【摘要】：研究背景:原發(fā)性肝癌是常見惡性腫瘤之一,嚴重威脅人類健康,全球惡性腫瘤致死率逐年下降的前提下,原發(fā)性肝癌的死亡率連年上升。我國是原發(fā)性肝癌發(fā)病大國,且每年死亡人數(shù)約有46萬左右,占全球肝癌患者總死亡人數(shù)的1/2。因此,找尋肝癌治療分子靶點和靶向治療藥物具有重要現(xiàn)實意義。信號傳導與轉(zhuǎn)錄激活因子3(Signal Transducer and Activator of Transcription3,STAT3)與多種腫瘤的發(fā)生、發(fā)展密切相關(guān),STAT3的過度表達和持續(xù)活化與參與70%的人實體瘤形成,STAT3是現(xiàn)階段抗腫瘤靶向藥物研究的主要生物分子靶點之一。TTF1是從長白山珍珠梅中分離得到的抗腫瘤單體成分,以生物體可降解材料硬脂酸為載體,制備了更易吸收的粒徑在190nm左右的珍珠梅黃酮納米粒(TTF1-NP)。TTF1-NP能夠通過調(diào)控細胞凋亡相關(guān)蛋白,影響腫瘤細胞周期,抑制多種腫瘤細胞的增殖,具有進一步深入研究的價值。研究目的:本文主要研究珍珠梅黃酮納米粒(TTF1-NP)對人肝癌細胞STAT3的調(diào)控作用,探討TTF1-NP調(diào)控STAT3對肝癌細胞血管形成、侵襲轉(zhuǎn)移、細胞凋亡等生物學行為的影響,同時檢測TTF1-NP調(diào)控STAT3影響肝癌細胞生物學行為作用的分子機制,為TTF1-NP的研究和開發(fā)提供理論依據(jù)。研究方法:通過體內(nèi)、體外實驗檢測TTF1-NP對不同種人肝癌細胞的增殖作用及對STAT3和p-STAT3蛋白表達的影響;利用EMSA技術(shù)檢測TTF1-NP對人肝癌HepG2細胞STAT3與其靶DNA結(jié)合活性的影響;分別利用HUVEC細胞與人肝癌HepG2細胞共培養(yǎng)、HE和Hochest染色、Transwell、細胞劃痕實驗、流式雙染凋亡檢測術(shù)檢測TTF1-NP對人肝癌HepG2細胞的血管形成、侵襲和轉(zhuǎn)移和細胞凋亡的作用,應用western blot技術(shù)檢測相關(guān)功能蛋白的表達;同時分別構(gòu)建低表達STAT3和過表達STAT3的人肝癌HepG2細胞株檢測TTF1-NP對人肝癌HepG2細胞生物學行為的作用和分子機制。研究結(jié)果:1.本研究通過MTT實驗檢測發(fā)現(xiàn),TTF1-NP對人肝癌HepG2、Hep3B、PLC/PRF/5和SMMC-7721細胞增殖有劑量和時間依賴性抑制作用,其中對HepG2細胞增殖抑制作用最明顯;TTF1-NP作用48 h抑制人肝癌Hep3B、PLC/PRF/5、SMMC-7721和HepG2細胞增殖的IC50值,分別為:121.5 μmol/L、119.4μmol/L、147.8 μmol/L和99.3 μmol/L;TTF1-NP(5 μmol/kg,10 μmol/kg,20 μmol/kg)對裸鼠移植瘤體積的增長有抑制作用,生長抑制率(%)分別為:48.9±4.7、52.9±3.5、58.8±5.4。2.免疫化學染色檢測發(fā)現(xiàn),TTF1-NP對人肝癌HepG2細胞及HepG2細胞構(gòu)建的裸鼠移植瘤的STAT3和p-STAT3蛋白表達有抑制作用;western blot檢測結(jié)果顯示,TTF1-NP抑制STAT3和p-STAT3蛋白表達,有劑量依賴性。EMSA檢測發(fā)現(xiàn),TTF1-NP可以抑制STAT3與靶DNA的結(jié)合,有劑量依賴性。3.TTF1-NP可以抑制HUVEC細胞小管形成,抑制HepG2細胞VEGF、KDR和bFGF蛋白的表達;減少人肝癌HepG2細胞的侵襲數(shù)量、縮短遷移距離,抑制MMP2和MMP9蛋白表達;誘導人肝癌HepG2細胞凋亡,抑制survivin蛋白活化,促進cleaved caspase3 蛋白表達。4.分別構(gòu)建低表達和過表達STAT3的人肝癌HepG2細胞,檢測發(fā)現(xiàn)TTF1-NP對低表達STAT3的人肝癌HepG2細胞生物學行為抑制作用及相關(guān)功能蛋白表達調(diào)控作用不明顯;TTF1-NP對過表達STAT3的人肝癌HepG2細胞生物學行為抑制作用及相關(guān)功能蛋白表達調(diào)控作用明顯,有統(tǒng)計學意義。研究結(jié)論:1.TTF1-NP通過抑制STAT3的磷酸化活化,抑制STAT3與其靶DNA的結(jié)合。2.TTF1-NP通過抑制STAT3調(diào)控人肝癌HepG2細胞增殖、血管生成、侵襲和轉(zhuǎn)移、細胞凋亡等生物學行為。3.TTF1-NP抑制STAT3調(diào)控人肝癌HepG2細胞增殖、血管生成、侵襲和轉(zhuǎn)移、細胞凋亡等生物學行為是通過影響VEGF、KDR、bFGF、MMP2、MMP9、survivin和cleaved caspase3蛋白表達實現(xiàn)的。
[Abstract]:Background: Primary liver cancer is one of the most common malignant tumors, which is a serious threat to human health. China is a major developing country of primary liver cancer, and the number of deaths per year is about 4.6 million, accounting for 1/ 2 of the total number of deaths in the global liver cancer. Therefore, it is of great practical significance to find the target of the treatment of the liver cancer and to target the medicine. The signal transduction and activator of Transcription3 (STAT3) are closely related to the occurrence and development of various tumors, and the overexpression and continuous activation of STAT3 are related to the formation of 70% of human solid tumors. TTF1 is an anti-tumor monomer component separated from the pearl plum of Changbai Mountain, and the biological degradable material stearic acid is used as a carrier to prepare the pearl-plum flavone nano-particle (TTF1-NP) with a particle size of about 190nm. and can inhibit the proliferation of a plurality of tumor cells, and has the value of further research. Objective: To study the effect of TTF1-NP on the expression of STAT3 in human liver cancer cells, and to investigate the