论文标题:基因重组TK-IRES-Endostatin腺相关病毒双靶点治疗膀胱癌的实验研究
Experimental Study on the Gene Therapy of Bladder Cancer with rAAV-TK-IRES-Endostatin
论文作者
论文导师 韩瑞发,论文学位 博士,论文专业 外科学
论文单位 天津医科大学,点击次数 100,论文页数 172页File Size18932K
2007-05-01论文网 http://www.lw23.com/lunwen_90490107/
bladder cancer; Adeno-associated virus; suicide gene; endostatin;gene therapy
膀胱癌是泌尿系统最常见的恶性肿瘤，手术治疗是其主要手段，手术后若不给予其他治疗，其近期复发率为60％～90％，远期复发率几乎为100％，局部放疗和全身性化疗因其毒副作用严重和抗药性目前在临床中几乎不被使用。膀胱腔生物免疫治疗是一种有效的佐剂和给药途径，但大约30％～45％的患者对此治疗无反应，且因为反复经尿道插入导尿管给药、严重的毒性反应、抗药性和较低的药物利用度而影响了临床治疗疗程和效果。 自杀基因疗法又称为药物敏感基因疗法，即将前药转换酶基因(自杀基因)直接转染肿瘤细胞，该基因编码特殊的酶，可将原先对哺乳动物细胞无毒性的前药在肿瘤细胞中代谢为有抗肿瘤作用的有效产物，该产物作为DNA合成的核苷替代物掺入到DNA中，干扰细胞DNA的合成，从而引起这些肿瘤细胞的凋亡。由于人体细胞中缺乏催化前体药物的酶系统，所以前体药物只有在被感染的细胞中被激活，只对自杀基因呈阳性的细胞起诱导凋亡的作用，而不损害患者的正常细胞。肿瘤细胞转入自杀基因后，前体药物对患者治疗所产生的毒性将被限定在转化细胞或它的微环境中，而不至于产生显著的系统毒性，基于这一优点，自杀基因治疗仍然是肿瘤基因治疗的热点之一。单纯疱疹病毒胸苷激酶基因(herpes simplex virus thymidine kinase，HSV-TK)是研究最多的自杀基因，其前体药物是环氧鸟苷(GCV)。HSV-TK催化GCV磷酸化生成GCV-MP，该步骤在GCV代谢形成GCV-TP的过程中是限速步骤。GCV-TP抑制DNA聚合酶活性，阻止DNA复制，从而杀死肿瘤细胞。 膀胱癌是血管生成依赖性的，新生血管容易造成瘤细胞逸入血管形成血行转移。抑制肿瘤血管生成是近年来治疗膀胱癌的一个新热点。内皮抑素(Endostatin)是目前已知作用最强的肿瘤血管生成抑制剂，且依据我们的动物实验研究未发现造成心脑血管缺血等副作用。它在美国1999年进入Ⅰ期临床试验，两年后实验结束，肯定内皮抑素能够有效减缓肿瘤的生长速度并且副作用极少。鉴于以上成果，内皮抑素在M．D．Anderson肿瘤中心已进入大规模临床试验。 腺相关病毒载体具有逆转录病毒和腺病毒的许多优点，并且与其它几种病毒相比，腺相关病毒载体具有安全性好、无致病性、免疫原性低、物理性质稳定、感染细胞谱广，可介导外源基因长期稳定表达等优点，被视为最有前途的基因治疗载体之一，成为目前基因治疗载体研究的热点。目前腺相关病毒载体基因治疗研究已在代谢性疾病和遗传性疾病中广泛开展，但其在膀胱肿瘤基因治疗中的研究尚少。 随着分子生物学技术的发展，90年代基因治疗作为肿瘤的全新第五代辅助疗法已成为人类征服癌症的最有前景的方法，但必须指出的是，目前困扰基因治疗应用进程和疗效的关键问题是：(1)载体的选择：选择高表达、低免疫原性、无插入致畸及生物安全性好的载体是基因治疗的核心技术；(2)基因的选择：肿瘤的发生、发展是一个多基因、多通道调控的过程，因此，采用针对单一靶点调控肿瘤细胞分化、加速凋亡过程的基因治疗其抗肿瘤作用有限。 为此，本课题采用生物安全性高、低免疫原性的基因重组腺相关病毒载体载体技术平台，将构建国际公认的抗肿瘤血管生成作用最强的内皮抑素基因和自杀基因非融合重组新型AAV载体，加入信号肽，利用AAV对膀胱癌细胞的高度亲和力，使感染后膀胱癌细胞持续表达并分泌内皮抑素抑制血管生成饿死肿瘤细胞、使肿瘤在缺血的状态下增强其对前药及自杀基因的敏感性，协同双靶点治疗膀胱癌，达到治疗与预防肿瘤复发的靶向作用，以期建立高效、靶向、低毒副作用，协同作用强的双靶点基因输送及表达系统。 第一部分含自杀基因及内皮抑素基因非融合重组腺相关病毒表达载体的构建 目的：应用基因工程和分子生物学技术，采用pIRES载体构建携带有自杀基因和内皮抑素基因的非融合AAV载体，充分发挥每个目的基因的生物学效应，为以后的进一步实验奠定载体基础。 方法：1．利用携带HSV-TK基因的腺病毒穿梭质粒pAdTrack-CMV-TK，PCR扩增TK序列至pMD-19T simple载体，分别用ClaI、XbaI两步法双酶切pMD-19Tsimple-TK及pAAV-ES，构建pAAV-TK。所构建质粒用酶切、PCR和测序等方法鉴定，准确无误后，进行下一步实验。 2．利用携带IRES基因的质粒pIRES-MCS，携带TK基因的质粒pAAV-TK，携带ES基因的质粒pAAV-ES及pCMV-MCS和pAAV-MCS，PCR分别扩增IRES、TK、ES序列至pMD-19T simple载体，构建过渡质粒pCMV-TK-IRES-ES，用酶切、PCR和测序等方法鉴定所构建质粒正确与否。 3．分别双酶切pCMV-TK-IRES-ES及pAAV-MCS，胶回收pAAV-MCS大片段和TK-IRES-ES小片段，选取克隆后，小量提取质粒，用ClaI、XbaI双酶切鉴定pAAV-TK-IRES-ES。 4．利用PstI、NotI双酶切和PCR两种方法来鉴定质粒pAAV-TK、pAAV-TK-IRES-ES中的ITR序列是否存在。 结果：1．提取pAdTrack-CMV-TK质粒后，通过电泳、PCR及测序证实TK序列为正确序列。PCR扩增带ClaI、EcoRI位点的TK基因片段，胶回收PCR产物并连接至pMD-19T simple载体后构建pMD-19Tsimple-TK质粒，用ClaI、EcoRI双酶切鉴定pMD-19Tsimple-TK，1％琼脂糖凝胶电泳示：酶切出的片段大小为1.1kb左右，符合预期，并将酶切正确的pMD-19Tsimple-TK克隆TK片段送交测序，与原序列完全一致。