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DNp73α及恩度?介导特异性杀伤肿瘤内皮细胞效应的研究
The Study of Specific Cytotoxicity Against Tumor-derived Endothelial Cells Induced by DNp73α Recombinant Adenovirus and Endostar? in Vitro
【作者】 蒋力;
【导师】 蒋耀光;
【作者基本信息】 第三军医大学 , 外科学, 2009, 博士
【摘要】 背景和目的:肿瘤是极大危害人类健康的疾病之一,肿瘤的发生是多因素、多阶段、多步骤的复杂过程。分子生物学研究表明,肿瘤的发生与癌基因激活、抑癌基因失活密切相关,这些基因改变的结果最终导致肿瘤细胞生长的无限制性,所以单独针对某个基因的作用对于抑制肿瘤的发生和发展并无明显的治疗意义。肿瘤血管是肿瘤形成、生长及转移的基础,破坏少量的肿瘤血管即可导致大片区域的肿瘤细胞缺血坏死,因此特异性杀伤肿瘤血管内皮细胞已成为肿瘤治疗的重要策略之一【1】。目前,肿瘤血管内皮靶向性杀伤的主要方法有两种:1.通过特异性作用于肿瘤血管内皮细胞的化学药物抑制肿瘤血管形成及生长;2.通过T细胞特异性杀伤来实现,这也是机体对肿瘤免疫的主要途经。内皮抑素(Endostatin,ES)是1997年Reilly等【2】从小鼠血管内皮瘤( EOMA)细胞培养液中提取的一种内源性血管生成抑制剂,为胶原ⅩⅧC末端水解片段,在小鼠体内对肿瘤诱导的血管生成具有几乎完全的抑制作用,并显示很强的抗肿瘤活性。研究表明,内皮抑素能特异性抑制血管内皮细胞增殖【3】,抑制内皮细胞的迁移,诱导内皮细胞凋亡和细胞周期阻滞【4-5】。反复使用内皮抑素不会使癌细胞产生抗药性,并且没有明显的毒副作用。美国食品药品管理局(FDA)于1999年底批准了由毕赤酵母表达的重组人内皮抑素作为肿瘤治疗药物进行I期临床试验;2001年又进行II期临床试验。然而,人重组内皮抑素半衰期短、复性及大量生产较为困难的特点限制了内皮抑素的临床应用。2005年,中国科学家在ES的基础上改变了其氨基酸序列,在ES母体上创造性的添加了9个氨基酸(MGGSHHHHH)使该蛋白的药用性能和疗效得到显著提高,该药即为恩度(Endostar)。恩度是一种新型的抗血管生成药物,相对于ES,不仅使稳定性提高、纯度明显增加、半衰期延长,而且生物活性增加【6】。但恩度对于肿瘤血管内皮细胞的作用效果及具体机制尚不十分清楚,恩度对肿瘤血管内皮细胞是否具有特异性的抑制作用,这一作用是如何发挥,都需要进一步深入细致的工作来研究探讨。在人体的免疫反应过程中,外来的抗原物质需经过处理并呈递给具有分泌细胞激素功能之辅助性T细胞,才能进一步活化以B细胞和T细胞为中心的体液免疫及细胞免疫反应。故有效激活特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL),是实现肿瘤主动免疫治疗的关键。人树突状细胞(Dendritic cell,DC)起源于造血干细胞(hemopoieticstemcell)。是机体功能最强的专职抗原呈递细胞(Antigen presenting cells, APC),它能高效地摄取、加工处理和递呈抗原,能高水平的表达与抗原提呈有关的MHC-Ⅰ类和MHC-Ⅱ类分子,并可高水平的表达多种共刺激分子及某些粘附分子,所以,DC细胞可摄取肿瘤内皮特异性相关抗原,吞噬处理凋亡或坏死肿瘤细胞等颗粒性抗原,并具有“交叉提呈(cross-presentation)”外源性抗原的能力。通过DC对肿瘤内皮抗原的高效提呈作用,激发机体的特异性抗肿瘤免疫反应成为肿瘤免疫治疗的一个热门课题,而为DC疫苗寻找特异性的肿瘤抗原、探索最佳的加载途径成为目前研究的一大方向。DNp73α由p53家族的成员p73基因经位于第3内含子的启动子启动,经过不同于p73的方式剪切,所生成N’端缺失的蛋白。DNp73α在正常组织细胞及血管内皮中不表达,但在肺癌、乳腺癌、肾母细胞癌、宫颈癌以及成肝癌等肿瘤内皮细胞中高表达。并对野生型P53和P73产生竞争性抑制其作用,促进肿瘤血管内皮增生和毛细血管的形成。是肿瘤生物治疗潜在的分子靶点。但目前鲜有针对DNp73α靶点抗血管生物治疗的研究报道。在本研究中我们通过设计含有DNp73α全长cDNA的重组腺病毒,并将其转染修饰DC,进而诱导针对高表达DNp73α的肿瘤源性血管内皮细胞的特异CTL反应,同时探讨恩度对肿瘤血管内皮细胞是否具有特异性的抑制作用及相关的抗血管形成机制。为后续的动物及临床实验研究提供依据。研究方法:1.采用AdEasy系统构建携带HA标记含人全长DNp73αcDNA的复制缺陷型重组腺病毒Adv-DNp73α,即:从pcDNA3.0-DNp73α-HA中酶切出DNp73α基因,并定向克隆插入pDC315-EGFP Vector中构建成腺病毒穿梭质粒;pDC315-EGFP Vector-DNp73α经PmeI线性化后电穿孔法转化到含有腺病毒骨架质粒pAdEasy-1的E. coli BJ5183菌株内同源重组。筛选正确的重组质粒,PacI线性化后脂质体法转染293T细胞包装成重组病毒颗粒;并在293T细胞中反复扩增。携带绿色荧光标记的Adv-EGFP作为实验对照。2.采用胰酶消化柱状离心法分离人脐静脉内皮获得HUVEC,并通过与A549上清共培养诱导生成TDEC细胞,通过MTT法、细胞迁徙实验、流式细胞仪等检测TDEC细胞生物学特性的改变。3.人脐带血经梯度离心法和细胞因子IL-4、GM-CSF诱导培养的树突状细胞。通过增强离心法使DNP73α重组腺病毒有效转染DC,并通过追踪绿色荧光蛋白的表达,检测不同感染复数(M.O.I.)的转染效率以及腺病毒转染对细胞的毒性,来确定合适的感染复数;3.用RT-PCR和Western Blot检测Adv-DNp73α转染48小时后DC胞内DNp73α的mRNA和蛋白表达;4.采用尼龙毛柱法分离纯化同源T细胞。纯化的T细胞在96孔板中与经丝裂霉素C预处理的各种DC共培养5天。结束培养前8小时加入1μCi/孔的3H胸腺嘧啶脱氧核苷。5.RT-PCR对比分析TDEC/DNp73α细胞、TDEC细胞和HUVEC细胞中DNp73αmRNA的表达水平;并检测不同效应细胞(细胞毒性T淋巴细胞CTL、普通T细胞以及淋巴因子活化的杀伤细胞LAK)针对不同DNp73α靶细胞(TDEC/DNp73α细胞、TDEC细胞和HUVEC细胞)的杀伤效应。6.采用人肺腺癌细胞A549培养上清作为条件培养基诱导培养人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVEC),并鉴定条件培养基诱导培养后的细胞(Tumor-derived Endothelial Cells,TDEC)是否具有肿瘤血管内皮细胞的特性。7.利用TDEC作为研究模型,检测恩度对HUVEC和TDEC细胞生物学特性影响的差异;建立裸鼠移植瘤模型,观察恩度的治疗效应并分析其机制。8.统计分析:数据采用x±SD表示;对于正态分布数据,组间比较采用t检验。非正态分布数据采用秩和检验分析。p<0.05认为有显著统计学差异,p<0.01认为差异非常显著。