雙氫青蒿素對(duì)C2株藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin抑制作用的研究

劉阿倩，王 洋，林志強(qiáng)，張亞粉，田喜鳳，余 源

·實(shí)驗(yàn)研究·

雙氫青蒿素對(duì)C2株藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin抑制作用的研究

劉阿倩，王 洋，林志強(qiáng)，張亞粉，田喜鳳，余 源

目的 建立實(shí)時(shí)熒光定量RT-PCR( real- time quantitative, RT- PCR)檢測(cè)C2株藍(lán)氏賈第鞭毛蟲(chóng)(Giardialamblia)Delta giardin基因mRNA表達(dá)量的方法，分析雙氫青蒿素(Dihydroartemisinin, DHA)對(duì)體外C2株藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin基因mRNA表達(dá)水平的影響。方法 分別采用100 μg/mL、200 μg/mL的雙氫青蒿素改良型TYI-S-33培養(yǎng)基培養(yǎng)C2株藍(lán)氏賈第鞭毛蟲(chóng)，以不含藥物組作為陰性對(duì)照，分別培養(yǎng)2 h、4 h、8 h、12 h，提取總RNA，逆轉(zhuǎn)錄合成cDNA，實(shí)時(shí)熒光定量PCR檢測(cè)Delta giardin基因mRNA表達(dá)情況。結(jié)果 100 μg/mL雙氫青蒿素培養(yǎng)2 h、4 h、8 h、12 h后，Delta giardin基因mRNA相對(duì)表達(dá)量分別為0.44、0.26、0.25、0.02；200 μg/mL雙氫青蒿素培養(yǎng)2 h、4 h、8 h、12 h后，Delta giardin基因mRNA相對(duì)表達(dá)量分別為0.30，0.26，0.11，0.02。藥物對(duì)照組中C2株藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin基因mRNA表達(dá)量明顯低于陰性對(duì)照組。結(jié)論 雙氫青蒿素對(duì)C2株藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin基因mRNA的表達(dá)具有明顯的抑制作用，提示雙氫青蒿素對(duì)藍(lán)氏賈第鞭毛蟲(chóng)具有明顯的防治效果。

C2株藍(lán)氏賈第鞭毛蟲(chóng); 雙氫青蒿素; 實(shí)時(shí)熒光定量RT-PCR; Delta giardin

藍(lán)氏賈第鞭毛蟲(chóng)(Giardialamblia，簡(jiǎn)稱賈第蟲(chóng))是一種世界性分布的胃腸道寄生蟲(chóng)，主要寄生于人和多種哺乳動(dòng)物的小腸內(nèi)并引起以腹瀉為主要癥狀的藍(lán)氏賈第鞭毛蟲(chóng)病(giardiasis, 簡(jiǎn)稱賈第蟲(chóng)病)[1]。賈第蟲(chóng)雙核、具有鞭毛，地理分布廣泛，有多種動(dòng)物宿主，因此可通過(guò)糞便、被污染的水源、食物進(jìn)行傳播。本病已被列為全世界危害人類健康的十種主要寄生蟲(chóng)病之一，在發(fā)達(dá)國(guó)家和發(fā)展中國(guó)家均有廣泛流行[2-5]。有研究表明藍(lán)氏賈第鞭毛蟲(chóng)能夠通過(guò)其腹吸盤(pán)吸附于宿主腸上皮細(xì)胞，是其致病的關(guān)鍵因素。Crossley和Holberton[6-7]首次提出賈第素是賈第蟲(chóng)細(xì)胞骨架的特有成分，主要分為4大類, 即α、β、γ和δ賈第素[8-10]。腹吸盤(pán)的主要成分包括骨架蛋白，因此，賈第蟲(chóng)的骨架結(jié)構(gòu)與致病性密切相關(guān)，針對(duì)藍(lán)氏賈第鞭毛蟲(chóng)骨架蛋白進(jìn)行新藥物的研發(fā)對(duì)賈第蟲(chóng)病的有效防治具有重要的意義。前期研究采用蛋白質(zhì)組學(xué)技術(shù)觀察到雙氫青蒿素對(duì)體外培養(yǎng)的藍(lán)氏賈第鞭毛蟲(chóng)滋養(yǎng)體蛋白質(zhì)具有明顯的損傷作用，其中包括細(xì)胞骨架蛋白[11]。為了進(jìn)一步研究雙氫青蒿素對(duì)藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin的抑制作用，本研究采用含有雙氫青蒿素的改良TYI-S-33培養(yǎng)基培養(yǎng)C2株藍(lán)氏賈第鞭毛蟲(chóng)，實(shí)時(shí)熒光定量RT-PCR檢測(cè)雙氫青蒿素對(duì)C2株藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin的抑制作用。

