亚砷酸钠诱导细胞死亡的分子机制研究

发布时间：2018-05-28 18:06

本文选题：亚砷酸钠 + 酵母　；参考：《山西大学》2011年博士论文

【摘要】：砷是常见的、对人类健康危害比较严重的环境污染物之一,可通过皮肤、呼吸道和消化道等途径进入人体,导致身体组织器官发生病变,从而引起多种疾病和癌症的发生。因此,砷毒性研究已经成为大家关注的焦点之一。砷化物能抑制细胞分裂,诱导微核形成,导致部分细胞死亡,但砷化物的毒性作用机制不是很清楚。酵母具有遗传背景简单,生长周期短等优点,是研究真核生物细胞学机制的模式生物。已有研究证明,砷化物可以诱导酵母细胞凋亡,并且伴随着胞内活性氧(ROS)水平的升高,但是关于砷诱导酵母细胞凋亡的调控机制研究尚处在起步阶段,尤其是动物体内相对保守的凋亡抑制基因BCL-2和CED-9在此过程中的作用尚未见报道。本文以酵母细胞为主要材料,研究了亚砷酸钠诱导细胞死亡的作用机制。主要实验结果如下：浓度为1-7 mmol/L的亚砷酸钠可抑制酵母细胞生长,并具有剂量依赖性,其中7 mmol/L亚砷酸钠几乎完全抑制了细胞的生长和分裂。亚砷酸钠可诱导酵母细胞死亡,细胞死亡率随处理浓度的升高和作用时间的延长逐渐升高。利用ROS荧光指示剂DCFH-DA, Ca2+荧光指示剂Fluo-3AM和NO荧光指示剂DAF-FM DA检测胞内ROS、Ca2+和NO水平,发现亚砷酸钠可诱导酵母胞内ROS, Ca2+和NO水平显著升高。一定浓度的抗氧化剂(0.5 mmol·L-1抗坏血酸AsA和200 U·mL-1过氧化氢酶CAT), Ca2+干扰剂(0.5 mmol·L-1 Ca2+螯合剂EGTA和0.1 mmol·L-1 Ca2+通道抑制剂LaCl3)及NO消除剂(0.2 mmol·L-1 NO清除剂c-PTIO和1 mmol·L-1 NO生成抑制剂NaN3)均能降低亚砷酸钠诱导的细胞死亡率,说明亚砷酸钠诱导的酵母细胞死亡与胞内ROS, Ca2+和NO水平的升高有关。以转有凋亡抑制基因BCL-2, CED-9及空载体(对照菌)的酵母为材料,研究了亚砷酸钠对细胞的毒性作用。在不含亚砷酸钠的培养基中,转有BCL-2和CED-9的酵母菌与对照菌生长曲线一致,无明显差异。培养24 h后,3 mmol/L和7 mmol/L处理组中,转基因酵母相对OD600显著高于对照菌,而且细胞死亡率显著低于对照菌,说明亚砷酸钠诱导的酵母细胞死亡可被BCL-2和CED-9调控。经DAPI染色后,处理组细胞出现了染色质凝集,细胞核形状不规则,核碎片等凋亡特征,说明亚砷酸钠可诱导酵母细胞发生凋亡亚砷酸钠诱导酵母细胞线粒体膜电位(△Ψm)下降,胞质中细胞色素c (cyt c)含量升高；2μmol·L-1泛caspase抑制剂Z-Asp-CH2-DCB可以明显降低亚砷酸钠引起的酵母细胞死亡率,说明亚砷酸钠诱导的酵母细胞死亡依赖于线粒体和caspase途径。抗氧化剂AsA和CAT, Ca2+干扰剂EGTA和LaCl3, NO消除剂c-PTIO和NaN3均可抑制亚砷酸钠引起的酵母细胞△Ψm的改变和cyt c含量的升高。酵母菌经°3 mmol/L亚砷酸钠处理6h后,转基因酵母胞内Ca2+水平与对照菌无显著差异,胞内NO水平及cyt c含量显著低于对照菌,△Ψm高于对照菌；转有BCL-2的酵母胞内ROS水平显著低于对照菌,而转有CED-9的酵母胞内ROS水平与对照菌无显著差异。研究结果表明,亚砷酸钠可抑制酵母细胞生长和分裂,诱导细胞死亡。酵母细胞经亚砷酸钠胁迫后可见典型的凋亡特征,而且caspase抑制剂和凋亡抑制基因BCL-2和CED-9均可以明显降低亚砷酸钠引起的细胞死亡率,说明亚砷酸钠可诱导酵母细胞凋亡。砷胁迫期间,酵母胞内ROS、Ca2+和NO水平显著升高,线粒体膜通透性增加,△Ψm下降,cyt c释放,进而激活下游的caspase途径,引起细胞凋亡。CED-9可通过抑制胞内NO的生成,△甲m的改变和cyt c的释放来降低亚砷酸钠引起的酵母细胞死亡率,而BCL-2还可通过降低胞内ROS水平抑制亚砷酸钠引起的酵母细胞死亡。将大蒜金属硫蛋白AsMT2b构建于酵母表达载体pPIC9K或带有酵母金属硫蛋白启动子PCUP1的表达载体pCUP9K,转化毕赤酵母。随机挑取阳性转化子,提取基因组DNA,经PCR法验证可知工程菌构建成功。由于所构建的工程菌带有AsMT2b与GFP的融合蛋白,经诱导表达后,细胞发出绿色荧光；用SDS凝胶电泳检测蛋白表达,可见与目的蛋白大小相符的条带,说明蛋白可正常表达,获得工程菌pPIC9K-AsMT2b-GFP和pCUP9K-AsMT2b-GFP。以工程菌pCUP9K-AsMT2b-GFP和对照菌pCUP9K为材料,研究AsMT2b在亚砷酸钠胁迫酵母细胞过程中的作用。细胞生长率及死亡率检测结果显示,50μM-1000μM的亚砷酸钠可抑制毕赤酵母细胞生长和分裂,诱导部分细胞死亡。在含有相同浓度亚砷酸钠的处理组中,转有AsMT2b的工程菌细胞死亡率显著低于对照菌,但与对照菌的OD600无显著差异。此外,我们以工程菌pPIC9K-AsMT2b-GFP和pCUP9K-AsMT2b-GFP,对照菌pPIC9K, pCUP9K和GS115为材料,采用平板点样法研究酵母细胞在含有一定浓度As3+,Cu2+,Cr6+和Cd2+的固体培养基中的生长情况。