【作者】 张勇；

【导师】 靳风烁；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 膀胱肿瘤是我国最常见的泌尿系统恶性肿瘤,具有多发、高复发性的特点,且随着复发,其恶性程度也呈增高趋势,最终发生浸润转移而危及生命。如何提高膀胱肿瘤的治疗效果是目前泌尿外科领域未解决的难点和一大课题。随着对肿瘤发病机制研究的深入,人们逐渐认识到肿瘤的生长、浸润和转移都需要依赖新生血管的形成。研究显示,当肿瘤长至1~2mm,即需要有新生血管供应,否则肿瘤细胞因缺血缺氧出现死亡,并由此提出了治疗肿瘤的新途径—抗血管生成治疗,为实体肿瘤的治疗提供了新的思路,并形成了抗肿瘤血管生成疗法。与传统的肿瘤治疗相比较,抗肿瘤血管生成策略有较多的优势:作用强,易于到达血管靶细胞,不产生耐药性,无明显毒副作用。虽然近年来抗肿瘤血管生成治疗的研究取得了令人瞩目的成果,国外已有多种此类药物进入临床应用或实验研究,显示出良好的效果,但还存在明显的不足之处:①这些治疗分子都是从外界施加的,作用时间短,必需持续给药;②由于这些分子可溶性方面的限制,需要大剂量静脉给药,使得毒性大为增加。血管内皮生长因子(VEGF)是目前已知的最重要促血管生成因子。多数肿瘤组织和肿瘤细胞株均过量表达VEGF。VEGF主要通过与高亲和力酪氨酸激酶受体血管内皮生长因子受体-2(VEGFR-2)结合而发挥生物学活性,VEGF/VEGFR-2信号转导通路被证实在肿瘤血管生成过程中起关键性作用。通过阻断该信号转导通路,可以达到抑制肿瘤血管生成,从而抑制肿瘤的生长和转移的目的。根据这一原理,我们设计了阻断VEGF与其受体相互作用的新的治疗方案:构建含VEGFR-2(Flk1)胞外段和IgG2a Fc段融合基因的腺病毒载体Ad-Flk1-Fc,转染膀胱肿瘤细胞和血管内皮细胞,使之表达分泌游离的融合Flk1-Fc,后者在肿瘤细胞和新生血管内皮细胞生长的微环境中可捕获血液中的VEGF,阻断细胞自/旁分泌产生的VEGF与VEGFR-2的结合,从而抑制血管生成和肿瘤的生长,为临床治疗膀胱肿瘤提供高效、安全新方法。主要研究内容和结果如下:第一部分腺病毒载体Ad-Flk1-Fc的构建与表达1.腺病毒载体Ad-Flk1-Fc的构建:扩增并提取含有Flk1-Fc片段的质粒pCD-Flk1-Fc,用XbaⅠ、BamHⅠ进行双酶切,产物用琼脂糖凝胶电泳,显示得到约5.1kp的酶切片段,与目的基因理论值相符。将腺病毒穿梭质粒载体pshuttle-CMV双酶切线性化后,插入腺病毒穿梭载体质粒pshuttle-CMV巨细胞病毒启动子下游,构建腺病毒穿梭质粒载体pShuttle-CMV-Flk1-Fc。用Pme I线性化后,转化到含有AdEasy-1的BJ5183感受态细菌中,与其中的AdEasy-1进行同源重组,得到重组腺病毒质粒pAd--Flk1-Fc。2.腺病毒载体Ad-Flk1-Fc的包装:重组腺病毒质粒pAd-Flk1-Fc转染人胚肾293细胞,转染48h后在荧光显微镜下可观察到细胞内的绿色荧光,表明质粒转染成功,外源基因开始表达,培养一天后开始收集上清,得到复制缺陷型腺病毒Ad--Flk1-Fc。用得到的质粒重复感染HEK293A,大量扩增腺病毒Ad-Flk1-Fc。用氯化铯梯度离心法纯化重组腺病毒,利用半数组织培养感染剂量法测得重组腺病毒最终的滴度为2×109PFU /ml。3.腺病毒Ad-Flk1-Fc的表达与鉴定:用包装后的腺病毒转染膀胱肿瘤BTT739细胞,提取细胞中的总蛋白,用抗Flk1抗体进行Western blot鉴定,结果显示有分子量为165kD的蛋白表达,与Flk1-Fc融合蛋白的理论分子量相同。将Ad-Flk1-Fc转染COS-7细胞后,进行免疫细胞化学染色检测Flk-1表达,Ad-Flk1-Fc转染细胞为阳性,而对照组则为阴性,进一步证实融合蛋白Flk1-Fc的表达。以上结果显示,实验成功构建了腺病毒载体Ad-Flk1-Fc,并在膀胱肿瘤BTT739等真核细胞中表达融合蛋白Flk1-Fc。第二部分Ad-Flk1-Fc在体外对血管内皮细胞(HUVEC)的作用1. Ad-Flk1-Fc细胞培养上清液对HUVEC的生长抑制作用:首先体外培养HUVEC,用Ad-Flk1-Fc转染膀胱肿瘤BTT739细胞培养上清液(即融合蛋白Flk1-Fc)处理离体培养的HUVEC,并设置Ad-Fc、pAd及PBS对照组,采用内皮细胞增殖抑制实验(MTT法),接种48h后,对照组细胞增殖迅速,Ad-Flk1-Fc组细胞数量较对照组有明显减少,与空白对照组相比HUVEC生长抑制率分别为Ad-Flk1-Fc组(53.08%)、Ad-Fc组(13.86%)、pAd组(14.38%)。结果显示Ad-Flk1-Fc细胞培养上清液对HUVEC细胞生长有明显抑制作用,而其它三组的HUVEC细胞的生长无抑制作用(p<0.01)。2. Ad-Flk1-Fc细胞培养上清液诱导HUVEC凋亡的作用:流式细胞仪检测Ad-Flk1-Fc细胞培养上清液对血管内皮细胞凋亡的影响,实验分组同上述,检测结果显示Ad-Flk1-Fc组的凋亡率为42.93%,明显大于Ad-Fc组(20.36%)、pAd组(19.45%)及PBS组(18.58%)。其可以显著诱导HUVEC凋亡(p<0.01)。3. Ad-Flk1-Fc细胞培养上清液抑制HUVEC迁移的作用:通过Transwell试验观察Ad-Flk1-Fc细胞培养上清液对HUVEC在Transwell中的迁移的影响,实验分组同上,Ad-Flk1-Fc组的抑制率为53.19%,而Ad-Fc组和pAd组分别为12.0%及2.4%,上清液对HUVEC迁移有明显的抑制作用(p<0.01)。4. Ad-Flk1-Fc细胞培养上清液抑制HUVEC管状化的作用:在Matrigel胶中观察发现,Ad-Flk1-Fc细胞培养上清液对HUVEC内皮小管管腔样结构的形成有显著的抑制作用。以上结果提示:Ad-Flk1-Fc转染膀胱肿瘤BTT739细胞培养上清液(融合蛋白Flk1-Fc)在体外对HUVEC的生长、迁移及管状化具有明显的抑制效应,而对HUVEC凋亡有明显的诱导作用,为Ad-Flk1-Fc后期的开发和应用奠定了理论基础。第三部分pAd- Flk1-Fc对可移植性小鼠膀胱肿瘤治疗效应的研究1. Ad-Flk1-Fc的抑瘤效应:T739小鼠48只,随机分4组,12只/组。每组再分为两个亚组,分别为动态组和病理组。动态组主要观察:①肿瘤体积变化:每周测两次,至其中一组小鼠死亡达到一半时停止测量;②生存率:观察记录荷瘤小鼠死亡情况,观察期2个月。病理组小鼠第21天时处死,剥离肿瘤,测量体积,并称重,计算瘤重和体积抑制率后进行病理检查。