富马毒素B1诱导细胞凋亡及机制初步研究

发布时间：2018-01-16 17:13

  本文关键词：富马毒素B1诱导细胞凋亡及机制初步研究　出处：《四川大学》2007年硕士论文　论文类型：学位论文

  更多相关文章： 富马毒素 细胞凋亡 Fas FasL

【摘要】： 研究背景及目的：富马毒素(Fumonisins)是一类主要由串珠镰刀菌(Fusarium moniliforme)产生的真菌毒素，1988年由Gelderblom，-W-C等发现。已经确认的富马毒素有FA1、FA2、FB1、FB2、FB3、FB4、FC1、HFB1(水溶性FB1)等十余种，其中富马毒素B1(FumonisinB1，FB1)是该毒素产生毒性作用的主要原因。FB1广泛存在于人、动物食入的粮食、饲料中，进入人、动物体内后可对人、动物产生损害，不同动物和不同器官对富马毒素的反应不同，可表现为组织损伤、细胞凋亡。 Ross，-P-F等经过分析发现，每克饲料中FB1的浓度为1mg～126mg可导致马脑白质软化症(ELEM)，浓度为1mg-330mg／克饲料可导致猪肺水肿(PPM)；Schmelz，-E-M等经过实验证明10mmol／L FB1可导致HT29细胞出现凋亡。Tolleson等以电镜观察、DNA琼脂糖凝胶电泳发现，FB1可诱导体外培养的角化上皮细胞、HET-1A细胞、CV-1细胞发生凋亡。Tsunoda，-M用0，0．25，0．75，2．25，6．75 mg FB1／kg／天的剂量注射五组雄性BALB／c鼠连续五天，在鼠的肝脏和肾脏可出现与剂量有关的凋亡。 细胞凋亡又称之为程序性细胞死亡，是细胞在基因调控下的自主死亡过程。现在认为细胞凋亡涉及生命活动的众多领域，并不是可有可无的事件，同细胞的分裂、分化一样是最基本的生物现象，是机体生存和发育的基础。细胞凋亡与免疫性疾病、肿瘤的发生发展有关系。凋亡的特点是在细胞核和细胞质存在特异性的形态改变，染色质在特定位点发生有规律的断裂。这种细胞死亡的模式可以平衡组织细胞的分裂，是维持生物体自身稳定的一种重要方式。因而了解细胞凋亡的机制有着重要的作用。FB1致细胞凋亡的作用已经有较多的证据，并已经得到证实，但其引起细胞凋亡的机制目前仍然不清楚，了解FB1导致凋亡的机制对明确其作用原理有着重要的作用。 Fas在介导细胞凋亡的过程中有重要的作用，Fas位于细胞膜，也称APO-1或CD95，，在胸腺细胞、激活的淋巴细胞、病毒感染细胞、部分肿瘤细胞等细胞中有表达，细胞类型不同，表达程度可能有差异。在过氧化氢诱导人脐静脉内皮细胞凋亡的研究中发现，细胞凋亡率增加，Fas表达增加。Fas可以通过与其配体FasL相结合，诱发多种细胞产生凋亡，如Jurket细胞、胸腺淋巴细胞等。本研究拟在FB1诱导细胞出现凋亡的基础上，进一步观察凋亡时Fas／FasL mRNA表达的变化，为明确FB1诱导细胞凋亡的机制打下基础 本研究分两个部分： 第一部分：目的诱导肝癌细胞凋亡。方法体外培养肝癌细胞，取对数生长期细胞消化计数，种入细胞约4×10~5／孔，待其细胞数量升至约6×10~6。加入1μmol／ml FB1 10ul，30ul，40ul，60ul，80ul，使培养液中所含的毒素的终浓度为5μmol／L，15μmol／L，20μmol／L，30μmol／L，40μmol／L，作用24小时后，收集细胞，利用流式细胞仪测定其凋亡率；吖啶橙染色观察出现凋亡的肝癌细胞形态变化。结果5μmol／L，15μmol／L，20μmol／L，30μmol／L，40μmol／L FB1均诱导肝癌细胞出现凋亡，为下一步研究其诱导凋亡的机制打下了基础。 第二部分：目的观察凋亡细胞组的Fas／FasL mRNA表达的变化。方法体外培养肝癌细胞，取对数生长期细胞消化计数，种入细胞约4×10~5／孔，待其细胞数量升至约6×10~6。加入1μmol／ml FB1 10ul，30ul，40ul，60ul，80ul使培养液中所含毒素的浓度为5μmol／L，15μmol／L，20μmol／L，30μmol／L，40μmol／L FB1，作用24小时后，收集细胞，提取总RNA，逆转录成cDNA，利用荧光定量PCR检测Fas mRNA和FasL mRNA表达的变化。结果5μmol／L FB1，15μmol／L FB1实验组与对照组相比FasmRNA表达未见明显增加(p＞0．05)；20μmol／L FB1，30μmol／L FB1，40μmol／L FB1实验组与对照组相比FasmRNA表达有不同程度增加，与对照组相比差异有显著性(p＜0．05)；30μmol／L FB1实验组FasmRNA的表达达到最高，与20μmol／L FB1，40μmol／L FB1浓度实验组相比差异有显著性(p＜0．05)。各实验组FasLmRNA与对照组相比表达均有一定程度降低，差异有显著性(p＜0．05)。该结果为进一步明确其诱导细胞凋亡的机制提供了理论依据。结论：利用5-40μmol／L的FB1作用于体外培养的肝癌细胞，成功诱导了凋亡。较高浓度的FB1诱导肝癌细胞凋亡时有FasmRNA表达的增加；各实验组FasLmRNA均无表达的增加并且降低，说明在凋亡的过程中有Fas的参与，但未证实FasL参与凋亡。
[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.
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