人参皂苷Rg3抗黑色素瘤作用机制的研究

发布时间：2018-04-22 07:05

本文选题：Rg3 + 黑色素瘤　；参考：《大连医科大学》2015年博士论文

【摘要】：一、目的与意义黑色素瘤是全世界皮肤肿瘤相关性死亡的主要原因(80%)。它是一种恶性程度较高,临床预后较差的恶性肿瘤。IV期黑色素瘤患者的中位生存时间少于1年,而5年生存期少于10%。黑色素瘤的发病率在世界范围内逐年增加,它的致死率的增长速度高于其他任何形式的恶性肿瘤。传统的晚期转移性恶性黑色素瘤的治疗包括:化疗(达卡巴嗪、替莫唑胺)、生物治疗(干扰素α-2b)及药物靶向治疗(vemurafenib和ipilimumab)等。由于这些治疗的肿瘤反应率低,肿瘤耐药的产生及严重的毒副作用而限制了它们的应用。因此,对于黑色素瘤的治疗仍然需要寻找新的治疗策略,新的靶点及新的治疗药物。人参是一个古老的中草药,它具有滋补、强心、抗炎、抗肿瘤和免疫刺激的作用。20(R)-人参皂苷Rg3是从人参中提取的单体成分,具有广泛的药理学活性和治疗作用。报道显示Rg3在结肠癌、胃癌、乳腺癌及肝癌中具有抑制肿瘤生长的作用;Rg3在前列腺癌和结肠癌中具有增加多西他赛和顺铂化疗敏感性的作用;Rg3在肝癌细胞中具有抗肿瘤血管形成和诱导凋亡的作用。目前,参一胶囊(其主要成分为Rg3)已经在中国被批准为治疗临床晚期非小细胞肺癌的辅助药物。蛋白质转录后的修饰与肿瘤发生发展密切相关,其中乙酰化和糖基化也成为抗恶性黑色素瘤的研究热点。然而Rg3抗黑色素瘤的作用机制是否与调控蛋白质的乙酰化和糖基化目前还不清楚。乙酰化和去乙酰化是调控基因表达的表观遗传学过程。异常的去乙酰化酶(HDACs)的表达与肿瘤的生长、侵袭和转移密切相关。例如,HDAC6在人类乳腺癌和前列腺癌中过表达,HDAC2在宫颈癌和胃癌中过表达,HDAC8在儿童神经母细胞瘤中过表达。HDAC3的过表达也在许多恶性肿瘤中被报导,如肺癌、结肠癌、胃癌和前列腺癌。有研究认为HDAC3的表达与p53的乙酰化与肿瘤的生长有关。然而Rg3抗恶性黑色素瘤生长的机制是否与调节HDAC3和p53乙酰化的表达需要进一步探索。蛋白的糖基化在肿瘤病理生理过程中发挥重要的作用,包括肿瘤增殖、侵袭、血源性转移和血管形成。岩藻糖基化是蛋白糖基化的一种重要形式。岩藻糖基转移酶(FUTs)是催化合成岩藻糖的关键酶。其中FUT4是FUTs家族的成员之一,是催化合成α1,3-岩藻糖Le Y的关键酶。文献报道显示FUT4在很多肿瘤中过表达,例如在结肠癌、胃癌和肺癌中。过表达FUT4能够促进细胞的增殖、转移和抗凋亡。然而Rg3在恶性黑色素瘤生长诱导凋亡中的作用与潜在的调节FUT4表达与EGFR/MAPK和NF-κB信号通路目前仍不清楚。本研究通过Rg3对HDAC3和FUT4表达调控的研究,分析Rg3在黑色素瘤中抑制肿瘤生长诱导凋亡及其调控的相关信号通路,进一步探讨Rg3介导的抗黑色素瘤作用的分子机制,以期为临床治疗黑色素瘤提供科学依据。二、实验方法1、收集2003至2010年在大连医科大学附属第一医院病理科存档的石蜡组织标本且临床资料完整的恶性黑色素瘤患者32例。采用免疫组化法测定所有病理标本中HDAC3的表达情况,并对所有病例进行随访。分析HDAC3与患者临床病理特征和预后的相关性。应用SPSS16.0统计软件进行数据分析,以P0.05为显著性标准。表达的组间差异比较采用χ2检验;2、利用CCK-8、MTT法及细胞克隆形成实验分析黑色素瘤细胞的增殖能力;3、利用脂质体瞬时转染技术将HDAC3 si RNA、FUT4 si RNA转染至人黑色素瘤细胞中,下调HDAC3和FUT4的表达;4、采用Real-time PCR的方法检测HDAC3、PCNA、FUT4和p65基因的表达;5、采用Western blot的方法检测HDAC3、FUT4、Le Y、Caspase-3、-8、-9、Bcl-2、Bax、Survivn、NF-κB和EGFR/MAPK信号通路等相关蛋白的表达;6、利用免疫}D化和免疫荧光检测HDAC3、FUT4、PCNA和p65的表达;7、采用流式细胞仪分析细胞周期及细胞凋亡;8、采用凝胶电泳迁移率(EMSA)方法检测NF-κB与DNA的结合情况;9、使用荧光素酶报告基因检测p53和NF-κB的活性情况;10、使用染色质免疫共沉淀(Ch IP)方法检测NF-κB与FUT4启动子区的结合情况。三、结果(一)人参皂苷Rg3抑制黑色素瘤的增殖与下调HDAC3,增加p53乙酰化的相关性研究1、HDAC3过表达与淋巴结转移及肿瘤分期有关;2、Rg3抑制黑色素瘤的生长;3、Rg3降低HDAC3的表达;4、下调HDAC3表达抑制细胞的增殖;5、Rg3和下调HDAC3的表达增加p53的乙酰化和转录活性;6、Rg3下调HDAC3改变细胞周期相关蛋白的表达;7、Rg3在体内抑制裸鼠肿瘤的生长。(二)人参皂苷Rg3抑制黑色素瘤增殖与下调FUT4/Le Y介导的EGFR/MAPK通路的失活的相关性研究1、Rg3抑制A375黑色素瘤细胞的生长;2、Rg3降低FUT4和Le Y的表达;3、下调FUT4的表达抑制A375细胞的增殖和减少Le Y的表达;4、下调FUT4的表达减少EGFR/MAPK通路的磷酸化;5、Rg3在体内抑制裸鼠肿瘤的生长。(三)人参皂苷Rg3诱导黑色素瘤凋亡与抑制FUT4表达通过靶向调节NF-κB/p65信号通路机制的研究1、Rg3抑制人黑色素瘤细胞的增殖;2、Rg3诱导黑色素瘤细胞的凋亡;3、Rg3抑制NF-κB信号通路的活性;4、Rg3下调FUT4的表达抑制NF-κB连接到FUT4启动子区;5、Rg3诱导凋亡通过抑制NF-κB信号通路及FUT4的下调;6、Rg3抑制黑色素瘤裸鼠肿瘤生长诱导凋亡。四、结论(一)人参皂苷Rg3抑制黑色素瘤的增殖与下调HDAC3,增加p53乙酰化的相关性研究1、Rg3具有抑制黑色素瘤裸鼠动物模型和黑色素瘤细胞生长的作用;2、Rg3下调HDAC3表达具有剂量和时间的依赖性;3、使用si RNA或HDAC3潜在的抑制剂(MS-275),下调HDAC3表达抑制黑色素瘤细胞的增殖;4、Rg3抑制黑色素瘤增殖通过抑制HDAC3的表达增加p53的乙酰化和转录活性。(二)人参皂苷Rg3抑制黑色素瘤增殖与下调FUT4/Le Y介导的EGFR/MAPK通路失活的相关性研究1、Rg3抑制黑色素瘤的生长与下调FUT4/Le Y有关;2、岩藻糖基转移酶FUT4与调控的糖抗原Le Y表达有关;3、Rg3抑制黑色素瘤增殖通过抑制FUT4/Le Y表达及其下游的EGFR/MAPK信号通路。(三)人参皂苷Rg3诱导黑色素瘤凋亡与抑制FUT4表达通过靶向调节NF-κB/p65信号通路机制的研究1、Rg3具有抑制黑色素瘤裸鼠动物模型和黑色素瘤细胞生长,诱导凋亡的作用;2、Rg3诱导细胞凋亡通过下调NF-κB信号通路蛋白的表达及NF-κB DNA连接/转录活性;3、NF-κB直接调控FUT4的启动子区,影响FUT4的表达;4、Rg3诱导凋亡通过抑制NF-κB信号通路介导的下调FUT4的表达。
