本文關(guān)鍵詞：以VEGFR-2為靶點(diǎn)的卵巢癌核素顯像及抑制卵巢癌細(xì)胞活性的研究，由筆耕文化傳播整理發(fā)布。

        研究目的卵巢癌是女性常見的生殖器官惡性腫瘤,因其早期的臨床癥狀不明顯,缺乏有效的早期診斷手段,75%的卵巢癌患者發(fā)現(xiàn)時(shí)已為晚期,大多已經(jīng)產(chǎn)生盆腹腔轉(zhuǎn)移而失去手術(shù)切除的機(jī)會,5年生存率僅為20%-30%,是婦科腫瘤中死亡率最高的惡性疾病,而早期卵巢癌患者的5年生存率可達(dá)90%。目前卵巢癌的治療方案復(fù)發(fā)率和耐藥率高,生存期沒有得到有效的提高。因此尋找一個(gè)特異性靶點(diǎn)進(jìn)行早期診斷及有效治療是當(dāng)前卵巢癌研究迫切需要解決的問題。研究發(fā)現(xiàn),血管內(nèi)皮生長因子受體-2／血管內(nèi)皮生長因子(vascular endothelial growth factor receptor-2/vascular endothelial growth factor, VEGFR-2/VEGF)信號通路參與腫瘤周圍新生內(nèi)皮細(xì)胞的遷移、增殖和生存,在腫瘤周圍新生血管生成過程中起到重要的作用。VEGF有多種蛋白形式,其中VEGF165的促血管生成作用最顯著。VEGFR-2是介導(dǎo)VEGF165發(fā)揮促血管生成作用的主要受體,在VEGF的信號轉(zhuǎn)導(dǎo)及血管內(nèi)皮生成中起主導(dǎo)作用,并且在卵巢癌腫瘤組織中高表達(dá)。本研究擬建立卵巢癌荷瘤裸鼠模型,采用131I-VEGF165進(jìn)行放射自顯影顯像來探討VEGFR-2是否可以作為卵巢癌核素顯像的新靶點(diǎn)。ISO-1[(S,R)-3-(4-羥苯基)-4,5-二氫-5-異(?)唑乙酸甲酯]是特異性的巨噬細(xì)胞移動(dòng)抑制因子(macrophage migration inhibitory factor, MIF)酶抑制劑,可以選擇性地結(jié)合MIF互變異構(gòu)酶的活性位點(diǎn),明顯抑制MIF活性,但I(xiàn)SO-1在卵巢癌中是否可以通過影響MIF的活性來調(diào)控VEGFR-2的水平目前尚未見報(bào)道。本研究旨在通過觀察不同濃度ISO-1對人卵巢癌細(xì)胞增殖、侵襲及對VEGFR-2的作用,為以VEGFR-2為靶點(diǎn)進(jìn)行卵巢癌治療奠定實(shí)驗(yàn)基礎(chǔ)。研究方法采用放射性碘化標(biāo)記Iodogen法合成131I-VEGF165,用SephadexG25柱純化,利用紙層析法測定其標(biāo)記率及放化純,同時(shí)進(jìn)行卵巢癌細(xì)胞對131I-VEGF165的攝取實(shí)驗(yàn)。建立荷人卵巢癌裸鼠模型,待腫瘤生長至1.0cm×1.0cm左右用于顯像。荷瘤裸鼠尾靜脈注射131I-VEGF165后分別于3h、6h和8h進(jìn)行放射自顯影顯像和體內(nèi)分布實(shí)驗(yàn),RT-PCR、Western blot和免疫組化法檢測腫瘤組織中VEGFR-2的表達(dá)。不同濃度的ISO-1作用于人卵巢癌細(xì)胞SKOV3、A2780,對照組以相應(yīng)濃度的稀釋液處理。MTT法檢測卵巢癌細(xì)胞增殖：L-多巴色素甲酯法測定卵巢癌細(xì)胞MIF互變異構(gòu)酶活性；微孔遷移法檢測卵巢癌細(xì)胞體外侵襲；卵巢癌細(xì)胞凋亡的檢測；RT-PCR檢測卵巢癌細(xì)胞mRNA的表達(dá)；細(xì)胞免疫化學(xué)檢測卵巢癌細(xì)胞蛋白的表達(dá)。