小量提取pAAV-TK质粒后，用ClaI、XbaI双酶切，1％琼脂糖凝胶电泳结果显示克隆酶切出的片段大小都为1.1kb左右，证明构建pAAV-TK成功。 2．提取pAAV-ES和pIRES-MCS质粒PCR扩增后，可获得大约700bp和650bp的ES和IRES基因片段，经基因测序证实为正确序列。分别双酶切鉴定pMD-19Tsimple-ES／IRES，1％琼脂糖凝胶电泳示：克隆酶切出的片段大小符合正确克隆，将酶切正确的pMD-19Tsimple-ES／IRES克隆ES／IRES片段送交测序，与原序列完全一致。用ClaI、EcoRI两步法双酶切pMD-19Tsimple-TK及pCMV，连接pCMV大片段和pMD-19Tsimple-TK小片段，用ClaI、EcoRI双酶切鉴定pCMV-TK，酶切出的片段大小为1.1kb左右构建pCMV-TK成功。用XbaI、EcoRI两步法双酶切pMD-19Tsimple-IRES及pCMV-TK，连接pCMV-TK大片段和pMD-19Tsimple-IRES小片段，用XbaI、EcoRI双酶切鉴定pCMV-TK-IRES，酶切出的片段大小为600bp左右，构建pCMV-TK-IRES成功。XbaI、BamI两步法双酶切pMD-19Tsimple-ES及pCMV-TK-IRES，胶回收pCMV-TK-IRES大片段和pMD-19Tsimple-ES小片段，选取克隆后，小量提取质粒，用XbaI、BamHI双酶切鉴定pCMV-TK-IRES-ES，酶切出的片段大小为700bp左右，为正确克隆，过渡质粒pCMV-TK-IRES-ES构建成功。 3．分别用两步法双酶切pCMV-TK-IRES-ES及pAAV-MCS，胶回收pAAV-MCS大片段和TK-IRES-ES小片段，选取克隆后，小量提取质粒，用ClaI、XbaI双酶切鉴定pAAV-TK-IRES-ES，酶切出的片段大小为2.3kb左右，为正确克隆，构建pAAV-TK-IRES-ES成功。 4．用PstI、NotI分别双酶切pAAV-TK、pAAV-TK-IRES-ES，2％琼脂糖凝胶电泳示：pAAV-TK，pAAV-TK-IRES-ES中均有137bp左右的条带切出，PCR产物1％凝胶电泳示大小在均3kb左右，符合预期，说明pAAV-TK-IRES-ES，pAAV-TK上两重组质粒中均有ITR序列。 第二部分重组腺相关病毒的包装、纯化、浓缩及鉴定 目的：包装、纯化、浓缩及鉴定含有治疗基因片段的重组腺相关病毒颗粒。 方法：1．利用AAV-Helper free system包装系统，pRC、pHelper三质粒电穿孔和磷酸钙共沉淀转染AAV-293细胞包装腺相关病毒，并用“氯仿-PEG／NaCl沉淀-氯仿抽提”来进行AAV病毒的粗纯化、冰乙醇沉淀法浓缩腺相关病毒。 2．通过Philips Tcnai-20电子显微镜扫描和透射观察重组腺相关病毒的形态及其从细胞核到细胞表面的分泌过程；采用AVSachTM ELISA法测定病毒的rAAV2颗粒滴度；采用SDS-PAGE和HPLC鉴定病毒及其纯度。 结果：1．电转化或者磷酸钙转化72小时后原先贴壁生长的AAV-293细胞由于病毒的大量扩增，293细胞死亡，变圆漂起。当90％的细胞变圆漂起时，提示病毒扩增完毕，可以进入分离纯化阶段。 2．病毒样品经过负染色后，电子显微镜扫描时可见较高浓度的病毒颗粒，有一定程度聚集，大小约为20nm。经过电转化或者磷酸钙转化72小时后的AAV-293细胞，切片后投射电子显微镜观察，可见病毒从细胞核到细胞表面的分泌。在SDS-PAGE电泳后考马斯亮蓝染色可见3条特征性条带；AVSachTM ELISA法测定rAAV病毒颗粒滴度达2×10~(10)v．p／mL，浓缩后可达到2×10~(11-12)v．p／mL。 第三部分rAAV-TK-IRES-ES双靶点膀胱癌基因治疗的体外实验研究 目的：鉴定人脐静脉内皮细胞；鉴定重组腺相关病毒对膀胱肿瘤细胞的转染影响；鉴定体外杀伤膀胱肿瘤细胞的效果。 方法：1．利用HE染色、硝酸银染色、免疫组化及电子显微镜对人脐静脉内皮细胞进行鉴定。 2．采用携带绿色荧光蛋白报告基因的rAAV-EGFP转染膀胱肿瘤T24细胞，来确定重组腺相关病毒对该细胞的转染情况及转染效率；通过提取经rAAV-ES、rAAV-MCS、rAAV-TK及rAAV-TK-IRES-ES转染后的细胞基因组，PCR分别扩增ES、TK及TK-IRES-ES(TIE)基因片段来鉴定重组腺相关病毒的活性；同时比较转染rAAV-TK前后T24细胞的形态、细胞周期等的变化来明确rAAV-TK对该细胞的影响。 3．通过rAAV-ES、rAAV-TIE重组腺相关病毒体外转染T24膀胱肿瘤细胞，鉴定培养上清液中内皮抑素的浓度；同时，研究内皮抑素对人脐静脉内皮细胞(HUVEC)细胞生长的影响及诱导凋亡的检测。 4．通过MTT法来检测rAAV-TK、rAAV-TIE／GCV系统对T24膀胱肿瘤细胞杀伤的剂量和时间依赖性；通过通过JC-1、流式细胞仪等方法来检测该系统对T24细胞凋亡的诱导作用。 结果：1．硝酸银染色后光镜观察到银颗粒沉淀在内皮细胞边缘，使内皮细胞出现典型的多边形，细胞间锯齿状镶嵌紧密排列；透射电镜观察到内皮细胞具有Webel-Plade小体(W-P小体)的特征性细胞器；HUVEC八因子相关抗原免疫组化可见细胞胞质中有棕黄色颗粒，以上说明实验细胞为HUVEC细胞。 2．rAAV-EGFP转染T24细胞72小时后，在荧光显微镜下可以看到大量细胞表达报告蛋白，绿色荧光分布在整个细胞中，以胞核最为集中，说明在T24细胞中可以表达携带的外源基因EGFP。流式细胞仪检测结果提示最适MOI值为1×10~6v．p．