1 材料與方法

1.1 材料 雙氫青蒿素粉劑(原藥批號(hào)010904)為北京豐臺(tái)科技園生物技術(shù)公司藤海寧女士惠贈(zèng)；C2株藍(lán)氏賈第鞭毛蟲(chóng)由本實(shí)驗(yàn)室液氮保存；M-MLV 逆轉(zhuǎn)錄酶，RNase inhibitor，Random Primers購(gòu)自Promega公司；dNTP，SYBR?Premix Ex TaqTMReal-Time PCR試劑盒購(gòu)自TaKaRa(大連)公司；引物由生工生物工程(上海)公司合成；組織/細(xì)胞基因組RNA提取試劑盒購(gòu)自Invitrogen；焦碳酸二乙酯(DEPC)購(gòu)自上海生工生物技術(shù)有限公司。

1.2 方法

1.2.1 藍(lán)氏賈第鞭毛蟲(chóng)復(fù)蘇培養(yǎng) 將液氮內(nèi)凍存的C2株藍(lán)氏賈第鞭毛蟲(chóng)復(fù)蘇，置含改良TYI-S-33培養(yǎng)基的4 mL硼酸硅培養(yǎng)管內(nèi)，于37 ℃培養(yǎng)。48～72 h后，蟲(chóng)體即呈對(duì)數(shù)生長(zhǎng)期。選取蟲(chóng)體生長(zhǎng)旺盛的培養(yǎng)管，置4 ℃冰浴，15 min后取出，在雙手掌間多次滾搓培養(yǎng)管，使貼壁生長(zhǎng)的蟲(chóng)體完全自管壁脫落。用血球計(jì)數(shù)板計(jì)數(shù)蟲(chóng)數(shù)，再用培養(yǎng)基將蟲(chóng)液濃度調(diào)為6×106～10×106個(gè)滋養(yǎng)體/mL[12]，傳代培養(yǎng)。

1.2.2 藥物培養(yǎng) 培養(yǎng)48 h后選取蟲(chóng)體貼滿瓶壁的培養(yǎng)管，藥物組培養(yǎng)基內(nèi)分別加入100 μg/mL 、200 μg/mL雙氫青蒿素，對(duì)照組培養(yǎng)基內(nèi)不加藥；37 ℃分別培養(yǎng)2 h、4 h、8 h、12 h后，收集蟲(chóng)體，用血球計(jì)數(shù)板計(jì)數(shù)蟲(chóng)數(shù)，用培養(yǎng)基將蟲(chóng)液濃度調(diào)至1×107個(gè)滋養(yǎng)體/mL；取1mL蟲(chóng)液，用PBS(pH7.4)清洗3次，離心10 min(4 000 r/min)，棄上清，留沉淀。

1.2.3 總RNA提取及cDNA的合成 離心分離的蟲(chóng)體細(xì)胞中加入細(xì)胞裂解液Trizol(Invitrogen)1 mL，反復(fù)顛倒后迅速吸入經(jīng)焦碳酸二乙酯(DEPC)處理過(guò)的eppendorf離心管中，按照說(shuō)明書(shū)提取總RNA，紫外分光光度法鑒定總RNA的純度。

建立20 μL反轉(zhuǎn)錄體系：RNA模板，5 μL，M-MLV 1 μL，Random Primers(100 ng/μL) 1 μL，RNase inhibitor 1 μL，10 mmol/L dNTP 1 μL，5×反應(yīng)緩沖液4 μL，DEPC水7 μL；反轉(zhuǎn)錄條件：70 ℃變性5 min，42 ℃反轉(zhuǎn)錄60 min，即得cDNA，-80 ℃保存?zhèn)溆谩?/p>

