本文選題：硫化氫 + 甲醛　； 參考：《南華大學(xué)》2011年碩士論文

【摘要】：【研究背景與目的】 甲醛(formaldehyde, FA)是一種具有揮發(fā)性的有毒物質(zhì),其神經(jīng)毒性越來越受到人們的關(guān)注。甲醛暴露可直接造成中樞神經(jīng)系統(tǒng)的損害。同時,人類機體自身也可產(chǎn)生甲醛,內(nèi)源性甲醛慢性損傷可能是神經(jīng)退行性疾病發(fā)病的一個重要機制。因此,深入探討甲醛神經(jīng)毒性的防治措施具有重要意義。 硫化氫(hydrogen sulfide, H_2S)廣泛參與機體多種生理和病理過程,被認(rèn)為是繼一氧化氮和一氧化碳之后的第三類氣體信號分子。近年來,內(nèi)源性H_2S對神經(jīng)功能的調(diào)節(jié)作用已得到公認(rèn),特別是其抗氧化、神經(jīng)保護的功能倍受關(guān)注。 越來越多的證據(jù)顯示氧化應(yīng)激是甲醛暴露時產(chǎn)生的最關(guān)鍵效應(yīng)。是否可以用H_2S來防治甲醛的神經(jīng)毒性呢?值得深入研究。為此,本研究將以甲醛損害PC12細(xì)胞為甲醛神經(jīng)毒性的細(xì)胞模型,探討H_2S對甲醛神經(jīng)毒性的拮抗作用及其機制。 【方法】 胎盤藍拒染法觀察PC12細(xì)胞的存活率;Elisa法檢測細(xì)胞上清液中乳酸脫氫酶(lactate dehydrogenase, LDH)的含量,評價PC12細(xì)胞的損傷程度;Hoechst 33258染色后,熒光顯微鏡下觀察PC12細(xì)胞核染色體形態(tài)改變及PI染色流式細(xì)胞儀(flow cytometry, FCM)檢測細(xì)胞凋亡;羅丹明123 (Rhodamine123, Rh123)染色后,FCM檢測細(xì)胞線粒體膜電位(mitochondrial membrane potential, MMP);JC-1染色后,熒光顯微鏡下觀察細(xì)胞內(nèi)MMP變化; DCFH-DA染色后,熒光顯微鏡及FCM檢測細(xì)胞內(nèi)活性氧(reactive oxydren species, ROS)水平;Western Blot檢測PC12細(xì)胞內(nèi)Bcl-2和對氧磷酶-1 (Paraoxonase-1, PON-1)的蛋白表達狀況以及細(xì)胞色素c (cytochrome c, Cyt-c)的釋放;分光光度計法檢測PC12細(xì)胞內(nèi)PON-1活性。 【結(jié)果】 1.甲醛對PC12細(xì)胞的毒性作用及機制 甲醛(60、120、240μmol/L)處理PC12細(xì)胞24 h后,能濃度依賴性地抑制PC12細(xì)胞的活力、促進細(xì)胞LDH的釋放。PC12細(xì)胞經(jīng)120μmol/L甲醛處理24 h后,細(xì)胞變成圓形或橢圓形,而且可見部分細(xì)胞游離于孔壁呈懸浮狀態(tài),并有大量的胞核呈現(xiàn)為強熒光的凋亡細(xì)胞;PI染色FCM定量分析亦表明甲醛(120μmol/L)作用24 h可顯著誘導(dǎo)PC12細(xì)胞凋亡。上述結(jié)果表明甲醛對PC12細(xì)胞具有毒性作用。 120μmol/L甲醛作用9 h后,細(xì)胞MMP下降;PC12細(xì)胞經(jīng)60、120、240μmol/L甲醛作用24 h后,Bcl-2蛋白表達水平呈濃度依賴性地降低,Cyt-c的釋放呈濃度依賴性地增強,表明甲醛可通過激活細(xì)胞線粒體凋亡通路誘導(dǎo)細(xì)胞凋亡和神經(jīng)毒性。 120μmol/L甲醛處理PC12細(xì)胞9 h后,細(xì)胞內(nèi)ROS顯著升高;甲醛(60、120、240μmol/L)作用24 h后,PC12細(xì)胞PON-1的蛋白表達及其酶活性呈濃度依賴性地降低,提示甲醛的神經(jīng)毒性與其抑制PON-1蛋白表達及其酶活性,進而促進ROS積累有關(guān)。 2.硫化氫對甲醛損傷PC12細(xì)胞的保護作用及機制 200μmol/L H_2S預(yù)處理30 min可明顯升高120、240μmol/L甲醛作用24 h后的PC12細(xì)胞活力,200、400μmol/L H_2S預(yù)處理30 min可明顯升高120μmol/L甲醛作用24 h后的PC12細(xì)胞活力。200μmol/L H_2S預(yù)處理PC12細(xì)胞30 min能顯著降低120μmol/L甲醛處理24 h后對細(xì)胞LDH釋放的促進作用、對細(xì)胞形態(tài)的損害作用、對細(xì)胞凋亡的誘導(dǎo)作用。這些結(jié)果表明H_2S對甲醛損傷PC12細(xì)胞具有拮抗作用。 200μmol/L H_2S預(yù)處理30 min可顯著抑制120μmol/L甲醛對PC12細(xì)胞MMP的降低作用、對Bcl-2表達的下調(diào)作用和對Cyt-c釋放的促進作用,表明H_2S可阻止甲醛激活細(xì)胞線粒體凋亡通路。 200μmol/L H_2S預(yù)處理30 min可顯著減輕120μmol/L甲醛對PC12細(xì)胞內(nèi)ROS積累的誘導(dǎo)作用、對細(xì)胞PON-1表達及其活性的抑制作用,而且200μmol/L H_2S可顯著增強PON-1的活性,表明H_2S可抑制ROS積累,阻止甲醛抑制PON-1的表達和活性,并能上調(diào)PON-1的活性。 200μmol/L PON-1的特異性抑制劑二羥基喹啉(2-Hydroxy-4-methylquinoline, 2-HQ)預(yù)處理30 min可顯著降低200μmol/L H_2S對甲醛(120μmol/L, 24h)促進PC12細(xì)胞內(nèi)ROS積累、誘導(dǎo)細(xì)胞凋亡、降低PC12細(xì)胞活力的拮抗作用,表明H_2S可通過阻止甲醛抑制PON-1的表達和活性、增強PON-1的活性而降低細(xì)胞ROS的積累,進而阻止甲醛激活細(xì)胞線粒體凋亡通路而實現(xiàn)其抗甲醛神經(jīng)毒性作用。 【結(jié)論】 1.甲醛對PC12細(xì)胞具有細(xì)胞毒性作用,其機理可能與其抑制細(xì)胞PON-1蛋白的表達和活性,從而促進細(xì)胞內(nèi)ROS的積累,進而激活細(xì)胞線粒體凋亡通路有關(guān)。 2.硫化氫可拮抗甲醛對PC12細(xì)胞的毒性作用,其機理可能與其增強細(xì)胞PON-1活性、減輕甲醛對PON-1蛋白表達和活性的抑制作用,從而降低細(xì)胞內(nèi)ROS含量,并進一步抑制甲醛激活細(xì)胞線粒體凋亡通路有關(guān)。
[Abstract]:[research background and purpose]
Formaldehyde (FA) is a volatile toxic substance, and its neurotoxicity is attracting more and more attention. Formaldehyde exposure can directly cause damage to the central nervous system. At the same time, the human body can also produce formaldehyde. The chronic damage of endogenous formaldehyde may be an important mechanism for the pathogenesis of neurodegenerative disease. Therefore, it is of great significance to explore the prevention and cure measures of formaldehyde neurotoxicity.
