【作者】 梁树辉；

【导师】 丁杰；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 【背景】血管生成是肿瘤发展过程中的重要条件之一,肿瘤组织微血管生成的进程、性质和密度直接关系到肿瘤生长、侵袭、转移的能力及预后,肿瘤血管诊断与治疗逐渐成为研究热点。然而,有关的肿瘤血管检测手段和治疗药物尚不令人满意,这主要与缺乏肿瘤血管特异性靶向分子及相关技术手段的不足有关。研究表明,肿瘤血管具有分子异质性,鉴定这些异质性分子并研究其作用,检测其表达水平并动态监测其变化规律,有可能为肿瘤血管靶向性诊断与治疗提供特异性靶向分子。目前肿瘤血管生成的评价主要靠免疫组化染色进行MVD计数,难以从功能上评价血管生成活性,而近年发展的分子成像技术有望实现肿瘤血管生成的可视化定量检测及动态实时追踪。受体靶向性核素内放射治疗与肿瘤血管抑制治疗相结合的肿瘤血管放射受体治疗,成为近年研究的一个热点,有望解决肿瘤血管抑制治疗中的一些问题。分子成像技术及放射受体治疗均有赖于高亲和力的特异性靶向分子。前期工作中,我们通过噬菌体随机肽库筛选获得胃癌血管内皮细胞特异结合肽GEBP11,并对其结合活性进行了初步鉴定。【目的】1、进一步深入鉴定该短肽与胃癌血管内皮细胞的结合特性及其体内胃癌血管靶向性;2、利用该短肽制备同位素探针,进行荷人胃癌裸鼠SPECT成像规律及受体放射治疗的初步研究;3、明确GEBP11短肽对胃癌血管生成的影响,并初步探讨其作用机制。【方法】1、通过蓝色噬斑形成实验及GEBP11、IN11噬菌体在荷瘤裸鼠体内的竞争抑制实验检测噬菌体体内归巢的特异性;2、免疫荧光技术检测GEBP11在荷瘤裸鼠体内的结合特异性;3、免疫细胞化学或荧光检测GEBP11在Co-HUVECs中的定位及内化;4、免疫组织荧光检测GEBP11在人胃癌组织中的结合特异性;5、采用直接法⁹⁹Tc^mO₄^-标记GEBP11,采用NBS法¹³¹I标记GEBP11,纸层析法测定标记率、放射化学纯度等;6、放射自显影鉴定GEBP11同位素探针的结合活性;7、受体放射配基结合分析实验鉴定受体亲和力及受体细胞密度;8、同位素示踪技术检测¹³¹I-GEBP11在荷瘤裸鼠体内的生物学分布;9、SPECT成像技术进行荷人胃癌裸鼠体内⁹⁹Tc^m-GEBP11显像;10、MTT细胞增殖实验、荷瘤鼠抑瘤实验评价¹³¹I-GEBP11对内皮细胞及荷瘤鼠肿瘤的抑制能力;11、免疫组织化学染色计数瘤组织MVD;12、病理学HE染色、血液血细胞分析及生化指标检测观察肝脏损伤及骨髓抑制情况;13、Matrigel管状结构形成实验、鸡胚绒毛尿囊膜血管生成抑制实验、小鼠体内Matrigel Plug诱导血管生成实验分析GEBP11对血管生成的影响;14、MTT细胞增殖实验、细胞周期及凋亡分析、细胞侵袭迁移实验、细胞粘附实验探讨GEBP11抑制血管生成的细胞机制;15、基因表达谱芯片技术筛选GEBP11短肽作用Co-HUVECs后的差异表达基因。【结果】1、GEBP11体内胃癌血管靶向性及其结合特性的进一步鉴定IN11噬菌体在肿瘤组织回收的滴度显著高于对照噬菌体或其在对照组织中回收的滴度,而对照噬菌体在各组织之间的差别不大。GEBP11短肽对IN11噬菌体向荷瘤鼠肿瘤组织的归巢具有抑制作用,并且,随着GEBP11短肽浓度的升高,移植瘤内的归巢噬菌体数量逐渐下降,抑制率逐渐增高。荷瘤鼠体内荧光共定位分析显示,肿瘤组织中GEBP11短肽与抗Ⅷ因子抗体分布一致,而对照肽或在心脏、肌肉等对照组织无明显着色。免疫细胞化学或荧光染色显示,GEBP11短肽在Co-HUVECs胞膜及核周胞浆着色,而在HUVECs、GES细胞、胃癌SGC7901细胞及AGS细胞上呈阴性或弱阳性反应。激光共聚焦免疫荧光染色结果显示,4℃孵育时GEBP11主要结合于内皮细胞胞膜,给予内化条件37℃孵育后,结合于胞膜受体的GEBP11短肽可被内化入胞内。免疫组织荧光染色结果显示,GEBP11短肽在人胃癌组织上与CD31抗体着色位置一致,而对照肽URP无阳性反应,慢性胃炎组织上亦未见明显着色。2、GEBP11核素探针在荷人胃癌裸鼠体内的SPECT成像直接法将⁹⁹Tc^mO₄^-标记于GEBP11,纸层析法测定其标记率达90-98%,比活度大于100Ci /mmol,⁹⁹Tc^m-GEBP11在体内外具有良好的稳定性。NBS法将¹³¹I标记于GEBP11,纸层析法测定其标记率达90%,比活度大于10Ci /mmol,具有良好的稳定性。放射自显影结果显示,⁹⁹Tc^m-GEBP11及¹³¹I-GEBP11与Co-HUVECs的结合明显强于HUVECs,而对照标记肽⁹⁹Tc^m-URP或¹³¹I-OXT在Co-HUVECs上无明显结合。受体放射配基结合分析实验结果显示,相同⁹⁹Tc^m-GEBP11标记肽浓度时,Co-HUVECs与标记肽的结合量高于HUVECs,均呈饱和曲线。Scatchard作图示GEBP11与Co-HUVECs、HUVECs的Kd值分别为1.972nM、2.489nM,两种细胞受体密度分别为5.7×105/细胞、3.3×105/细胞。放射性同位素示踪技术检测¹³¹I-GEBP11在荷瘤裸鼠体内的生物学分布结果显示,¹³¹I-GEBP11注入体内2小时后,肿瘤内放射活性高出大多数器官,随时间的延迟,肿瘤内放射性活性下降相对较慢,致使它与其它非肿瘤器官的相对比值逐渐增高,最高比值可达15以上。¹³¹I-GEBP11在体内多数器官清除较快,肾脏内放射性最高,清除时间长。SPECT成像结果显示,⁹⁹Tc^m-GEBP11在体内相对聚集于肿瘤部位,随时间延长渐强,T/NT逐渐增高,12h后开始高于心血池本底,18-24h肿瘤灶显示更加清晰,是比较理想的显像时段。⁹⁹Tc^m-URP对照组SPECT显像无肿瘤部位放射性浓聚现象。3、¹³¹I-GEBP11对荷人胃癌裸鼠移植瘤的抗肿瘤效应体外细胞杀伤实验显示,¹³¹I-GEBP11对内皮细胞增殖有抑制作用,呈浓度依赖性;Na¹³¹I对内皮细胞无特异性杀伤作用;GEBP11浓度达10μg/ml时开始表现出对内皮细胞增殖的抑制作用。抑瘤实验结果显示,eADM肿瘤抑制率最高,为64.9%,¹³¹I-GEBP11次之,为56.3%,Na¹³¹I、GEBP11无明显抑瘤作用。¹³¹I-GEBP11、eADM及GEBP11组可明显延长生存期。¹³¹I-GEBP11治疗组伴有血小板降低、肝功损伤等副反应,但明显轻于表阿霉素。