韋睿, 吳杰, 曾偉, 張瑋

(昆明醫(yī)科大學(xué)第一附屬醫(yī)院，昆明650032)

SGK-1在缺氧誘導(dǎo)小鼠垂體瘤細(xì)胞AtT-20增殖、凋亡中的表達(dá)及作用探討

韋睿, 吳杰, 曾偉, 張瑋

(昆明醫(yī)科大學(xué)第一附屬醫(yī)院，昆明650032)

目的 觀察血清和糖皮質(zhì)激素調(diào)節(jié)蛋白激酶1(SGK-1)在缺氧誘導(dǎo)小鼠垂體瘤細(xì)胞AtT-20增殖、凋亡中的表達(dá)，并探討其在這一過(guò)程中的作用。方法 將AtT-20細(xì)胞隨機(jī)分為觀察組和對(duì)照組，觀察組分別加入50、100、200 μmol/L CoCl2(模擬缺氧)，對(duì)照組不處理，采用MTT法檢測(cè)24、48、72、96 h時(shí)細(xì)胞增殖的A570值；將200 μmol/L CoCl2加入AtT-20細(xì)胞，培養(yǎng)0、24、48、72、96 h時(shí)采用流式細(xì)胞術(shù)測(cè)算細(xì)胞凋亡率，分別采用半定量PCR法和Western blot法檢測(cè)SGK-1 mRNA及蛋白。將AtT-20細(xì)胞隨機(jī)分為4組，A、B組轉(zhuǎn)染干擾質(zhì)粒SGK-1 siRNA，C、D組轉(zhuǎn)染對(duì)照質(zhì)粒siCon，轉(zhuǎn)染24 h后A、C 組加入200 μmol/L CoCl2，B、D組不處理，分別采用MTT法和流式細(xì)胞術(shù)檢測(cè)24、48、72、96 h時(shí)細(xì)胞增殖的A570值及細(xì)胞凋亡率。結(jié)果 缺氧處理72 h后，觀察組隨著CoCl2濃度增加及作用時(shí)間延長(zhǎng)，AtT-20細(xì)胞增殖的A570值逐漸降低，與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P均<0.05)。與0 h時(shí)比較，處理24、48 h時(shí)細(xì)胞凋亡率無(wú)明顯變化，處理72、96 h時(shí)細(xì)胞凋亡率增加(P均<0.05)；與0 h時(shí)比較，處理24、48時(shí)AtT-20細(xì)胞SGK-1 mRNA及蛋白表達(dá)量均增加(P均<0.05)，72 h后其表達(dá)量無(wú)顯著變化(P均>0.05)。A組缺氧后各時(shí)點(diǎn)細(xì)胞增殖的A570值均低于其余各組，C組缺氧后72、96 h細(xì)胞增殖的A570值高于A組而低于B、C組(P均<0.05)。A組缺氧后各時(shí)點(diǎn)細(xì)胞凋亡率均高于其余各組，C組缺氧后72 h細(xì)胞凋亡率低于A組而高于B、C組(P均<0.05)。結(jié)論 缺氧可抑制AtT-20細(xì)胞增殖、促進(jìn)其凋亡，該過(guò)程中SGK-1表達(dá)增加可保護(hù)細(xì)胞免受缺氧導(dǎo)致的損傷。

腦缺氧；垂體瘤細(xì)胞；血清和糖皮質(zhì)激素調(diào)節(jié)蛋白激酶；氯化鈷；基因干擾；細(xì)胞凋亡；細(xì)胞增殖

在大鼠乳腺癌細(xì)胞中，有學(xué)者通過(guò)糖皮質(zhì)激素誘導(dǎo)轉(zhuǎn)錄表達(dá)的差異篩選發(fā)現(xiàn)了一種新的絲氨酸/蘇氨酸蛋白激酶，當(dāng)細(xì)胞受到糖皮質(zhì)激素或血清刺激時(shí)該蛋白表達(dá)迅速升高，所以將其命名為血清和糖皮質(zhì)激素誘導(dǎo)蛋白激酶(SGK)[1]。有研究[2]表明，SGK在細(xì)胞受到外界刺激后對(duì)維持其存活起到重要作用。目前，SGK在腎臟[3]、心血管[4]、子宮等外周器官的作用研究比較多，但在中樞神經(jīng)系統(tǒng)的作用相關(guān)研究較少。早期有研究[5]發(fā)現(xiàn)，在腦損傷部位SGK-1 mRNA 表達(dá)升高,提示SGK可能參與軸突再生。動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)，前腦局灶缺血可導(dǎo)致大腦皮質(zhì)中SGK-1表達(dá)明顯增加,提示SGK作為一種即早基因,在全腦缺血應(yīng)激條件下迅速被誘導(dǎo)、轉(zhuǎn)錄及表達(dá)[6～10]。2014年9月～2016年3月，我們觀察了SGK-1在缺氧誘導(dǎo)小鼠垂體瘤細(xì)胞AtT-20增殖、凋亡中的表達(dá)變化，以初步闡釋SGK-1在腦缺血過(guò)程中的重要作用。