小鼠皮下种植等基因小鼠膀胱肿瘤细胞BTT739的同时,注射Ad-Flk1-Fc2×109 PFU,2次/wk,共2 wk、并设置Ad-Fc、pAd及PBS对照组。结果显示:Ad-Flk1-Fc治疗组荷瘤鼠的平均瘤重和平均体积均显著低于Ad-Fc、pAd及PBS对照组;而肿瘤抑制率和荷瘤鼠生存时间则均显著大于其它三组。2. Ad-Flk1-Fc对肿瘤血管生成的抑制效应:Ad-Flk1-Fc治疗后进行膀胱肿瘤病理检查,有明显的肿瘤细胞坏死。根据CD31抗原标记肿瘤内皮细胞计算MVD,在所有的膀胱肿瘤组织中都能计数到MVD,阳性染色多位于肿瘤细胞之间,微血管形态不一,呈星状、裂隙状或分支状。肿瘤巢内多呈片状或灶状分布。计数结果表明Ad-Flk1-Fc组MVD值明显低于其它三组(P<0.01)。采用原位末端标记(TUNEL)法检测肿瘤细胞凋亡情况,Ad-Flk1-Fc治疗组明显高于其它三组(P<0.01)。3. Ad- Flk1-Fc对非肿瘤血管新生的抑制效应:BTT739小鼠24只,同上随机均分为4组,于皮下注射含0.4ug/ml小鼠VEGF的Matrigel胶诱导血管新生,然后同上分别注射Ad-Flk1-Fc、Ad-Fc、pAd及PBS。再利用携带荧光素的Isolectin B4对内皮细胞进行染色及定量检测,Ad-Flk1-Fc治疗组的荧光强度明显低于其它三组。以上结果显示Ad-Flk1-Fc对移植性小鼠膀胱肿瘤及肿瘤的血管生成具有抑制效应,并延长荷瘤鼠的生存期。结论:1.成功构建了腺病毒载体Ad-Flk1-Fc,并在膀胱肿瘤BTT739等真核细胞中表达融合蛋白Flk1-Fc。2. Ad-Flk1-Fc转染膀胱肿瘤BTT739细胞培养上清液(即融合蛋白Flk1-Fc)在体外对HUVEC的生长、迁移及管状化具有明显的抑制效应,而对HUVEC凋亡有明显的诱导作用。3. Ad-Flk1-Fc对移植性小鼠膀胱肿瘤具有抑瘤效应,并延长荷瘤鼠的生存期,其抑瘤机制主要是抑制了肿瘤的血管生成。本研究为新型抗膀胱肿瘤药物Ad-Flk1-Fc的开发和应用奠定了良好的基础。更多还原

【Abstract】 In China, bladder tumor is the commonest malignant tumor of the urinary system, which is characterized by multiple tumors and high relapse rates. In addition, recurrent tumors tend to be increasingly malignant and, finally, tumor infiltration and metastasis may threaten life. How to increase the efficacy of bladder tumor treatment is a challenging topic in urology. With research advances in tumorigenesis, it has been known that tumor growth, infiltration and metastasis depend on neovascularization. It was shown that a tumor of 1~2mm in diameter requires blood supplies by new vessels to prevent tumor cell death from ischemia and hypoxia. Based on these findings, a new anti-angiogenesis treatment has been proposed to treat solid tumors. Compared to conventional tumor treatments, the anti-angiogenesis strategy has many advantages, such as high efficacy, easy access to target vascular cells, and no resistance or obvious toxic and side effects. Although great research advances in anti-angiogenesis treatment have been achieved over recent years and many anti-angiogenesis drugs under clinical trials and experimental studies have shown sound effects, such drugs have obvious drawbacks:①these treatment molecules are all administered, and must be administered continually due to their short action durations;②these molecules need to be administered intravenously at high doses due to their limited solubility, thus increasing drug toxicity substantially.Vascular endothelial growth factor (VEGF) is the most important pro-angiogenesis factor yet known so far. Most tumor tissues and tumor cell strains overexpress VEGF. VEGF exerts its biological action primarily through binding to high-affinity tyrosine kinase receptor VEGFR-2, and the VEGF/VEGFR-2 signal transduction pathway plays a key role in tumor angiogenesis. Blocking this pathway may suppress tumor angiogenesis and, subsequently, suppress tumor growth