／cell；FV10-ASW激光共聚焦电子显微镜观察rAAV2感染细胞时，可见病毒颗粒首先与细胞膜受体结合，然后逐步进入细胞核；病毒颗粒感染T24细胞72小时后，提取细胞基因组，PCR扩增目的片段，1％凝胶电泳可见大约1.1kb、2.8kb和700bp的TK、TIE、ES基因片段，测序后与预期序列一致，说明重组腺相关病毒具有生物活性，可以转染T24细胞；转HSV-TK基因T24细胞传代培养后，提取细胞基因组，PCR产物1％琼脂糖凝胶电泳及测序结果均正确，说明转HSV-TK基因T24细胞可以稳定携带TK基因；透射电镜及流式细胞仪提示细胞转染病毒前后形态及周期没有变化，说明重组腺相关病毒rAAV-TK对细胞没有影响。 3．分别用含有人内皮抑素的重组腺相关病毒(rAAV-ES、rAAV-TIE)和空病毒(rAAV-MCS)转染T24膀胱肿瘤细胞培养72小时，收集培养液上清，Endostatin浓度各组分别为：rAAV-ES组(60.08ng／mL)、rAAV-TIE组(58.14ng／mL)、rAAV-MCS组(0 ng／mL)，说明rAAV-ES、rAAV-TIE病毒转染肿瘤细胞后可以同等分泌内皮抑素。重组腺相关病毒(rAAV-ES、rAAV-TIE)转染细胞的培养液上清处理HUVEC细胞72h后，流式细胞仪检测结果显示，rAAV-ES、rAAV-TIE两组凋亡率分别是37.02％和32.14％，明显高于空病毒转染组(7.90％)和空白对照组(0.30％)。说明基因转染rAAV-ES、rAAV-TIE细胞产生的内皮抑素均具有诱导内皮细胞凋亡的生物活性。 4．重组腺相关病毒(rAAV-TK、rAAV-TIE)转染T24细胞72h后，流式细胞仪检测结果显示，rAAV-TK、rAAV-TIE两组凋亡率分别是34.12％和36.91％，明显高于空病毒转染组和空白对照组，说明基因转染rAAV-TK、rAAV-TIE／GCV系统均可以诱导膀胱肿瘤细胞凋亡。 第四部分rAAV-TK-IRES-ES双靶点基因治疗的裸鼠体内实验研究 目的：构建裸鼠膀肮肿瘤模型，分析rAAV-TK-IRES-ES双靶点基因治疗膀胱癌的体内效果。 方法：1．T24细胞悬液接种于裸鼠右侧肩胛区皮下，SPF条件下饲养，观察皮下肿瘤成瘤情况。 2．取已成瘤裸鼠25只，随机分为5组：对照组、空病毒组(rAAV-MCS)、rAAV-TK组、rAAV-ES组、rAAV-TIE组，每组5只，治疗4周后，颈椎脱臼处死荷瘤裸鼠，取出移植瘤，10％中性福尔马林固定，石蜡切片，准备常规HE染色及组织免疫组化及计算MVD。 3．治疗4周后，用摘眼球放血方法收集裸鼠血液标本，静置2 h，5000rpm离心15 min，吸取血清，双夹心ELISA法检测血清中Endostatin浓度。 结果：1．将膀胱肿瘤细胞T24细胞接种于裸鼠皮下，大约3周后，有25只接种部位长处肿瘤，成瘤率约为93％。 2．瘤内注射rAAV-ES、rAAV-TK、rAAV-TIE大约9天后，肿瘤生长受到显著抑制，治疗结束后：各组肿瘤的体积分别是：rAAV-ES组(0.75±0.08)cm~3、rAAV-TK组(0.71±0.11)cm~3、rAAV-TIE组(0.52±0.09)cm~3、rAAV-MCS组(1.27±0.13)cm~3和空白对照组(1.24±0.17)cm~3；rAAV-ES、rAAV-TK、rAAV-TIE 3组分别与阳性对照与阴性对照比较，差异均有显著性(p均＜0.05)；rAAV-TIE组与rAAV-ES组、rAAV-TK组比较，差异均有显著性(p均＜0.05)；rAAV-ES组与rAAV-TK组比较，差异没有显著性(p＞0.05)。 3．用免疫组织化方法标记微血管内皮细胞，计数微血管密度，结果显示各组裸鼠移植瘤MVD分别：rAAV-ES组(18.72±2.53)个／HP、rAAV-TK组(21.74±4.62)个／HP、rAAV-TIE组(12.73±1.78)个／HP、rAAV-MCS组(52.38±6.46)个／HP和空白对照组(49.94±7.17)个／HP；治疗组显著低于空白对照组和空病毒组(p＜0.05)；rAAV-TIE组较rAAV-ES组和rAAV-TK组显著降低，组间差异有显著统计学意义(p＜0.05)。 4．ELISA结果显示荷瘤裸鼠血清中的内皮抑素分别如下：rAAV-ES组(38.52±6.53)μg／L、rAAV-TIE组(40.33±7.48)μg／L；而rAAV-TK组、rAAV-MCS和空白对照组裸鼠血清中未检测到内皮抑素的表达。 结论：我们成功构建重组rAAV-TK-IRES-ES腺相关病毒，体外和体内实验表明rAAV-TK-IRES-ES可有效抑制膀胱癌的血管生成和肿瘤的生长，可有效双靶点基因治疗膀胱肿瘤，为膀胱癌原位基因治疗提供一种有效的辅助方法。
Bladder transitional cell carcinoma(BTCC) is one of the most commonurological malignant diseases. Its recurrent rate is almost 60% to 90% after operationalthough as a major method to prevent this tumor. Local radiotherapy and systematicchemotherapy are rarely used due to their severe side effects and drug resistance.Nowadays, intravesical biotherapy and immunotherapy instillation are regarded as aneffective adjuvant method, but almost 30% to 45% patients had no response to thistherapy, furthermore, the clinical effect was limited by its severe side effects,drugresistance and fewer drug taking. Numerous scholars are paying close attentions to suicide gene treatment