1 材料與方法

1.1 細(xì)胞培養(yǎng)與缺氧誘導(dǎo) 小鼠垂體瘤細(xì)胞AtT-20購(gòu)自中國(guó)科學(xué)院昆明細(xì)胞庫(kù)，培養(yǎng)于含10%胎牛血清的RPMI1640培養(yǎng)基中；每1～2天換液1次，3～4 d離心傳代1次。用CoCl2刺激細(xì)胞模擬缺氧環(huán)境[11]:用50～200 μmol/L的CoCl2刺激細(xì)胞96 h，將細(xì)胞置于5% CO2培養(yǎng)箱中37 ℃培養(yǎng)。

1.2 缺氧對(duì)細(xì)胞增殖、凋亡影響的觀察

1.2.1 細(xì)胞增殖情況觀察 采用MTT法。取對(duì)數(shù)生長(zhǎng)期的AtT-20細(xì)胞，以1×104/mL的密度接種于96孔板中，接種體積為200 μL/孔。培養(yǎng)8 h穩(wěn)定后隨機(jī)分為觀察組和對(duì)照組，觀察組分別加入50、100、200 μmol/L CoCl2，對(duì)照組不處理，每個(gè)孔設(shè)置5個(gè)復(fù)孔。孵育24、48、72、96 h后，每孔中加入5 mg/mL的MTT(Sigma公司)溶液20 μL，繼續(xù)培養(yǎng)4 h后輕輕吸去上清；加入150 μL DMSO(上海生工生物工程股份有限公司)，震蕩15 min，用酶標(biāo)儀測(cè)定570 nm的吸光度，以A570值表示細(xì)胞的增殖情況。

1.2.2 細(xì)胞凋亡情況觀察 采用流式細(xì)胞術(shù)。取對(duì)數(shù)生長(zhǎng)期的AtT-20細(xì)胞，以1×104/mL的密度接種于96孔板中，接種體積為200 μL/孔。培養(yǎng)8 h穩(wěn)定后，加入200 μmol/L CoCl2；分別于孵育0、24、48、72、96 h時(shí)，用PBS洗滌2次，進(jìn)行Annexin V/PI雙染色(南京凱基生物科技發(fā)展有限公司)，上流式細(xì)胞檢測(cè)凋亡細(xì)胞，計(jì)算細(xì)胞凋亡率。

1.3 缺氧對(duì)細(xì)胞SGK-1表達(dá)影響的觀察

1.3.1 SGK-1 mRNA 檢測(cè) 采用PRC半定量法。細(xì)胞接種及處理同1.2.2，使用RNA Simple總RNA提取試劑盒(北京天根生化科技有限公司)提取各組細(xì)胞總RNA，定量后用PrimeScriptTM1st Strand cDNA Synthesis Kit(大連寶生物工程有限公司)合成cDNA第一鏈；以cDNA第一鏈為模板進(jìn)行PCR反應(yīng)，用1.5%瓊脂糖凝膠電泳檢測(cè)PCR產(chǎn)物并對(duì)其進(jìn)行半定量，以2-ΔΔct計(jì)算其相對(duì)表達(dá)量。SGK-1上游引物5′-CTCCGCCAAGTCCCTCTCAACAAAT-3′，下游引物5′-CTCATACAGGACAGCCCCAAGACAC-3′，產(chǎn)物長(zhǎng)度644 bp；GAPDH上游引物為5′-AATGCATCCTGCACCACCAA-3′，下游引物為5′-GTAGCCATATTCATTGTCATA-3′，產(chǎn)物長(zhǎng)度513 bp；擴(kuò)增反應(yīng)：95 ℃預(yù)變性10 s；60 ℃ 60 s，95 ℃ 15 s，40個(gè)循環(huán)；99 ℃退火60 s。