and metastasis. Based on this, we designed a new treatment to block VEGF binding to its receptors: adenovirus vector (Ad-Flk1-Fc) carrying a fusion gene comprising the extracellular domain of VEGF R-2 (Flk1) and IgG2a Fc was constructed, and used to transfect bladder tumor cells and endothelial cells. The transfected cells expressed and secreted free Flk1-Fc, which can capture blood VEGF in the microenvironment in which tumor cells and new endothelial cells grow, thus blocking autocrine/paracrine VEGF binding to VEGF R-2 and suppressing angiogenesis and tumor growth. This method may serve as an effective, safe treatment for bladder tumor. The study and main results are described below.Part 1 Construction and expression of adenovirus vector Ad-Flk1-Fc1. Construction of adenovirus vector Ad-Flk1-Fc: pCD-Flk1-Fc plasmid containing Flk1-Fc fragment was amplified and extracted, and digested with Xba I and BamH I. Agarose gel electrophoresis of the digestion products resulted in a band of approximately 5.1kb in length, which was consistent with the expected length of the target gene. Adenovirus shuttle plasmid pshuttle-CMV was linearized by digestion with the two endonucleases and inserted into downstream of pshuttle-cytomegalovirus promoter to construct the adenovirus shuttle plasmid vector pShuttle-CMV-Flk1-Fc. The obtained vector was linearized by digestion with Pme I, and transformed into competent bacteria containing AdEasy-1BJ5183 to perform homologous combination with AdEasy-1. Finally, recombinant adenovirus plasmid pAd--Flk1-Fc was obtained.2. Packaging of adenovirus vector Ad-Flk1-Fc: Recombinant adenovirus plasmid pAd-Flk1-Fc was used to transfect human embryonic kidney 293 cells. At 48h after transfection, green fluorescence was observed under fluorescence microscopy in the cells, suggesting successful plasmid transfection and expression of the exogenous gene. After one day of culture, the supernatants were collected, and replication-defective adenovirus Ad--Flk1-Fc was obtained. HEK293A cells were infected with the obtained plasmid repeatedly to amplify adenovirus Ad-Flk1-Fc. Recombinant adenoviruses were purified by cesium chloride gradient centrifugation, and the final titer of recombinant adenovirus was determined by the half culture tissue infection method to be 2×109PFU /ml.3. Expression and identification of adenovirus vector Ad-Flk1-Fc: The packaged adenoviruses were used to transfect bladder tumor cells BTT739, and total cellular protein was extracted for Western blotting using Flk1 antibody. The results indicated expression of an 165kD protein, which was in agreement with the theoretical molecular weight of Flk1-Fc fusion protein. After transfection of COS-7 cells with Ad-Flk1-Fc, Flk-1 expression was detected by immunocytochemical staining. The results indicated that