免疫组化染色计数MVD显示,与NS组对照,¹³¹I-GEBP11、GEBP11肿瘤组织MVD计数明显降低。4、GEBP11短肽抑制血管生成的作用及机制Matrigel管状结构形成实验、鸡胚绒毛尿囊膜血管生成抑制实验、小鼠体内Matrigel Plug诱导血管生成实验结果显示,GEBP11能抑制内皮细胞的管状结构形成能力、抑制CAM及小鼠体内血管生成。MTT细胞增殖实验、细胞周期及凋亡分析、细胞侵袭迁移实验、细胞粘附实验显示,GEBP11短肽对Co-HUVECs及HUVECs的增殖表现出不同程度的抑制作用,对Co-HUVECs抑制作用更强,GEBP11能诱导内皮细胞的凋亡,而对细胞周期无明显影响。GEBP11对内皮细胞的基质降解和迁移有抑制作用,对内皮细胞的粘附也有抑制作用。采用Trizol法抽提细胞总RNA,经鉴定RNA质量合格。经探针标记、芯片杂交、染色及图像采集等实验步骤,结果显示芯片的杂交信号强度较高,pm_mean（探针信号均值）大于200,neg_control_mean（阴性对照均值）小于10（图44）,共有30000以上个杂交点。表达差异2倍以上定为差异表达基因,分析发现,与Co-HUVECs相比,GEBP11处理后的Co-HUVECs中上调表达的基因有2104个,下调表达的基因有1202个;与HUVECs相比,Co-HUVECs中上调表达的基因有194个,下调表达的基因有579个。【结论】1、GEBP11具有活体内胃癌血管靶向结合的能力,它与胃癌血管内皮细胞膜受体特异结合后可被内化。2、⁹⁹Tc^m-GEBP11 SPECT成像能较好地显示体内肿瘤灶,¹³¹I-GEBP11具有明显的抗肿瘤效应及较弱的毒副作用,为开发肿瘤血管成像核素探针及受体放射治疗新药奠定了基础。3、GEBP11具有抑制胃癌血管生成的作用,这可能通过抑制ECs增殖、基质降解及迁移、粘附能力实现。GEBP11作用于内皮细胞可引起多种基因表达变化,这为探讨GEBP11抑制血管生成的分子机制提供了重要线索。更多还原

【Abstract】 【Background】Angiogenesis and vasculature play important roles in the growth and metastasis of solid tumors. The progress, character and density of microvasculature are related to growth, invasion, metastatic and prognosis of tumor. Microvascular diagnosis and anti-vascular therapy have become an intensive studies field in cancer research. However, the means to detect tumor vasculature and anti-vascular drugs are not satisfactory. This could be explained by the deficiency of molecules targeting to tumor vasculature. It was found that tumor vessels had characteristics different from normal vessels, which would provide molecules targeting possibility for diagnosis and therapeutics for these heterogeneity molecules in tumor vasculatures. Now MVD count is the popular used methods to estimate the vasculatures in tumor, which, however, cannot evaluate the functions of them. Molecular imaging technology developed in the last several years provides the possibility to do it. And the radiation therapeutics targeting the receptors in tumor vasculatures hopes to promote anti-vascular therapy. These two means rely on efficient targeting molecules. In our previous works, we have found a peptide GEBP11 targeting to the vascular endothelial cells in gastric cancer by panning phage library.【Objectives】1. To identify the ability of GEBP11 to target the vasculature of gastric cancer in vivo.. 2. To make the isotope probes of GEBP11 and perform SPECT imaging and targeting therapy of gastric cancer in vivo.. 3. To identify the effects of GEBP11 on angiogenesis in gastric cancer and approach the possible mechanism.【Methods】1. The blue phage plaque formation assay and competitive binding assay in vivo. were performed to investigate the homing specificity of the IN11 phage which displayed the peptide GEBP11. 2. Immunofluorescent staining was used to study the binding specificity of synthesized peptide GEBP11. 