去甲斑蝥素对人绒癌和皮肤癌的作用及其机制研究

发布时间：2018-04-15 23:34

本文选题：去甲斑蝥素 + 绒癌　；参考：《重庆医科大学》2017年博士论文

【摘要】：去甲斑蝥素(Norcantharidin,NCTD)是通过去除斑蝥素分子中2,3-甲基所得的化合物,是我国首先人工合成并拥有独立知识产权的新型治癌药物。与斑蝥素相比,它具有更低的毒性,且具有独特的升白细胞作用。已有的研究表明,去甲斑蝥素可以保护正常肝细胞免受因脂多糖(LPS)导致的损伤;减弱肾小管间质纤维化,保护肾脏;还对人身体免疫系统具有良好的调节作用。除了以上的特点之外,去甲斑蝥素还具有较强的抗癌活性,它对多种肿瘤具有良好的治疗效果。这些肿瘤包括卵巢癌、前列腺癌、宫颈癌、肝癌等。利用抗癌药提升肿瘤细胞内的活性氧水平从而诱导细胞凋亡被认为是一条潜在的抗癌途径。活性氧的升高可以诱导细胞凋亡与自噬,使细胞死亡。关于NCTD对人绒癌及皮肤癌方面的研究仍然知之甚少。我们在前期实验研究中发现,NCTD可诱导人绒癌JEG-3细胞与人皮肤鳞状细胞癌A431细胞活性氧明显升高,至于它能否诱导这两种癌细胞凋亡及自噬等情况,值得进一步研究。第一部分:NCTD对人绒癌JEG-3细胞的增殖、凋亡和自噬的影响,以及与5-FU化疗增敏作用目的:探讨NCTD对人绒癌JEG-3细胞的增殖能力、凋亡和自噬的影响及分子机制。此外,NCTD对绒癌主要化疗药5-FU是否具有增敏作用。方法:mtt实验检测细胞增殖的变化,流式细胞术检测细胞增殖周期,annexinv-fitc荧光双染法检测细胞凋亡,dapi染色法和hochest33258染色法检测凋亡细胞形态,电镜观察细胞凋亡与自噬。我们还使用相关试剂盒检测凋亡、活性氧水平、膜电位、自噬阳性细胞、mda及caspase-3,-9酶活性变化,最后用定量pcr方法检测抗氧化基因的mrna表达,western-blot法检测p21、cyclinb1、fas、fasl、bcl-2、bax、mtor、p62、beclin1、lc3-Ⅱ蛋白的表达。结果:1.nctd对人绒癌jeg-3细胞生长增殖有明显的抑制作用;nctd能引起jeg-3细胞g2期阻滞,其分子机制可能是通过增加p21蛋白表达和下调cyclinb1蛋白而引起的。2.nctd能提升jeg-3细胞中ros水平,同时细胞内总的sod酶活性降低,mda含量升高。定量pcr结果提示,抗氧化酶sod1、prdx1、prdx2、prdx3以及细胞因子il-6mrna表达随着nctd浓度的增加而降低,提示了nctd作用jeg-3细胞,诱导ros水平有可能是通过下调抗氧化物相关基因表达进行的。3通过hochest33258和dapi染色、电镜观察细胞形态及流式细胞术检测凋亡率及膜电位,结果提示了nctd明显诱导jeg-3细胞凋亡,同时细胞中bcl-2蛋白表达降低,而bax、fas、fasl蛋白表达增加4.为了探索活性氧水平升高是nctd诱导jeg-3细胞凋亡的主要原因,我们采用活性氧清除剂n-乙酰半胱氨酸(nac)与nctd共同作用进行反向求证。结果发现nac可逆转nctd诱导jeg-3细胞凋亡,其机制与nac能降低caspase-3、9酶活性有关。5.与单独使用nctd或5-fu比较,nctd与5fu联合使用,对jeg-3细胞具有更明显的抑制效果,其机制与增加caspase-3、9酶活性相关。6.nctd对jeg-3细胞自噬影响的实验中,我们发现,nctd能诱导jeg-3细胞自噬,其机制与mtor、akt、p62、lc3-Ⅱ及beclin1蛋白表达变化相关。为了进一步论证自噬与活性氧的关系,我们用nac与nctd共同处理细胞,发现nac可以明显减轻nctd诱导的自噬,同时使自噬相关蛋白beclin1、p62表达改变,结果提示了ros升高是nctd诱导jeg-3细胞自噬的主要原因。结论:nctd主要通过提高jeg-3细胞活性氧水平,诱导细胞凋亡、自噬,其机制可能与nctd能改变jeg-3细胞内的p21、cyclinb1、fas、fasl、bcl-2、bax、mtor、p62、beclin1、lc3-Ⅱ蛋白表达有关。另外,nctd可以抑制jeg-3细胞增殖,使细胞周期阻滞在g2期,可以对5-fu治疗绒癌具有增敏作用。本实验为nctd治疗人绒癌提供了重要的实验依据,进一步拓展了nctd作为抗癌药的应用领域。第二部分:nctd对人皮肤鳞状细胞癌a431细胞的增殖、凋亡及转移的影响,目的:探讨nctd对人皮肤鳞状细胞癌a431细胞的增殖能力、凋亡和侵袭转移的影响及分子机制。方法:mtt实验检测细胞增殖的变化,流式细胞术观察细胞周期变化,annexinv-fitc荧光双染法检测细胞凋亡,以及使用dapi染色法检测凋亡细胞形态。我们还使用相关试剂盒检测细胞活性氧水平、膜电位、sod、mda及caspase-3,-9酶活性变化。另外,我们使用小室实验检测细胞侵袭转移能力,用划痕修复法检测细胞的迁移能力,western-blot法检测p21、cyclinb1、fas、fasl、bcl-2、bax、vegf、mmp-2、mmp-9等蛋白表达。结果:1.nctd对a431细胞生长增殖有明显的抑制作用,引起a431细胞g2期阻滞。其分子机制可能是通过增加p21蛋白表达和下调cyclinb1蛋白而引起的。2.nctd能升高ros水平,诱导a431细胞凋亡率增加,同时细胞中bcl-2蛋白表达降低,而bax、fas、fasl蛋白表达增加。3.为了探索是否由于活性氧水平升高诱导a431细胞凋亡,我们采用活性氧清除剂n-乙酰半胱氨酸(nac)与nctd共同作用。发现nac可减少nctd诱导的凋亡,其机制与nac能降低caspase-3,9酶活性有关。提示nctd通过增加ros水平,诱导a431细胞凋亡。5.transwel小室、划痕修复、黏附及western-blot等实验发现,nctd对a431细胞的侵袭迁移和黏附能力均有明显的抑制,其机制可能是与nctd降低细胞内的vegf及mmp-2,-9蛋白表达水平有关。结论:nctd可明显抑制a431细胞的增殖和转移能力,并且可以通过活性氧途径诱导细胞凋亡。这些研究结果为nctd治疗皮肤癌提供了重要的实验依据,进一步扩大了nctd作为抗癌药的应用领域。
[Abstract]:Norcantharidin (Norcantharidin, NCTD) is the compound removal of cantharidin molecule 2,3- methyl income, is a new type of drugs in our country first synthetic and independent intellectual property rights. Compared with cantharidin, which has lower toxicity, and has leukogenic unique role. Previous studies show that to norcantharidin can protect cells from normal liver by lipopolysaccharide (LPS) induced injury; reduced renal interstitial fibrosis, protect the kidney; also has the good effect on human body immune system. In addition to the above features, norcantharidin has strong anticancer activity, it has a good curative effect on a variety of tumors. These tumors including ovarian cancer, prostate