Ad-Flk1-Fc transfected cells were stained positive and the control group negtive, suggesting Flk1-Fc expression by COS-7 cells.These results indicated the successful construction of adenovirus vector Ad-Flk1-Fc and expression of fusion protein Flk1-Fc in eukaryotic cells such as bladder tumor cells BTT739.Part 2 Effect of Ad-Flk1-Fc fusion protein on experimental in vitro angiogenesis of HUVECs1. Suppression of HUVEC growth by Ad-Flk1-Fc: Cultured HUVECs were treated with cellular culture supernatant of Ad-Flk1-Fc, or not treated with it(control). Control groups were set up with Ad-Fc、pAd and PBS. Endothelial cell proliferation suppression evaluation (MTT assay) indicated that after 48h culture, cells proliferated quickly in the control group, and the cell number decreased in the Ad-Flk1-Fc group cells, when compared to that in the control group. Compared with the blank group, HUVEC growth suppression rate was 56.3% in the Ad-Flk1-Fc group, 6.68% in the Ad-Fc group, and 4.23% in the pAd group. The results indicated significant suppression of HUVEC growth by Ad-Flk1-Fc, while no suppression in the other three groups (p<0.01).2. Induction of HUVEC apoptosis by Ad-Flk1-Fc: The effect of cellular culture supernatant of Ad-Flk1-Fc on endothelial cell apoptosis was investigated by flow cytometry. The groups were set as describe above. The results indicated that the apoptotic rate was significantly higher in the Ad-Flk1-Fc group than the Ad-Fc group (45.33% vs 21.49%), the pAd group (45.33% vs 17.4%) and the PBS group (45.33% vs 18.32%). The results suggest that Ad-Flk1-Fc induced HUVEC apoptosis significantly (p<0.01).3. Suppression of HUVEC migration by Ad-Flk1-Fc: The effect of cellular culture supernatant of Ad-Flk1-Fc on HUVEC migration in Transwell was observed, The groups were set as describe above. The suppression rate of HUVEC migration was 72.23% in the Ad-Flk1-Fc group, 13.70% in the Ad-Fc group, and 17.13% in the pAd group, suggesting significant suppression of HUVEC migration (p<0.01).4. Suppression of formation of HUVEC tubules by Ad-Flk1-Fc: It was found that cellular culture supernatant of Ad-Flk1-Fc significantly suppressed the formation of HUVECs tubules in Matrigel.These results suggest that Ad-Flk1-Fc suppresses the growth, migration and tubule formation of HUVECs significantly in vitro, and induces HUVEC apoptosis significantly, which provides a theoretical basis for future development and application of Ad-Flk1-Fc.Part 3 Therapeutic effect of recombinant adenovirus plasmid pAd- Flk1-Fc on transplantable mouse bladder tumor1. Tumor-suppressing effect of Ad-Flk1-Fc: BTT739 bearing mice models were established successfully. 