王霞，董文斌，李清平，康蘭，雷小平，翟雪松，趙帥(四川醫(yī)科大學(xué)附屬第一醫(yī)院，四川瀘州 646000)

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高氧對(duì)人肺泡上皮細(xì)胞凋亡的影響及其作用機(jī)制

王霞，董文斌，李清平，康蘭，雷小平，翟雪松，趙帥(四川醫(yī)科大學(xué)附屬第一醫(yī)院，四川瀘州 646000)

摘要：目的觀察高氧對(duì)人肺泡上皮細(xì)胞(HPAEC)凋亡的影響，并探討其機(jī)制。方法 取對(duì)數(shù)生長(zhǎng)期HPAEC細(xì)胞，隨機(jī)分為高氧組和對(duì)照組。對(duì)照組細(xì)胞不作處理，放置于50 mL/L CO2培養(yǎng)箱中培養(yǎng)；高氧組細(xì)胞換液1次后，以3 L/min速度通入含900 mL/L O2和50 mL/L CO2高純混合氣，通入時(shí)間10 min；兩組細(xì)胞均培養(yǎng)24 h。采用流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率，倒置相差顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化，免疫共聚焦檢測(cè)細(xì)胞組蛋白去乙?；?SIRT1)轉(zhuǎn)位情況，Western blotting法檢測(cè)細(xì)胞SENP1蛋白、乙?；痯53蛋白。結(jié)果高氧組和對(duì)照組細(xì)胞凋亡率分別為24.77%±2.17%、5.33%±2.60%，兩組比較，P<0.05。高氧組細(xì)胞從正常的長(zhǎng)梭形、多角形變成圓形、橢圓形，細(xì)胞間的間隙增寬，懸浮的細(xì)胞較多；對(duì)照組細(xì)胞大多為長(zhǎng)梭形、多角形，貼壁較好，細(xì)胞間的間隙較小，懸浮的細(xì)胞較少。高氧組和對(duì)照組細(xì)胞SIRT1蛋白轉(zhuǎn)位率分別為88.89%、16.23%，兩組比較，P<0.05。高氧組和對(duì)照組細(xì)胞SENP1蛋白相對(duì)表達(dá)量分別為0.76±0.12、0.67±0.02，乙酰化p53蛋白相對(duì)表達(dá)量分別為0.81±0.07、0.52±0.03，兩組比較，P均<0.05。結(jié)論 高氧可促進(jìn)HPAEC細(xì)胞凋亡，其機(jī)制可能是高氧誘導(dǎo)HPAEC細(xì)胞SENP1蛋白表達(dá)增加，進(jìn)而SIRT1蛋白從胞核轉(zhuǎn)位到胞質(zhì)，使乙?；痯53蛋白表達(dá)增加。

關(guān)鍵詞：高濃度氧；人肺泡上皮細(xì)胞；組蛋白去乙?；福籗ENP1蛋白；乙?；痯53蛋白

近年隨著科技的進(jìn)步，越來越多的極度早產(chǎn)兒和極低出生體質(zhì)量?jī)盒掖嫦聛?，但是由于長(zhǎng)期吸入高濃度氧，患兒可發(fā)生肺損傷，嚴(yán)重者甚至發(fā)生支氣管肺發(fā)育不良(BPD)[1,2]。目前，高氧性肺損傷的機(jī)制尚不清楚，最近研究的熱點(diǎn)主要集中在氧化應(yīng)激方面[3,4]，而NAD+依賴性的組蛋白去乙?；?SIRT1)在氧化應(yīng)激及DNA損傷修復(fù)中發(fā)揮重要作用[5]。本研究在前期實(shí)驗(yàn)基礎(chǔ)上，通過建立高氧損傷人肺泡上皮細(xì)胞(HPAEC)模型[6]，探討SIRT1轉(zhuǎn)位是否介導(dǎo)肺泡上皮細(xì)胞發(fā)生凋亡，從而為減輕高氧肺損傷尋找新的治療靶點(diǎn)。

