以VEGFR-2为靶点的卵巢癌核素显像及抑制卵巢癌细胞活性的研究

  本文关键词：以VEGFR-2为靶点的卵巢癌核素显像及抑制卵巢癌细胞活性的研究，由笔耕文化传播整理发布。

        研究目的卵巢癌是女性常见的生殖器官恶性肿瘤,因其早期的临床症状不明显,缺乏有效的早期诊断手段,75%的卵巢癌患者发现时已为晚期,大多已经产生盆腹腔转移而失去手术切除的机会,5年生存率仅为20%-30%,是妇科肿瘤中死亡率最高的恶性疾病,而早期卵巢癌患者的5年生存率可达90%。目前卵巢癌的治疗方案复发率和耐药率高,生存期没有得到有效的提高。因此寻找一个特异性靶点进行早期诊断及有效治疗是当前卵巢癌研究迫切需要解决的问题。研究发现,血管内皮生长因子受体-2／血管内皮生长因子(vascular endothelial growth factor receptor-2/vascular endothelial growth factor, VEGFR-2/VEGF)信号通路参与肿瘤周围新生内皮细胞的迁移、增殖和生存,在肿瘤周围新生血管生成过程中起到重要的作用。VEGF有多种蛋白形式,其中VEGF165的促血管生成作用最显著。VEGFR-2是介导VEGF165发挥促血管生成作用的主要受体,在VEGF的信号转导及血管内皮生成中起主导作用,并且在卵巢癌肿瘤组织中高表达。本研究拟建立卵巢癌荷瘤裸鼠模型,采用131I-VEGF165进行放射自显影显像来探讨VEGFR-2是否可以作为卵巢癌核素显像的新靶点。ISO-1[(S,R)-3-(4-羟苯基)-4,5-二氢-5-异(?)唑乙酸甲酯]是特异性的巨噬细胞移动抑制因子(macrophage migration inhibitory factor, MIF)酶抑制剂,可以选择性地结合MIF互变异构酶的活性位点,明显抑制MIF活性,但ISO-1在卵巢癌中是否可以通过影响MIF的活性来调控VEGFR-2的水平目前尚未见报道。本研究旨在通过观察不同浓度ISO-1对人卵巢癌细胞增殖、侵袭及对VEGFR-2的作用,为以VEGFR-2为靶点进行卵巢癌治疗奠定实验基础。研究方法采用放射性碘化标记Iodogen法合成131I-VEGF165,用SephadexG25柱纯化,利用纸层析法测定其标记率及放化纯,同时进行卵巢癌细胞对131I-VEGF165的摄取实验。建立荷人卵巢癌裸鼠模型,待肿瘤生长至1.0cm×1.0cm左右用于显像。荷瘤裸鼠尾静脉注射131I-VEGF165后分别于3h、6h和8h进行放射自显影显像和体内分布实验,RT-PCR、Western blot和免疫组化法检测肿瘤组织中VEGFR-2的表达。不同浓度的ISO-1作用于人卵巢癌细胞SKOV3、A2780,对照组以相应浓度的稀释液处理。MTT法检测卵巢癌细胞增殖：L-多巴色素甲酯法测定卵巢癌细胞MIF互变异构酶活性；微孔迁移法检测卵巢癌细胞体外侵袭；卵巢癌细胞凋亡的检测；RT-PCR检测卵巢癌细胞mRNA的表达；细胞免疫化学检测卵巢癌细胞蛋白的表达。实验结果131I-VEGF165的标记率为90.47%,放化纯为94.17%,标记物免疫活性好。细胞摄取实验表明卵巢癌细胞在与131I-VEGF165混合孵育0.5、1、2、4和24h后,卵巢癌细胞SKOV3对131I-VEGF165的摄取率均明显高于对照Na131的摄取(P＜0.05); RT-PCR、Western blot和免疫组化结果表明在移植瘤部位VEGFR-2的mRNA、蛋白均特异性高表达；体内分布实验显示荷瘤裸鼠在尾静脉注射131I-VEGF165后T/NT比值分别为2.72±0.19(3h),4.85±0.81(6h)和3.31±0.57(8h)；放射自显影显像表明荷瘤小鼠在尾静脉注射I31I-VEGF165后3h移植瘤部位开始有放射性浓聚,6h移植瘤可清晰显像,这与131I-VEGF165在荷瘤裸鼠移植瘤部位有较高的T／NT比值相一致。ISO-1能显著抑制人卵巢癌细胞SKOV3、A2780的增殖及其MIF互变异构酶活性(P＜0.05)；50μmol/L ISO-1作用于卵巢癌细胞SKOV3、A278024h后,其穿透聚碳酸酯膜的能力显著降低(P＜0.05),且细胞凋亡明显,同时卵巢癌细胞的VEGFR-2、VEGF的mRNA和蛋白水平显著降低(P＜0.05)。结论放射性碘化标记的131I-VEGF165制备简便、性能稳定,能够特异性靶向聚集于肿瘤部位,且在靶组织中清除缓慢,放射自显影图像质量好,提示VEGFR-2可作为卵巢癌早期诊断的特异性靶点；ISO-1可抑制卵巢癌细胞的增殖及侵袭,能显著下调VEGFR-2、VEGF的表达。为临床卵巢癌以VEGFR-2为靶点的早期特异性诊断和靶向治疗奠定了实验基础。

