鄒湘輝,劉博聰,2,莊東紅,查廣才,吳云影

(1.韓山師范學(xué)院生物系,潮州 521041;2.汕頭大學(xué)生物系,汕頭 515063)

3-羥基丁酸衍生物對(duì)PC12細(xì)胞凋亡的保護(hù)作用*

鄒湘輝1,劉博聰1,2,莊東紅1,查廣才1,吳云影1

(1.韓山師范學(xué)院生物系,潮州 521041;2.汕頭大學(xué)生物系,汕頭 515063)

目的 觀察3-羥基丁酸甲酯和3-羥基丁酸乙酯對(duì)PC12缺血清誘導(dǎo)凋亡細(xì)胞的保護(hù)作用。方法化學(xué)法降解聚羥基丁酸酯制備3-羥基丁酸甲酯和3-羥基丁酸乙酯。培養(yǎng)PC12細(xì)胞,以含血清培養(yǎng)組細(xì)胞為陰性對(duì)照組,缺血清培養(yǎng)組細(xì)胞為陽(yáng)性對(duì)照組,以在缺血清條件下添加不同濃度3-羥基丁酸甲酯或3-羥基丁酸乙酯細(xì)胞為樣品組,通過(guò)形態(tài)學(xué)觀察法、噻唑藍(lán)(MTT)法檢測(cè)各組細(xì)胞形態(tài)及活性變化。結(jié)果與陰性對(duì)照組比較,陽(yáng)性對(duì)照組細(xì)胞出現(xiàn)大量凋亡,形態(tài)變小變細(xì),活性降低。樣品處理組細(xì)胞活性不同程度提高,低濃度(0.01和0.001 mg·mL-1)3-羥基丁酸甲酯和3-羥基丁酸乙酯對(duì)凋亡細(xì)胞有保護(hù)作用,濃度為1 mg·mL-1時(shí)效果最好。結(jié)論通過(guò)化學(xué)法能夠制備高純度3-羥基丁酸甲酯和3-羥基丁酸乙酯,上述兩種樣品對(duì)缺血清誘導(dǎo)凋亡的PC12細(xì)胞有保護(hù)作用。

3-羥基丁酸;3-羥基丁酸甲酯;3-羥基丁酸乙酯;PC12細(xì)胞;細(xì)胞凋亡

3-羥基丁酸甲酯和3-羥基丁酸乙酯是3-羥基丁酸衍生物,3-羥基丁酸是人體脂肪酸代謝后產(chǎn)生的3種酮體之一,通常人體血液和組織中濃度約0.1 mmol·L-1,在糖饑餓情況下可以作為腦部的一種應(yīng)急性能源使用[1]。已有研究表明,3-羥基丁酸與糖尿病、體內(nèi)能量代謝紊亂等有著密切聯(lián)系[2]。在以往的研究中發(fā)現(xiàn),聚羥基脂肪酸酯(polyhydroxyalkanoates,PHA)在組織工程和材料學(xué)方面具有十分突出的應(yīng)用潛力,目前PHA的主要應(yīng)用是作為一種環(huán)境友好型生物可降解塑料。3-羥基丁酸作為高分子PHA的一種最普遍的降解產(chǎn)物,受到越來(lái)越多的關(guān)注[3],3-羥基丁酸和通過(guò)對(duì)聚羥基丁酸酯(polyhydroxybutyrate,PHB)進(jìn)行醇解衍生化生成3-羥基丁酸甲酯和-3羥基丁酸乙酯能夠促進(jìn)神經(jīng)膠質(zhì)細(xì)胞、成骨細(xì)胞等的生長(zhǎng)[3]。筆者前期的工作發(fā)現(xiàn),3-羥基丁酸及其衍生物具有增強(qiáng)大鼠腦部神經(jīng)遞質(zhì)表達(dá)和提高大鼠認(rèn)知功能的作用[4]。筆者在本實(shí)驗(yàn)中采用缺血清誘導(dǎo)PC12細(xì)胞凋亡,研究3-羥基丁酸甲酯和3-羥基丁酸乙酯對(duì)凋亡細(xì)胞的保護(hù)作用。