1材料與方法

1.1細(xì)胞、儀器及試劑HPAEC細(xì)胞購自廣州吉妮歐生物科技有限公司。LX71型倒置相差顯微鏡購自O(shè)lympus公司，F(xiàn)ORMA311型CO2培養(yǎng)箱、胎牛血清、DMEM高糖培養(yǎng)基購自Gibco公司，ANNEXIN V流式細(xì)胞凋亡檢測(cè)試劑盒購自凱基生物公司，單克隆小鼠抗SIRT1抗體購自博士德生物公司，羅丹明標(biāo)記山羊抗小鼠IgG購自ZSGB-BIO公司，DAPI購自碧云天生物公司，抗熒光淬滅封片液購自碧云天生物公司，單克隆小鼠抗SENP1抗體與單克隆抗乙酰化p53lys(382)抗體購自Abcam公司，ECL化學(xué)發(fā)光試劑購自北京普利萊基因技術(shù)有限公司。

l.2HPAEC細(xì)胞培養(yǎng)先將HPAEC細(xì)胞復(fù)蘇，加入含有10%胎牛血清的DMEM高糖培養(yǎng)液，放入37 ℃、含5%CO2培養(yǎng)箱中培養(yǎng)。待細(xì)胞生長(zhǎng)至對(duì)數(shù)生長(zhǎng)期時(shí)，用0.25%的胰蛋白酶進(jìn)行消化，傳代培養(yǎng)。

1.3HPAEC細(xì)胞分組及高氧干預(yù)取對(duì)數(shù)生長(zhǎng)期的細(xì)胞，消化后傳代接種于培養(yǎng)瓶中，隨機(jī)分為高氧組、對(duì)照組。對(duì)照組不作處理，放置于50 mL/L CO2培養(yǎng)箱中培養(yǎng)；高氧組換液1次后，以3 L/min的速度通入含900 ml/L O2和50 mL/L CO2高純混合氣，通入時(shí)間為10 min，參照我們前期高氧模型建立的方法[6]。分別培養(yǎng)24 h(高氧組細(xì)胞干預(yù)24 h后，使用測(cè)氧儀檢測(cè)培養(yǎng)瓶中氧濃度，如果氧濃度<90%則棄去該標(biāo)本)，之后收集HPAEC細(xì)胞。

1.4HPAEC細(xì)胞凋亡情況觀察兩組細(xì)胞培養(yǎng)24 h后，收集細(xì)胞，用流式細(xì)胞儀檢測(cè)，按照凱基ANNEXIN V-FITC細(xì)胞凋亡檢測(cè)試劑盒說明書操作。

1.5HPAEC細(xì)胞形態(tài)學(xué)觀察倒置相差顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化，并照相。

1.6HPAEC細(xì)胞SIRT1蛋白轉(zhuǎn)位情況觀察將HPAEC細(xì)胞消化后，按細(xì)胞密度為2×104/孔接種于6孔板中蓋玻片上培養(yǎng)，同步化細(xì)胞，兩組均加入1 mL培養(yǎng)基，高氧組另通入高濃度氧，分別培養(yǎng)24 h；多聚甲醛固定細(xì)胞10 min；0.3%Triton X-100打孔10 min；5%封閉血清在37 ℃封閉30 min；將稀釋的抗體SIRT1(終濃度為1∶100)滴加在細(xì)胞爬片上，4 ℃冰箱孵育過夜，在避光情況下將羅丹明標(biāo)記的山羊抗小鼠IgG(終濃度為1∶50)，37 ℃濕盒中孵育60 min；用PBS洗滌3次，滴加DAPI復(fù)染，免疫共聚焦顯微鏡下觀察并照相；每張細(xì)胞爬片截取5個(gè)視野，每組8張爬片，并照相，計(jì)算細(xì)胞轉(zhuǎn)位率。