cancer, cervical cancer, liver cancer and so on. The use of anticancer drugs enhance the ROS level in tumor cells to induce apoptosis is considered to be a potential anti-cancer Way. Increased ROS can induce cell apoptosis and autophagy, cause cell death. Research on human choriocarcinoma and skin cancer NCTD remains poorly understood. We found in the initial experiments, NCTD induced human choriocarcinoma JEG-3 cells and the activity of human skin squamous cell carcinoma A431 cell oxygen increased significantly, as it can in these two kinds of cancer cell apoptosis and autophagy, is worthy of further study. The first part: NCTD on the proliferation of human choriocarcinoma JEG-3 cells, apoptosis and autophagy, and the sensitivity of objective and 5-FU increased chemotherapy: To investigate the proliferation ability of NCTD on human choriocarcinoma JEG-3 cells, apoptosis and autophagy and its molecular mechanism in addition. Whether or not, NCTD has sensitizing effect on choriocarcinoma main chemotherapy drug 5-FU. Methods: the changes of cell proliferation by MTT assay, the cell cycle was detected by flow cytometry, annexinv-fitc double fluorescent staining method to detect cell Apoptosis, DAPI staining and Hochest33258 staining method to detect cell apoptosis, apoptosis and autophagy were observed. We also use the related kit for detection of apoptosis, ROS level, membrane potential, autophagy positive cells, MDA and Caspase-3, the changes of -9 activity, expression finally quantitative PCR method for detection of antioxidant genes mRNA, detection p21, Western-blot cyclinB1, Fas, FasL, Bcl-2, Bax, mTOR, p62, Beclin1, expression of lc3- protein. Results: the proliferation of 1.nctd significantly inhibited the growth of human choriocarcinoma JEG-3 cells; NCTD can cause JEG-3 cell arrest in G2 phase and its molecular mechanism may be through increasing p21 protein expression and down-regulation of cyclinB1 protein caused by.2.nctd can improve the level of ROS in JEG-3 cells, and SOD enzyme activity in total cell decreased, MDA content increased. The quantitative PCR results suggest that antioxidant enzymes SOD1, Prdx1, prdx2, prdx3 and il-6mrn cytokines The expression of a decreased with the increase of NCTD concentration, suggesting that JEG-3 cells induced by NCTD, ROS levels may be.3 by Hochest33258 and DAPI staining by down regulating the expression of antioxidant related gene expression, electron microscope and detect the apoptosis rate and membrane potential of flow cytometry, the results revealed that NCTD significantly induced apoptosis in JEG-3 cells at the same time, the expression of Bcl-2 protein in the cells decreased, while Bax, Fas, FasL protein expression increased 4. to explore increased levels of reactive oxygen species is a major cause of NCTD induced apoptosis in JEG-3 cells, we use the active oxygen scavenger of n- acetylcysteine (NAC) reverse proof and NCTD together. The results showed that NAC could reverse NCTD induced apoptosis in JEG-3 cells the mechanism, and NAC can reduce the activity of caspase-3,9.5. and the use of NCTD or 5-FU alone, the combined use of NCTD and 5FU, has more obvious inhibition on JEG-3 cells The effect, mechanism and increase caspase-3,9 activity of.6.nctd in autophagy of JEG-3 cells in the experiment, we found that NCTD could induce autophagy