1 材料與方法

1.1 試劑及設(shè)備 PHB(清華大學(xué)生命科學(xué)學(xué)院微生物實(shí)驗(yàn)室提供,該材料為其實(shí)驗(yàn)室微生物發(fā)酵產(chǎn)物,純度:97.5%);達(dá)爾伯克必需基本培養(yǎng)液(Dulbecco's Modified Eagle Medium,DMEM)高糖培養(yǎng)液(批號(hào): 1154412)購(gòu)自美國(guó)Gibco公司;噻唑藍(lán)(thiazole blue, MTT,批號(hào):C18H16N5SBr)購(gòu)自美國(guó)Sigma-Aldrich公司;胎牛血清購(gòu)自四季青公司(批號(hào):130620);二甲亞砜(dimethyl sulfoxide,DMSO)購(gòu)自天津希恩思公司; Shimadzu氣質(zhì)聯(lián)用(GasChromatograph-Mass Spectrometer,GC-MS)儀(GC-MSQP5050A);OLYMPUS 1X7/1X51倒置顯微鏡。

1.2 3-羥基丁酸甲酯及3-羥基丁酸乙酯的制備及鑒定 稱取PHB 5 g,溶于50 mL三氯甲烷,轉(zhuǎn)移至圓底燒瓶。安裝上水浴冷凝管,并放入恒溫水浴鍋,緩慢加熱攪拌溶解。另量取甲醇100 mL放入150 mL錐形瓶,同時(shí)加入濃硫酸2 mL常溫?cái)嚢杌旌?將上述乙醇-濃硫酸溶液加入PHB三氯甲烷溶液圓底燒瓶,混合。溫度控制在約90℃。反應(yīng)時(shí)間48 h。反應(yīng)結(jié)束后,抽濾除雜。將濾液轉(zhuǎn)移至250 mL分液漏斗,加入純化水25 mL,靜置分層,收集下層有機(jī)相,加入5%碳酸氫鈉(NaHCO3)25 mL中和1次,純化水25 mL洗滌1次,分液漏斗分離,有機(jī)相用無(wú)水硫酸鈉干燥,過(guò)濾出硫酸鈉。有機(jī)相用旋轉(zhuǎn)蒸發(fā)儀蒸發(fā)三氯甲烷并干燥,減壓蒸餾得到產(chǎn)物。通過(guò)以上過(guò)程制備的產(chǎn)物用GC-MS分析其組成和比例。

3-羥基丁酸乙酯制備及鑒定與3-羥基丁酸甲酯制備方法相同,將其中的甲醇更換成乙醇。通過(guò)以上過(guò)程制備的產(chǎn)物用GC-MS分析其組成和比例。

1.3 PC12細(xì)胞的培養(yǎng)及損傷模型的建立 PC12細(xì)胞用含10%胎牛血清的DMEM高糖培養(yǎng)液于37℃、5%二氧化碳(CO2)細(xì)胞培養(yǎng)箱中培養(yǎng),當(dāng)細(xì)胞覆蓋瓶底面積約80%時(shí),0.25%胰蛋白酶消化離心,完全培養(yǎng)液重懸后進(jìn)行繼代培養(yǎng)。取對(duì)數(shù)期生長(zhǎng)期細(xì)胞用于分組實(shí)驗(yàn)。

陰性對(duì)照組更換新鮮含5%胎牛血清的高糖DMEM培養(yǎng)液,陽(yáng)性對(duì)照組更換新鮮不含血清的DMEM高糖培養(yǎng)液,3-羥基丁酸甲酯保護(hù)組更換新鮮不含胎牛血清但3-羥基丁酸甲酯終濃度為1,0.1, 0.01,0.001 mg·mL-1DMEM高糖培養(yǎng)液,3-羥基丁酸乙酯保護(hù)組更換新鮮不含胎牛血清但3-羥基丁酸乙酯終濃度為1,0.1,0.01,0.001 mg·mL-1DMEM高糖培養(yǎng)液。繼續(xù)培養(yǎng)24 h。

1.4 細(xì)胞形態(tài)學(xué)觀察 倒置顯微鏡下觀察各組細(xì)胞形態(tài)并攝像。

1.5 MTT法檢測(cè)細(xì)胞活性 各組細(xì)胞每孔加入5 mg·mL-1MTT 20 μL,孵育4 h后去上清液,每孔加入DMSO 150 μL,水平搖床震蕩10 min,酶標(biāo)儀[芬蘭/雷勃(美國(guó)熱電),型號(hào):Multiskan MK3]492 nm波長(zhǎng)下測(cè)定吸光度(A)值,調(diào)零孔加入DMSO 150 μL為空白對(duì)照。