1.7HPAEC細(xì)胞SENP1、乙酰化p53蛋白檢測(cè)采用Western blotting法。兩組細(xì)胞培養(yǎng)干預(yù)24 h(細(xì)胞生長(zhǎng)80%融合)，胰酶消化后PBS沖洗，離心并移去上清。加入200 μL裂解混合液，冰浴30 min，離心取上清，電泳、轉(zhuǎn)膜，加入一抗，再加入顯色的二抗，以TBST清洗，使用ECL進(jìn)行結(jié)果的顯影，然后用Labworks4.6分析目的條帶的灰度值。

2結(jié)果

高氧組和對(duì)照組細(xì)胞凋亡率分別為24.77%±2.17%、5.33%±2.60%，兩組比較，P<0.05。高氧組細(xì)胞從正常的長(zhǎng)梭形、多角形變成圓形、橢圓形，細(xì)胞間的間隙增寬，懸浮的細(xì)胞較多；對(duì)照組細(xì)胞大多為長(zhǎng)梭形、多角形，貼壁較好，細(xì)胞間的間隙較小，懸浮的細(xì)胞較少。高氧組和對(duì)照組細(xì)胞SIRT1蛋白轉(zhuǎn)位率分別為88.89%(96/108)、16.23%(25/154)，兩組比較，P<0.05。高氧組和對(duì)照組細(xì)胞SENP1蛋白相對(duì)表達(dá)量分別為0.76±0.12、0.67±0.02，乙?；痯53蛋白相對(duì)表達(dá)量分別為0.81±0.07、0.52±0.03，兩組比較，P均<0.05。

3討論

近年來，早產(chǎn)兒的出生率逐年增高，早產(chǎn)兒中大部分會(huì)發(fā)生夭折，氧療是提高早產(chǎn)兒存活率的有效方法，但是長(zhǎng)時(shí)間高濃度吸氧可導(dǎo)致肺損傷，從而導(dǎo)致BPD的發(fā)生[7]?；钚匝醮?ROS)增多所致的氧化應(yīng)激損傷是早產(chǎn)兒高氧肺損傷發(fā)生的主要機(jī)制[8]，而SIRT1蛋白能顯著降低ROS水平和促進(jìn)細(xì)胞生存[9]。

SIRT1蛋白是一種NAD+依賴性脫乙酰酶，作為哺乳動(dòng)物生命周期相關(guān)蛋白，其主要功能是調(diào)節(jié)細(xì)胞氧化應(yīng)激反應(yīng)和調(diào)控生命周期。SIRT1蛋白主要定位于細(xì)胞核，在細(xì)胞能量代謝、DNA損傷修復(fù)、細(xì)胞周期控制、抑制細(xì)胞凋亡、抗氧化逆境和延長(zhǎng)細(xì)胞壽命方面發(fā)揮重要的調(diào)控作用，是機(jī)體內(nèi)抗氧化應(yīng)激的關(guān)鍵蛋白[10,11]。Yang等[12]用紫外線輻射、過氧化氫誘導(dǎo)人肺腺癌細(xì)胞后發(fā)現(xiàn)，SIRT1蛋白可發(fā)生翻譯后修飾，在734位賴氨酸上發(fā)生小泛素樣修飾蛋白(SUMO)修飾。該修飾決定著SIRT1蛋白在細(xì)胞核的定位。這是一個(gè)動(dòng)態(tài)的、可逆的過程，其去SUMO修飾的過程則由SENP1介導(dǎo)[13,14]。SIRT1蛋白主要存在于細(xì)胞核，少量存在于細(xì)胞質(zhì)，當(dāng)細(xì)胞應(yīng)激時(shí)SIRT1蛋白能被SENP1去SUMO化，使p53蛋白的第382位賴氨酸殘基去乙?；瘻p少，抑制p53與靶DNA順式原件結(jié)合，進(jìn)而抑制p53促凋亡活性[15～18]。本研究顯示，高氧組細(xì)胞生長(zhǎng)較差，細(xì)胞間隙增寬，懸浮細(xì)胞較多，細(xì)胞的凋亡率增加，這與我們的前期實(shí)驗(yàn)[6]結(jié)果一致，即高氧可以誘導(dǎo)細(xì)胞凋亡。本研究還顯示，高氧組SENP1蛋白表達(dá)、SIRT1轉(zhuǎn)位率、乙?；痯53表達(dá)均較對(duì)照組升高，這與Yang等[12]結(jié)果一致。提示高氧可誘導(dǎo)細(xì)胞凋亡，其凋亡的機(jī)制與SIRT1蛋白相關(guān)。