所有数据采用统计学软件SPSS10.0分析。结果:1.采用定向克隆以及同源重组在293T细胞内产生了具有转染能力的腺病毒颗粒;通过反复感染,获得较高滴度腺病毒液;2.分离人脐静脉内皮获得HUVEC,并通过与A549上清共培养诱导生成TDEC细胞,证实TdEC有较强的细胞迁徙能力,增殖活跃,S期、G2-M细胞比例远大于HUVEC细胞,并特异性的表达肿瘤内皮相关抗原TEM1/TEM8。3.人肺腺癌细胞A549培养上清作为条件培养基能诱导HUVEC衍生为具有肿瘤血管内皮细胞特性的TDEC。4.较低浓度的恩度对HUVEC没有明显的抑制作用,而对TDEC则具有显著的生长抑制作用,该抑制作用具有时间和浓度依赖性。同时,较低浓度的恩度能显著降低TDEC细胞迁徙活性,诱导细胞凋亡及阻滞TDEC细胞于G0/S期,但对于HUVEC的作用并不显著。在体研究中,瘤内注射一定浓度的恩度能抑制移植瘤生长,其机制可能是通过诱导肿瘤细胞凋亡,升高肿瘤内Ca2+浓度,抑制肿瘤内新生血管的形成。5.从人脐静脉血中培养出DC,采用M.O.I 200的病毒液转染48小时后,DC的转染效率达到80%以上,并且细胞具有较高活力。6.DNp73α特异性CTL能有效裂解TDEC/DNp73α细胞、少量裂解TDEC细胞;而HUVEC细胞几乎不裂解。同时这种特异性CTL杀伤TDEC/DNp73α细胞的水平高于TDEC及HUVEC细胞。而对比研究DNp73α特异的CTL、LAK和正常T细胞分别针对TDEC/DNp73α细胞、TDEC细胞和HUVEC细胞的杀伤作用,发现DNp73α特异性的CTL针对TDEC/DNp73α细胞杀伤效应接近高于LAK的能力;LAK同样可以有效的杀伤TDEC细胞。结论:HUVEC可以通过与A549细胞上清共培养诱导生成TDEC,生成的TDEC具有肿瘤源性血管内皮细胞相关特性,其迁徙增殖能力显著强于HUVEC,可以作为体外抗肿瘤血管内皮效应研究的模型。恩度可以抑制肿瘤血管内皮细胞的生长和肿瘤血管的形成而不损伤正常的血管内皮细胞,因此这种抑制作用是具有肿瘤特异性的,在肿瘤的临床治疗中具有疗效好、副作用小的特点,有着理想的临床应用前景。。DNp73α2.2修饰的DC,可以诱导生成DNp73α特异性CTL,有效裂解DNp73α高表达的TDEC/DNp73α细胞。
【Abstract】 Background:Lung cancer is one of the most aggressive carcinoma.,it becomes the leading cause of cancer-related deaths. Tumorigenesis involves a complicated multi-step process and it is made clear by molecular biology’s researchs that tumorigenesis are in connections with activations of proto-oncogenes and inactivation of various antioncogenes at different stages. These gene changes will eventually lead to limitlessness of tumor cell growth. In this process, interaction based on one correlated gene has not significant efficiency. Tumor microvessels are essential for the growth and migration of malignant tumors. Targeting tumor microvessels by inhibiting formation or destructing existing vessels can block the nutrition supplies and migration path. Therefore, antio-angiogenesis therapies have become an important strategy in cancer therapy【1】. The main methods of specific cytotoxicity against tumor-derived endothelial cells at present included: 1. Chemical medicines that have specific inhibitory effect on endothelial cells; 2. Though specific cytotoxicity of T cells, which is the main way to immune response against cancer cells.Endostatin was originally isolated from the culture medium of murine endothelioma (EOMA) as an endogenous angiogenesis inhibitor by Reilly et al.【2】. It is the c-terminal fragment of collegen X VIIIC, and it demonstrated a nearly complete inhibition of tumor-induced angiogenesis, conveying a high anti-tumor activity. Numerous studies have shown that endostatin can specifically inhibit the proliferation of blood vessel endothelial cells【3】, block their migration, and induce apoptosis and cell cycle arrest【4-5】, while having no inhibitory effect on smooth muscle cells or fibroblasts. It is interesting that long-term use of endostatin does not induce drug resistance in cancer cells. In addition, it has low cellular toxicity. In 1999, a phase I trial of Pichia pastoris expressed human recombinant endostatin as cancer therapeutic was approved by FDA. In 2001, a phase II trial was conducted. However, human recombinant endostatin has a