    ObjectiveOvarian cancer is one of the most common gynecologic cancers. Its early clinical symptoms is not obvious, and early detection lack of reliable method,75%of ovarian cancers were diagnosed in late stage and had abdominal metastasis, which had lost the opportunity of surgical ablation. The five-year survival rate is only20%-30%. Ovarian cancer is the leading cause of death from gynecological malignancy. But the five-year survival rate of ovarian cancer patients diagnosed in early stage is up to90%. The standard therapy is cytoreductive surgery followed by cisplatin-based chemotherapy. But the relapse rate and resistance rate is high, survival has not been effectively improved. Therefore, to seek a specific target for early diagnosis and effective treatment of ovarian cancer has become a hot research. Studies have revealed that the vascular endothelial growth factor receptor-2/vascular endothelial growth factor (VEGFR-2/VEGF) signaling pathway involves in cell migration, proliferation and survival of nascent endotheliocyte around tumor, and plays a central role in tumor vasculature of ovarian cancer. VEGF has many forms of protein, and the most significant proangiogenic is VEGF165. VEGFR-2is one of the main receptors that mediated VEGF165promote angiogenesis, and it plays a central role in signal transduction and vascular endothelium generation. Moreover, VEGFR-2is highly expressed in ovarian cancer tumor tissue. This study plans to establish ovarian tumor-bearing models in nude mouse, to investigate whether VEGFR-2could be used as a diagnostic biomarker for ovarian cancer using autoradiography imaging method with131I-VEGFi65.(S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) is a competitive inhibitor of tautomerase activity as well as an specific inhibitor of MIF. It can selectively combined with the active site of MIF tautomeric isomerase, and significantly inhibit MIF activity. But it has not been reported that whether ISO-1can regulate the VEGFR-2level through effect the activities of MIF in ovarian cancer. This study investigate the effect of different concentrations of ISO-1on the proliferation、invasion and VEGFR-2level of ovarian cancer cells, and provide experimental evidence for the VEGFR-2-targeted treatment of ovarian cancer.MethodsVEGF165was radioiodinated with Na131I by Iodogen method,131I-VEGF165were separated from free iodine using Sephadex G-25columns. The radiochemical purity of radioiodinated VEGF165was got by paper chromatography. At the same time, the uptakes of I31I-VEGF165by ovarian cancer cells were analyzed. The ovarian tumor-bearing models were established, and when the tumor volume reached1cm3(6weeks after inoculation), the tumor-bearing nude mice were used for autoradiography imaging studies. The tumor targeting and body distribution of ovarian tumor-bearing models was analyzed after injection of131I-VEGF165for3h、6h and8h. Using RT-PCR, Western blot and immunohistochemical assay of tumor tissue to detect VEGFR-2expression.The human ovarian cancer cell lines SKOV3and A2780were treated with series concentrations of ISO-1. MTT assay was used to measure the cell proliferation. The tautomerase activity of MIF was evaluated by using L-dopachromemethyl ester. Microporous migration assay w as performed to determine the effect of ISO-1on the invasion of SKOV3and A2780cells. Fluorescence was used to test apoptosis. The expression of mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression of protein was detected by cell immunochemistry.ResultsThe labeled rate of131I-VEGF,65was90.47%, radiochemical purity was94.17%, and the immune activity of markers was well. The uptake of131I-VEGF165by human ovarian cancer cells SKOV3was much higher than that of control group (Na131I) at0.5、1、2、4and24h (P<0.05).The target-to-non-target (T/NT) ratios were2.72±0.19(3h),4.85±0.81(6h), and3.31±0.57(8h).RT-PCR and Western Blot revealed thatVEGF-165, VEGFR-2were detected in the ovarian carcinoma tissues and SKOV3cells not in the controls. Wholebody autoradiography showed that uptake in well-perfused organs at3h and clear tumor localization from6h onward, these findings were in accordance with the high T/NT ratio.ISO-1inhibited the proliferation and the MIF tautomerase activities of SKOV3and A2780cells obviously (P<0.05).After treated with50μmol/L ISO-1for24h, the ability to penetrate the polycarbonate film is significantly reduced (P<0.05), apoptosis is obviously. meanwhile, the mRNA and protein level of VEGFR-2and VEGF is also reduced (P<0.05)ConclusionsVEGF165was easily to be iodinated with131I by Iodogen method, and131I-VEGF165was stable performance.131I-VEGF165can targeting gathered at the tumor site, and the autoradiography imagings were clearly. Based on our experience, the tumor uptake of131I-VEGF165measured by wholebody autoradiography reflects tumor VEGFR-2expression level in vivo. ISO-1can down-regulate the expression of VEGFR-2and VEGF, thus inhibit the proliferation and invasion of ovarian cancer cells. It provides the experimental evidence for the targeted therapy of VEGFR-2for ovarian cancer.

以VEGFR-2为靶点的卵巢癌核素显像及抑制卵巢癌细胞活性的研究

目录4-5CATALOGUE5-7中文摘要7-10ABSTRACT10-13符号说明14-16第一部分 以VEGFR-2为靶点的卵巢癌核素显像的研究16-38    前言16-18    材料与方法18-32    实验结果32-34    讨论34-37    结论37-38第二部分 以VEGFR-2为靶点的抑制卵巢癌细胞活性的实验研究38-51    前言38-40    材料与方法40-46    实验结果46-48    讨论48-51    结论51全文小结51-52附表及附图52-63参考文献63-69致谢69-70攻读学位期间发表的学术论文目录70-71学位论文评阅及答辩情况表71

  本文关键词：以VEGFR-2为靶点的卵巢癌核素显像及抑制卵巢癌细胞活性的研究，，由笔耕文化传播整理发布。