in JEG-3 cells, and the mechanism of mTOR, Akt, p62, lc3- II and Beclin1 protein expression changes. In order to further demonstrate the relationship between autophagy and active oxygen, we use NAC and NCTD CO treatment of cells, found that NAC can alleviate NCTD induced autophagy, and autophagy related protein Beclin1, expression of p62, the results suggest that the increase in ROS is the main reason of NCTD induced autophagy in JEG-3 cells. Conclusion: NCTD mainly through JEG-3 cells increased levels of reactive oxygen species, apoptosis, autophagy, and its mechanism may be NCTD can change the JEG-3 cells in p21, cyclinB1, Fas, FasL, Bcl-2, Bax, mTOR, p62, Beclin1, lc3- II protein expression. In addition, NCTD can inhibit the proliferation of JEG-3 cells, induce cell cycle arrest in G2 phase, can be 5 -fu treatment of choriocarcinoma have a sensitizing effect. It provides an important experimental basis for the experimental treatment of human choriocarcinoma NCTD, to further expand the application field of NCTD as anticancer drugs. The second part: NCTD on the proliferation of human skin squamous cell carcinoma A431 cells, influence, apoptosis and metastasis Objective: To investigate the proliferation ability of NCTD in squamous cell carcinoma human skin A431 cells, apoptosis and metastasis and molecular mechanisms. Methods: the changes of cell proliferation by MTT assay, flow cytometry cell cycle, annexinv-fitc fluorescence method to detect cell apoptosis, and to use DAPI staining method to detect the morphology of apoptotic cells. We also use the kit ROS the level of detection of membrane potential, SOD, MDA and Caspase-3, the changes of -9 activity. In addition, we use assay cell invasion and metastasis, cell migration by scratch repair method, The detection of p21, Western-blot cyclinB1, Fas, FasL, Bcl-2, Bax, VEGF, MMP-2, expression of MMP-9 protein. Results: the proliferation of 1.nctd significantly inhibited the growth of A431 cells, A431 induced G2 cell cycle arrest. Its molecular mechanism may be through increasing the expression of p21 protein and cyclinB1 protein caused by.2.nctd modulation to increase the level of ROS, induced A431 cell apoptosis rate increased, while the cells in the lower expression of Bcl-2, Bax, Fas, FasL increased the expression of.3. in order to explore whether due to elevated levels of reactive oxygen species induced apoptosis in A431 cells, we use the active oxygen scavenger of n- acetylcysteine (NAC) together with NCTD. It is found that NAC can decrease apoptosis induced by NCTD, and the mechanism of NAC on activity of caspase-3,9. NCTD by increasing the level of ROS, A431 cell apoptosis induced by.5.transwel cell adhesion, scratch repair, discovery and Western-blot experiment, NCTD has inhibited the invasion of A431 cells migration and adhesion obviously, its mechanism may be related with the decrease of VEGF and NCTD in MMP-2 cells, the expression level of -9 protein. Conclusion: NCTD can inhibit the proliferation and metastasis of A431 cells, and can induce cell apoptosis through ROS pathway. These results provide an important experimental basis for NCTD treatment of skin cancer, to further expand the application field of NCTD as anticancer drugs.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96

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