1.3.2 SGK-1蛋白檢測(cè) 采用Western blot法[12]。細(xì)胞接種及處理同1.2.2，用RIPA裂解液裂解細(xì)胞，12 000 r/min離心取上清；用Bradford法定量后行SDS-聚丙烯酰胺凝膠電泳，每孔上樣量為30 g總蛋白。電轉(zhuǎn)移至PVDF膜，并用3% BSA封閉2 h；用SGK-1抗體(美國(guó)Invitrogen公司)孵育4 h，PBS緩沖液沖洗3次；二抗孵育2 h， 用PBST緩沖液沖洗膜3次，每次沖洗10 min，最后再用PBS緩沖液沖洗1次，結(jié)合HRP底物ECL發(fā)光液用BIO-RAD Chemical XRS+顯影系統(tǒng)顯影得到條帶圖像。圖像的蛋白條帶灰度用Image J 3.0軟件定量，以目標(biāo)蛋白與對(duì)應(yīng)內(nèi)參灰度比值為目標(biāo)蛋白的相對(duì)表達(dá)量。

1.4 降SGK-1表達(dá)對(duì)缺氧環(huán)境下細(xì)胞增殖、凋亡影響的觀察

1.4.1 細(xì)胞分組與轉(zhuǎn)染處理 取對(duì)數(shù)生長(zhǎng)期的AtT-20細(xì)胞，以1×104/mL的密度接種于96孔板中，接種體積為200 μL/孔。培養(yǎng)8 h穩(wěn)定后隨機(jī)分為4組，A、B組轉(zhuǎn)染干擾質(zhì)粒SGK-1 siRNA(Santa Cruz公司)，C、D組轉(zhuǎn)染對(duì)照質(zhì)粒siCon(Santa Cruz公司)，使用HiPerFect(Qiagen公司)轉(zhuǎn)染細(xì)胞24 h。然后A、C 組均加入200 μmol/L CoCl2，B、D組不予處理。

1.4.2 細(xì)胞增殖、凋亡觀察 取培養(yǎng)24、48、72、96 h 的細(xì)胞，分別按照1.2.1、1.2.2的方法觀察各組細(xì)胞增殖、凋亡情況。

2 結(jié)果

2.1 缺氧對(duì)AtT-20細(xì)胞增殖的影響 缺氧處理72 h后，觀察組隨著CoCl2濃度增加及作用時(shí)間延長(zhǎng)，AtT-20細(xì)胞增殖的A570值逐漸降低，與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P均<0.05)。見(jiàn)表1。

表1 兩組細(xì)胞增殖情況比較±s)

注：與對(duì)照組同時(shí)點(diǎn)比較，*P<0.05；與觀察組50 μmol/L同時(shí)點(diǎn)比較，#P<0.05；與觀察組100 μmol/L同時(shí)點(diǎn)比較，△P<0.05 。

2.2 缺氧對(duì)AtT-20細(xì)胞凋亡的影響 200 μmol/L的CoCl2處理細(xì)胞0、24、48、72、96 h時(shí)，細(xì)胞凋亡率分別為10.32%±3.15%、11.26%±3.02%、11.32%±3.25%、19.87%±4.31%、24.41%±5.09%；與0 h時(shí)比較，處理24、48 h時(shí)細(xì)胞凋亡率無(wú)明顯變化，處理72、96 h時(shí)細(xì)胞凋亡率增加(P均<0.05)。

2.3 缺氧對(duì)AtT-20細(xì)胞SGK-1表達(dá)的影響 200 μmol/L 的CoCl2處理24、48時(shí)AtT-20細(xì)胞SGK-1表達(dá)均增加(P均<0.05)，72 h時(shí)其表達(dá)量與未處理(0 h)的細(xì)胞基本持平(P>0.05)。見(jiàn)表2。

表2200 μmol/L 的CoCl2處理不同時(shí)間AtT-20細(xì)胞SGK-1表達(dá)比較

注：與0 h時(shí)比較，*P<0.05。

2.4 SGK-1表達(dá)對(duì)缺氧環(huán)境下細(xì)胞增殖和凋亡的影響

2.4.1 各組細(xì)胞增殖情況比較 A組缺氧后各時(shí)點(diǎn)細(xì)胞增殖的A570值均低于其余各組，C組缺氧后72、96 h細(xì)胞增殖的A570值高于A組而低于B、C組(P均<0.05)。見(jiàn)表3。