thesedays and gene engineering is used to transduce suicide gene into tumor cells. Suicidegene included in virus or bacterial genome, encodes special enzyme. Nontoxicprodrug can be metabolized by the latter, then become venenous production and leadto tumor cell"s death. Therefore, the treatment is called "virus oriented enzymolysisprodrug medication" or "molecular chemotherapy". Thymidine kinase gene treatmentis one of most used suicide gene treatments. The enzyme metabolizes nuclide analoginto diphosphorylation and then becomes triphosphorylation which against tumor andselectively metabolizes GCV. GCV becomes triphosphorylation and then interferesand intercepts DNA normal synthesis and cellular proliferation. In the treatment fortumor, herpes simplex virus-thymidine kinase gene is considered to be the mosteffective and also licensed into clinical trial only. It can play a part in preventing DNA chain elongating, so that selectively kill and wound suicide gene cells inmultiplicative division period. Vascular formation is regulated by stimulating factors and inhibiting factors, andmany stimulators and inhibitors have been identified by now. Among all the inhibitors,endostatin has been testified as the strongest one. It could inhibit the proliferation ofendothelial cells, induce apoptosis of tumor cells, inhibit tumor growth and metastasiseffectively, and has no side effect. So endostatin maybe the most preferred agent fortumor anti-angiogenic therapy. In 1999, it has been used to tumor patients as thefirst stage clinical experiment in US, after two years" observation, it was proved thatendostatin therapy can inhibit tumor growth and progression with little side effects. Adeno-associated virus (AAV) offers many desirable features of retrovirusestherapy. The recombinant AAV vectors can transducer both dividing andnon-dividing cells in vitro and in vivo. Efficient and long-term transduction in vivoand the lack of both cytotoxicity and cellular immune responses are the hallmarkfeatures of AAV mediated gene transfer. AAV was regarded as one of the extremevectors in gene therapy in future. Although AAV vectors have been extensively usedin gene therapy for genetic and metabolic diseases, few studies have used this vectorsystem for bladder cancer gene therapy. With the development of molecular biology, gene therapy are now regarded as awholly new and promising method to conquer human carcinoma. But, we have tosolve two key cruxes: a) vector choose, to select a kind of high expressed, low levelantigen and safety vector is the major aim toward this. b) therapeutic