[Abstract]:Objective and significance melanoma is the main cause of death associated with skin tumor in the world (80%). It is a high malignant and clinically poor malignant tumor of.IV melanoma with a median survival time of less than 1 years, while the incidence of less than 10%. melanoma in the 5 year survival period increases year by year in the world. The rate of death is higher than any other form of malignant tumor. The treatment of traditional advanced metastatic malignant melanoma includes chemotherapy (Dhaka basazine, temozolomide), biological therapy (interferon alpha -2b) and drug targeting therapy (vemurafenib and ipilimumab). Serious toxic and side effects restrict their application. Therefore, the treatment of melanoma still needs to find new therapeutic strategies, new targets and new therapeutic drugs. Ginseng is an ancient Chinese herbal medicine, which has the effects of nourishing, heart, anti-inflammatory, anti-tumor and immune stimulation,.20 (R) - ginsenoside Rg3 is extracted from ginseng It has been reported that Rg3 plays a role in inhibiting tumor growth in colon, gastric, breast and liver cancers; Rg3 has the effect of increasing the chemosensitivity of docetaxel and cisplatin in prostate and colon cancer; Rg3 has antitumor angiogenesis and induction in liver cancer cells. The effect of apoptosis. Currently, Shen Yi capsule (its main component is Rg3) has been approved as an auxiliary drug in the treatment of advanced non small cell lung cancer in China. The posttranscriptional modification of protein is closely related to the development of tumor. Acetylation and glycosylation also become a hot spot in the study of malignant melanoma. However, Rg3 is anti melanoma. It is not clear whether the mechanism of action and the regulation of acetylation and glycosylation of proteins is still unclear. Acetylation and deacetylation are epigenetic processes regulating gene expression. The expression of abnormal deacetylase (HDACs) is closely related to tumor growth, invasion and metastasis. For example, HDAC6 is overexpressed in human breast and prostate cancer, HDAC2 Overexpression of HDAC8 in children with cervical cancer and gastric cancer, overexpression of.HDAC3 in children with neuroblastoma is also reported in many malignant tumors, such as lung cancer, colon cancer, gastric cancer, and prostate cancer. Studies have suggested that the expression of HDAC3 and the acetylation of p53 are related to the growth of the tumor. However, the mechanism of Rg3 for the growth of malignant melanoma is whether or not. The expression of HDAC3 and p53 acetylation needs further exploration. Protein glycosylation plays an important role in the pathophysiological process of tumor, including tumor proliferation, invasion, hematogenous metastasis and angiogenesis. Fucoylation is an important form