表3 各組細(xì)胞增殖情況比較±s)

注：與B、D組比較，*P<0.05；與C組比較，#P<0.05。

2.4.2 各組細(xì)胞凋亡情況比較 A組缺氧后各時(shí)點(diǎn)細(xì)胞凋亡率均高于其余各組，C組缺氧后72 h細(xì)胞凋亡率低于A組而高于B、C組(P均<0.05)。見(jiàn)表4。

表4 各組細(xì)胞凋亡率比較

注：與B、D組比較，*P<0.05；與C組比較，#P<0.05。

3 討論

足夠的血液和氧氣供應(yīng)對(duì)維持腦的正常功能非常重要，腦缺血可導(dǎo)致腦組織死亡。因此，探索腦缺血后細(xì)胞凋亡以及保護(hù)自身細(xì)胞的機(jī)制，對(duì)于理解和治療腦缺血相關(guān)疾病有相當(dāng)重要的意義。SGK1是一種絲氨酸/蘇氨酸蛋白激酶，具有廣泛的生理功能，其表達(dá)及活性增加參與多種疾病病理過(guò)程的發(fā)生和發(fā)展[13～15]。研究發(fā)現(xiàn)，SGK1在全腦缺血再灌注損傷的大鼠海馬細(xì)胞中表達(dá)明顯增加，而在損傷的神經(jīng)元細(xì)胞中也有上調(diào)趨勢(shì)；推測(cè)SGK-1表達(dá)可能參與維持腦細(xì)胞存活的過(guò)程，從而減少腦缺血引起的腦損傷。有研究在細(xì)胞水平上發(fā)現(xiàn)，腎上腺素受體介導(dǎo)的抗凋亡信號(hào)與SGK-1有關(guān)[16～18]。因此，有充分的理由推測(cè)SGK-1表達(dá)可能參與腦缺血過(guò)程中對(duì)腦細(xì)胞的保護(hù)，我們的研究正是基于這些前期基礎(chǔ)和推測(cè)開(kāi)展的。

我們用CoCl2刺激細(xì)胞模擬細(xì)胞缺氧，并檢測(cè)缺氧對(duì)垂體瘤細(xì)胞AtT-20增殖的影響；發(fā)現(xiàn)短期(24～48 h)的低氧刺激對(duì)細(xì)胞增殖并沒(méi)有太明顯的影響，甚至可以對(duì)細(xì)胞增殖有一定的促進(jìn)作用，而長(zhǎng)期的低氧刺激則明顯抑制了細(xì)胞的增殖。長(zhǎng)期低氧刺激抑制細(xì)胞增殖是在預(yù)料之中的，而短期刺激導(dǎo)致的促細(xì)胞增殖則可能是因?yàn)榈脱醮碳ふT導(dǎo)的其他因子導(dǎo)致了細(xì)胞增殖的加快，也可以理解為細(xì)胞對(duì)低氧刺激做出的一種應(yīng)激反應(yīng)。對(duì)細(xì)胞凋亡的研究也發(fā)現(xiàn)，CoCl2刺激在短時(shí)間并不會(huì)明顯誘導(dǎo)細(xì)胞凋亡，而在刺激72 h后則可明顯誘導(dǎo)細(xì)胞凋亡。對(duì)SGK-1在受到CoCl2刺激缺氧后表達(dá)的研究發(fā)現(xiàn)，CoCl2刺激在24、48 h均可以誘導(dǎo)SGK-1表達(dá)，而隨著時(shí)間的延長(zhǎng)SGK-1回復(fù)到正常水平。這些結(jié)果都說(shuō)明，SGK-1可能在低氧誘導(dǎo)早期為維持細(xì)胞生存起到一定作用。為了驗(yàn)證上述推測(cè)，我們采用了RNA干擾的方法降低SGK-1在細(xì)胞內(nèi)的表達(dá)。如果我們的推測(cè)正確，干擾SGK-1表達(dá)則必將加劇低氧誘導(dǎo)的增殖抑制，并增加細(xì)胞凋亡率。而我們的結(jié)果與推測(cè)非常吻合，這說(shuō)明SGK-1在AtT-20受到低氧刺激后，能夠迅速表達(dá)上調(diào)，從而激活相關(guān)信號(hào)通路，維持細(xì)胞生存并抑制細(xì)胞凋亡。

