巴西苏木素诱导膀胱癌T24细胞程序性坏死的初步研究

发布时间：2018-04-13 08:20

本文选题：巴西苏木素 + 膀胱癌　；参考：《山西大学》2017年硕士论文

【摘要】：膀胱癌是泌尿系统中的主要癌症,具有发病率高和易复发等特点。手术后进行膀胱灌注是术后防复发的重要手段,课题组合作单位研制的膀胱灌注药物“苏复宁洗液”能有效的预防膀胱癌术后复发。课题组研究发现,苏复宁洗液的主效成分巴西苏木素(Brazilin)能有效的致死膀胱癌T24细胞。但目前尚缺乏对巴西苏木素作为膀胱癌治疗药物的广谱性、安全性及其致死膀胱癌细胞的死亡机制的系统研究。在本文中我们以不同来源膀胱细胞为对象,检测巴西苏木素作为膀胱癌治疗药物的广谱性和安全性;通过检测巴西苏木素对膀胱癌T24细胞中的叠加增殖性质、乳酸脱氢酶、钙离子和线粒体膜电位等生理生化性质的影响,进一步明确巴西苏木素对膀胱癌细胞的毒性;检测不同死亡方式抑制剂对巴西苏木素致死T24细胞效应的影响,结合半胱氨酸蛋白酶(Caspase)酶活性测定及透射电镜形态学分析,证明巴西苏木素通过程序性坏死致死膀胱癌T24细胞;进一步利用巴西苏木素作用T24细胞6 h后的数字基因表达谱数据分析及RT-qPCR(Real-time qPCR),提出巴西苏木素是通过RIP1/RIP3/MLKL介导的细胞程序性坏死途径致死T24细胞。具体研究有以下四部分内容:1.利用低血清实验检测巴西苏木素对不同类型膀胱癌细胞系的影响,发现巴西苏木素能有效致死T24、5637和J82细胞,证明巴西苏木素作为膀胱癌治疗药物具备一定的广谱性。比较巴西苏木素对正常膀胱上皮细胞系SV-HUC-1及膀胱上皮细胞癌细胞系T24、5637的细胞毒性,发现巴西苏木素在浓度效应和时间效应方面对膀胱上皮细胞SV-HUC-1的致死效应小于膀胱癌T24细胞和5637细胞。说明巴西苏木素作为膀胱癌药物具有一定的药物安全性。2.检测巴西苏木素对膀胱癌T24细胞一些生理生化性质的影响,发现巴西苏木素能够抑制T24细胞增殖、阻碍其不断重叠生长;引起细胞膜通透性改变、促进乳酸脱氢酶释放;引起细胞内参与众多信号传导的钙离子浓度显著升高;导致参与细胞代谢的线粒体膜电位降低。进一步明确了巴西苏木素对膀胱癌细胞的毒性效应,为巴西苏木素的药物开发提供了实验依据。3.通过不同死亡方式抑制剂处理,半胱氨酸蛋白酶活性测定及形态学观察探究巴西苏木素致死T24细胞的死亡方式。研究发现凋亡抑制剂z-VAD-fmk不能抑制巴西苏木素的药性,结合半胱氨酸蛋白酶(Caspase)家族酶活测定,证明巴西苏木素不通过Caspase诱导的凋亡信号致死T24细胞。而自噬小体抑制剂3-MA和程序性坏死抑制剂Nec-1均能在一定程度上抑制巴西苏木素对T24细胞的致死效应。透射性电镜观察发现巴西苏木素引起T24细胞的细胞膜破裂、线粒体崩裂、细胞核弥散等程序性坏死形态学特征,这些数据表明,巴西苏木素通过诱导程序性坏死致死膀胱癌T24细胞。4.半致死浓度的巴西苏木素作用T24细胞6 h后的数字基因表达谱数据显示程序性坏死关键基因RIP1/MLKL超表达,RT-qPCR实验证明巴西苏木可诱导T24细胞中程序性坏死关键基因RIP1/RIP3/MLKL表达显著上调,初步探明巴西苏木素是通过RIP1/RIP3/MLKL介导的细胞程序性坏死途径致死T24细胞。
[Abstract]:Bladder cancer is the major cancer in the urinary system, and has a high incidence of recurrence and characteristics. After the operation of bladder is an important means of postoperative recurrence, intravesical drug development research group unit of Funing lotion Su "can effectively prevent bladder cancer after relapse. The study group found the main topic. The effective component of Funing lotion Brazil Su hematoxylin (Brazilin) can effectively kill T24 cells of bladder cancer. But there is still a lack of Brazil hematoxylin as broad-spectrum drugs for the treatment of bladder cancer, study mechanism of death safety and mortality of bladder cancer cells. In this paper we use different sources of bladder cells as the object. Brazil hematoxylin as broad-spectrum detection and safety of drugs for the treatment of bladder cancer through the detection of Brazil; hematoxylin is added to the proliferation properties of bladder cancer T24 cells in lactate dehydrogenase, and mitochondrial calcium Effect of membrane potential and other physiological and biochemical properties, to further clarify the toxicity of Brazil hematoxylin on bladder cancer cell death detection; effects of different inhibitors on Brazil hematoxylin lethal effect of T24 cells combined with cysteine protease (Caspase) analysis of enzyme activity assay and transmission electron microscope morphology, proved Brazil hematoxylin by programmed necrosis of bladder cancer death T24 cells; the further use of Brazil hematoxylin T24 cells after 6 h