本文選題：氯化鑭　切入點(diǎn)：MDCC-MSB1　出處：《東北農(nóng)業(yè)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 許多研究表明,一定濃度稀土具有顯著的抑癌作用。隨著稀土在醫(yī)藥、生物化學(xué)等領(lǐng)域研究的不斷深入,其在生命科學(xué)中的地位日益重要。雞MD的許多特性與哺乳動物腫瘤的發(fā)生有相似之處,被作為研究腫瘤性疾病的良好模型。本課題以體外培養(yǎng)MDCC-MSB1細(xì)胞系為研究對象,在培養(yǎng)體系中加入不同濃度LaCl_3,應(yīng)用流式細(xì)胞術(shù)、原位末端標(biāo)記、DNA電泳分析、熒光染色、電子顯微鏡等方法,從細(xì)胞超微結(jié)構(gòu)、細(xì)胞內(nèi)游離鈣離子濃度([Ca~(2+)]i)、染色體DNA損傷、細(xì)胞周期、線粒體膜電位(Δψm)、Caspase-3、Caspase-9活性及CaM、Caspase3 mRNA表達(dá)水平等方面,研究LaCl_3誘導(dǎo)MDCC-MSB1凋亡的形態(tài)學(xué)改變、生物化學(xué)改變及可能的調(diào)控機(jī)制,為臨床應(yīng)用LaCl_3治療MD及其他腫瘤性疾病提供理論依據(jù)。研究結(jié)果表明: 1應(yīng)用MTT比色法、二硝基苯肼比色法檢測MDCC-MSB1細(xì)胞的活性和培養(yǎng)上清液中LDH活性,結(jié)果表明各濃度組LaCl_3均可以抑制MDCC-MSB1細(xì)胞增殖,應(yīng)用PI染色流式細(xì)胞術(shù)檢測法檢檢測細(xì)胞周期變化,RT-PCR方法檢測CaMm RNA表達(dá)水平,結(jié)果表明LaCl_3可誘導(dǎo)細(xì)胞發(fā)生G0/G1期阻滯,從而抑制MDCC-MSB1增殖,且抑制率具有劑量依賴性。 2倒置顯微鏡下觀察MDCC-MSB1細(xì)胞生長情況,HE染色、AO/EB雙熒光染色、透射電鏡觀察不同濃度下LaCl_3誘導(dǎo)MDCC-MSB1細(xì)胞凋亡的形態(tài)學(xué)變化,流式細(xì)胞術(shù)檢測磷脂酰絲氨酸(PS),結(jié)果表明LaCl_3可誘導(dǎo)MDCC-MSB1細(xì)胞凋亡,且凋亡率具有劑量依賴性。 3應(yīng)用瓊脂糖凝膠電泳、堿性SCGE、TUNEL法檢測LaCl_3對MDCC-MSB1細(xì)胞DNA的損傷作用,結(jié)果證實(shí)LaCl_3通過引起MDCC-MSB1細(xì)胞[Ca~(2+)]i增加,激活內(nèi)源性核酸內(nèi)切酶導(dǎo)致DNA在核小體間斷裂途徑誘導(dǎo)細(xì)胞凋亡。 4通過對細(xì)胞內(nèi)抗氧化功能的檢測,表明LaCl_3能夠誘導(dǎo)MDCC-MSB1細(xì)胞NOS活性與NO含量升高,ROS含量增高,證實(shí)LaCl_3能夠破壞機(jī)體抗氧化防御系統(tǒng)及清除自由基的功能,使細(xì)胞發(fā)生氧化損傷,這可能是LaCl_3誘導(dǎo)MDCC-MSB1細(xì)胞發(fā)生凋亡的機(jī)制之一。 5應(yīng)用Fura-2/AM熒光法檢測[Ca~(2+)]i,流式細(xì)胞術(shù)檢測Δψm,比色法檢測Caspase-3、Caspase-9活性,RT-PCR方法檢測caspase-3 mRNA表達(dá)水平。結(jié)果表明LaCl_3可導(dǎo)致MDCC-MSB1細(xì)胞Δψm下降,Caspase-3、Caspase-9活性上升,Caspase-3 mRNA表達(dá)增加,證實(shí)LaCl_3通過激活Caspase通路導(dǎo)致MDCC-MSB1細(xì)胞凋亡。 LaCl_3能抑制MDCC-MSB1細(xì)胞的生長并誘導(dǎo)其凋亡。高濃度LaCl_3可明顯抑制腫瘤細(xì)胞生長,低濃度LaCl_3對腫瘤細(xì)胞的生長無明顯抑制作用,其抑制作用隨LaCl_3濃度的增加而增強(qiáng)。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R363

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