本文選題：亞砷酸鈉 + 酵母��； 參考：《山西大學(xué)》2011年博士論文

【摘要】：砷是常見的、對人類健康危害比較嚴(yán)重的環(huán)境污染物之一,可通過皮膚、呼吸道和消化道等途徑進(jìn)入人體,導(dǎo)致身體組織器官發(fā)生病變,從而引起多種疾病和癌癥的發(fā)生。因此,砷毒性研究已經(jīng)成為大家關(guān)注的焦點(diǎn)之一。砷化物能抑制細(xì)胞分裂,誘導(dǎo)微核形成,導(dǎo)致部分細(xì)胞死亡,但砷化物的毒性作用機(jī)制不是很清楚。酵母具有遺傳背景簡單,生長周期短等優(yōu)點(diǎn),是研究真核生物細(xì)胞學(xué)機(jī)制的模式生物。已有研究證明,砷化物可以誘導(dǎo)酵母細(xì)胞凋亡,并且伴隨著胞內(nèi)活性氧(ROS)水平的升高,但是關(guān)于砷誘導(dǎo)酵母細(xì)胞凋亡的調(diào)控機(jī)制研究尚處在起步階段,尤其是動物體內(nèi)相對保守的凋亡抑制基因BCL-2和CED-9在此過程中的作用尚未見報道。本文以酵母細(xì)胞為主要材料,研究了亞砷酸鈉誘導(dǎo)細(xì)胞死亡的作用機(jī)制。主要實(shí)驗(yàn)結(jié)果如下： 濃度為1-7 mmol/L的亞砷酸鈉可抑制酵母細(xì)胞生長,并具有劑量依賴性,其中7 mmol/L亞砷酸鈉幾乎完全抑制了細(xì)胞的生長和分裂。亞砷酸鈉可誘導(dǎo)酵母細(xì)胞死亡,細(xì)胞死亡率隨處理濃度的升高和作用時間的延長逐漸升高。利用ROS熒光指示劑DCFH-DA, Ca2+熒光指示劑Fluo-3AM和NO熒光指示劑DAF-FM DA檢測胞內(nèi)ROS、Ca2+和NO水平,發(fā)現(xiàn)亞砷酸鈉可誘導(dǎo)酵母胞內(nèi)ROS, Ca2+和NO水平顯著升高。一定濃度的抗氧化劑(0.5 mmol·L-1抗壞血酸AsA和200 U·mL-1過氧化氫酶CAT), Ca2+干擾劑(0.5 mmol·L-1 Ca2+螯合劑EGTA和0.1 mmol·L-1 Ca2+通道抑制劑LaCl3)及NO消除劑(0.2 mmol·L-1 NO清除劑c-PTIO和1 mmol·L-1 NO生成抑制劑NaN3)均能降低亞砷酸鈉誘導(dǎo)的細(xì)胞死亡率,說明亞砷酸鈉誘導(dǎo)的酵母細(xì)胞死亡與胞內(nèi)ROS, Ca2+和NO水平的升高有關(guān)。 以轉(zhuǎn)有凋亡抑制基因BCL-2, CED-9及空載體(對照菌)的酵母為材料,研究了亞砷酸鈉對細(xì)胞的毒性作用。在不含亞砷酸鈉的培養(yǎng)基中,轉(zhuǎn)有BCL-2和CED-9的酵母菌與對照菌生長曲線一致,無明顯差異。培養(yǎng)24 h后,3 mmol/L和7 mmol/L處理組中,轉(zhuǎn)基因酵母相對OD600顯著高于對照菌,而且細(xì)胞死亡率顯著低于對照菌,說明亞砷酸鈉誘導(dǎo)的酵母細(xì)胞死亡可被BCL-2和CED-9調(diào)控。經(jīng)DAPI染色后,處理組細(xì)胞出現(xiàn)了染色質(zhì)凝集,細(xì)胞核形狀不規(guī)則,核碎片等凋亡特征,說明亞砷酸鈉可誘導(dǎo)酵母細(xì)胞發(fā)生凋亡 亞砷酸鈉誘導(dǎo)酵母細(xì)胞線粒體膜電位(△Ψm)下降,胞質(zhì)中細(xì)胞色素c (cyt c)含量升高；2μmol·L-1泛caspase抑制劑Z-Asp-CH2-DCB可以明顯降低亞砷酸鈉引起的酵母細(xì)胞死亡率,說明亞砷酸鈉誘導(dǎo)的酵母細(xì)胞死亡依賴于線粒體和caspase途徑�？寡趸瘎〢sA和CAT, Ca2+干擾劑EGTA和LaCl3, NO消除劑c-PTIO和NaN3均可抑制亞砷酸鈉引起的酵母細(xì)胞△Ψm的改變和cyt c含量的升高。酵母菌經(jīng)°3 mmol/L亞砷酸鈉處理6h后,轉(zhuǎn)基因酵母胞內(nèi)Ca2+水平與對照菌無顯著差異,胞內(nèi)NO水平及cyt c含量顯著低于對照菌,△Ψm高于對照菌；轉(zhuǎn)有BCL-2的酵母胞內(nèi)ROS水平顯著低于對照菌,而轉(zhuǎn)有CED-9的酵母胞內(nèi)ROS水平與對照菌無顯著差異。 