of protein glycosylation. Fucose transferase (FUTs) is the key to catalyze the synthesis of fucose. FUT4 is one of the members of the FUTs family and is the key enzyme to catalyze the synthesis of alpha 1,3- fucose Le Y. The literature has reported that FUT4 is overexpressed in many tumors, such as colon, gastric and lung cancer. Over expressed FUT4 can promote cell proliferation, metastasis and anti apoptosis. However, Rg3 is in the growth induced apoptosis of malignant melanoma. The expression of EGFR/MAPK and NF- kappa B signaling pathway with potential regulatory FUT4 is still unclear. Through the study of the regulation of the expression of HDAC3 and FUT4 in Rg3, this study analyzed the signaling pathway of Rg3 in the inhibition of tumor growth induced apoptosis and its regulation in melanoma, and further explored the molecular mechanism of Rg3 mediated anti melanoma. In order to provide a scientific basis for clinical treatment of melanoma. Two, method 1, 32 cases of malignant melanoma were collected from 2003 to 2010 in the pathology department of the first hospital of the First Affiliated Hospital of Dalian Medical University and 32 cases with complete malignant melanoma. The expression of HDAC3 in all the diseased specimens was measured by immunohistochemistry and all the diseases were detected. Follow up. Analyze the correlation between HDAC3 and the patient's clinicopathological features and prognosis. Using the SPSS16.0 statistical software to analyze the data with P0.05 as the significant standard. The difference of the expression between the groups was compared with the x 2 test; 2, the proliferation ability of melanoma cells was analyzed by CCK-8, MTT and cell clone formation; 3, using liposome transient. HDAC3 Si RNA and FUT4 Si RNA were transfected into human melanoma cells to reduce the expression of HDAC3 and FUT4. 4, Real-time PCR method was used to detect HDAC3, PCNA, and expressions. Expression of the protein; 6, the expression of HDAC3, FUT4, PCNA and p65 by immunization}D and immunofluorescence; 7, the cell cycle and apoptosis were analyzed by flow cytometry; 8, the binding of NF- kappa B and DNA was detected by gel electrophoresis mobility (EMSA) method; 9, the activity of p53 and NF- kappa B was detected by luciferase reporter gene; 10, use Chromatin immunoprecipitation (Ch IP) method was used to detect the combination of NF- kappa B and FUT4 promoter region. Three. Results (1) ginsenoside Rg3 inhibits melanoma proliferation and down-regulation of HDAC3, increases the correlation of p53 acetylation, 1, HDAC3 overexpression is related to lymph node metastasis and tumor staging; 2, Rg3 inhibits the growth of melanoma; 3, Rg3 reduces HDAC3 4, down regulated the proliferation of HDAC3 expression inhibition cells; 5, Rg3 and down regulated HDAC3 increased the acetylation and transcriptional activity of p53; 6, Rg3 reduced HDAC3 to change