1.2.4 引物設(shè)計(jì) NCBI網(wǎng)站檢索藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin(XM_001707397.1)和GAPDH (XM_001704991.1)基因序列(http://www.ncbi.nlm.nih.gov/gene/5700349)，采用Primer Premier 5軟件分別設(shè)計(jì)合成藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin(ATCC 50803)及GAPDH(內(nèi)參基因)特異性引物 (表1)。

表1 Delta giardin和GAPDH引物序列

1.2.5 實(shí)時(shí)熒光定量PCR檢測(cè)Delta giardin基因mRNA的相對(duì)表達(dá)量 采用Corbett實(shí)時(shí)熒光定量PCR儀，分別以Delta giardinS/Delta giardinA; GAPDHS/GAPDHA為引物進(jìn)行實(shí)時(shí)熒光定量PCR檢測(cè)。藥物培養(yǎng)組及陰性對(duì)照組每個(gè)樣品均做2個(gè)重復(fù)，以各組逆轉(zhuǎn)錄合成的cDNA作為模板，磷酸甘油醛脫氫酶基因(GAPDH)為內(nèi)參基因，建立20 μL反應(yīng)體系： 2×SYBR MIX 10 μL，cDNA 1 μL，Prime S 1 μL，Prime A 1 μL，DEPC H2O 7 μL，反應(yīng)條件：95 ℃變性15 s，60 ℃復(fù)性15 s， 72 ℃延伸30 s。Delta Delta CT法分析檢測(cè)結(jié)果，確定Delta giardin基因mRNA的相對(duì)表達(dá)量。

2 結(jié) 果

2.1 總RNA提取結(jié)果 嚴(yán)格按照試劑盒操作說(shuō)明進(jìn)行，利用Trizol提取藍(lán)氏賈第鞭毛蟲(chóng)總RNA，各組RNAA260nm/A280nm值均在1.8～2.0范圍內(nèi)，總RNA提取質(zhì)量符合實(shí)驗(yàn)要求。

2.2 Real-Time PCR檢測(cè)結(jié)果 分別得到Delta giardin基因Real-time PCR擴(kuò)增曲線、熔解曲線(圖1-2)和GAPDH基因Real-time PCR擴(kuò)增曲線、熔解曲線(圖3-4)，融解曲線只有單峰值, 排除了非特異性擴(kuò)增。Delta Delta CT法分析各組Delta giardin基因mRNA的相對(duì)表達(dá)量。△循環(huán)閾值(cycle threshold, Ct) =樣品Ct均值-內(nèi)參照Ct均值，△△Ct = △Ct-(隨機(jī)陰性對(duì)照樣品Ct均值-該樣品內(nèi)參照Ct均值)，以2-△△Ct表示樣品中目的基因初始cDNA相對(duì)表達(dá)量[13]。研究結(jié)果表明(表2)，培養(yǎng)基內(nèi)加入濃度為100 μg/mL的雙氫青蒿素，分別培養(yǎng)藍(lán)氏賈第鞭毛蟲(chóng)2 h、4 h、8 h、12 h后，Delta giardin基因mRNA相對(duì)表達(dá)量為0.44，0.26，0.25，0.02；培養(yǎng)基內(nèi)加入濃度為200 μg/mL的雙氫青蒿素，分別培養(yǎng)藍(lán)氏賈第鞭毛蟲(chóng)2 h、4 h、8 h、12 h后，Delta giardin基因mRNA相對(duì)表達(dá)量為0.30，0.26，0.11，0.02。藥物組Delta giardin基因mRNA均低于對(duì)照組，表明雙氫青蒿素對(duì)C2株藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin基因表達(dá)具有明顯的抑制作用，抑制作用隨著藥物濃度的增高和作用時(shí)間的延長(zhǎng)而增強(qiáng)。

1-2: DHA 200 μg/mL for 2 h; 3-4: DHA 200 μg/mL for 4 h; 5-6: DHA 200 μg/mL for 8 h; 7-8: DHA 200 μg/mL for 12 h; 9-10: DHA 100 μg/mL for 2 h; 11-12: DHA 100 μg/mL for 4 h; 13-14: DHA 100 μg/mL for 8 h; 15-16: DHA 100 μg/mL for 12 h; 17-18: Negative control.

