【作者】 陈蓓；

【导师】 吴开春； 王新；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 【背景】自1971年Folkman教授提出血管生成是实体肿瘤生长和转移的必要条件之一后,肿瘤血管靶向性治疗已逐渐成为目前抗肿瘤综合治疗领域研究的热点。大量实验证实,肿瘤血管与正常血管相比存在“异质性”,某些在正常血管不表达或仅微量表达的分子在肿瘤血管表达上调。将这类特异性分子作为肿瘤血管抑制治疗的靶标应用于临床,有可能在提高抗肿瘤治疗疗效的同时,降低药物的毒副作用。近年来,研究者们已陆续发现了一些肿瘤血管异质性分子;而噬菌体肽库筛选技术的出现与完善又使这一领域的研究向前迈进了一大步。目前,这类异质性分子中有一部分已进入临床Ⅰ/Ⅱ期实验(如RGD、NGR、DMXAA、AVE8062等),并在乳腺癌、卵巢癌、前列腺癌等多种肿瘤的综合治疗中取得了可喜的疗效。然而,到目前为止,在胃癌研究领域尚未筛选出这类血管特异性靶分子。我们先前利用体内噬菌体呈现肽技术,从小鼠肾包膜下人胃腺癌移植瘤模型中筛选出一段能特异性与人胃癌血管内皮结合的环形九肽CGNSNPKSC,命名为GX1。此后我们通过免疫组化实验,证实了GX1噬菌体能够与人胃癌组织及小鼠移植瘤内的血管特异性结合,且其亲和力在低分化胃癌组织中明显高于中、高分化组织。我们还构建了放射性核素99TcmO4-标记的GX1,并将99Tcm-GX1从尾静脉注入荷瘤裸鼠体内,ECT显像的结果显示99Tcm-GX1具有良好的体内肿瘤血管靶向性,再次证实了GX1与胃癌血管内皮的特异性结合能力。目前,该短肽序列已获国家发明专利。然而,要将GX1短肽作为胃癌血管靶向性治疗的候选分子应用于临床,还必须对其生物学特性有更全面深入的了解,要阐明GX1除具有胃癌血管靶向性外,是否还具有其他生物学功能,而其可能的作用机制又是什么。本课题就将围绕以上问题展开。【目的】1.构建肿瘤血管内皮细胞体外培养模型,作为后续GX1功能及机制实验的体外研究平台;同时利用该模型验证GX1短肽本身(而非GX1呈现噬菌体)与胃癌血管内皮细胞的亲和力。2.一方面,通过体外实验探讨GX1对胃癌血管内皮细胞的增殖、迁移等生物学特性的影响;另一方面,通过体内实验分析GX1对肿瘤血管生成及肿瘤生长的作用,从而阐明GX1的生物学功能。3.在上述实验的基础上,通过基因芯片等技术分析GX1产生生物学功能的潜在作用机制,鉴定GX1具体影响的信号通路及相关分子,为GX1的临床应用研究提供理论依据,同时也为寻找GX1的受体提供参考信息。【方法】1.利用条件培养基共培养技术,建立胃癌血管内皮细胞体外培养模型(co-HUVEC),同时用化学方法直接合成GX1短肽及对照肽(Pep2)。在体外通过细胞化学染色方法,检测GX1短肽对共培养的co-HUVEC的结合特异性,从而验证GX1短肽对胃癌血管内皮细胞的靶向性。2.通过体外MTT、微管形成、损伤刮擦等实验研究GX1对血管内皮细胞和胃癌细胞增殖能力、微管形成能力、迁移能力等生物学特性的影响;通过体内鸡尿囊膜实验研究GX1对新血管生成的作用;通过裸鼠体内移植瘤实验分析不同给药模式下GX1对肿瘤生长的影响。3.利用流式细胞学技术,分析GX1对其靶细胞的细胞周期分布、凋亡情况等方面的影响,为探讨GX1的具体作用机制提供实验参考。4.利用基因芯片技术,分析GX1作用后肿瘤血管内皮细胞基因表达的变化情况;通过生物信息学分析,筛选与GX1功能相关的具体差异基因及可能影响的信号通路;通过western blot实验检测基因芯片中筛选出的部分差异基因的蛋白表达水平(包括Bcl-2、BAX、caspase3、caspase8),验证芯片结果的可靠性,同时进一步鉴定GX1可能影响的下游分子。【结果】1.成功构建了co-HUVEC的体外培养模型。成功合成了GX1短肽(CGNSNPKSC)和对照肽Pep2(CNKSPSGNC),经验证所合成环肽氨基酸序列正确,纯度>95%。细胞化学染色证实,与Pep2和PBS相比,GX1短肽可特异性与肿瘤血管内皮细胞co-HUVEC结合,证实了GX1短肽确实具有胃癌血管靶向性。2.体外实验结果显示,GX1可抑制HUVEC和co-HUVEC的体外增殖,且其抑制作用呈剂量依赖性;同时GX1对共培养的肿瘤血管内皮细胞的抑制率(约60%)明显高于对正常血管内皮细胞的抑制率(25%-45%)(P<0.05);此外,GX1还可降低co-HUVEC的微管形成能力。但GX1对胃癌细胞的增殖及迁移能力均无明显影响。3.鸡胚体内血管生成实验显示,与对照肽相比,GX1可显著抑制鸡尿囊膜上新血管的生成(新生微血管数分别为20.0±1.6和10.6±1.0)(P<0.05),提示GX1具有体内血管生成抑制作用。在裸鼠体内成瘤实验中,全身或局部给予GX1后,实验组荷瘤裸鼠均表现出肿瘤生长受抑制的趋势,提示GX1可在一定程度上抑制体内肿瘤的生成。4.流式细胞学分析显示,与Pep2相比,GX1可显著诱导肿瘤血管内皮细胞的凋亡(凋亡率分别为1.8%和10.5%)(P<0.05);而GX1对细胞周期分布则无明显影响。5.基因芯片分析显示,在检测的39200个基因中,GX1显著上调了248条基因的表达,同时显著下调了137条基因的表达。其中主要包括细胞凋亡相关基因、细胞代谢相关基因、部分癌基因及抑癌基因等。Western blot检测了其中Bcl-2、BAX、caspase3、8等凋亡相关蛋白的表达水平,发现GX1可上调靶细胞中BAX的表达、增强caspase3的活性,同时可抑制Bcl-2的表达,而对caspase8无明显影响。【结论】1.条件培养基共培养模型可在体外成功模拟肿瘤微环境,使共培养的血管内皮细胞出现肿瘤血管内皮的特性,适宜用于肿瘤血管异质性分子的相关研究。2. GX1短肽除具有胃癌血管靶向性外,还可通过诱导胃癌血管内皮细胞凋亡,在体内外发挥抑制胃癌血管生成的作用,并可在体内表现出一定的肿瘤生长抑制作用,提示我们可将GX1作为胃癌血管靶向性治疗的候选分子应用于临床研究。3.对GX1的作用机制研究显示,GX1可能是通过影响Bcl-2/BAX通路,上调了caspase蛋白的活性,促进了靶细胞的凋亡,最终发挥对肿瘤血管的抑制作用。这一结果为GX1的临床应用研究,及GX1受体的筛选和鉴定提供了参考信息。更多还原

【Abstract】 【Background】In 1971, Folkman laid the groundwork for research on therapies that target tumor vascularization by proposing that angiogenesis is necessary for tumor growth and invasion, and this system could be a legitimate target for anti-cancer agents. Subsequent studies showed that tumor vessels expressed a bunch of specific molecules which were hardly expressed in normal vessels. The discovery of such heterogeneity of tumor vasculature might lead to the investigation and application of these specific molecules in tumor vascular targeted therapy, so as to improve the efficiency