结果发现,在金属离子胁迫下,转有AsMT2b的工程菌生长情况明显好于对照菌,说明AsMT2b的表达可以提高酵母细胞对As3+, Cu2+, Cr6+和Cd2+的抗性；在4种不同金属离子的胁迫下,工程菌pCUP9K-AsMT2b-GFP生长状况明显优于pPIC9K-AsMT2b-GFP,说明As3+, Cu2+,Cr6+和Cd2+对pCUP9K-AsMT2b-GFP均有一定的诱导作用。AsMT2b的表达可降低亚砷酸钠引起的酵母细胞死亡率,提高酵母细胞对As3+, Cu2+, Cr6+和Cd2+的耐受性,推测AsMT2b可通过螫合金属离子降低金属离子引起的细胞死亡率。砷化物可抑制蚕豆和洋葱根尖细胞分裂,诱导微核率和MDA含量升高；高浓度的砷可导致植物根尖细胞死亡,出现核固缩现象。在一定浓度范围内,微核率与砷浓度间呈显著正相关,微核率在一定程度上能够反映水体中的砷污染程度。因此,植物根尖微核技术可用于检测环境中砷污染的水体。
[Abstract]:Arsenic is one of the most common environmental pollutants that are more serious to human health. It can enter the human body through skin, respiratory and digestive tract, resulting in pathological changes of body tissues and organs, resulting in a variety of diseases and cancer. Therefore, arsenic toxicity research has become one of the focus of attention. Arsenic can inhibit cells. Division, induced micronucleus formation, leading to some cell death, but the toxicity mechanism of arsenic compounds is not very clear. Yeast has the advantages of simple genetic background and short growth cycle. It is a model organism to study the mechanism of eukaryotes cytology. OS) levels are rising, but the regulatory mechanism of arsenic induced apoptosis in yeast cells is still in its infancy, especially the role of the relative conserved apoptosis suppressor gene BCL-2 and CED-9 in this process has not yet been reported. In this paper, the mechanism of sodium arsenite induced cell death was studied with yeast cells as the main material. The main experimental results are as follows:
Sodium arsenite with a concentration of 1-7 mmol/L inhibited the growth of yeast cells and was dose-dependent. 7 mmol/L sodium arsenite almost completely inhibited the growth and division of cells. Sodium arsenite could induce the death of yeast cells. The cell death rate increased with the increase of treatment concentration and the prolongation of action time. The use of ROS fluorescent indicator. DCFH-DA, Ca2+ fluorescent indicator Fluo-3AM and NO fluorescent indicator DAF-FM DA were used to detect intracellular ROS, Ca2+ and NO levels. It was found that sodium arsenite could induce intracellular ROS, Ca2+ and NO levels increased significantly. The agent EGTA and 0.1 mmol. L-1 Ca2+ channel inhibitor LaCl3) and NO elimination agents (0.2 mmol L-1 NO scavenger c-PTIO and 1 mmol L-1) can reduce the cell mortality induced by sodium arsenite.
