上清,0.45μm滤器过滤,25000rpm离心90min,沉淀溶于Hanks溶液,-80℃贮存备用。将梯度稀释的病毒液,加入293T细胞中,72h后置于荧光显微镜下观察GFP的表达情况,计数发荧光细胞数目,根据病毒用量和稀释度计算滴度。二、慢病毒介导的FGW-KDRP-CD/TK融合基因系统对LOVO细胞及ECV304细胞的体外杀伤作用1.重组慢病毒体外感染及鉴定细胞LOVO、ECV304、LS174T分别加入200μl的重组慢病毒悬液（100MOI）培养72小时后,在荧光显微境下观察GFP基因的表达,可见80-90%有绿色荧光存在,三种细胞具有相似的感染率。通过RT-PCR检测发现,除LS174T细胞外,LOVO、ECV304细胞检测有目的基因的表达。2.重组慢病毒对各细胞的体外抑制效应将200μl的重组慢病毒悬液（100MOI）感染各细胞株,加入不同浓度的前药培养72h后MTT法检测细胞生长抑制率。结果:感染慢病毒FGW-KDRP-CD/TK的LOVO及ECV304细胞对前药具有较高的敏感性,转基因细胞的存活率随前药浓度的增加而递减。当前药浓度为GCV=100mg/L,5-FC=160mg/L时,LOVO存活率为（5.40±1.85）%,ECV304存活率为（5.26±1.82）%。而感染慢病毒的LS174T细胞对前药不敏感,LS174T存活率仍有（95.02±2.82）%,与其他各组细胞相比差异有显著性意义（F=6567.806,P＜0.001）。感染FGW-KDRP-CD/TK的LOVO、ECV304及LS174T细胞,单用浓度100mg/L的GCV时,生存率分别为（31.96±1.62）%、（29.10±2.86）%、（96.57±1.89）%,单用浓度为160mg/L的5-FC,生存率分别为（29.60±1.91）%、（25.23±2.05）%、（96.20±1.91）%,二者联合时,各细胞生存率分别为（5.40±1.85）%、（5.26±1.82）%、（95.02±2.82）%,提示联合用药比单一用药对LOVO细胞和ECV304细胞均有较强杀伤效应（F=803.123,P＜0.001;F=375.99,P＜0.001;F=1.555,P=0.226）。3、旁观者效应将感染慢病毒的细胞与未感染细胞以不同混合培养,观察到该体系明显的旁观者效应,其随转基因细胞所占比例的增加而明显增强。当转基因细胞的比率为40%时,LOVO和ECV304细胞分别有（32.40±1.29）%、（29.95±1.96）%的存活,而LS174T细胞存活率仍在（97.74±1.74）%,三者比较差异均有显著性意义（F=5180.938,P＜0.001）。4、重组慢病毒对LOVO细胞及ECV304细胞凋亡的影响转基因LOVO细胞给以前药（GCV:1mg/L;5-FC:40mg/L）处理24h后,流式细胞仪检测发现:治疗组LOVO细胞加药24h后凋亡率为27.09±1.45%,明显高于对照组凋亡率2.96±0.34%（P=0.007）。转基因ECV304细胞给以前药（GCV:1mg/L;5-FC:40mg/L）处理24h后,流式细胞仪检测发现:治疗组ECV304细胞加药24h后凋亡率为10.85±1.50%,明显高于对照组凋亡率2.25±0.55%（P=0.035）。5、前药处理前后LOVO细胞的电镜观察透射电镜下见部分细胞出现细胞体收缩、染色质边聚,有的胞核形态不规则,核发生碎裂,胞浆内可见多个电子密度增强的核碎片,有的可见凋亡小体、（或整个凋亡细胞）被吞噬和降解的现象。部分细胞出现细胞器溶解等坏死征象。三、慢病毒介导的FGW-KDRP-CD/TK自杀基因系统对大肠癌LOVO细胞裸鼠模型的体内抑瘤效应1.荷瘤裸鼠肿瘤生长情况接种肿瘤细胞后10d左右,出现皮下肿瘤结节。裸鼠成瘤达0.5cm后开始治疗,治疗结束时肿瘤标本的称重结果及抑瘤率:Ⅰ组:（21.34±5.72）mg,95.59%:Ⅱ组:（466.61±67.89）mg,3。58%;Ⅲ组:（451.91±48.29）mg,6.61%;Ⅳ组:空白对照组（483.91±51.60）mg。可见,组间瘤重有显著性差异（F=165.805,P＜0.001）,Ⅰ组明显缩小,第Ⅱ、Ⅲ、Ⅳ组肿瘤生长情况无统计学意义。2.肿瘤病理变化及肿瘤组织目的基因表达的检测前药加慢病毒治疗组肿瘤组织切片中可见片状坏死区,而且这些区域可见大量炎性细胞浸润。RT-PCR检测发现重组慢病毒转基因荷瘤动物肿瘤组织内有CD/TK融合基因表达。3.治疗后裸鼠重要脏器的组织学观察为了解该治疗系统对裸鼠有无毒副作用,取治疗组小裸鼠心、肝、脾、肺、肾行常规病理检查未见明显异常。4.裸鼠可视化大肠癌动物模型成功的建立了大肠癌裸鼠皮下移植瘤可视化动物模型,LT-9MACIMSYSPULS整体荧光成像系统下,可满意观察到GFP绿色荧光,其优点:①早期发现肿瘤形成;②动态观察肿瘤生长及转移情况等。结论1.本实验首先以慢病毒为载体,成功地构建含KDR启动子驱动的CD/TK自杀基因系统的重组慢病毒,重组慢病毒的滴度达6×10¹⁰pfu/ml;2.重组慢病毒对LOVO细胞、ECV304细胞及LS174T细胞均具有较高的转染率,且对三者转染率相似;3.重组慢病毒能将KDR启动子驱动的CD/TK融合基因转入LOVO细胞及ECV304细胞中并进行有效表达,使该系统对LOVO细胞及ECV304细胞具有满意的靶向杀伤作用,这种杀伤作用具有浓度依赖性,并且联合应用两种前药较单用一种前药更为有效;4.该治疗体系的作用机制为对靶细胞的直接杀伤作用及旁观者效应,表现为处理后靶细胞的凋亡及坏死;5.应用LOVO细胞可成功建立大肠癌移植瘤裸鼠普通动物模型及可视化动物模型,并能方便地检测肿瘤的生长、转移;6.重组慢病毒转基因荷瘤动物肿瘤组织内可表达CD/TK融合基因产物,使该治疗体系具有满意的体内抑瘤效应,表现为肿瘤的生长抑制及肿瘤组织坏死;7.KDR启动子驱动的CD/TK融合基因系统治疗后,荷瘤裸鼠心、脾、肝、肺、肾等器官无明显组织学变化,提示该系统对荷瘤裸鼠无系统毒副作用。更多还原

【Abstract】 Background:In China, the incidence of colon cancer is thrice more than it’s twenty years ago as one in million,accompanying with it’s rank changed from sixth to second in all cities’ cancers.This incidence ascends rapidly at 4% annual growth rate,while no fundamental changes were seen in cure rate.It’s urged to find new therapies since the traditional treatments had their limitations.Suicide gene therapy is an important strategy in cancer gene treatment,especially the selection and recombination of suicide gene,the optimization of vector system,the gene targeting transfer expression, and combined suicide gene therapy can increase the specific expression of suicide gene and enhance the effect to kill tumor cells.In the suicide gene therapy ,suicide gene is imported into the cancer cells and translated into enzymes which can convert the atoxic prodrug into cytotoxic metabolites,inducing the suicide of targeting cells and the death of cancer cells, meanwhile killing the non-transfected cancer cells through the bystander effect. The most investigated suicide gene system which approved into clinic trials by FDA in US are Herpes simplex virus type 1 thymidine kinase（HSV-TK）/ganciclovir（GCV） and E. coli cytosine deaminase （CD）/5-fluorocytosine （5-FC）,the anticancer effect of them have been approved in many tumors in vivo and vitro.The HSV-TK gene therapy has been used in prostate cancer clinical trials in Japan,Holland and Mexico.As shown in recent trials, in situ gene therapy was safe and effective in treating human prostate cancer, with good anticancer biologic activity.HSV-TK/GCV and CD/5-FC systems are most investigated in the suicide gene therapy. Some HSV-TK/GCV and CD/5-FC clinical treatment projects have been validated by FDA.Due to the different sensitivity of cancer cells to the suicide gene therapy,elevating drug resistance treated with only one system, complementarity in the mechanisms of CD/5-FC and HSV-TK/GCV, the combination of these two systems can not only increase the lethal effect of cancer cells, improving the treatment effect, but also enlarge the oncotherapy spectrum, reducing the drug resistance.It’s well known that although the suicide gene therapy of cancer is popular, the low efficiency of purpose gene targeting and gene transfer are the major problems this method meets.The retroviral and adenoviral vectors were wildly used to transfect gene, compared with other viral vectors, the retroviral vector has low virus titer, only infects the division cells, while can contain the size of exogenous gene segment not more than 8kb.When the adenovirus infect cells, their DNA can not combine to chromatosomes but liberate in the nucleus, so it can not reach the long steady expression, and will cause the immune response when being used repeatedly, which limited the usage of these two viral vectors. The features of lentiviral vector such as infecting both division and non-division cells, holding larger segment size of exogenous gene , expressing long in vivo, lower immune response, better safety, producing high titer of virus in host cells at large range have been popular in the current gene therapy research. Meanwhile, this strategy will be more improved when the suicide gene can selectively affected and expressed in the cancer cells not normal cells. The cancer specific regulatory element such as erbB-2,α- hALA promoter were used to promote suicide gene therapy in breast cancer, AFP promoter was used in liver cancer, CEA promoter in intestine cancer and so on. Each specific promoter was used to treat specific type of targeting cancer cells in those treating protocols, while most tumor grows rapidly causing the nourishing vessel cannot supply all the tumor cells which prevents the effect of prodrugs. The suitable promoter can be selected to target the tumor vessel endothelial cells（VECs） and the tumor cells so that it can kill tumor cells and target destroy tumor nourishing vessels which leads to the tumor cells ischemic necrosis in the supplying area, highly amplifying the effect of the suicide gene. Amounts of studies indicated that KDR was expressed at low levels in normal human tissues, while highly expressed in VECs and most tumors cells. The expression of KDR was positively correlated with the VECs renewing rate and tumor malignancy which not only stimulated the division, proliferation of VECs, but also induced blood vessel hyperplasia, spured the growth and metastasis of the tumor cells. The expression of KDR in tumors is obviously higer than the normal endothelial cells, the genesis of vessels plays a key role in the growth and metastasis of tumor .So the application of KDR as a target is a novel theoretical support to diagnosis and treatment of tumor. KDR was reported highly expressed in gastric cancer ,colon cancer, rectal cancer, ovarian cancer, breast cancers and correlated with their biological behaviors, higher when the tumor grading ascends.Based on these studies, to infect ECV304 and LOVO colon cancer cells by lentiviral KDR promoted