of anti-tumor treatment, as well as to minimize the side effects.Since then, many studies have been done, and the application of phage display technique significantly facilitates the researches in this field. Currently, several candidate molecules, including RGD, NGR, DMXAA, AVE8062, etc. are in their phaseⅠ/Ⅱclinical trials, and promising results are observed in various kinds of tumors, such as breast cancer, ovarian cancer and prostate carcinoma. However, no such vascular targeted molecule had been identified in gastric cancer until GX1 was found in our lab.By using in vivo phage display technology, a cyclic 9-mer peptide CGNSNPKSC homing specifically to vasculature of human gastric adenocarcinoma was obtained. Immunohistochemical staining showed positive signal of GX1-displaying phage in the vascular endothelium of human gastric cancer, and GX1 expression level was negatively correlated with tumor differentiation, while no positive staining was observed in heart, liver, muscle, spleen or normal gastric tissues. In another study, GX1 was labeled with 99TcmO4- for ECT scanning to detect the in vivo distribution of 99Tcm-GX1. The imaging results showed that GX1 could concentrate in tumor xenograft in nude mice. All these results indicated that GX1 was a novel vascular target of human gastric cancer. However, there is still a lot of work to be done before GX1 become possible for clinical anti-tumor therapy as a tumor vascular targeted agent. The present study is to investigate the bioactivities and relevant molecular mechanisms of GX1 other than its targeting effect.【Objectives】1. To establish a co-culture model of tumor vascular endothelial cells for verification of the targeting effects of GX1 peptide (not the peptide displaying phage), and also as a platform for further tests.2. To study the in vitro effects of GX1 on proliferation, migration and other characteristics of vascular endothelial cells, and also to clarify the in vivo role of GX1 in tumor formation and angiogenesis.3. To investigate the possible mechanisms underlying the bioactivities of GX1, and to identify the specific signaling pathway and relevant molecules that may be regulated by GX1, so as to provide experimental evidence for clinical application of this targeted peptide.【Methods】1. HUVEC were co-cultured with tumor conditioned medium to establish the co-culture system of tumor endothelial cells (co-HUVEC). GX1 peptide, together with a control peptide (Pep2) was chemically synthesized and purified. Immunocytochemical staining was used to detect the binding specificity of GX1 to co-HUVEC.2. GX1 effects on cell proliferation, migration, vascular formation and other characteristics were analyzed by in vitro MTT assay, tube formation assay, wound healing test, etc. Chorioallantoic membrane (CAM) assay was used to observe GX1 effects on in vivo angiogenesis. Tumor formation test in nude mice bearing human gastric adenocarcinoma xenografts was performed with different drug administration modes of GX1.3. Cell cycle distribution and cell apoptosis induced by GX1 treatment were detected by flow cytometry to look into the possible mechanisms of GX1 effects on tumor angiogenesis.4. Microarray analysis was performed to detect the changes of gene expression induced by GX1 treatment in co-HUVEC. Bioinformatics analysis was used to identify the specific signaling pathway and downstream molecules which might be involved in GX1 functions.Western blot was used to determine the expression level of several genes significantly changed by GX1 treatment, including Bcl-2, BAX, caspase3 and caspase8, so as to verify the data from microarray analysis. 