effects of TTF1-NP on angiogenesis, invasion and metastasis and apoptosis of human liver cancer cells. The molecular mechanism of the effect of TTF1-NP on the biological behavior of liver cancer cells was also tested, and the theoretical basis for the research and development of TTF1-NP was provided. Methods: The effects of TTF1-NP on the proliferation and expression of STAT3 and p-STAT3 protein were measured in vivo and in vitro. The effect of TTF1-NP on the binding activity of STAT3 and its target DNA was detected by EMSA technique, and HUVEC cells were used to co-culture with human liver cancer HepG2 cells. The effect of TTF1-NP on the formation, invasion, metastasis and apoptosis of human hepatocellular carcinoma HepG2 cells was detected by HE and Hochest staining, Transwell, cell scratch test and flow-type double-dye apoptosis test. The expression of relevant functional protein was detected by western blot. The effect of TTF1-NP on the biological behavior of human hepatocellular carcinoma HepG2 cells and the molecular mechanism were constructed. Study Results: 1. The effects of TTF1-NP on the proliferation of HepG2, Hep3B, PLC/ PRF/ 5 and SMMC-7721 cells of human liver cancer HepG2, Hep3B, PLC/ PRF/ 5 and SMMC-7721 were detected by MTT assay. The inhibitory effect of TTF1-NP on the proliferation of HepG2 cells was most significant, and the effect of TTF1-NP on the proliferation of Hep3B, PLC/ PRF/ 5, SMMC-7721 and HepG2 cells was inhibited by TTF1-NP. TTF1-NP (5. mu.mol/ kg, 10. mu.mol/ kg, 20. mu.mol/ kg) of TTF1-NP (5. mu.mol/ kg, 10. mu.mol/ kg, 20. mu.mol/ kg) had an inhibitory effect on the growth of the volume of the nude mice. The growth inhibition rate (%) was 48. 9, 4. 7, 52. 9, 3. 5, 58. 8 and 5. 4. 2, respectively. Immunochemical staining showed that TTF1-NP had an inhibitory effect on the expression of STAT3 and p-STAT3 protein in nude mice with HepG2 cells and HepG2 cells. The results of western blot showed that TTF1-NP inhibited the expression of STAT3 and p-STAT3 proteins and had a dose-dependent manner. EMSA showed that TTF1-NP could inhibit the binding of STAT3 and target DNA, and dose-dependent. TTF1-NP could inhibit the formation of small tube of HUVEC cells, inhibit the expression of VEGF, KDR and bFGF in HepG2 cells, reduce the number of invasion of HepG2 cells, shorten the migration distance, and inhibit the expression of MMP2 and MMP9 proteins. The apoptosis of human hepatoma HepG2 cells was induced, the activation of survivin was inhibited, and the expression of clear caspase3 was promoted. It was found that the inhibitory effect of TTF1-NP on the biological behavior of HepG2 cells with low expression of STAT3 and related functional protein expression was not obvious. The effect of TTF1-NP on the biological behavior and the expression of related functional proteins of human liver cancer HepG2 cells overexpressing STAT3 was significant, and it was of statistical significance. Conclusion: 1. TTF1-NP inhibits the phosphorylation and activation of STAT3 and inhibits the binding of STAT3 to its target DNA. The biological behavior of invasion and metastasis and cell apoptosis is realized by the expression of VEGF, KDR, bFGF, MMP2, MMP9, survivin and clear caspase3.
【學位授予單位】：延邊大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.7

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