2 結(jié)果

2.1 PHB醇解產(chǎn)物的鑒定分析 通過(guò)醇解PHB制得3-羥基丁酸甲酯和3-羥基丁酸乙酯,經(jīng)過(guò)GC-MS檢測(cè)后各產(chǎn)物鑒定分析結(jié)果見圖1,2和表1,2所示。

1.3-羥基丁酸乙酯;2.3-羥基丁酸乙酯圖1 PHB乙醇解產(chǎn)物GC-MS分析1.ethyl-3-hydroxybutyrate;2.ethyl-3-hydroxybutyrateFig.1 GC-MS analysis on ethanolysis product of PHB

表1 PHB乙醇解產(chǎn)物成分Tab.1 Component of ethanolysis product of PHB

1.3-羥基丁酸甲酯;2.3-羥基丁酸甲酯圖2 PHB甲醇解產(chǎn)物GC-MS分析1.methyl-3-hydroxybutyrate;2.methyl-3-hydroxybutyrateFig.2 GC-MS analysis on methanolysis product of PHB

表2 PHB甲醇解產(chǎn)物成分Tab.2 Component of methanolysis product of PHB

由表1,2可以看出,通過(guò)醇解方法對(duì)PHB進(jìn)行降解可以制備高純度3-羥基丁酸甲酯和3-羥基丁酸乙酯,產(chǎn)物在GC-MS分析下分別有兩個(gè)不同的出峰時(shí)間。查閱數(shù)據(jù)庫(kù)后分析可知,是制備過(guò)程中同種產(chǎn)物出現(xiàn)左旋和右旋兩種不同構(gòu)型形成的。

2.2 細(xì)胞形態(tài) 見圖3,4。陰性對(duì)照組細(xì)胞長(zhǎng)勢(shì)良好,單個(gè)細(xì)胞呈現(xiàn)粗壯多變性,有3或4個(gè)突起,折光性高,細(xì)胞生長(zhǎng)均勻且密度較大。陽(yáng)性對(duì)照組細(xì)胞呈細(xì)絲線形或圓形欲浮起狀,兩端無(wú)法看見類似陽(yáng)性對(duì)照組細(xì)胞的突起,折光性低,細(xì)胞生長(zhǎng)不均勻且密度較小。在兩種樣品實(shí)驗(yàn)組中可以看出在各個(gè)劑量下的細(xì)胞形態(tài)與陽(yáng)性對(duì)照比較較粗壯,折光性恢復(fù),無(wú)明顯細(xì)胞突起,但細(xì)胞數(shù)目明顯較模型對(duì)照組多。在3-羥基丁酸甲酯保護(hù)組中,0.01 mg·mL-13-羥基丁酸甲酯組細(xì)胞形態(tài)較陽(yáng)性對(duì)照組有較大變化,細(xì)胞寬度明顯加大,少數(shù)細(xì)胞還能觀察到細(xì)胞突起存在,折光性較好。1 mg·mL-13-羥基丁酸乙酯保護(hù)組細(xì)胞寬度加大,折光性好,細(xì)胞突觸數(shù)量多,細(xì)胞數(shù)目也較陽(yáng)性對(duì)照組多。

2.3 細(xì)胞活性檢測(cè) 經(jīng)過(guò)缺血清誘導(dǎo)處理后,陽(yáng)性對(duì)照組細(xì)胞活性較陰性對(duì)照組差,而添加了不同劑量3-羥基丁酸甲酯和3-羥基丁酸乙酯的細(xì)胞活性均有不同程度提高,3-羥基丁酸甲酯和3-羥基丁酸乙酯在1 mg·mL-1時(shí)均呈現(xiàn)出最好的保護(hù)作用,劑量為0.1 mg·mL-1時(shí)保護(hù)作用不明顯,劑量為0.01和0.001 mg·mL-1時(shí)均具有一定保護(hù)作用(表3)。