【Results】1. The co-culture system of tumor vascular endothelial cells (co-HUVEC) was successfully established, and GX1 and control peptide were synthesized and purified with the purity over 95%. GX1 was shown by immunocytochemical staining to bind specifically to co-HUVEC, while no positive staining was observed in Pep2 or PBS groups, indicating that GX1 could selectively target gastric cancer vasculature.2. MTT assay showed that compared with control peptide, GX1 significantly inhibited the cell proliferation of HUVEC and co-HUVEC in a dose-dependent manner, and the inhibition rate in co-HUVEC was higher than that of HUVEC (60% vs. 25%-45%, P<0.05). In tube formation assay, GX1 significantly hampered the micro-tube formation of co-HUVEC, while no such effect was seen in Pep2 treated cells. On the other hand, GX1 showed no significant effects on cell proliferation or migration ability in tumor cells.3. CAM assay exhibited that, compared with Pep2, GX1 remarkably suppressed the neovascularization on CAM in chicken embryo (micro-vessel count: 20.0±1.6 vs. 10.6±1.0, P<0.05), suggesting that GX1 could inhibit in vivo angiogenesis. In in vivo tumor formation assay, GX1, either administered intravenously or subcutaneously, partly delayed tumor growth in comparison with Pep2 or PBS.4. Flow cytometry analysis showed that compared with Pep2, GX1 increased the proportion of apoptotic cells in co-HUVEC (1.8% vs. 10.5%, P<0.05), while no significant changes in cell cycle distribution was detected in GX1 treated co-HUVEC.5. Microarray data exhibited that, among the total of 39200 tested genes, 248 genes were notably upregulated, while 137 genes were downregulated by GX1 treatment. These genes were classified as genes associated with cell apoptosis, cell metabolism, oncogenes and tumor suppressor genes, etc. Western blot results confirmed the upregulated protein level of BAX and caspase3, but a downregulation of Bcl-2 after GX1 treatment of co-HUVEC. There was no significant change of caspase8 activity.【Conclusions】1. Tumor microenvironment can be effectively mimicked by in vitro co-culture system. HUVEC co-cultured with tumor conditioned medium exhibit some of the characteristics of tumor endothelial cells, and could be used in research of tumor vascular targeted molecules.2. Besides the tumor vascular targeting ability, GX1 is also able to inhibit tumor angiogenesis in vitro and in vivo, as well as partly suppress tumor growth in vivo possibly by induction of cell apoptosis of tumor endothelial cells.3. GX1 might induce cell apoptosis via regulation of Bcl-2/BAX signaling pathway and activation of caspase family members. These results may facilitate the development of GX1 as a novel candidate for vascular targeted therapy of human gastric cancer, and may also provide valuable information for identification of GX1 receptors.更多还原