CD/TK fusion gene lentivirus and to observe the lethal effects in vitro of this therapy can not only overcome the shortage of using single suicide gene and the limited effect of some promoters which only available on specific tumor cells, but also exert the advantages of lentiviral vector, provide the basis for further study on this gene targeted therapy in colon cancer.Note: this study was supported by Natural science foundation of Guangdong province （013072）. Objective:To investigate the selective killing effect of CD/TK double suicide genes drivbed by KDR promoter mediated by lentivirus on colon tumor cells. Firstly, the FUGW lentiviral vector was used to construct the recombinant lentivirus carrying double suicide gene medicated by KDR promoter which were used to infect ECV304 cells and LOVO cells. To determine the efficiency of infection, detect the expression of transgene, treat transgenic cells with prodrug （5-FC and /or GCV） and observe the sensitivity. At last, to build the nude mouse models with transplanted subcutaneous tumor on the base of LOVO cell line and to observe the tumor suppression of this therapy in vivo.Methods:1 .The construction of the recombinant lentivirus.The Ubiqutin promoter in the FUGW lentiviral vector was digested by enzyme, KDR promoter,CD gene sequence,TK gene sequence amplified by PCR were subcloned into the intermedial vector pcDNA3 to construct pcDNA3-KDRP-CD/TK, then the KDRP-CD/TK fragment was digested with restriction enzyme and inserted into the FGW, the lentiviral recombinant plasmid FGW- KDRP-CD/TK with CD/TK fusion gene regulated by the KDR promoter was constructed then.Then recombinant plasmid was packaged in the 293T cells, further amplified and purified, the package and cloning of recombinant virus were observed through the expression of green fluorescent protein（GFP） in 293T cells by fluorescent microscope, the recombinant lentivirus were identified by PCR.2. The lethal effect of KDRP-CD/TK fusion gene system mediated by lentivirus to LOVO and ECV304 cells in vitro.The recombinant lentiviral KDRP-CD/TK was used to infect LOVO, ECV304,LS174T（control） cells, the expression of objective gene was observed. The tumor cell survival rate, the bystander effect were detected under the treatment of prodrug 5-FC/GCV, the cell apoptosis and necrosis were observed through flow cytometry and transmission electron microscope respectively.3. The anti-tumor effect of FGW-KDRP-CD/TK fusion gene system mediated by lentivirus to the colon cancer LOVO cell athymic mouse model in vivo.The LOVO cell line in exponential phase of growth was vaccinated subcutaneously into the left axillary area of BALB/C female, 4 to 6 weeks old athymic mouse, to construct the colon cancer model. When the diameter of the tumor reached to 0.5cm, the tumor bearing nude mice were divided into group （ie viruses +5-FC+GCV,viruses only, 5-FC+GCV only,blank）at random. The growth of tumor was measured every five days, the weight of tumor and the tumor inhibitory rate were calculated, the tumor growth state was observed, the anti-tumor effect of the therapy was observed in vivo. After treatment, the CD/TK gene expression in tissue was detected, the routine pathological examination of tumor was made, and major organs such as heart, liver, spleen,lung,kidney were observed to find whether the pathological changes existed.Results:1. The identify of the recombinant lentiviral plasmids.1.1 The identify of the recombinant lentiviral plasmids KDRP-CD/TK.During the construction of recombinant lentiviral plasmid KDRP-CD/TK, the enzyme cutting and sequencing were made to identify. The CD ,TK fragment and KDR were united to T vector （pMD18-T） respectively and send to Shanghai Shenyou biologic technique LtD for sequencing, results were shown correct. The