48 BTT739 bearing mice were randomized to 4 groups (n=12). Each group was divided into two subgroups, i.e., the dynamic group and the pathology group. The observations of the dynamic group included①Changes in tumor size: The tumor size was measured twice weekly until 50% of mice in a group died;②survival rate: The survival of tumor-bearing mice was observed for 2 months. Mice of the pathology group were sacrificed on day 21, and the tumors were isolated, and determined for size and weight. After calculation of the tumor weight and size suppression rates, the tumors were subject to pathological examination. Mice were implanted with homologous tumor cells BTT739 subcutaneously; meanwhile, Ad-Flk1-Fc (2×109 PFU) was injected twice per week for 2 weeks. Control groups were set using Ad-Fc, pAd and PBS. The average tumor weight and volume were significantly lower in the Ad-Flk1-Fc treatment group than the three control groups, and the tumor suppression rate and survival time of tumor-bearing mice were significantly higher in the Ad-Flk1-Fc treatment group than the three control groups.2. Suppression of tumor angiogenesis by Ad-Flk1-Fc: After Ad-Flk1-Fc treatment, pathological examination revealed obvious necrosis of bladder tumor cells. MVD was calculated according to CD31 antigen-labeled tumor endothelial cells. MVD was obtained in all bladder tumor tissues, and positive stains were mostly located between tumor cells. Microvessels were polymorphous, being asterisk, slit-shaped or branched. Microvessels were patchy or focal in tumor nests. MVD was significantly lower in the Ad-Flk1-Fc treatment group than the three control groups (P<0.01). Tumor cell apoptosis assay by TUNEL indicated an apoptotic rate higher in the Ad-Flk1-Fc treatment group than the three control groups (P<0.01) 3. Suppression of non-tumor angiogenesis by Ad-Flk1-Fc: 24 BTT739 bearing mice were randomized as above to 4 groups, and were injected subcutaneously with 0.4ug/ml VEGF Matrigel, Ad-Flk1-Fc, Ad-Fc, pAd and PBS, respectively. Then, endothelial cells were stained and quantified with fluorescein-carrying Isolectin B4, and the fluorescence intensity was significantly lower in the Ad-Flk1-Fc treatment group than the three control groups.The above results show that Ad-Flk1-Fc suppresses transplanted mice bladder tumor and tumor angiogenesis and prolongs the survival of tumor-bearing mice.Conclusions:1. Adenovirus vector Ad-Flk1-Fc was constructed successfully and the fusion protein Flk1-Fc was expression in eukaryotic cells such as bladder tumor cells BTT739.2. Flk1-Fc fusion protein suppressed the in vitro growth, migration and tubule formation of HUVECs significantly and induced HUVEC apoptosis significantly.3. Ad-Flk1-Fc suppressed transplanted mice bladder tumor and prolonged the survival of tumor-bearing mice mainly through suppressing tumor angiogenesis.This study provides a good basis for developing and applying the novel drug Ad-Flk1-Fc for bladder tumor treatment.更多还原