The toxic effects of sodium arsenite on the cells were studied with the yeast of BCL-2, CED-9 and the empty body (control bacteria). In the medium without sodium arsenite, the growth curves of BCL-2 and CED-9 were consistent with the control bacteria, and there was no significant difference. After 24 h culture, the 3 mmol/L and 7 mmol/L treatment groups were genetically modified. The relative OD600 of yeast was significantly higher than that of the control bacteria, and the cell death rate was significantly lower than that of the control bacteria. It indicated that the yeast cell death induced by sodium arsenite could be regulated by BCL-2 and CED-9. After DAPI staining, the cells in the treatment group appeared chromatin agglutination, irregular nucleus shape, nuclear debris and other apoptotic characteristics, indicating that sodium arsenite could induce yeast cells. Apoptosis
Sodium arsenite induced the decrease of mitochondrial membrane potential (delta m) and the increase of cytochrome c (cyt c) in the cytoplasm, and the 2 u mol. L-1 pan caspase inhibitor Z-Asp-CH2-DCB could significantly reduce the yeast cell mortality caused by sodium arsenite, indicating that the death of sodium arsenite induced yeast cells is dependent on mitochondria and caspase pathway. AsA and CAT, Ca2+ interfering agent EGTA and LaCl3, NO elimination agent c-PTIO and NaN3 could inhibit the change of delta m and Cyt C content of yeast cells caused by sodium arsenite. After the yeast was treated with 3 mmol/L arsenite, there was no significant difference between the yeast cell and the control bacteria. In the control bacteria, the delta m was higher than the control, and the intracellular ROS level of the yeast transferred with BCL-2 was significantly lower than that of the control bacteria, while the intracellular ROS level of the yeast transferred to CED-9 had no significant difference with the control bacteria.
The results show that sodium arsenite can inhibit the growth and division of yeast cells and induce cell death. The typical apoptosis characteristics can be seen in yeast cells after sodium arsenite stress, and both caspase inhibitor and apoptosis suppressor gene BCL-2 and CED-9 can significantly reduce the cell mortality caused by sodium arsenite, indicating that sodium arsenite can induce yeast. Cell apoptosis. During arsenic stress, the intracellular ROS, Ca2+ and NO levels increased significantly, the mitochondrial membrane permeability increased, delta m decreased, Cyt C released, and then activated the downstream caspase pathway, causing apoptosis.CED-9 to reduce the yeast cell death caused by sodium arsenite by inhibiting the formation of intracellular NO, the change of delta m and cyt c. However, BCL-2 can also inhibit yeast cell death induced by sodium arsenite by lowering intracellular ROS levels.
Garlic metallothionein AsMT2b was constructed by yeast expression vector pPIC9K or expression vector pCUP9K with yeast metallothionein promoter PCUP1, and Pichia pastoris was transformed into Pichia pastoris. The positive transformants were randomly selected and genomic DNA was extracted. The engineering bacteria were constructed successfully by PCR method. The engineered bacteria were constructed with the fusion protein of AsMT2b and GFP, After induced expression, the cell emitted green fluorescence, and the protein expression was detected by SDS gel electrophoresis. The protein could be found to be in line with the size of the target protein, indicating that the protein could be expressed normally. The engineering bacteria pPIC9K-AsMT2b-GFP and pCUP9K-AsMT2b-GFP. were used as the engineering bacteria pCUP9K-AsMT2b-GFP and the control bacteria pCUP9K as the material, and the AsMT2b was stressed by sodium arsenite stress. The results of cell growth and mortality detection showed that sodium arsenite at 50 M-1000 mu M inhibited the growth and division of Pichia pastoris cells and induced partial cell death. In the treatment group containing the same concentration of sodium arsenite, the death rate of the engineered bacteria with AsMT2b was significantly lower than that of the control bacteria, but with the control bacteria In addition, we studied the growth conditions of yeast cells in the solid medium containing a certain concentration of As3+, Cu2+, Cr6+ and Cd2+ with the engineering bacteria pPIC9K-AsMT2b-GFP and pCUP9K-AsMT2b-GFP and the control bacteria pPIC9K, pCUP9K and GS115 as materials. The results showed that under the stress of metal ions, the yeast cells were converted to AsMT2b. The growth of engineering bacteria was better than that of the control bacteria, indicating that the expression of AsMT2b could improve the resistance of yeast cells to As3+, Cu2+, Cr6+ and Cd2+. Under the stress of 4 different metal ions, the growth of pCUP9K-AsMT2b-GFP of the engineering bacteria was obviously superior to that of pPIC9K-AsMT2b-GFP, indicating that As3+, Cu2+, Cr6+ and Cd2+ were certain to lure pCUP9K-AsMT2b-GFP. The expression of.AsMT2b can reduce the mortality of yeast cells caused by sodium arsenite and improve the tolerance of yeast cells to As3+, Cu2+, Cr6+ and Cd2+. It is speculated that AsMT2b can reduce the cell mortality caused by metal ions by chelating metal ions.
Arsenic can inhibit the cell division of Vicia Vicia and onion root tip cells, induce micronucleus rate and increase the content of MDA. High concentration of arsenic can lead to the death of plant root tip cells and nuclear condensation. In a certain concentration range, the micronucleus rate is positively correlated with arsenic concentration, and the micronucleus rate can reflect the degree of arsenic pollution in water to a certain extent. The root tip micronucleus technology can be used to detect arsenic polluted water in the environment.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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