【摘要】：目的：研究ST誘導(dǎo)的DNA損傷與細(xì)胞內(nèi)溶酶體膜穩(wěn)定性以及ROS水平的關(guān)系，旨在探討ST遺傳毒性的機(jī)制。 方法：實(shí)驗(yàn)體系選定Hep G2細(xì)胞，為了評(píng)價(jià)ST的遺傳毒性，首先通過(guò)利用單細(xì)胞凝膠電泳（SCGE）實(shí)驗(yàn)(彗星實(shí)驗(yàn)）來(lái)檢測(cè)HepG2細(xì)胞DNA的損傷狀況。為了進(jìn)一步研究ST遺傳毒性發(fā)生的途徑，我們檢測(cè)了細(xì)胞中ROS的水平，通過(guò)2’，7’ 二氫二氯熒光素（DCFH）法來(lái)完成，用熒光色素吖啶橙（Acridine orange，AO）測(cè)定細(xì)胞內(nèi)溶酶體膜的穩(wěn)定性，，用免疫組化技術(shù)分析ST誘導(dǎo)DNA中產(chǎn)生的8羥基脫氧鳥(niǎo)苷（8-OHdG）的水平；同時(shí)，分別采用氯化銨（NH4CL）和N-乙酰半胱氨酸（N-Acetylcysteine,NAC）干預(yù)ST所導(dǎo)致的DNA損傷。應(yīng)用SPSSv11.5統(tǒng)計(jì)軟件包對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行統(tǒng)計(jì)分析。 結(jié)果：HepG2細(xì)胞在經(jīng)過(guò)0.5~2μg/ml的ST染毒處理1h后，細(xì)胞溶酶體膜穩(wěn)定性發(fā)生改變，通透性增加，細(xì)胞內(nèi)ROS水平增加，8-OHdG表達(dá)水平增加，DNA鏈發(fā)生斷裂，細(xì)胞形成拖尾，呈彗星樣，且其尾長(zhǎng)同對(duì)照組相比增長(zhǎng)明顯并且呈劑量依賴關(guān)系。與此同時(shí)，分別用10mM的NH4CL和NAC對(duì)HepG2細(xì)胞進(jìn)行1h的預(yù)處理后，ST所引起的DNA損傷鏈斷裂幾乎完全被抑制。溶酶體內(nèi)部的酸性環(huán)境維持了溶酶體膜的穩(wěn)定性，經(jīng)10mM的NH4CL預(yù)處理降低了細(xì)胞內(nèi)的PH值后，ST所引起的Hep G2細(xì)胞溶酶體膜穩(wěn)定性得到很好的保護(hù)；NAC是已知有效的抗氧化劑，在經(jīng)過(guò)10mM的NAC預(yù)處理1h的干預(yù)試驗(yàn)后，明顯降低了細(xì)胞內(nèi)ROS產(chǎn)生的水平。 結(jié)論：雜色曲霉素會(huì)導(dǎo)致Hep G2細(xì)胞DNA鏈發(fā)生斷裂，對(duì)DNA造成損傷，具有DNA損傷毒性。其作用機(jī)制可能與溶酶體途徑以及氧化應(yīng)激途徑有關(guān)。在ST的毒性作用下，溶酶體膜穩(wěn)定性遭到破壞，從而釋放一些酸性水解酶，導(dǎo)致DNA鏈斷裂；ST也可能通過(guò)引起HepG2細(xì)胞發(fā)生氧化應(yīng)激，導(dǎo)致細(xì)胞內(nèi)ROS水平升高，DNA氧化損傷標(biāo)志物8-OHdG表達(dá)增強(qiáng)，從而對(duì)DNA造成氧化性損傷。此外，NAC作為一種有效的抗氧化劑，減輕了ST對(duì)HepG2細(xì)胞所致的DNA鏈斷裂，說(shuō)明ST所引起的DNA損傷也可能是氧化性的損傷。
[Abstract]:Aim: to study the relationship between DNA damage induced by ST and lysosome membrane stability and ROS level in order to explore the mechanism of ST genotoxicity. Methods: in order to evaluate the genotoxicity of HepG2 cells, single cell gel electrophoresis (SCGE) assay (comet assay) was used to detect the damage of DNA in HepG2 cells. In order to further study the way of genotoxicity of ST, we detected the level of ROS in the cells, which was performed by the (DCFH) method of 2, 7', dichlorofluorescein dichloride, and the fluorescent pigment acridine orange (Acridine orange, was used. AO) was used to determine the stability of lysosome membrane and the level of 8-hydroxy-deoxyguanosine (8-OHdG) in DNA induced by ST was analyzed by immunohistochemistry. At the same time, ammonium chloride (NH4CL) and N-acetylcysteine (NAC) were used to interfere with DNA damage induced by ST, respectively. The experimental results were analyzed by SPSSv11.5 software package. Results: after HepG2 cells were treated with 0.5 ~ 2 渭 g / ml ST for 1 h, the membrane stability of lysosome changed, permeability increased, intracellular ROS level increased, 8-OHdG expression increased, DNA strand breaks, and cells formed tail-dragging. The tail length was significantly increased in a dose-dependent manner compared with the control group. At the same time, after pretreatment of HepG2 cells with NH4CL and NAC of 10mM for 1 h, the damage chain breaks of DNA induced by ST were almost completely inhibited. The lysosome inner acidic environment maintained the stability of lysosome membrane. After pretreatment with 10mM's NH4CL, the intracellular PH value was decreased, the lysosome membrane stability induced by ST was well protected from lysosome membrane in Hep G2 cells. NAC is known to be an effective antioxidant. After 1 h of pretreatment with NAC of 10mM, the level of intracellular ROS production was significantly decreased. Conclusion: the DNA strand breaks of Hep G2 cells can be induced by heterochromic aspergillus sp., which can damage DNA and have the toxicity of DNA damage. The mechanism may be related to lysosome pathway and oxidative stress pathway. Under the toxicity of ST, the stability of lysosome membrane was destroyed, and some acid hydrolases were released, which led to DNA strand break. ST may also cause oxidative damage to HepG2 cells by inducing oxidative stress in HepG2 cells, resulting in an increase in intracellular ROS level and an increase in the expression of 8-OHdG, an oxidative damage marker of DNA, thus causing oxidative damage to DNA. In addition, as an effective antioxidant, NAC alleviates ST-induced DNA strand breaks in HepG2 cells, suggesting that ST-induced DNA damage may also be oxidative damage.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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