short half life; its refolding and large-scale production is relatively difficult. Therefore, the clinical application of endostatin has been limited. In 2005, Chinese scientists invented a new protein named endostar, which is endostatin with an addition of a 9-amino acid peptide (MGGSHHHHH). The stability and purity of endostar are greatly enhanced, and the half life is prolonged. Most importantly, the biological activity is increased【6】. However, the effect and mechanism of endostar on tumor endothelial cells remains unknown, and whether this effect is specific to tumor-derived endothelial cells also needs to investigate.Among varied biotherapy patterns, immunotherapy is the most attractive one. It was confirmed that specific anti-tumor immune response was done by activated T lymphocyte, and exotic antigens had to be processed and presented to helper t cells. So it is the key point to activate the specific cytotoxic T lymphocyte (CTL). Dendritic cells (DC) originated from hemopoieticstemcell,and are the most potent professional antigen-presenting cells and only ones initiating primary T lymphocyte. It is capable of uptaking (endocytosis and phagocytosis), presenting tumor associated antigen in the context of both MHC classⅠand classⅡin association with costimulatory molecules. Much attention has been directed to the problem of how and what antigens should be pulsed to DC in their clinical application as immunotherapy.The DNp73αprotein, a protein of the p53 family, was recently identified to be generated from the p73 gene under an alternative promoter in intron 3, and thus DNp73αprotein lacks a transactivation domain presenting in p73. DNp73αis not expressed in normal tissues but overexpressed in lung cancer, ovarian cancer, vulval cancer, neuroblastoma and breast cancer cell lines; it acts as a potent transdominant inhibitor of the wild-type p53 and the transactivation-competent TAp73. Several studies found that positive expression of DNp73αwas a significant independent factor for predicting a poor prognosis, and considered it an important molecular target for effective therapy. However, few studies have reported experimental manipulations targeting DNp73α.This study was aimed to investigate the effects of dendritic cells transfected with recombined adenovirus vector encoding DNp73αon induction of immunity against DNp73α-overexpressing tumor cells, simultaneously,we studied wheather endostar has specific cytotoxicity against tumor-derived endothelial cells and its antiangiogenesis’mechanism,which provide evidences for subsequent animal and clinical research.Methods:1. A recombinant replication-deficient adenovirus vector carrying a HA tagged full-length human DNp73αcDNA driven by a CMV promoter, was prepared using the AdEasy system and referred to as Adv-DNp73α. In brief, human DNp73αcDNA was cloned into adenovirus transfer vector pAdTrack-CMV. The resulting plasmid, pDC315-EGFP Vector-DNp73α, was then linearized with PmeI and transformed into E. coli strain BJ5183 containing pAdEasy-1, the viral DNA plasmid, for homologous