珍珠梅黄酮纳米粒抑制STAT3调控肝癌细胞生物学行为作用的研究
发布时间：2019-01-18 07:35
【摘要】：研究背景:原发性肝癌是常见恶性肿瘤之一,严重威胁人类健康,全球恶性肿瘤致死率逐年下降的前提下,原发性肝癌的死亡率连年上升。我国是原发性肝癌发病大国,且每年死亡人数约有46万左右,占全球肝癌患者总死亡人数的1/2。因此,找寻肝癌治疗分子靶点和靶向治疗药物具有重要现实意义。信号传导与转录激活因子3(Signal Transducer and Activator of Transcription3,STAT3)与多种肿瘤的发生、发展密切相关,STAT3的过度表达和持续活化与参与70%的人实体瘤形成,STAT3是现阶段抗肿瘤靶向药物研究的主要生物分子靶点之一。TTF1是从长白山珍珠梅中分离得到的抗肿瘤单体成分,以生物体可降解材料硬脂酸为载体,制备了更易吸收的粒径在190nm左右的珍珠梅黄酮纳米粒(TTF1-NP)。TTF1-NP能够通过调控细胞凋亡相关蛋白,影响肿瘤细胞周期,抑制多种肿瘤细胞的增殖,具有进一步深入研究的价值。研究目的:本文主要研究珍珠梅黄酮纳米粒(TTF1-NP)对人肝癌细胞STAT3的调控作用,探讨TTF1-NP调控STAT3对肝癌细胞血管形成、侵袭转移、细胞凋亡等生物学行为的影响,同时检测TTF1-NP调控STAT3影响肝癌细胞生物学行为作用的分子机制,为TTF1-NP的研究和开发提供理论依据。研究方法:通过体内、体外实验检测TTF1-NP对不同种人肝癌细胞的增殖作用及对STAT3和p-STAT3蛋白表达的影响;利用EMSA技术检测TTF1-NP对人肝癌HepG2细胞STAT3与其靶DNA结合活性的影响;分别利用HUVEC细胞与人肝癌HepG2细胞共培养、HE和Hochest染色、Transwell、细胞划痕实验、流式双染凋亡检测术检测TTF1-NP对人肝癌HepG2细胞的血管形成、侵袭和转移和细胞凋亡的作用,应用western blot技术检测相关功能蛋白的表达;同时分别构建低表达STAT3和过表达STAT3的人肝癌HepG2细胞株检测TTF1-NP对人肝癌HepG2细胞生物学行为的作用和分子机制。研究结果:1.本研究通过MTT实验检测发现,TTF1-NP对人肝癌HepG2、Hep3B、PLC/PRF/5和SMMC-7721细胞增殖有剂量和时间依赖性抑制作用,其中对HepG2细胞增殖抑制作用最明显;TTF1-NP作用48 h抑制人肝癌Hep3B、PLC/PRF/5、SMMC-7721和HepG2细胞增殖的IC50值,分别为: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)对裸鼠移植瘤体积的增长有抑制作用,生长抑制率(%)分别为:48.9±4.7、52.9±3.5、58.8±5.4。2.免疫化学染色检测发现,TTF1-NP对人肝癌HepG2细胞及HepG2细胞构建的裸鼠移植瘤的STAT3和p-STAT3蛋白表达有抑制作用;western blot检测结果显示,TTF1-NP抑制STAT3和p-STAT3蛋白表达,有剂量依赖性。EMSA检测发现,TTF1-NP可以抑制STAT3与靶DNA的结合,有剂量依赖性。3.TTF1-NP可以抑制HUVEC细胞小管形成,抑制HepG2细胞VEGF、KDR和bFGF蛋白的表达;减少人肝癌HepG2细胞的侵袭数量、缩短迁移距离,抑制MMP2和MMP9蛋白表达;诱导人肝癌HepG2细胞凋亡,抑制survivin蛋白活化,促进cleaved caspase3 蛋白表达。4.分别构建低表达和过表达STAT3的人肝癌HepG2细胞,检测发现TTF1-NP对低表达STAT3的人肝癌HepG2细胞生物学行为抑制作用及相关功能蛋白表达调控作用不明显;TTF1-NP对过表达STAT3的人肝癌HepG2细胞生物学行为抑制作用及相关功能蛋白表达调控作用明显,有统计学意义。研究结论:1.TTF1-NP通过抑制STAT3的磷酸化活化,抑制STAT3与其靶DNA的结合。2.TTF1-NP通过抑制STAT3调控人肝癌HepG2细胞增殖、血管生成、侵袭和转移、细胞凋亡等生物学行为。3.TTF1-NP抑制STAT3调控人肝癌HepG2细胞增殖、血管生成、侵袭和转移、细胞凋亡等生物学行为是通过影响VEGF、KDR、bFGF、MMP2、MMP9、survivin和cleaved caspase3蛋白表达实现的。
[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.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7
[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.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7
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