本文關(guān)鍵詞：富馬毒素B1誘導(dǎo)細(xì)胞凋亡及機(jī)制初步研究　出處：《四川大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

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【摘要】： 研究背景及目的：富馬毒素(Fumonisins)是一類主要由串珠鐮刀菌(Fusarium moniliforme)產(chǎn)生的真菌毒素，1988年由Gelderblom，-W-C等發(fā)現(xiàn)。已經(jīng)確認(rèn)的富馬毒素有FA1、FA2、FB1、FB2、FB3、FB4、FC1、HFB1(水溶性FB1)等十余種，其中富馬毒素B1(FumonisinB1，F(xiàn)B1)是該毒素產(chǎn)生毒性作用的主要原因。FB1廣泛存在于人、動(dòng)物食入的糧食、飼料中，進(jìn)入人、動(dòng)物體內(nèi)后可對人、動(dòng)物產(chǎn)生損害，不同動(dòng)物和不同器官對富馬毒素的反應(yīng)不同，可表現(xiàn)為組織損傷、細(xì)胞凋亡。 Ross，-P-F等經(jīng)過分析發(fā)現(xiàn)，每克飼料中FB1的濃度為1mg～126mg可導(dǎo)致馬腦白質(zhì)軟化癥(ELEM)，濃度為1mg-330mg／克飼料可導(dǎo)致豬肺水腫(PPM)；Schmelz，-E-M等經(jīng)過實(shí)驗(yàn)證明10mmol／L FB1可導(dǎo)致HT29細(xì)胞出現(xiàn)凋亡。Tolleson等以電鏡觀察、DNA瓊脂糖凝膠電泳發(fā)現(xiàn)，F(xiàn)B1可誘導(dǎo)體外培養(yǎng)的角化上皮細(xì)胞、HET-1A細(xì)胞、CV-1細(xì)胞發(fā)生凋亡。Tsunoda，-M用0，0．25，0．75，2．25，6．75 mg FB1／kg／天的劑量注射五組雄性BALB／c鼠連續(xù)五天，在鼠的肝臟和腎臟可出現(xiàn)與劑量有關(guān)的凋亡。 細(xì)胞凋亡又稱之為程序性細(xì)胞死亡，是細(xì)胞在基因調(diào)控下的自主死亡過程�，F(xiàn)在認(rèn)為細(xì)胞凋亡涉及生命活動(dòng)的眾多領(lǐng)域，并不是可有可無的事件，同細(xì)胞的分裂、分化一樣是最基本的生物現(xiàn)象，是機(jī)體生存和發(fā)育的基礎(chǔ)。細(xì)胞凋亡與免疫性疾病、腫瘤的發(fā)生發(fā)展有關(guān)系。凋亡的特點(diǎn)是在細(xì)胞核和細(xì)胞質(zhì)存在特異性的形態(tài)改變，染色質(zhì)在特定位點(diǎn)發(fā)生有規(guī)律的斷裂。這種細(xì)胞死亡的模式可以平衡組織細(xì)胞的分裂，是維持生物體自身穩(wěn)定的一種重要方式。因而了解細(xì)胞凋亡的機(jī)制有著重要的作用。FB1致細(xì)胞凋亡的作用已經(jīng)有較多的證據(jù)，并已經(jīng)得到證實(shí)，但其引起細(xì)胞凋亡的機(jī)制目前仍然不清楚，了解FB1導(dǎo)致凋亡的機(jī)制對明確其作用原理有著重要的作用。 Fas在介導(dǎo)細(xì)胞凋亡的過程中有重要的作用，F(xiàn)as位于細(xì)胞膜，也稱APO-1或CD95，，在胸腺細(xì)胞、激活的淋巴細(xì)胞、病毒感染細(xì)胞、部分腫瘤細(xì)胞等細(xì)胞中有表達(dá)，細(xì)胞類型不同，表達(dá)程度可能有差異。在過氧化氫誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞凋亡的研究中發(fā)現(xiàn)，細(xì)胞凋亡率增加，F(xiàn)as表達(dá)增加。Fas可以通過與其配體FasL相結(jié)合，誘發(fā)多種細(xì)胞產(chǎn)生凋亡，如Jurket細(xì)胞、胸腺淋巴細(xì)胞等。本研究擬在FB1誘導(dǎo)細(xì)胞出現(xiàn)凋亡的基礎(chǔ)上，進(jìn)一步觀察凋亡時(shí)Fas／FasL mRNA表達(dá)的變化，為明確FB1誘導(dǎo)細(xì)胞凋亡的機(jī)制打下基礎(chǔ) 本研究分兩個(gè)部分： 第一部分：目的誘導(dǎo)肝癌細(xì)胞凋亡。