recombination. The recombinant adenoviral construct was then cleaved with PacI and transfected into 293T cells to produce viral particles. The same adenoviral vector backbone carrying the marker gene EGFP, Adv-EGFP, was used as the control vector. Titers of the viral stocks were determined with a plaque-forming assay.2. We obtained HUVEC by trypsinization and centrifugation method and cultured Human umbilical vein endothelial cells (HUVEC) and differentiated them into Tumor-derived Endothelial Cells(Td-ECs) after co-cultured with supernatants of A549 cells,and examined the anti-angiogenesis effect of endostar on Td-ECs in vitro with the methods of MTT, cell migration assay, flow cytometry and TUNEL assay.For futher study of Endostar’s effect,3. Immature dendritic cells generated in the presence of interleukin-4 and granulocyte/macrophage colony-stimulating factor from human umbilical cord blood were transfected with the adenovirus vector with centrifugal force method. Following up expression of GFP, we detected the infective efficiency of varied M.O.I. recombined adenovirus. Combined with viability of DC evaluated through FCM, we then ascertained the optimal infection programmer and M.O.I..4. RT-PCR and Western Blot were use to detect the level of DNp73αmRNA and protein in the transfected DC to ensure the overexpression of DNp73α.5. Autologous T cells were isolated and purified using nylon wool columns. The purified cells were cultured in 96-well U-bottom plates in the presence of cytomycin-C-pretreated transfected or untransfected DC as stimulators for 5 days. During the last 8 h of incubation, 1μCi/well of [3H] thymidine was added. Assays were performed in triplicate. And then the cells were harvested and [3H] thymidine incorporated into T cells was measured by a beta counter.6. T cells (1×106) were co-cultured with Adv-DNp73αmodified DC (5×104) for 72 h to induce cytotoxic T lymphocytes (CTLs). Then the CTLs were collected and used as the effector cells in CTL assays. TDEC/DNp73αcells, HUVEC cells, and TDEC cells, as the target cells with different DNp73αespression levels determined by real time PCR, were placed in 96-well tissue culture plates at 1×104 cells per well respectively, and co-cultured with effector cells (CTLs) at varied E/T (effect cells : target cells) ratio of 1:10, 1:20, 1:40 and 1:80. The cytotoxic activities were determined by CytoTox non-radioactive cytotoxicity assay. Normal DC and Adv-EGFP transfected DC were used as the control.7. Cultured Human umbilical vein endothelial cells (HUVEC) and differentiated them into Tumor-derived Endothelial Cells(Td-ECs) after co-cultured with supernatants of A549 cells,and examined the anti-angiogenesis effect of endostar on Td-ECs in vitro .8. Established human-xenograft-nude mouse model, Endostar was injected directly to tumors as local treatment and conservative management of tumor growth was close observed in the future days.9. For a single comparison of two groups, Student’s t test was used to evaluate the significance of differences. If the data distribution was not