3. Immunocytochemical or immunofluorescent staining was performed to identify the location and internalization of GEBP11 on Co-HUVECs. 4. Immunohistofluorescent staining was used to identify the binding specificity of GEBP11 in gastric cancer tissues. 5. GEBP11 was labeled with ⁹⁹Tc^mO₄^- using direct labeling method, and was labeled with ¹³¹I using NBS labeling method. Then the labeled peptides were validated for radiochemical yield, specific activity, and in vitro. stability. 6. The bioactivity of ⁹⁹Tc^m-GEBP11 or ¹³¹I-GEBP11 was validated by cell receptor autoradiography in cultured HUVECs. 7. Receptor binding assay was performed in vitro. to analyze quantitatively the binding specificity and affinity of GEBP11 to HUVEC, the receptor intensity on HUVEC, and the difference of affinity of GEBP11 binding to Co-HUVEC and HUVEC. 8. The radioactivity of all organs of nude mice injected with ¹³¹I-GEBP11 was determined and the radioactivity of all organs per g （%ID/g） was calculated to analyze the specific distribution of ¹³¹I-GEBP11. 9. Gamma camera images of nude mice bearing tumor xenografts of human gastric carcinoma injected with ⁹⁹Tc^m-GEBP11 was obtained 0.5-24 h after being injected to identify the targeting ability of labeled peptide to tumor tissues. 10. MTT assay on HUVECs and antitumor assay in nude mice bearing tumor xenografts of human gastric cancer were performed to evaluate the effects of ¹³¹I-GEBP11 on HUVECs and xenografts. 11. Immunohistochemical staining was used for MVD counting in tumor. 12. H.E. staining, blood cells analysis and blood biochemical indicator analysis were performed to investigate the injury of liver and bone marrow. 13. Tube formation assay in matrigel, CAM angiogenesis assay and angionenesis induced by matrigel in mice were performed to identify the effects of GEBP11 on angiogenesis. 14. The cellular mechanisms of angiogenesis inhibition of GEBP11 were clarified by proliferation assay, cellular cycle and apoptosis analysis, invasion and migration assay and adherency assay. 15. The differential expression genes in Co-HUVECs treated by GEBP11 or not were screened by microarray.【Results】1. The binding ability of GEBP11 targeting to gastric cancer vasculature in vivo. and its binding characteristicsThe titer of IN11 phage recovered from tumor tissue was higher than that of control phage or IN11 phage but from control tissue. GEBP11 peptide inhibited homing effect of the IN11 phage to tumor tissue in nude mice bearing human gastric cancer xenografts, and the inhibition intensity was related to the concentrate of GEBP11. By immunohistofluorescent staining, GEBP11 was co-located with anti-FⅧantibody in tumor tissue. However, URP or