方法體外培養(yǎng)肝癌細(xì)胞，取對數(shù)生長期細(xì)胞消化計(jì)數(shù)，種入細(xì)胞約4×10~5／孔，待其細(xì)胞數(shù)量升至約6×10~6。加入1μmol／ml FB1 10ul，30ul，40ul，60ul，80ul，使培養(yǎng)液中所含的毒素的終濃度為5μmol／L，15μmol／L，20μmol／L，30μmol／L，40μmol／L，作用24小時(shí)后，收集細(xì)胞，利用流式細(xì)胞儀測定其凋亡率；吖啶橙染色觀察出現(xiàn)凋亡的肝癌細(xì)胞形態(tài)變化。結(jié)果5μmol／L，15μmol／L，20μmol／L，30μmol／L，40μmol／L FB1均誘導(dǎo)肝癌細(xì)胞出現(xiàn)凋亡，為下一步研究其誘導(dǎo)凋亡的機(jī)制打下了基礎(chǔ)。 第二部分：目的觀察凋亡細(xì)胞組的Fas／FasL mRNA表達(dá)的變化。方法體外培養(yǎng)肝癌細(xì)胞，取對數(shù)生長期細(xì)胞消化計(jì)數(shù)，種入細(xì)胞約4×10~5／孔，待其細(xì)胞數(shù)量升至約6×10~6。加入1μmol／ml FB1 10ul，30ul，40ul，60ul，80ul使培養(yǎng)液中所含毒素的濃度為5μmol／L，15μmol／L，20μmol／L，30μmol／L，40μmol／L FB1，作用24小時(shí)后，收集細(xì)胞，提取總RNA，逆轉(zhuǎn)錄成cDNA，利用熒光定量PCR檢測Fas mRNA和FasL mRNA表達(dá)的變化。結(jié)果5μmol／L FB1，15μmol／L FB1實(shí)驗(yàn)組與對照組相比FasmRNA表達(dá)未見明顯增加(p＞0．05)；20μmol／L FB1，30μmol／L FB1，40μmol／L FB1實(shí)驗(yàn)組與對照組相比FasmRNA表達(dá)有不同程度增加，與對照組相比差異有顯著性(p＜0．05)；30μmol／L FB1實(shí)驗(yàn)組FasmRNA的表達(dá)達(dá)到最高，與20μmol／L FB1，40μmol／L FB1濃度實(shí)驗(yàn)組相比差異有顯著性(p＜0．05)。各實(shí)驗(yàn)組FasLmRNA與對照組相比表達(dá)均有一定程度降低，差異有顯著性(p＜0．05)。該結(jié)果為進(jìn)一步明確其誘導(dǎo)細(xì)胞凋亡的機(jī)制提供了理論依據(jù)。結(jié)論：利用5-40μmol／L的FB1作用于體外培養(yǎng)的肝癌細(xì)胞，成功誘導(dǎo)了凋亡。較高濃度的FB1誘導(dǎo)肝癌細(xì)胞凋亡時(shí)有FasmRNA表達(dá)的增加；各實(shí)驗(yàn)組FasLmRNA均無表達(dá)的增加并且降低，說明在凋亡的過程中有Fas的參與，但未證實(shí)FasL參與凋亡。
[Abstract]:Background and purpose: fumonisins (Fumonisins) is a kind of mainly by Fusarium moniliforme (Fusarium moniliforme) of mycotoxins produced by Gelderblom in 1988, -W-C, etc. found. Fumonisin has confirmed that there are FA1, FA2, FB1, FB2, FB3, FB4, FC1, HFB1 (water soluble FB1) more than ten kinds, the fumonisin B1 (FumonisinB1, FB1) is the main cause of the toxicity of.FB1 toxin exists widely in human and animal food into food, feed, animal can enter, after damage to people, animal, animal and different organs of different fumonisins react differently, can be expressed as the organization injury, cell apoptosis.