gene select, aswe all known, the occurrence of tumor is an multi-controlled pathway progress. So,the gene therapeutic effect was limited by only one gene selection. Therefore, in our study, we aims to combined the endostatin and suicide genetogether by pIRES vector via recombined AAV, which was regarded as one of the extreme vectors in gene therapy in future. Enhanced the effect of TK on the basis oftumor anti-angiogenic gene therapy of endostatin by double targeted at these twogenes and then established the express way of the therapeutic gene with little sideeffects. Part 1 Construction of the non-melted plasmid AAV contain TK and endostatin gene Objective: To construct the non-melted plasmid AAV contain TK and endostatingene via pIRES vector. Methods:1. PCR TK segment from pAdTrack-CMV-TK and then cloned it tovector pMD-19T simple. Digested pMD-19Tsimple-TK and pAAV-ES by ClaI,XbaIand then construct pAAV-TK and verified by sequence indentification. 2. PCR IRES,TK,ES framents from pIRES-MCS, pAAV-TK, pAAV-ES and thencloned these genes into vector pMD-19T simple to construct pCMV-TK-IRES-ES.verified by sequence indentification and enzyme digestion. 3. Double digested pCMV-TK-IRES-ES and pAAV-MCS, connection fragmentsto construct pAAV-TK-IRES-ES and then digested by ClaI and XbaI to verification. 4. To avoid the lost of ITR, we identified pAAV-TK and pAAV-TK-IRES-ESthisby PCR and enzyme digestion. Results: 1. Extract pAdTrack-CMV-TK, PCR TK fragment and verified byelectrophoresis. Digested by ClaI and XbaI, the fragment is about 1.1kb, whichestablished the successfully construction of pAAV-TK. 2. Extract pAAV-ES and pIRES-MCS and then PCR them, we got two framentsabout 700bp and 650bp which are in accordance with correct sequences. By digeseting pMD-19Tsimple-TK and pCMV and connectded them by T4 ligase, wesucessfully constructed pCMV-TK. Also, By digeseting pMD-19Tsimple-IRES andpCMV-TK and connectded them by T4 ligase, we sucessfully constructedpCMV-TK-IRES. Digested pCMV-TK-IRES-ES by XbaI and BamHI, we got fragmentabout 700bp, which is the correct size. So, we successfully constructedpCMV-TK-IRES-ES. 3. Digested pCMV-TK-IRES-Es and pAAV-MCS, and then connected themtogether by T4 ligase, and then digested its clone by ClaI and XbaI, the fragment isabout 2.3kb. We successfully constructed pAAV-TK-IRES-ES. 4. When pAAV-Tk and pAAV-TK-IRES-ES were digested by PstI and NotI, wegot 137bp fragment by electrophoresis. The PCR product were about 3kb, which wereas expected. Part 2 The package, purification, concentration and idenfication of recombination AAV