the expression of cell cycle related proteins; 7, Rg3 inhibits the growth of nude mice in vivo. (two) ginsenoside Rg3 inhibits melanoma proliferation and down-regulation of FUT4/Le Y mediated EGFR/MAPK passage Correlation study of inactivation of the road 1, Rg3 inhibits the growth of A375 melanoma cells; 2, Rg3 reduces the expression of FUT4 and Le Y; 3, down regulation of FUT4 inhibits the proliferation of A375 cells and reduces the expression of Le Y; 4, down-regulation of FUT4 expression reduces the phosphorylation of EGFR/MAPK pathway; (three) induction of ginsenoside induction in vivo Melanoma apoptosis and inhibition of FUT4 expression through targeted regulation of NF- kappa B/p65 signaling pathway mechanism 1, Rg3 inhibits the proliferation of human melanoma cells; 2, Rg3 induces apoptosis of melanoma cells; 3, Rg3 inhibits the activity of NF- kappa B signaling pathway; 4, Rg3 downregulation FUT4 expression inhibits NF- kappa B to connect to the promoter region; 5, induces apoptosis passing through Inhibition of NF- kappa B signaling pathway and down regulation of FUT4; 6, Rg3 inhibits the growth of melanoma tumor growth induced apoptosis. Four. Conclusion (1) ginsenoside Rg3 inhibits the proliferation of melanoma and down regulated HDAC3, and increases the correlation of p53 acetylation 1. Rg3 has the effect of inhibiting the growth of melanoma model and melanoma cells in melanoma, and 2, Rg3 downregulation H DAC3 expression has a dose and time dependence; 3, the use of Si RNA or HDAC3 potential inhibitor (MS-275), down regulation of HDAC3 expression to inhibit the proliferation of melanoma cells; 4, Rg3 inhibits the proliferation of melanoma by inhibiting the expression of HDAC3 and increases p53 acetylation and transcriptional activity. (two) ginsenoside Rg3 inhibits melanoma proliferation and downregulation FUT4/Le Y Correlation study mediated inactivation of EGFR/MAPK pathway 1, Rg3 inhibition of melanoma growth is related to down-regulation of FUT4/Le Y; 2, fucose transferase FUT4 is associated with the regulated sugar antigen Le Y expression; 3, Rg3 inhibits the proliferation of melanoma by inhibiting FUT4/Le Y expression and downstream EGFR /MAPK signal pathway. (three) ginsenoside induces melanin Apoptosis and inhibition of FUT4 expression through the targeting mechanism of targeting NF- kappa B/p65 signaling pathway 1, Rg3 can inhibit the growth of melanoma nude mice and melanoma cell growth and induce apoptosis; 2, Rg3 induced apoptosis by down-regulation of NF- kappa B signaling pathway protein and NF- kappa B DNA connection / transcriptional activity; 3, NF- kappa B The promoter region of FUT4 controls the expression of FUT4. 4, Rg3 induces apoptosis to inhibit FUT4 expression through inhibition of NF- kappa B signaling pathway.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.5

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