Hydrogen sulfide (H_2S) is widely involved in various physiological and pathological processes of the body. It is considered to be the third kind of gas signal after nitric oxide and carbon monoxide. In recent years, the regulation of endogenous H_2S has been recognized, especially its anti oxidation, and the function of neuroprotection has attracted much attention.
More and more evidence shows that oxidative stress is the most important effect of formaldehyde exposure. Is it possible to use H_2S to prevent the neurotoxicity of formaldehyde? It is worth further study. Therefore, this study will explore the antagonism and mechanism of H_2S to formaldehyde neurotoxicity by using formaldehyde to damage the PC12 cell as a formaldehyde neurotoxicity.
[method]
The survival rate of PC12 cells was observed by placental blue staining; the content of lactate dehydrogenase (LDH) in cell supernatant was detected by Elisa method and the damage degree of PC12 cells was evaluated. After Hoechst 33258 staining, the morphological changes of chromosomes in PC12 and PI staining flow cytometer (flow cytometry, FCM) were observed under the fluorescence microscope. After staining with Luo Danming 123 (Rhodamine123, Rh123), the mitochondrial membrane potential (mitochondrial membrane potential, MMP) was detected by FCM. After JC-1 was stained, the MMP changes in the cells were observed under the fluorescence microscope. After DCFH-DA staining, the fluorescence microscope and FCM detected the intracellular reactive oxygen species. T detected the expression of Bcl-2 in PC12 cells and the expression of -1 (Paraoxonase-1, PON-1) and the release of cytochrome c (cytochrome c, Cyt-c), and the spectrophotometer was used to detect the PON-1 activity in the PC12 cells.
[results]
The toxic effect of 1. formaldehyde on PC12 cells and its mechanism
After the treatment of 24 h by formaldehyde (60120240 mol/L), the activity of PC12 cells can be inhibited in a concentration dependent manner, which promotes the release of.PC12 cells from LDH by 120 mu mol/L formaldehyde to treat 24 h, and the cells become round or oval, and some cells are suspended in the wall of the pore, and a large number of nuclei appear to be strong fluorescent apoptosis. PI staining FCM quantitative analysis also showed that formaldehyde (120 u mol/L) action of 24 h could significantly induce apoptosis of PC12 cells. The above results showed that formaldehyde had toxic effects on PC12 cells.
After the action of 120 mu mol/L formaldehyde for 9 h, the cell MMP decreased, and the expression level of Bcl-2 protein decreased in a concentration dependent manner after the 24 h action of 60120240 mu mol/L formaldehyde. The release of Cyt-c increased in a concentration dependent manner, indicating that formaldehyde could induce apoptosis and neurotoxicity by activating the apoptosis pathway of mitochondria.