總之，高氧誘導(dǎo)SENP1蛋白表達(dá)，促使SIRT1蛋白發(fā)生去SUMO化，發(fā)生核質(zhì)轉(zhuǎn)位，去乙?；钚詼p低，對(duì)p53去乙?；钚詼p低，乙?；痯53相對(duì)增加，從而激活下游的凋亡通路而介導(dǎo)細(xì)胞凋亡。

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Effects of hyperoxia on apoptosis of human alveolar epithelial cells and the mechanism

WANGXia,DONGWenbin,LIQingping,KANGLan,LEIXiaoping,ZHAIXuesong,ZHAOShuai

(TheFirstAffiliatedHospitalofSichuanMedicalUniversity,Luzhou646000,China)

Abstract:ObjectiveTo observe the influence of hyperoxia on the apoptosis of human alveolar epithelial cells (HPAEC), and to explore its mechanism. MethodsThe HPAEC cells in the logarithmic phase were randomly divided into the hyperoxia group and control group. The cells of the control group were not processed and were placed in 50 mL/LCO2incubator. After one time of liquid change, the cells of the hyperoxia group were exposed to a mixture of O2(900 mL/L) and CO2(50mL/L) for 10 minutes with speed of 3 L/min. After 24 hours, the apoptosis rates of the two groups were detected by flow cytometry, the morphological changes were observed by the inverted phase contrast microscope, the transposition of histone deaceylase (SIRT1) was detected by immunofluorescence and the expression of SENP1 and acetylated p53 proteins were measured by Western blotting. ResultsThe apoptosis rates of the hyperoxia group and control group were 24.77%±2.17% and 5.33%±2.60%, respectively (P<0.05). In the hyperoxia group, the shapes of cells changed from normal long spindle, polygon into the circle or ellipse, the distance between cells was enlarged, and the suspension cells were more. In the control group, HPAEC cells were in good condition, closely to each other and the suspension cells were less. The transposition rates of SIRT1 protein in the hyperoxia group and control group were 88.89% and 16.23%, respectively (P<0.05). The expression levels of SENP1 protein in the two groups were separately 0.76±0.12 and 0.67±0.02 (P<0.05). The acetylated p53 protein expression of the two groups was 0.81±0.07 and 0.52±0.03, respectively (P<0.05).Conclusion Hyperoxia can promote HPAEC cell apoptosis, and its mechanism may be that the expression of SENP1 protein is increased, which promotes the transposition of SIRT1 protein from the nucleus to the cytoplasm, then the expression of acetylated p53 protein is increased.

Key words:hyperoxia; human alveolar epithelial cells; histone deaceylase; SENP1 protein; acetylated p53 protein

(收稿日期:2015-09-22)

中圖分類號(hào)：Q255

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1002-266X(2016)03-0014-03

doi：10.3969/j.issn.1002-266X.2016.03.005

通信作者簡(jiǎn)介：董文斌(1967-)，男，碩士，教授，主要研究方向?yàn)楦哐醴螕p傷。E-mail: DongWenbin2000@163.com

作者簡(jiǎn)介：第一王霞(1987-)，女，碩士，主要研究方向?yàn)楦哐醴螕p傷。E-mail: 13982400021@163.com

基金項(xiàng)目：國家自然科學(xué)基金資助項(xiàng)目(81571480)；中華兒科雜志第二屆雙鶴珂立蘇科研基金資助項(xiàng)目(cjp2011-009);

四川省教育廳科研基金資助項(xiàng)目(08ZA150)；四川省衛(wèi)生廳科研基金資助項(xiàng)目(90191)。