Objective: To package, purify, concentrate and idenfy of the recombinationAAV. Methods: 1. AAV-293 cells were co-transfected by electroporationusing pAAV -TK(or Paav-TIE), pRC and pHelper to package rAAV-TK and rAAVTIE. the cells were under Chloroform-NaCI sediment-Chloroform and ice alcoholto dissociate, purify and concentrate rAAV. 2. Viral particle of purified rAAV were assayed by AVSachTM ELISA. Use.SDS-PAGE Coomassie brilliant blue staining to observe capsid protein of rAAV. And use electric microscope to observe the size and the procedure of rAAV. Results: We obtained high quality of rAAV after dissociating and purifying. Theviral particlesof rAAV were 2×10~(11-12). Under SDS-PAGE and Coomassie brilliantblue staining, we could find three notable viral capsid protein strap of AAV Weobserved the viral particle under electron microscope and found that rAAV werelocated uniformly, the diameters were about 20 to25 nm and the figure werepolyhedron. The successful packaging of rAAV established the foundation for laterexperiment using this vector in vivo and in vitro. Part 3 Study on rAAV-TIE gene therapy of bladder cancer in vitro Objective: To observe the effect on apoptosis and growth of cell bladder cancercell by transfecting with rAAV-ES, rAAV-TK-IRES-ES, rAAV-TK; To indentify theHUVEC cell and the influence of bladder tumor cell by tranfected by rAAV-TK. Methods: 1.To indenfy the HUVEC cell by HE and AgNo_3 staining, IHC andelecric microscope. 2. To value the MOI by transfect the T24 cell by rAAV-EGFP; We identify thebiological effect of rAAV by extracting the gene of ES, TK and TK-IRES-ES(TIE)transfected by rAAV-ES, rAAV-MCS, rAAV-TK and rAAV-TK-IRES-ES; Also, weuse microscope and etc. to study the influence of T24 transfected by rAAV-TK. 3. T24 cell was transfected by rAAV-ES and rAAV-TIE, collected the serum andidentified the concentration of Endostatin by ELISA. A t the same time, we use FCMto value the apoptosis extent cultured by the serum which contain the Endostatin. 4. We study the dose and time effect of rAAV-TK and rAAV-TIE/GCV system by MTT test. And also we use JC-1 and FCM to observe the apoptosis effect of thissystem. Results: 1. After the HUVEC cell was stained by AgNO_3, we saw the silverparticles just like the stone road. When scaned by the electric microscope, we saw theW-P body, whichi was the charicteristic of HUVEC cell. 2. After the bladder tumor cell was transfected by rAAV-EGFP about 72 hours,lots of GFP was expressed in the core of cell. The MOI was about 1×10~6v.p./cell.The T24 cell was little affected after transfecting by rAAV-tk by FCM test. 3. The concentration of endostatin was tested by ELISA and just as followings indifferent groups: rAAV-ES group(60.08ng/mL)、rAAV-TIE group(58.14ng/mL),rAAV-MCS group(0 ng/mL). The cell in the rAAV-ES group and rAAV-TIE groupwere more easy to apoptosis than control group.