實(shí)驗(yàn)結(jié)果131I-VEGF165的標(biāo)記率為90.47%,放化純?yōu)?4.17%,標(biāo)記物免疫活性好。細(xì)胞攝取實(shí)驗(yàn)表明卵巢癌細(xì)胞在與131I-VEGF165混合孵育0.5、1、2、4和24h后,卵巢癌細(xì)胞SKOV3對131I-VEGF165的攝取率均明顯高于對照Na131的攝取(P＜0.05); RT-PCR、Western blot和免疫組化結(jié)果表明在移植瘤部位VEGFR-2的mRNA、蛋白均特異性高表達(dá)；體內(nèi)分布實(shí)驗(yàn)顯示荷瘤裸鼠在尾靜脈注射131I-VEGF165后T/NT比值分別為2.72±0.19(3h),4.85±0.81(6h)和3.31±0.57(8h)；放射自顯影顯像表明荷瘤小鼠在尾靜脈注射I31I-VEGF165后3h移植瘤部位開始有放射性濃聚,6h移植瘤可清晰顯像,這與131I-VEGF165在荷瘤裸鼠移植瘤部位有較高的T／NT比值相一致。ISO-1能顯著抑制人卵巢癌細(xì)胞SKOV3、A2780的增殖及其MIF互變異構(gòu)酶活性(P＜0.05)；50μmol/L ISO-1作用于卵巢癌細(xì)胞SKOV3、A278024h后,其穿透聚碳酸酯膜的能力顯著降低(P＜0.05),且細(xì)胞凋亡明顯,同時(shí)卵巢癌細(xì)胞的VEGFR-2、VEGF的mRNA和蛋白水平顯著降低(P＜0.05)。結(jié)論放射性碘化標(biāo)記的131I-VEGF165制備簡便、性能穩(wěn)定,能夠特異性靶向聚集于腫瘤部位,且在靶組織中清除緩慢,放射自顯影圖像質(zhì)量好,提示VEGFR-2可作為卵巢癌早期診斷的特異性靶點(diǎn)；ISO-1可抑制卵巢癌細(xì)胞的增殖及侵襲,能顯著下調(diào)VEGFR-2、VEGF的表達(dá)。為臨床卵巢癌以VEGFR-2為靶點(diǎn)的早期特異性診斷和靶向治療奠定了實(shí)驗(yàn)基礎(chǔ)。

    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為靶點(diǎn)的卵巢癌核素顯像及抑制卵巢癌細(xì)胞活性的研究

目錄4-5CATALOGUE5-7中文摘要7-10ABSTRACT10-13符號說明14-16第一部分 以VEGFR-2為靶點(diǎn)的卵巢癌核素顯像的研究16-38    前言16-18    材料與方法18-32    實(shí)驗(yàn)結(jié)果32-34    討論34-37    結(jié)論37-38第二部分 以VEGFR-2為靶點(diǎn)的抑制卵巢癌細(xì)胞活性的實(shí)驗(yàn)研究38-51    前言38-40    材料與方法40-46    實(shí)驗(yàn)結(jié)果46-48    討論48-51    結(jié)論51全文小結(jié)51-52附表及附圖52-63參考文獻(xiàn)63-69致謝69-70攻讀學(xué)位期間發(fā)表的學(xué)術(shù)論文目錄70-71學(xué)位論文評閱及答辯情況表71

  本文關(guān)鍵詞：以VEGFR-2為靶點(diǎn)的卵巢癌核素顯像及抑制卵巢癌細(xì)胞活性的研究，，由筆耕文化傳播整理發(fā)布。