研究結(jié)果表明,亞砷酸鈉可抑制酵母細(xì)胞生長和分裂,誘導(dǎo)細(xì)胞死亡。酵母細(xì)胞經(jīng)亞砷酸鈉脅迫后可見典型的凋亡特征,而且caspase抑制劑和凋亡抑制基因BCL-2和CED-9均可以明顯降低亞砷酸鈉引起的細(xì)胞死亡率,說明亞砷酸鈉可誘導(dǎo)酵母細(xì)胞凋亡。砷脅迫期間,酵母胞內(nèi)ROS、Ca2+和NO水平顯著升高,線粒體膜通透性增加,△Ψm下降,cyt c釋放,進(jìn)而激活下游的caspase途徑,引起細(xì)胞凋亡。CED-9可通過抑制胞內(nèi)NO的生成,△甲m的改變和cyt c的釋放來降低亞砷酸鈉引起的酵母細(xì)胞死亡率,而BCL-2還可通過降低胞內(nèi)ROS水平抑制亞砷酸鈉引起的酵母細(xì)胞死亡。 將大蒜金屬硫蛋白AsMT2b構(gòu)建于酵母表達(dá)載體pPIC9K或帶有酵母金屬硫蛋白啟動子PCUP1的表達(dá)載體pCUP9K,轉(zhuǎn)化畢赤酵母。隨機(jī)挑取陽性轉(zhuǎn)化子,提取基因組DNA,經(jīng)PCR法驗(yàn)證可知工程菌構(gòu)建成功。由于所構(gòu)建的工程菌帶有AsMT2b與GFP的融合蛋白,經(jīng)誘導(dǎo)表達(dá)后,細(xì)胞發(fā)出綠色熒光；用SDS凝膠電泳檢測蛋白表達(dá),可見與目的蛋白大小相符的條帶,說明蛋白可正常表達(dá),獲得工程菌pPIC9K-AsMT2b-GFP和pCUP9K-AsMT2b-GFP。以工程菌pCUP9K-AsMT2b-GFP和對照菌pCUP9K為材料,研究AsMT2b在亞砷酸鈉脅迫酵母細(xì)胞過程中的作用。細(xì)胞生長率及死亡率檢測結(jié)果顯示,50μM-1000μM的亞砷酸鈉可抑制畢赤酵母細(xì)胞生長和分裂,誘導(dǎo)部分細(xì)胞死亡。在含有相同濃度亞砷酸鈉的處理組中,轉(zhuǎn)有AsMT2b的工程菌細(xì)胞死亡率顯著低于對照菌,但與對照菌的OD600無顯著差異。此外,我們以工程菌pPIC9K-AsMT2b-GFP和pCUP9K-AsMT2b-GFP,對照菌pPIC9K, pCUP9K和GS115為材料,采用平板點(diǎn)樣法研究酵母細(xì)胞在含有一定濃度As3+,Cu2+,Cr6+和Cd2+的固體培養(yǎng)基中的生長情況。結(jié)果發(fā)現(xiàn),在金屬離子脅迫下,轉(zhuǎn)有AsMT2b的工程菌生長情況明顯好于對照菌,說明AsMT2b的表達(dá)可以提高酵母細(xì)胞對As3+, Cu2+, Cr6+和Cd2+的抗性；在4種不同金屬離子的脅迫下,工程菌pCUP9K-AsMT2b-GFP生長狀況明顯優(yōu)于pPIC9K-AsMT2b-GFP,說明As3+, Cu2+,Cr6+和Cd2+對pCUP9K-AsMT2b-GFP均有一定的誘導(dǎo)作用。AsMT2b的表達(dá)可降低亞砷酸鈉引起的酵母細(xì)胞死亡率,提高酵母細(xì)胞對As3+, Cu2+, Cr6+和Cd2+的耐受性,推測AsMT2b可通過螫合金屬離子降低金屬離子引起的細(xì)胞死亡率。 砷化物可抑制蠶豆和洋蔥根尖細(xì)胞分裂,誘導(dǎo)微核率和MDA含量升高；高濃度的砷可導(dǎo)致植物根尖細(xì)胞死亡,出現(xiàn)核固縮現(xiàn)象。在一定濃度范圍內(nèi),微核率與砷濃度間呈顯著正相關(guān),微核率在一定程度上能夠反映水體中的砷污染程度。因此,植物根尖微核技術(shù)可用于檢測環(huán)境中砷污染的水體。
[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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363

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