Ross -P-F, the analysis found that the concentration of FB1 in feed per gram of 1mg ~ 126mg can lead to equine Leukoencephalomalacia (ELEM), the concentration of 1mg-330mg / g diet can lead to pulmonary edema (PPM); Schmelz, -E-M 10mmol / L FB1 experiments proved that it can lead to the apoptosis of.Tolleson was observed by HT29 cells, DNA agarose gel electrophoresis, FB1 can induce cultured keratinized epithelial cells, HET-1A cells, apoptosis of CV-1 cells in.Tsunoda, -M with the dose of FB1 / kg / day 0,0.25,0.75,2.25,6.75 mg five groups of male BALB / C rats for five consecutive days, in rat liver and kidney can appear apoptosis is related to the dose.
Apoptosis is also known as programmed cell death, cell death process is independent in the regulation of genes. Now that many areas of apoptosis involves the activities of life, is not essential events with cell division, differentiation is the most basic biological phenomena, is the basis of the life and development of apoptosis and. Autoimmune diseases, the relationship between the occurrence and development of tumor. Apoptosis is characterized by specific changes in the nucleus and cytoplasm of morphology, chromatin break at specific sites occur regularly. The cell death mode can balance the tissue cell division is an important way to maintain the stability of the organism itself. Therefore, understanding the mechanism cell apoptosis with cell apoptosis induced by.FB1 important role has more evidence, and has been confirmed, but the cause of the mechanism of cell apoptosis is still It is not clear that understanding the mechanism of apoptosis induced by FB1 plays an important role in identifying the principles of its action.
There is an important role in Fas mediated apoptosis, Fas is located in the cell membrane, also known as APO-1 or CD95 in thymocytes, activated lymphocytes, virus infected cells, some tumor cells in the expression of different cell types, the degree of expression may be different. Found in the study of hydrogen peroxide induced apoptosis in human umbilical vein endothelial cells, cell apoptosis, increase the expression of Fas.Fas by binding to its ligand FasL phase, induce a variety of cell apoptosis, such as Jurket cells, thymus lymphocytes. The study on FB1 induced cell apoptosis on the basis of the changes of the expression of Fas / FasL mRNA to observe apoptosis, lay the basic mechanism of the FB1 induce apoptosis
This study is divided into two parts:
The first part: to induce apoptosis of hepatoma cells. Cultured hepatoma cells in vitro, cells in the logarithmic growth phase of digestion into the cell count, which is about 4 * 10~5 / hole, the cell number rose to about 6 * 10~6. with 1 mol / ml FB1 10ul, 30ul, 40ul, 60ul, 80ul, the final concentration of culture the liquid containing toxins in the 5 mol / L 15 mol / L 20 mol / L 30 mol / L 40 mol / L, after 24 hours, cells were collected by measuring the apoptosis rate by flow cytometry; acridine orange staining was used to observe the morphological changes of hepatic cancer cells apoptosis the results appear. 5 mol / L 15 mol / L 20 mol / L 30 mol / L 40 mol / L FB1 were induced hepatoma cell apoptosis, for further study the mechanism of apoptosis of the foundation.
絎簩閮ㄥ垎錛氱洰鐨勮瀵熷噵浜＄粏鑳?yōu)缁勭殑Fas錛廎asL mRNA琛ㄨ揪鐨勫彉鍖

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