GEBP11 in control tissues was not co-located with anti-FⅧantibody.Immunocytochemistry or immunofluorescence microscopy results showed that GEBP11 was stained on the membrane and perinuclear cytoplasm of Co-HUVECs, but not in HUVECs, GES cells, SGC7901 cells and AGS cells. We also found that GEBP11 peptide was abundant on membrane of ECs at 4℃, and they would be internalized into cytoplasm at 37℃. And the immunohistofluorescence microscopy results suggested that GEBP11 was co-located on the human gastric cancer tissues with anti-CD31 antibody, while URP or GEBP11 on the chronic gastritis tissues was not co-located with anti-CD31 antibody.2. Gamma camera images of nude mice bearing tumor xenografts of human gastric carcinoma injected with ⁹⁹Tc^m-GEBP11By direct labeling GEBP11 with ⁹⁹Tc^mO₄^-, we acquired high radiolabeling efficiency at 90-98%, as well as high specific activity beyond 100 Ci/mmol. in vitro. stability test indicated that ⁹⁹Tc^m-GEBP11 is fine in vitro. stability. By using NBS labeling method, we acquired high radiolabeling efficiency at 90% or more, as well as high specific activity beyond 10 Ci/mmol with ¹³¹I-GEBP11 being fine stability either. Receptor autoradiography showed that there were more obvious silver particles on Co-HUVECs than on HUVECs, which indicated that ⁹⁹Tc^m-GEBP11 or ¹³¹I-GEBP11 has fine biological activity. Receptor binding assay in vitro. showed that the binding of GEBP11 by Co-HUVECs was higher than that of HUVECs. The binding constant of ⁹⁹Tc^m-GEBP11 was calculated by Scatchard analysis. The Kd value of Co-HUVECs and HUVECs were determined to be 1.972 nM, 2.489 nM. The number of binding sites for the labeled peptide （receptor density） was estimated as 5.7×105 per Co-HUVEC and 3.3×105 per HUVEC, respectively.Biodistribution data of ¹³¹I-GEBP11 in nude mice bearing tumour xenografts of human gastric carcinoma showed that the radiotracer exhibited a quick decrease in radioactivity over time in blood and primary organs. Highest activity concentration was observed in kidneys. After 2 h p.i., the tumor radioactivity was higher than that in most of other organs; After 48h p.i., tumor accumulation in the tumour xenografts was 15 times higher than that in the intestine. The tumor/non-tumor ratios steadily increased over time, which showed that ¹³¹I-GEBP11 had the specifical targeting ability to tumor tissues in vivo.. Gamma camera images of nude mice bearing tumor xenografts of human gastric carcinoma injected with ⁹⁹Tc^m-GEBP11 showed the tumors could be visualized as early as 12 h and the activity was higher than that of heart until 24 h. The most clearly visualized imaging appeared at 18-24 h. Compared to ⁹⁹Tc^m-GEBP11, the radioactivity of tumor in nude mice injected