3 討論

3-羥基丁酸是最普遍的PHA降解產(chǎn)物,近年來(lái)對(duì)于3-羥基丁酸的生理作用的研究逐漸增多,包括為大腦提供能量[3],作為腦的谷氨酸抑制性神經(jīng)遞質(zhì)(γ-氨基丁酸)的代謝前體物[4],和作為細(xì)胞凋亡的抑制劑作用等[5]。目前利用生物方法來(lái)進(jìn)行3-羥基丁酸及其衍生物的合成很困難,且有關(guān)3-羥基丁酸甲酯和3-羥基丁酸乙酯在PC12細(xì)胞凋亡保護(hù)方面的研究還未見報(bào)道。筆者通過(guò)化學(xué)降解法成功制備得到3-羥基丁酸甲酯和3-羥基丁酸乙酯,具有很高的得率,再利用GC-MS對(duì)產(chǎn)物進(jìn)行檢測(cè),發(fā)現(xiàn)產(chǎn)物的純度也比較高,化學(xué)法降解PHA制備3-羥基丁酸衍生物作為一種簡(jiǎn)單的制備手性藥物分子的途徑具有推廣利用的價(jià)值。

A.陰性對(duì)照組;B.陽(yáng)性對(duì)照組;C.1 mg·mL-13-羥基丁酸乙酯組;D.0.1 mg·mL-13-羥基丁酸乙酯組;E.0.01 mg·mL-13-羥基丁酸乙酯組;F.0.001 mg·mL-13-羥基丁酸乙酯組圖4 3-羥基丁酸乙酯對(duì)缺血清誘導(dǎo)細(xì)胞凋亡保護(hù)作用A.negative control group;B.positive control group;C.1 mg·mL-1Ethyl-3-hydroxybutyrate group;D.0.1 mg·mL-1Ethyl-3-hydroxybutyrate group;E.0.01 mg·mL-1Ethyl-3-hydroxybutyrate group;F.0.001 mg·mL-1Ethyl-3-hydroxybutyrate groupFig.4 Protection of ethyl-3-hydroxybutyrate on the apoptosis of PC12 induced by FBS deficiency

表3 3-羥基丁酸衍生物對(duì)PC12細(xì)胞活性影響分析Tab.3 Effect of derivate of 3-hydroxybutyrate on the activity of PC12 cells ±s

表3 3-羥基丁酸衍生物對(duì)PC12細(xì)胞活性影響分析Tab.3 Effect of derivate of 3-hydroxybutyrate on the activity of PC12 cells ±s

與陽(yáng)性對(duì)照組比較,*1P＜0.05Compared with positive control group,*1P＜0.05

組別吸光度值(A492nm)組別吸光度值(A492nm)陰性對(duì)照組0.176±0.008*1,χ2=4.017陰性對(duì)照組0.127±0.003*1,χ2=3.115陽(yáng)性對(duì)照組0.112±0.002陽(yáng)性對(duì)照組0.087±0.001 1 mg·mL-13-羥基丁酸甲酯組0.149±0.007*1,χ2=3.3231 mg·mL-13-羥基丁酸乙酯組0.122±0.003*1,χ2=3.634 0.1 mg·mL-13-羥基丁酸甲酯組0.106±0.004,χ2=0.5680.1 mg·mL-13-羥基丁酸乙酯組0.086±0.003,χ2=0.115 0.01 mg·mL-13-羥基丁酸甲酯組0.127±0.003*1,χ2=2.1190.01 mg·mL-13-羥基丁酸乙酯組0.105±0.003*1,χ2=2.616 0.001 mg·mL-13-羥基丁酸甲酯組0.141±0.003*1,χ2=3.1350.001 mg·mL-13-羥基丁酸乙酯組0.118±0.004*1,χ2=3.213

