FasL联合siRNA沉默DcR3对人肝癌细胞凋亡及侵袭能力影响的相关研究

发布时间：2018-06-01 13:12

本文选题：肝癌 + 诱骗受体3　；参考：《首都医科大学》2017年博士论文

【摘要】：研究背景:肝细胞癌(HCC)是最常见的恶性肿瘤之一,是全球癌症死亡的第三大原因。最新资料显示,我国2015年肝癌发病人数约46.61万,死亡人数约42.20万。由于肝癌发病隐匿,诊断不及时,许多患者在发现肝癌时已经是中晚期,预后非常差,5年生存率不足5%。因此,研究肝癌的发病机制,寻找其新的诊断和治疗靶点,已经成为提高肝癌预防和治疗效果的当务之急。运用分子生物学手段抑制体内肿瘤相关基因的表达以达到肿瘤基因治疗的目的,是目前肿瘤治疗的热点,其中,能引起肿瘤细胞的凋亡的相关基因又是人们关注的焦点。Fas/Fas L系统是体内介导细胞凋亡的重要途径,在维持机体免疫自稳及肿瘤发生、发展过程中具有重要作用。Fas L有两种受体,Fas和Dc R3。Dc R3可以与Fas竞争性结合Fas L,从而抑制Fas L介导的细胞凋亡,继而引起疾病的发生发展,其中就包括了肿瘤。有关文献发现Fas L介导的细胞凋亡与Dc R3的表达水平有直接关系,Dc R3减低了Fas L介导的细胞坏死。在肝癌中,相关研究并未见报道。研究目的:利用稳定靶向敲低Dc R3表达的肝癌细胞系,通过体外、体内两方面实验,观察Fas L联合沉默Dc R3表达在肝癌细胞系中的作用,明确Fas L对Hep G2细胞的周期、凋亡、生长、克隆增殖与侵袭能力的影响,观察Fas L对Hep G2细胞MMP9、VEGF-C和VEGF-D的表达的影响。以期为肝癌肿瘤基因治疗手段提供实验基础和理论依据,也为其他恶性肿瘤的防治提供经验。研究结果:1.DcR3表达沉默后,加入FasL可导致肝癌细胞的凋亡增加,细胞周期改变,G0/G1期细胞增加,G2/M期细胞减少;2.Dc R3表达沉默后,加入Fas L后肝癌细胞的活力下降;3.下调Dc R3的表达后,加入Fas L后,肝癌细胞迁移、侵袭能力进一步下降;4.经Fas L处理后的肝癌细胞的MMP9、VEGF-C、VEGF-D的m RNA及蛋白表达水平明显下降;5.Fas L可致小鼠皮下成瘤增长速度显著减慢,瘤体相关转移侵袭因子的表达亦有下降。研究结论:1.Fas L联合Dc R3沉默可导致肝癌细胞凋亡增加。2.Fas L联合Dc R3沉默可使肝癌细胞生长速度减慢,迁移和侵袭能力下降。3.Fas L联合Dc R3沉默引起肝癌细胞中的MMP 9、VEGF-C、VEGF-D表达下降。4.Fas L联合Dc R3沉默可以作为肝癌治疗的一个新的研究方向和思路。
[Abstract]:Background: hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the third leading cause of cancer death worldwide. According to the latest data, the incidence of liver cancer in China in 2015 was about 466100 and the death toll was about 422000. Due to the occult incidence of liver cancer, diagnosis is not timely, many patients found liver cancer has been in the middle and late stage, the prognosis is very poor, 5-year survival rate is less than 5. Therefore, it is urgent to study the pathogenesis of liver cancer and find new targets for diagnosis and treatment of liver cancer. Using molecular biological methods to inhibit the expression of tumor-related genes in vivo in order to achieve the purpose of tumor gene therapy is a hot spot in tumor therapy at present. The genes associated with apoptosis of tumor cells are also the focus of attention. Fas- / FAS / FAS L system is an important way to mediate apoptosis in vivo, which maintains immune homeostasis and tumorigenesis. FAS L has two receptors, FAS and DC R3.Dc R3, which can compete with Fas to bind to Fas L, thus inhibit the apoptosis mediated by Fas L, and then cause the occurrence and development of the disease, including tumor. It was found that apoptosis mediated by Fas L was directly related to the expression level of DC R3. DcR3 reduced the cell necrosis mediated by Fas L. In liver cancer, there are no reports of related studies. Objective: to observe the role of Fas L combined with silencing DC R3 expression in hepatocellular carcinoma cell lines with stable targeting low DC R3 expression in vitro and in vivo, and to clarify the cell cycle and apoptosis of Hep G2 cells induced by Fas L. The effects of Fas L on the expression of VEGF-C and VEGF-D in Hep G2 cells were observed. In order to provide experimental and theoretical basis for gene therapy of liver cancer, and provide experience for the prevention and treatment of other malignant tumors. Results 1. After the expression of DcR3 was silenced, the apoptosis of hepatoma cells was increased after adding FasL, and the cell cycle change increased in G _ 0 / G _ 1 phase and decreased in G _ 2 / M phase. After silencing the expression of DcR3, the activity of hepatoma cells decreased after adding Fas L. After down-regulating the expression of DC R3 and adding Fas L, hepatoma cells migrated and the invasion ability decreased further. The expression level of m RNA and protein of VEGF-C VEGF-D in hepatoma cells treated with Fas L was significantly decreased. 5. FAS L significantly decreased the growth rate of subcutaneous tumor formation and the expression of tumor-associated metastasis and invasion factor in mice. Conclusion: 1. FAS L combined with DC R3 silencing can increase apoptosis of hepatoma cells. 2. FAS L combined with DC R3 silencing can slow down the growth rate of hepatoma cells. Decreased migration and invasion. 3. FAS L combined with DC R3 silencing can induce the decrease of VEGF-D expression in hepatocellular carcinoma cells. 4. FAS L combined with DC R3 silencing may be a new research direction and thought for the treatment of liver cancer.
【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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