圖1 Delta giardin mRNA Real-time PCR擴(kuò)增曲線

Fig.1 Amplification curve of Delta giardin mRNA

3 討 論

1-2: DHA 200 μg/mL for 2 h; 3-4: DHA 200 μg/mL for 4 h; 5-6: DHA 200 μg/mL for 8 h; 7-8: DHA 200 μg/mL for 12 h; 9-10: DHA 100 μg/mL for 2 h; 11-12: DHA 100 μg/mL for 4 h; 13-14: DHA 100 μg/mL for 8 h; 15-16: DHA 100 μg/mL for 12 h; 17-18: Negative control.

圖2 Delta giardin mRNA Real-time PCR熔解曲線

Fig.2 Melting curve of Delta giardin mRNA

1-2: DHA 200 μg/mL for 2 h; 3-4: DHA 200 μg/mL for 4 h; 5-6: DHA 200 μg/mL for 8 h; 7-8: DHA 200 μg/mL for 12 h; 9-10: DHA 100 μg/mL for 2 h; 11-12: DHA 100 μg/mL for 4 h; 13-14: DHA 100 μg/mL for 8 h; 15-16: DHA 100 μg/mL for 12 h; 17-18: Negative control.

圖3 GAPDH mRNA Real-timePCR擴(kuò)增曲線

Fig.3 Amplification curve of GAPDH mRNA

1-2: DHA 200 μg/mL for 2 h; 3-4: DHA 200 μg/mL for 4 h; 5-6: DHA 200 μg/mL for 8 h; 7-8: DHA 200 μg/mL for 12 h; 9-10: DHA 100 μg/mL for 2 h; 11-12: DHA 100 μg/mL for 4 h; 13-14: DHA 100 μg/mL for 8 h; 15-16: DHA 100 μg/mL for 12 h; 17-18: Negative control.

圖4 GAPDH mRNA Real-time PCR熔解曲線

Fig.4 Melting curve of GAPDH mRNA

表2 DHA作用后Delta giardin基因mRNA表達(dá)量分析結(jié)果

青蒿素(artemisinin)是從菊科蒿屬黃花蒿莖葉中提取的一種含過(guò)氧基團(tuán)的倍半萜內(nèi)酯, 經(jīng)過(guò)人工化學(xué)修飾、改進(jìn)后發(fā)展了多種衍生物，主要包括青蒿琥酯(artesunate) 、雙氫青蒿素(dihydroartemisinin)及蒿甲醚(artemether) 等[14]。雙氫青蒿素是青蒿素及其衍生物蒿甲醚和青蒿琥酯在體內(nèi)的有效活性代謝產(chǎn)物，為廣譜抗寄生蟲(chóng)藥物，對(duì)多種寄生性原蟲(chóng)有良好的殺滅作用[15-16]。在抗瘧疾、弓形蟲(chóng)、肺孢子蟲(chóng)肺炎、腫瘤，以及調(diào)節(jié)免疫和抗孕等方面具有廣泛的應(yīng)用[17-18]。

實(shí)時(shí)熒光定量PCR技術(shù)( real-time fluorescent quantitative PCR, FQ-PCR) 于1996年由美國(guó)Applied Biosystems公司推出，該技術(shù)在PCR反應(yīng)體系中加入熒光基團(tuán)，利用熒光信號(hào)積累實(shí)時(shí)監(jiān)測(cè)整個(gè)PCR進(jìn)程[19-20]。實(shí)時(shí)熒光定量RT-PCR ( real-time fluorescent quantitative reverse transcription-polymerase chain reaction, FQ RT-PCR )將實(shí)時(shí)熒光定量PCR與逆轉(zhuǎn)錄技術(shù)相結(jié)合，能夠?qū)崟r(shí)檢測(cè)記錄PCR擴(kuò)增產(chǎn)物的增加，從而準(zhǔn)確檢測(cè)相應(yīng)mRNA的含量[21-22]。