normal, the Mann-Whitney rank-sum test was employed for the nonparametric analysis. For all analyses, the level of significance was set at p<0.05. All statistical calculations were performed using the SigmaStat statistical software package SPSS10.0. Data are presented as the mean±SD.Results:1. Via directed cloning and homologous recombination, infection-competent adenovirus was generated in 293T package cell line. Following repeatedly infecting, high titer adenovirus particles yielded.2. Human umbilical vein endothelial cells (HUVEC) were cultured and differentiated to tumor-derived endothelial cells (TDECs) after co-culturing with conditioned medium from A549 cells, a lung cancer cell line. we could detect the mRNA of TEM1 and TEM8 in the Td-Ec with RT-PCR. Td-EC had more active cellular proliferation and cell migration capacity, G0/G1 and S phase proportion of Td-Ec was higher than HUVEC.3. After co-culturing with conditioned medium from A549 cells, a lung cancer cell line, HUVEC can be induced to TDEC which has the characteristics of tumor vessels endothelial cells.4. Endostar has a significant inhibition effect in time-and concentration-dependent for TDEC in vitro, at the same time, though Endostar at a certain concentration can inhibit cell migration process significantly, and induced G0/G1 and S phase arrest and cell apoptosis, under the same conditions, Endostar has little impact on HUVEC. In addition, a certain concentration of Endostar after local injection, further study details was provided that concentration of Ca2+ of the tumor be significantly higher than the contral group’s, and had significant anti- angiogenesis effect and tumor growth inhibition.5. The addition of purified Adv-DNp73αvector to the DC culture with centrifugal force method, at an M.O.I. of 200, resulted in a transduction efficiency of nearly 80% without significant cytotoxic effects.6.DC/Adv-DNp73αstimulated effector T cells could effectively lyse DNp73α-overexpressing TDEC/DNp73αcells but not the DNp73α-null TDEC and HUVEC cells. The untreated T cells had minimal lysis of all the tumor cells. LAK cells showed comparable lytic activity against TDEC/DNp73αcells with that of DC/Adv-DNp73αstimulated T cells, but demonstrated much more effective killing of TDEC cells than the DC/Adv-DNp73αstimulated T cells.Conclusions:After co-culturing with conditioned medium from A549 cells, a lung cancer cell line, HUVEC can be induced to TDEC which has the characteristics of tumor vessels endothelial cells,and its had more active properties of proliferation and cell migration.Endostar has a significant inhibition effect in time-and concentration-dependent for TDEC in vitro, at the same time, endostar has little impact on HUVEC, so, this inhibitory effects are some kind of specific cytotoxicity to tumor, and have significant efficacy and minimal side effects in clinical treatment. DNp73α-engineered DC can induce DNp73α-specific CTL against tumor cells over-expressing DNp73α.
【Key words】 DNp73α; TDEC; recombined adenovirus; dendritic cells; Endostar; cell migration;