the number of gene expression data analysis and RT-qPCR (Real-time qPCR), the Brazil hematoxylin is programmed cell necrosis pathway mediated by RIP1/RIP3/MLKL cells. The specific lethal T24 study has the following four parts: 1. the effect of low serum test of Brazil sun the different types of bladder cancer cell lines and found that Brazil hematoxylin can effectively lethal T245637 and J82 cells, hematoxylin as evidence of Brazil Drugs for the treatment of bladder cancer with a broad-spectrum. Brazil hematoxylin of normal bladder epithelial cell line SV-HUC-1 and bladder epithelial cell carcinoma cell line T245637 cells, found in Brazil hematoxylin in concentration and time effects on bladder epithelial cells SV-HUC-1 induced death effect is smaller than the bladder cancer T24 cells and 5637 cells in Brazil. Hematoxylin as has an impact on drug safety.2. detection of certain Brazil hematoxylin on some physiological and biochemical properties of bladder cancer T24 cells of bladder cancer drugs found in Brazil, hematoxylin can inhibit the proliferation of T24 cells, which hinder the continuous growth caused by overlapping; cell membrane permeability changes, promote the release of lactate dehydrogenase; intracellular calcium concentration in many signal transduction was significantly increased; lead to mitochondrial membrane potential in cell metabolism decreased. To further clarify the Brazil hematoxylin of bladder The toxic effect of bladder cancer cells, provides the experimental basis for.3. through different forms of death inhibitors for drug development in Brazil hematoxylin, cysteine protease activity was observed on Brazil hematoxylin lethal T24 cell death and apoptosis morphology. The study found that the inhibitor z-VAD-fmk can inhibit the growth of Brazil hematoxylin resistance, combined with the cysteine protease (Caspase) determination of enzyme family live, Brazil hematoxylin not through the apoptotic signal induced by Caspase and T24 cells. Lethal autophagosome inhibitor 3-MA and programmed necrosis inhibitor Nec-1 could inhibit the Brazil sum to a certain extent on T24 cells were observed by transmission electron microscope. The lethal effect of Brazil hematoxylin caused T24 cell membrane rupture. Mitochondrial crack, nucleus dispersion necroptosis morphological characteristics, these data suggest that Brazil hematoxylin induced through Guide necroptosis lethal T24 bladder cancer cell.4. semi lethal concentration Brazil hematoxylin T24 cells after 6 h the number of gene expression data showed programmed necrosis of key genes over expression of RIP1/MLKL, RT-qPCR proved that Brazil hematoxylin can induce programmed necrosis of T24 cells in the key gene RIP1/RIP3/MLKL expression was significantly up-regulated, preliminary study of Brazil wood in the programmed cell necrosis pathway mediated by RIP1/RIP3/MLKL cell death T24.

【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14

【参考文献】