綜上所述，本研究利用體外細(xì)胞缺氧模型，證明了SGK-1在腦缺血早期表達(dá)，起到保護(hù)腦細(xì)胞的作用。進(jìn)一步研究SGK-1在腦缺血中的作用以及相關(guān)信號(hào)通路，有利于更好地闡述腦缺血保護(hù)的相關(guān)機(jī)制，并指導(dǎo)臨床診療。

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Expression and role of SGK-1 in hypoxia-induced proliferation andapoptosis of mouse pituitary tumor cells AtT-20

WEIRui,WUJie,ZENGWei,ZHANGWei

(TheFirstAffiliatedHospitalofKunmingMedicalUniversity,Kunming650032,China)

Objective To observe the expression of serum and glucocorticoid-regulated kinase-1 (SGK-1) in mouse pituitary tumor cells AtT-20′s proliferation and apoptosis induced by hypoxia and to explore the role of SGK-1 in the process.Methods We classified AtT-20 cells into the observation group and control group randomly, then put 50, 100, 200 μmol/L CoCl2(for modulating the hypoxia) in the observation group while nothing was put in the control group, next, we tested the A570 value of cell proliferation at 0, 24, 48, 72, 96 h by MTT. We added 200 μmol/L CoCl2to AtT-20 cells, when the cells were cultured for 0, 24, 48, 72, 96 h, we calculated the apoptosis rate by flow cytometry, separately.Meanwhile, we detected the mRNA and protein of SGK-1 by semi quantitative PCR and Western blot. After that, we divided AtT-20 cells into 4 groups, cells in the groups A and B were transfected with interfering plasmid SGK-1 siRNA, and cells in the groups C and D were transfected with control interfering plasmid siCon. At 24 h after transfection, cells in the groups A and C were added with 200 μmol/L CoCl2, while cells in the groups B and D were not treated.Finally, we tested the A570 value and the apoptosis rate at 0, 24, 48, 72, 96 h by MTT and flow cytometry, respectively.Results At 72 h after hypoxia, A570 value of AtT-20 cells in the observation group decreased gradually with the increasing concentration and prolonged time, and difference was statistical significant as compared with that of the control group (allP<0.05). The apoptosis rate had no obvious change at 24, 48 h, and the apoptosis rate increased at 72, 96 h as compared with that at 0 h (allP<0.05). The mRNA and protein expression of SGK-1 increased at 24, 48 h (allP<0.05), but had no change at 72 h as compared with that at 0 h (allP>0.05). The A570 value in the group A at every time point after hypoxia was lower than that of the other groups, while the A570 value in the group C at 72, 96 h was higher than that of the group A and lower than that of groups B, C, meanwhile, significant difference was found between them (allP<0.05). The apoptosis rate in the group A at every time point after hypoxia was higher than that of the other groups, while the apoptosis rate in the group C at 72 h was lower than that of the group A and higher than that of groups B, C, meanwhile, significant difference was found between them (allP<0.05).Conclusion Hypoxia inhibits proliferation of AtT-20 cells and promotes its apoptosis, and the increasing expression of SGK-1 in the process protects cells from damage induced by hypoxia.

cerebral hypoxia; pituitary tumor cell; serum and glucocorticoid-regulated kinase (SGK); CoCl2; gene interference; cell apoptosis; cell proliferation

國(guó)家自然科學(xué)基金資助項(xiàng)目(81560319)；云南省應(yīng)用基礎(chǔ)研究面上項(xiàng)目(2016FB130) ；云南省衛(wèi)生科技計(jì)劃項(xiàng)目 (2016NS066)。

韋睿(1976-), 女, 主治醫(yī)師, 主要研究方向?yàn)榧痹\科疾病的診治。 E-mail: 769320515@qq.com

張瑋(1975-),男, 副主任醫(yī)師,博士碩士生導(dǎo)師，主要研究方向?yàn)槟X缺血與腦保護(hù)。E-mail: zhangwei7222@126.com

10.3969/j.issn.1002-266X.2017.18.005

R739.41

A

1002-266X(2017)18-0016-04

2017-02-23)