with ⁹⁹Tc^m-URP was constantly lower than that of heart.3. ¹³¹I-GEBP11 inhibited the growth of xenografts in nude miceThe cell kill assay in vitro. showed that ¹³¹I-GEBP11 could inhibit the growth of ECs at a radioactivity dependent manner. Na¹³¹I had no inhibition on ECs. GEBP11 began to inhibit the ECs’s growth when the concentrate of GEBP11 reached 10μg/ml. Antitumor assay in vivo. showed that the tumor control rate of eADM was highest at 64.9%, and that of ¹³¹I-GEBP11 was second at 56.3%. Compared with eADM and ¹³¹I-GEBP11, Na¹³¹I and GEBP11 had no inhibition effects on tumor. By life span analysis, ¹³¹I-GEBP11, eADM and GEBP11 groups could prolong the life span. PLT decrease and liver injure occurred in therapy groups with ¹³¹I-GEBP11, which was lower than that in eADM groups. Immunohistochemical staining showed that MVD counts in tumor in ¹³¹I-GEBP11 and GEBP11 groups were lower than that in the NS group.4. GEBP11 was identified to have ability to inhibit angiogenesisBy tube formation assay in matrigel, CAM angiogenesis assay and angionenesis induced by matrigel in mice, GEBP11 was identified to have ability in inhibiting angiogenesis. Proliferation assay, cellular cycle and apoptosis analysis, invasion and migration assay and adherency assay showed that GEBP11 could inhibit the proliferation of Co-HUVECs and HUVECs, induce the apoptosis of ECs, but not alter the cell cycle of ECs. Additionally, GEBP11 appeared to inhibit the ECM degradation, migration and adhere of ECs.The total RNA of HUVECs, Co-HUVECs and GEBP11-treated Co-HUVECs were extracted. The quality of extracted RNA was evaluated by agarose electrophoresis and analysis of Lab-on-chip. After microarray hybridization and data normalization, more than 30000 gene spots were detected. There were 1202 down-regulated genes and 2104 up-regulated genes in Co-HUVECs treated by GEBP11. And there were 579 down-regulated genes and 194 up-regulated genes in Co-HUVECs vs. HUVECs.【Conclusions】1. GEBP11 peptide has the ability in targeting to gastric cancer vasculature in vivo., and its binding to the receptor on EC membrane promotes its internalization ability into cytoplasm.2. The SPECT imaging of ⁹⁹Tc^m-GEBP11 can show the tumor mass clearly and ¹³¹I-GEBP11 has anti-tumor effect with weak toxicant and secondary effect, which is the foundation for them to develop nuclide probe or radiotherapeutics drugs targeting to tumor vasculature.3. GEBP11 can inhibit angiogenesis, which comes true probably through its inhibition effects on the proliferation, invasion, migration and adherence of ECs. GEBP11 induces the changed expression of many genes, which provides important clew for approaching the molecular mechanism of GEBP11 inhibiting angiogenesis.更多还原