After 120 mu mol/L formaldehyde treated PC12 cells for 9 h, the intracellular ROS increased significantly. After 24 h of formaldehyde (60120240 mu mol/L), the protein expression and enzyme activity of PON-1 in PC12 cells decreased in a concentration dependent manner. It suggested that the neurotoxicity of formaldehyde was related to the inhibition of the expression of PON-1 protein and the activity of the enzyme, thus promoting the accumulation of ROS.
Protective effect and mechanism of hydrogen sulfide on formaldehyde injured PC12 cells 2.
200 mu mol/L H_2S pretreatment 30 min can significantly increase the activity of PC12 cells after 120240 u mol/L formaldehyde effect 24 h, 200400 mu mol/L H_2S pretreatment 30 min can obviously increase the activity of 120 mu mol/L after 24 h PC12 cells 30 These results indicate that H_2S has an antagonistic effect on formaldehyde induced PC12 cells.
200 mol/L H_2S pretreatment 30 min could significantly inhibit the decrease of 120 mu mol/L on PC12 cell MMP, the down regulation of Bcl-2 expression and the promotion of Cyt-c release, indicating that H_2S can prevent the apoptosis pathway of formaldehyde activated mitochondria.
200 mu mol/L H_2S pretreatment 30 min could significantly reduce the induction of ROS accumulation in PC12 cells by 120 mu mol/L, and inhibit the expression and activity of PON-1 in cell, and 200 u mol/L H_2S significantly enhanced the activity of PON-1, indicating that H_2S could inhibit the accumulation of ROS, prevent the expression and activity of formaldehyde inhibited and up regulate the activity of PON-1.
The pre treatment of two hydroxyquinoline (2-Hydroxy-4-methylquinoline, 2-HQ), a specific inhibitor of 200 mol/L PON-1, could significantly reduce the inhibitory effect of 200 mu H_2S on the accumulation of formaldehyde (120 mu mol/L, 24h) in PC12 cells, inducing cell apoptosis and reducing the activity of PC12 cells. Activity can enhance the activity of PON-1 and decrease the accumulation of ROS, thereby preventing formaldehyde from activating mitochondrial apoptosis pathway and achieving its neuroprotective effect against formaldehyde.
[Conclusion]
1. formaldehyde has cytotoxic effect on PC12 cells. The mechanism may be related to the inhibition of the expression and activity of PON-1 protein in cell, thus promoting the accumulation of ROS in cells and activating the apoptosis pathway of cell mitochondria.
2. hydrogen sulfide can antagonize the toxic effect of formaldehyde on PC12 cells. Its mechanism may enhance the activity of PON-1, reduce the inhibitory effect of formaldehyde on the expression and activity of PON-1 protein, thus reduce the intracellular ROS content, and further inhibit the activation of the mitochondrial apoptosis pathway by formaldehyde.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

【參考文獻】

相關(guān)期刊論文 前5條

1 范衛(wèi),王法弟,賈曉東,金復(fù)生,金錫鵬;近十年國內(nèi)有關(guān)甲醛的環(huán)境與職業(yè)危害調(diào)查研究[J];環(huán)境與職業(yè)醫(yī)學(xué);2004年02期

2 李芳序;盧靜;許亞杰;童志前;聶春來;赫榮喬;;老年性癡呆發(fā)病過程中內(nèi)源性甲醛慢性損傷機制[J];生物化學(xué)與生物物理進展;2008年04期

3 ;The protective effect of vitamin E against oxidative damage caused by formaldehyde in the testes of adult rats[J];Asian Journal of Andrology;2006年05期

4 唐國華,龍治峰,賀修勝,唐朝克;石蠟切片厚度對流式細(xì)胞儀檢測細(xì)胞DNA的影響[J];中華病理學(xué)雜志;2004年06期

5 唐國華;何淑雅;賀修勝;謝紅衛(wèi);;流式細(xì)胞儀每秒進樣細(xì)胞數(shù)對細(xì)胞G_0/G_1期峰的熒光強度值及變異系數(shù)值的影響[J];中華病理學(xué)雜志;2005年12期

，

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