雖然有研究表明雙氫青蒿素對(duì)瘧原蟲(chóng)、弓形蟲(chóng)、血吸蟲(chóng)、肺孢子蟲(chóng)等多種寄生蟲(chóng)均有顯著的損傷效果，但其作用機(jī)制鮮有報(bào)道，為了進(jìn)一步研究其藥理機(jī)制，本研究采用實(shí)時(shí)熒光定量RT-PCR檢測(cè)雙氫青蒿素對(duì)C2株藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin的抑制作用。研究結(jié)果表明，雙氫青蒿素對(duì)藍(lán)氏賈第鞭毛蟲(chóng)Delta giardin基因的表達(dá)具有明顯的抑制作用，抑制作用與藥物濃度和作用時(shí)間成正比。此結(jié)果與藍(lán)氏賈第鞭毛蟲(chóng)骨架蛋白雙向電泳質(zhì)譜結(jié)果一致[23]。藍(lán)氏賈第鞭毛蟲(chóng)能夠通過(guò)其腹吸盤(pán)吸附于宿主腸上皮細(xì)胞，其骨架結(jié)構(gòu)與致病性密切相關(guān)。Delta giardin是藍(lán)氏賈第鞭毛蟲(chóng)骨架蛋白的主要成分，是其致病的關(guān)鍵因素，因此雙氫青蒿素對(duì)賈第蟲(chóng)Delta giardin基因的抑制作用可能會(huì)影響藍(lán)氏賈第鞭毛蟲(chóng)的感染過(guò)程，對(duì)藍(lán)氏賈第鞭毛蟲(chóng)的防治起到一定的效果，本研究也為進(jìn)一步闡明其藥理機(jī)制提供了有價(jià)值的參考資料。

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Yu Yuan,Email:yuyuan5188@163.com

Dihydroartemisinin inhibition on Delta giardin in C2Giardialamblia

LIU A-qian,WANG Yang,LIN Zhi-qiang,ZHANG Ya-fen,TIAN Xi-feng,YU Yuan

(NorthChinaUniversityofScienceandTechnology，Tangshan063000,China)

We established real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for determining the expression of Delta giardin mRNA to explore effects of dihydroartemisinin (DHA) on the expression level of Delta giardin mRNA in C2Giardialamblia.Giardialambliarespectively cultivated for 2, 4, 8, and 12 h respectively with modified TYI-S-33 medium which contained 100 μg/mL and 200 μg/mL DHA, while the negative control group performed in the same experimental condition without DHAs. All of the RNAs were extracted and cDNA were synthesized. The relative expressive quantity of Delta giardin mRNA was determined by real-time PCR and the results were 0.44, 0.26, 0.25, and 0.02 whenGiardialambliarespectively cultivated for 2 h, 4 h, 8 h, and 12 hours which contained 100 μg/mL DHA. The relative expression quantities of Delta giardin mRNA were 0.30, 0.26, 0.11, and 0.02 whenGiardialambliarespectively cultivated for 2 h, 4 h, 8 h, and 12 h which contained 200 μg/mL DHA. The expressive quantities of Delta giardin mRNA with DHA were significantly lower than that in the control group. It suggests that dihydroartemisinin has obvious inhibitory effects on the expression level of Delta giardin mRNA in C2Giardialamblia, and DHA has significant prevention and cure function to C2Giardialamblia.

C2Giardialamblia; dihydroartemisinin; real-time reverse transcription PCR; Delta giardin

國(guó)家自然科學(xué)基金(No. 31471954)、河北省青年科學(xué)基金(No. C2012401039)、河北聯(lián)合大學(xué)培養(yǎng)基金(No.GP201308)、唐山市科技支持計(jì)劃項(xiàng)目(No.12140209A-33)聯(lián)合資助

余源，Email:yuyuan5188@163.com

華北理工大學(xué)生命科學(xué)學(xué)院，唐山 063000

Supported by the National Natural Science Foundation of China (No. 31471954), the Youth Science Fund Project in Hebei Province (No. C2012401039), the Hebei United University Training Fund (No. GP201308), and the Scientific and Technological Support Projects from Tangshan (No. 12140209A-33)

10.3969/cjz.j.issn.1002-2694.2015.06.006

R382.2

A

1002-2694(2015)06-0522-05

2014-07-21；

2014-10-30