electrophoresis after enzyme restrictions of FGW-KDRP-CD/TK with Hind III and BamHI shew the product was 2.4kb （CDglyTK）, which was correct.1.2 The package of recombinant lentiviral plasmid in 293T cells and identification of virus titer.The 10μg transfer vector, 7.5μg CMVAR8.2 packaging plasmid, 5μg VSV-G amicula plasmid were added in 130μL 2mol/L CaCL₂, three plasmids were transiently cotransfected into 293T cells by calcium acid phosphate method, after 24h the GFP fluorescent expression was seen in fluorescent microscope, the supernatant was collected after 72h and filtered by 0.45μm filtrum, then centrifuged at 25000rpm for 90min, precipitate was solved in Hanks solution and stored at - 80℃for use. The gradient diluted virus solution was added into 293T cells, the expression of GFP was detected under fluorescent microscope after 72h, fluorescent cells was counted and the tite was calculated based on the virus dosage and dilution.2. the lethal effect of KDRP-CD/TK fusion gene system mediated by lentivirus to LOVO and ECV304 cells in vitro .2.1 The infection and identification of recombinant lentivirus in vitro.LOVO, ECV304, LS174T cells were added in the 200μl recombinant lentiviral suspension（100MOI） and cultured for 72h, the GFP gene expression was observed by fluorescent microscope, 80-90% cells had the green fluorescence, three types of cells shared similar infection rate. The expression of the objective gene was detected by RT-PCR in the LOVO,ECV304 cells except LS174T cells.2.2 The inhibition effect of recombinant lentivirus to cells in vitro.The 200μl recombinant lentiviral suspension（100MOI） was used to infect each cell lines, the different concentration of prodrugs were added and cultured for 72h, then the MTT was used to detect the inhibitory rate of cell growth.Results: the LOVO and ECV304 cells infected by lentiviral FGW-KDRP-CD/TK had higher sensitivity to the prodrug, the survival rate of the transgenic cells decreased when the concentration of prodrug increased. When the concentration of GCV and 5-FC were 100mg/L and 160mg/L respectively, the survival rate of LOVO cells was （5.40±1.85）%, the ECV304 was （5.26±1.82）%. While the LS174T cells infected by lentivirus were not sensitive to prodrug, the survival rate was （95.02±2.82）%, compared with other groups the significant difference existed （F=6567.806, P< 0.001） . The survival rate of FGW-KDRP-CD/TK infected LOVO,ECV304 and LS174T cells treated with 100mg/L GCV were （31.96±1.62） % ,（29.10±2.86） %,（96.57±1.89）% respectively; while treated with 160mg/L 5-FC the rates were （29.60±l .91）%、（25.23±2.05）%、（96.20±1.91）% respectively; and the combination of both drugs were （5.40±1.85）%、（5.26±1.82）%、（95.02±2.82）% respectively. This suggested that the combined use of two drugs had more lethal effect than either one in LOVO and ECV304 cells （F=803.123, P<0.001; F= 375.99, P<0.001; F=1.555, P=0.226） .2.3 Bystander effect.The obvious bystander effect could be observed in the different proportion mixed culture of the lentivirus infected cells and uninfected cells, the effect was obviously enhanced when the transgenic cells ratio increased. when this ratio was 40%, there were （32.40±1 .29）% LOVO cells and（29.95±1.96） % ECV304 cells existed respectively, while the survival ratio of LS174T cells was （97.74±1.74）%,the significant statistical difference was seen among them （F=5180.938,P<0.001） .2.4 The effect of recombinant lentivirus to the apoptosis in LOVO and ECV304 cells.The transgenic LOVO cells was dealt with prodrug for 24h（ GCV: 1mg/L; 5-FC: 40mg/L） and then observed with flow cytometry: the apoptosis rate of LOVO cells treated group was 27.09±1.45%, significantly higher than the ratio of 2.96±0.34% in control group（ P=0.007）. The same procedure was done in the ECV304 transgenic cells , the apoptosis rate of ECV304 cells treated group was 10.85±1.50%, significantly higher than the ratio of 2.25±0.55% in control group（ P=0.035）.2.5 The transmission electron microscopy observation of prodrug dealt LOVO cells Cell body construction,chromatin fringe, irregular nucleus shape, nuclear fragmentation, several enhanced electron desity nuclear fragments in cytoplasm, apoptosis body, or the phagocytosis and degradation of entire apoptosis could be found in some cells under the transmission electron microscopy. Some cells may exhibit the necrosis signs like organelles dissolve.3. The anti-tumor effect of FGW-KDRP-CD/TK fusion gene system mediated by lentivirus to the colon cancer LOVO cell athymic mouse model in vivo .3.1 The growth of cancer in tumor bearing nude mice.The subcutaneous tumor node was found in the 10d after tumor cells injection.When the diameter of node reached 0.5cm, the nude mouse received treatment and the weight of tumor sample and tumor inhibited rate were recorded as follows: group I （21.34±5.72）mg,95.59%; group II :（466.61±67.89）mg, 3.58%; groupIII （451.91±48.29）mg, 6.61%; group IV: （483.91±51.60） mg;the significant difference was seen between four groups （F =165.805,P<0.001） ,weight in groups I decreased obviously,tumor weights hadn’t difference between between II group,III group, IV group.3.2 Pathological changes and the detection of objective gene expression in tumor.Focal necrosis areas were seen infiltrated by amounts of inflammatory cells in the tumor tissue sections in group treated with prodrug and lentivirus. The expression of CD/TK fusion gene at mRNA levels was detected by RT-PCR in the recombinant lentivirus transgenic tumor bearing animal’s tumor tissue.3.3 The histological observation in the nude mouse’s major organs after treatment.In order to understand if the treatment system had toxic side effects to the nude mouse, the routine pathologic tests of heart, liver, spleen, lung,kidney were done in the treated group with no obvious abnormal findings.3.4 The visible colon cancer nude mice model. The visible subcutaneous transplanted colon cancer nude mice model was successfully constructed. The GFP green fluorescence could be observed under the LT-9MACIMSYSPULS general fluorescence imaging system. The features included:①Find the tumor as early as possible.②Observe the tumor growth and metastasis dynamically.Conclusions:1 .The KDRP promoted CD/TK suicide gene system of recombinant lentivirus was successfully constructed based on the lentiviral vector, the virus tite was 6×10¹⁰ pfu/ml2.The recombinant lentivirus could be highly transfected to LOVO cells, ECV304 cells and LS174T cells, the transfection efficiency was almost the same;3.The recombinant lentivirus could transfect the the KDRP promoted CD/TK fusion gene to LOVO cells and ECV304 cells and express effectively, with the satisfied targeting lethal effect which was concentration dependent. The combination of both prodrugs were more effective than either one;4.The mechanism of this therapy were direct tumoricidal activity and bystander effect to the targeting cells, causing the apoptosis and necrosis of target cells;5.The common and visible animal model of colon cancer transplanted nude mice could be constructed using LOVO cells, it’s convenient to detect the growth, transfer;6.CD/TK fusion gene product could be expressed in the tumor tissues of recombinant lentivirus transgenic tumor bearing animals, made the satisfied anti-tumor effect in vivo, such as tumor growth inhibiting and tissue necrosis;7.After the KDRP promoted CD/TK fusion gene therapy, no obvious histological changes were seen in heart, liver, kidney, lung,spleen of the tumor bearing nude mice, suggested that this system had no toxic side effects to the tumor bearing nude mice.更多还原