(rAAV-ES37.02%, rAAV-TIE32.14%) 4. After 72h of transfecting by rAAV-TK and rAAV-TIE/GCV system, theapoptosis were 34.12%(rAAV-TK) and 36.91%(rAAV-TIE). Part 4 The study of rAAV-TIE gene therapy of bladder cancer of nude mice in vivo Objective: To construct bladder cancer animal model using nude mice and studythe therapeutical effect of rAAV-TIE model in vivo. Methods: 1. Cell suspension of T24 cells were injected into thesubcutaneously of right scapular region of nude mice, The nude mice were raised under SPF condition and observed the xenograft tumor growth. 2. Bearing tumor nude mice were randomly divided into 5 groups: rAAV-MCSgroup, rAAV-Tk group,rAAV-ES group,rAAV-TIE group. 4 weeks later of treatment,the nude mice were killed by dislocate vertebrae cevicales. The xenografts tumorswere fixed for HE stain. The liver tissue and nephridial tissue were also fixed for HEstain. 3. Four weeks later, nude mice were sacrificed for blood sample and endostatinconcentration was assayed by ELISA. Results: 1. After 3 weeks of injected with T24 cells on nude mice, 25 showvisible tumor on the injected location. The rate of tumor formation is 93%. 2. After 9 days injected by rAAV-ES, rAAV-TK, rAAV-TIE, the tumor volumewere: rAAV-ES group(0.75±0.08)cm~3, rAAV-TK group (0.71±0.11)cm~3, rAAV-TIEgroup (0.52±0.09)cm~3, rAAV-MCS group(1.27±0.13) cm~3 and control group1.24±0.17)cm~3. 3. The MVD in the different groups were as followings: rAAV-ES group(18.72±2.53)/HP, rAAV-TK group (21.74±4.62)/HP, rAAV-TIE group(12.73±1.78)/HP, rAAV-MCS group (52.38±6.46)/HP and control group(49.94±7.17)个/HP 4. The endostatin concentration in the different groups were as followings:rAAV-ES group (38.52±6.53)μg/L and rAAV-TIE group(40.33±7.48)μg/L. 5. HE statin confirmed the tumor. The liver tissue and kidney tissue of eachgroup has no obviously cell degeneration and necrosis. Conclusions: We successfully constructed rAAV-TK and rAAV-TIE. Experiment in vitro and in vivo indicated that interested gene could express in hostcell mediated by rAAV. In vitro study shows that rAAV-TIE could inhibit tumorinduced angiogenesis and suppress both the initiation and the subsequent growth ofhuman bladder cancer. Gene therapy with rAAV-TIE may be an effective adjuvantmethod.
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