PC12細(xì)胞是大鼠腎上腺髓質(zhì)嗜鉻細(xì)胞瘤克隆化的細(xì)胞株,在正常情況下PC12細(xì)胞在形態(tài)上和生理生化上與腎上腺髓質(zhì)細(xì)胞相似,而在培養(yǎng)物中添加神經(jīng)生長(zhǎng)因子(nerve growth factor,NGF)后,PC12細(xì)胞具有神經(jīng)細(xì)胞的形態(tài)和相類似的代謝產(chǎn)物形成[9],目前PC12細(xì)胞正被廣泛地應(yīng)用于研究各種作用于神經(jīng)細(xì)胞的分化、受體及神經(jīng)遞質(zhì)釋放等的中樞神經(jīng)系統(tǒng)藥物的作用機(jī)制。細(xì)胞凋亡在20世紀(jì)70年代首先由HERR等[10]提出,細(xì)胞凋亡在組織和器官的發(fā)育或者多種病變過(guò)程中起著重要的作用。神經(jīng)細(xì)胞凋亡是許多中樞神經(jīng)系統(tǒng)疾病發(fā)生的最終原因,諸如老年癡呆癥、帕金森病和低氧缺血性腦損傷等,而在體外細(xì)胞培養(yǎng)過(guò)程中模型細(xì)胞凋亡的方式有很多種,缺血清誘導(dǎo)細(xì)胞凋亡是其中比較簡(jiǎn)便且行之有效的方法之一,具體做法是去除培養(yǎng)液中的血清,使細(xì)胞在一定時(shí)間內(nèi)處于無(wú)血清培養(yǎng)狀態(tài),細(xì)胞因缺乏必要的生長(zhǎng)因子和營(yíng)養(yǎng)物質(zhì)而發(fā)生凋亡[11]。本研究發(fā)現(xiàn),PC12細(xì)胞缺血清誘導(dǎo)凋亡組與正常的含血清培養(yǎng)組比較,細(xì)胞形態(tài)變化明顯,活性降低,凋亡增加,而在添加了不同濃度3-羥基丁酸甲酯或者3-羥基丁酸乙酯的實(shí)驗(yàn)組中,極低濃度的3-羥基丁酸甲酯和3-羥基丁酸乙酯都可以抑制缺血清誘導(dǎo)的PC12細(xì)胞凋亡,細(xì)胞數(shù)目增加,對(duì)細(xì)胞形態(tài)具有一定的修復(fù)作用,細(xì)胞活性顯著增強(qiáng),高濃度組細(xì)胞的活性可達(dá)到與陰性對(duì)照組接近。推測(cè)這兩種小分子可能在低濃度時(shí)作為某一種信號(hào)分子啟動(dòng)了細(xì)胞內(nèi)抑制細(xì)胞凋亡基因的表達(dá),在較高濃度時(shí)同時(shí)啟動(dòng)了凋亡基因的表達(dá)而表現(xiàn)出對(duì)細(xì)胞的保護(hù)作用減弱,但是隨著濃度的進(jìn)一步升高,可作為營(yíng)養(yǎng)成分促進(jìn)細(xì)胞的生存,詳細(xì)機(jī)制還有待進(jìn)一步研究。

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DOI 10.3870/yydb.2014.11.007

Protective Effect of 3-hydroxybutyrate Derivate on the Apoptosis of PC12

ZOU Xiang-hui1,LIU Bo-cong1,2,ZHUANG Dong-hong1,ZHA Guang-cai1,WU Yun-ying1
(1.Department of Biology,Hanshan Normal University,Chaozhou 521041,China;2.Department of Biology,Shantou University, Shantou 515063,China)

ObjectiveTo investigate the protective effect of 3-hydroxybutyratederivate(methyl-3-hydroxybutyrateand ethyl-3-hydroxybutyrate)on the apoptosis of PC12 induced by FBS deficiency.MethodsMethyl-3-hydroxybutyrateand ethyl-3-hydroxybutyratewere prepared by chemical degradation.PC12 cells were divided into different groups:medium with FBS as negative control,medium without FBS as positive control,medium with different concentrations of methyl-3-hydroxybutyrateor ethyl-3-hydroxybutyratewithout FBS as sample groups.The shape and viability of cells in each group were analyzed by optical microscope and MTT.ResultsCompared with the negative control,cells in the positive control group demonstrated a large number of apoptosis,smaller and thinner morphology,and lower activity.However,the activity of cells was improved in the sample groups.Low concentration(0.01 and 0.001 mg·mL-1)of methyl-3-hydroxybutyrateand ethyl-3-hydroxybutyrateshowed a protective effect on cell apoptosis,and 1 mg·mL-1had the best protection effect.ConclusionHigh purity methyl-3-hydroxybutyrateand ethyl-3-hydroxybutyratecan be produced by chemical method,and both chemicals present good effect on protecting the PC12 from apoptosis.

3-hydroxybutyrate;Methyl-3-hydroxybutyrate;Ethyl-3-hydroxybutyrate;PC12 cell;Apoptosis

R977;R965

A

1004-0781(2014)11-1427-04

2013-09-27

2013-10-25

*廣東省自然科學(xué)基金博士啟動(dòng)項(xiàng)目(10452104101004655);韓山師范學(xué)院青年基金項(xiàng)目(LQ200801)

鄒湘輝(1976-),男,湖南衡陽(yáng)人,副教授,博士,主要研究方向:細(xì)胞生物學(xué)。E-mail:zxh11043@126.com。