氯化镧诱导MDCC-MSB1细胞凋亡机制的研究

发布时间：2018-03-04 16:27

本文选题：氯化镧　切入点：MDCC-MSB1　出处：《东北农业大学》2008年硕士论文　论文类型：学位论文

【摘要】： 许多研究表明,一定浓度稀土具有显著的抑癌作用。随着稀土在医药、生物化学等领域研究的不断深入,其在生命科学中的地位日益重要。鸡MD的许多特性与哺乳动物肿瘤的发生有相似之处,被作为研究肿瘤性疾病的良好模型。本课题以体外培养MDCC-MSB1细胞系为研究对象,在培养体系中加入不同浓度LaCl_3,应用流式细胞术、原位末端标记、DNA电泳分析、荧光染色、电子显微镜等方法,从细胞超微结构、细胞内游离钙离子浓度([Ca~(2+)]i)、染色体DNA损伤、细胞周期、线粒体膜电位(Δψm)、Caspase-3、Caspase-9活性及CaM、Caspase3 mRNA表达水平等方面,研究LaCl_3诱导MDCC-MSB1凋亡的形态学改变、生物化学改变及可能的调控机制,为临床应用LaCl_3治疗MD及其他肿瘤性疾病提供理论依据。研究结果表明: 1应用MTT比色法、二硝基苯肼比色法检测MDCC-MSB1细胞的活性和培养上清液中LDH活性,结果表明各浓度组LaCl_3均可以抑制MDCC-MSB1细胞增殖,应用PI染色流式细胞术检测法检检测细胞周期变化,RT-PCR方法检测CaMm RNA表达水平,结果表明LaCl_3可诱导细胞发生G0/G1期阻滞,从而抑制MDCC-MSB1增殖,且抑制率具有剂量依赖性。 2倒置显微镜下观察MDCC-MSB1细胞生长情况,HE染色、AO/EB双荧光染色、透射电镜观察不同浓度下LaCl_3诱导MDCC-MSB1细胞凋亡的形态学变化,流式细胞术检测磷脂酰丝氨酸(PS),结果表明LaCl_3可诱导MDCC-MSB1细胞凋亡,且凋亡率具有剂量依赖性。 3应用琼脂糖凝胶电泳、碱性SCGE、TUNEL法检测LaCl_3对MDCC-MSB1细胞DNA的损伤作用,结果证实LaCl_3通过引起MDCC-MSB1细胞[Ca~(2+)]i增加,激活内源性核酸内切酶导致DNA在核小体间断裂途径诱导细胞凋亡。 4通过对细胞内抗氧化功能的检测,表明LaCl_3能够诱导MDCC-MSB1细胞NOS活性与NO含量升高,ROS含量增高,证实LaCl_3能够破坏机体抗氧化防御系统及清除自由基的功能,使细胞发生氧化损伤,这可能是LaCl_3诱导MDCC-MSB1细胞发生凋亡的机制之一。 5应用Fura-2/AM荧光法检测[Ca~(2+)]i,流式细胞术检测Δψm,比色法检测Caspase-3、Caspase-9活性,RT-PCR方法检测caspase-3 mRNA表达水平。结果表明LaCl_3可导致MDCC-MSB1细胞Δψm下降,Caspase-3、Caspase-9活性上升,Caspase-3 mRNA表达增加,证实LaCl_3通过激活Caspase通路导致MDCC-MSB1细胞凋亡。 LaCl_3能抑制MDCC-MSB1细胞的生长并诱导其凋亡。高浓度LaCl_3可明显抑制肿瘤细胞生长,低浓度LaCl_3对肿瘤细胞的生长无明显抑制作用,其抑制作用随LaCl_3浓度的增加而增强。
[Abstract]:Many studies have shown that a certain concentration of rare earths has a significant anti-cancer effect. With the further study of rare earths in medicine, biochemistry and other fields, Many characteristics of chicken MD are similar to the occurrence of mammalian tumors and have been used as a good model for the study of tumorous diseases. In this study, MDCC-MSB1 cell lines were cultured in vitro. Different concentrations of Lacl _ 3 were added into the culture system, and DNA damage was observed by flow cytometry, in situ end labeling DNA electrophoresis, fluorescence staining, electron microscopy, and so on, from the ultrastructure of cells, intracellular free calcium ion concentration ([Ca~(2)] ionomer, and chromosome DNA damage. The cell cycle, the activity of Caspase-3, Caspase-9 and the expression level of Caspase3 mRNA in LaCl_3 were studied to study the morphological changes, biochemical changes and possible regulatory mechanisms of MDCC-MSB1 apoptosis induced by LaCl_3. To provide a theoretical basis for the clinical application of LaCl_3 in the treatment of MD and other tumor diseases. 1 the activity of MDCC-MSB1 cells and the activity of LDH in culture supernatant were detected by MTT colorimetry and dinitrophenylhydrazine colorimetry. The results showed that LaCl_3 could inhibit the proliferation of MDCC-MSB1 cells. Pi staining flow cytometry was used to detect cell cycle changes and RT-PCR was used to detect the expression of CaMm RNA. The results showed that LaCl_3 could induce G _ 0 / G _ 1 phase arrest and thus inhibit the proliferation of MDCC-MSB1 in a dose-dependent manner. (2) the growth of MDCC-MSB1 cells was observed under inverted microscope. The apoptosis of MDCC-MSB1 cells induced by LaCl_3 was observed by transmission electron microscope (TEM). The results of flow cytometry showed that LaCl_3 could induce apoptosis of MDCC-MSB1 cells in a dose-dependent manner. 3 the damage of LaCl_3 to DNA of MDCC-MSB1 cells was detected by agarose gel electrophoresis and alkaline SCGE Tunel method. The results showed that LaCl_3 induced apoptosis of MDCC-MSB1 cells by increasing Ca~(2 I and activating endogenous nucleic acid endonuclease (endonuclease) to induce DNA to break between nucleosomes. The results showed that LaCl_3 could induce the increase of NOS activity and no content in MDCC-MSB1 cells. It was proved that LaCl_3 could destroy the antioxidant defense system and scavenge free radicals, thus causing oxidative damage to MDCC-MSB1 cells. This may be one of the mechanisms of MDCC-MSB1 cell apoptosis induced by LaCl_3. (5) Fura-2/AM fluorescence assay was used to detect [Ca~(2] I, flow cytometry to detect 螖蠄 m, colorimetric method to detect Caspase-3 caspase-9 activity and RT-PCR method to detect the expression of caspase-3 mRNA. The results showed that LaCl_3 could cause the decrease of 螖蠄 m in MDCC-MSB1 cells and the increase of Caspase-3 mRNA expression. It is confirmed that LaCl_3 induces apoptosis of MDCC-MSB1 cells by activating Caspase pathway. LaCl_3 could inhibit the growth of MDCC-MSB1 cells and induce its apoptosis. High concentration of LaCl_3 could significantly inhibit the growth of tumor cells, but low concentration of LaCl_3 had no obvious inhibitory effect on the growth of tumor cells, but the inhibitory effect was enhanced with the increase of LaCl_3 concentration.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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