醫(yī)藥學類文獻雙語版漢譯英

1、介導性shRNA能抑制肺癌細胞中l(wèi)ivin沉默基因的表達從而促進SGC-7901細胞凋亡背景由于腫瘤細胞抑制凋亡增殖,特定凋亡的抑制因素會對于發(fā)展新的治療策略提供一個合理途徑。Livin是一種凋亡抑制蛋白家族成員，在多種惡性腫瘤的表達中具有意義。但是, 在有關胃癌方面沒有可利用的數(shù)據(jù)。在本研究中,我們發(fā)現(xiàn)livin基因在人類胃癌中的表達并調(diào)查了介導的shRNA能抑制肺癌細胞中l(wèi)ivin沉默基因的表達，從而促進SGC-7901細胞凋亡。方法mRNA及蛋白質(zhì)livin基因的表達用逆轉(zhuǎn)錄聚合酶鏈反應技術(shù)及西方吸干化驗進行了分析。小干擾RNA真核表達載體具體到livin基因采用基因重組、測序核酸。然后

2、用Lipofectamin2000轉(zhuǎn)染進入SGC-7901細胞。逆轉(zhuǎn)錄聚合酶鏈反應技術(shù)和西方吸干化驗用來驗證的livin基因在SGC-7901細胞中使沉默基因生效。所得到的穩(wěn)定的復制品用G418來篩選。細胞凋亡用應用流式細胞儀(FCM)來評估。細胞生長狀態(tài)和5-FU的50%抑制濃度(IC50)和順鉑都由MTT比色法來決定。結(jié)果livin mRNA和蛋白質(zhì)的表達檢測40例中有19例(47.5%)有胃癌和SGC-7901細胞。沒有l(wèi)ivin基因表達的是在腫瘤鄰近組織和良性胃潰瘍病灶。相關發(fā)現(xiàn)在livin基因的表達和腫瘤的微小分化和淋巴結(jié)轉(zhuǎn)移一樣(P < 0.05)。4個小干擾RNA

3、真核表達矢量具體到基因重組的livin基因建立。其中之一,能有效地減少livin基因的表達,抑制基因不少于70%(P < 0.01)。重組的質(zhì)粒被提取和轉(zhuǎn)染到胃癌細胞。G418篩選所得到的穩(wěn)定的復制品被放大講究。當livin基因沉默,胃癌細胞的生殖活動明顯低于對照組(P < 0.05)。研究還表明,IC50上的5-Fu和順鉑在胃癌細胞的治療上是通過shRNA減少以及刺激這些細胞(5-Fu proapoptotic和順鉑)(P < 0.01)。結(jié)論livin基因在胃癌中的過分表達與腫瘤分化與淋巴結(jié)轉(zhuǎn)移建立聯(lián)系,建議了治療胃癌病例分子預后因素之一。S

4、hRNA可以抑制在SGC-7901細胞中的livin基因表達,誘導細胞凋亡。Livin可以作為治療胃癌凋亡的新目標。1介紹胃癌是世界上最常見的惡性腫瘤之一。大多數(shù)患者被診斷為這個疾病的階段,在最佳時間的機會錯失了手術(shù)治愈。盡管有很大改善,但處于晚期胃癌的化療患者的總體存活率仍然很低。癌癥細胞化療耐抗性可能導致手術(shù)失敗。在耐藥的原因中,抑制細胞凋亡的過程會起重要作用。癌細胞常有抗凋亡增長的特征1,介導其增加的阻力不同來刺激的細胞凋亡,如DNA損傷、缺氧、營養(yǎng)損失2、3。此外, 在臨床實踐中細胞凋亡抵抗被認為是腫瘤手術(shù)失敗的主要原因,因此許多化療藥物和/或放射線療法都是通過誘導凋亡腫瘤死亡實現(xiàn)的4

5、。酶抑制劑 (IAPs),是一種新型的凋亡蛋白抑制基因家族5,6,包括病毒感染,化療藥物, ,生長因子和腫瘤壞死因子-(TNF-凋亡信號通路)/ Fas信號通路7 - 9。IAPs是由一組有凋亡特性的結(jié)構(gòu)相關的蛋白質(zhì)構(gòu)成10，在預防腫瘤細胞凋亡方面可能扮演一個重要的角色,并已成為近年來研究的熱點。這個家庭的新成員是ML-IAP/livin/KIAP/BIRC7 (以下稱為livin)有兩個種類、livin和livin11-14。有證據(jù)表明, livin的過分表達能阻止 由多種刺激誘導的細胞凋亡12。有趣的是, livin基因被發(fā)現(xiàn)在腫瘤細胞中限制性表達,但是不存在或是很少數(shù)量存在于正常成人體組

6、織中11-15,并且通過允許惡性細胞,以避免凋亡細胞死亡的方式導致腫瘤形成,。所以抑制livin基因表達可能會呈現(xiàn)出一個有趣的治療策略。在目前的研究中,我們調(diào)查了livin的表達在胃cancinomas及其鄰近組織。livin的表達和臨床病理參數(shù)之間的關系進行了分析。此外,我們探索了在抑制livin基因表達的shRNA可行性和胃癌易感性的凋亡細胞由shRNA介導的 livin沉默基因。2患者和方法2.1 患者和腫瘤樣本胃癌中四十個病例及接受胃切除手術(shù)的患者收集到的胃癌組織中13個病例(患者年齡從2977歲)。其中良性胃潰瘍的13個病例(慢性淺表胃炎)患者在接受了胃內(nèi)視鏡檢查(患者年齡從3377

7、歲)。這些病例均來自南京醫(yī)科大學第一附屬醫(yī)院。胃癌患者被診斷為TNM級的14階段(UICC ，2002)。手術(shù)之后腫瘤標本就立即被凍結(jié)在液態(tài)氮中,儲存在-80直到使用為止。這是在所有的病人知情同意的情況下獲得的。2.2逆轉(zhuǎn)錄聚合酶鏈反應技術(shù)程序總共RNA(2毫克)提取冷凍組織反轉(zhuǎn)錄進行，最后的體積2微升是用100 pmol of oligo(dT)15和200U M-MLV與逆轉(zhuǎn)錄酶(promega、美國) ,根據(jù)制造商的說明。Aliquots對應的250微升cDNA被放大在PCR緩沖容器中，在最終的50微升中含25pmol / ml處理劑和1U聚合酶。每一個放大了35周期,一個周期的變性曲線

8、在30 s內(nèi)到達94。熱處理在30s內(nèi)59（livin and b-actin）擴大到30s內(nèi)72。沒有RNA的病例作為陰性對照物包含在RTPCR中。一系列常用的的 livin和-actin處理劑如下： livin / upstream，5-TCCACAGTGTGCAGGAGACT-3;livin/ downstream;5-TCCACAGTGTGCAGGAGACT-3;-actin upstream,5-ACGGCACAAAGACGATGGAC-3-actin downstream,5-AGCGCAAGTACTCCGTGTG-3。產(chǎn)品的尺寸分別為livin/是312/258 bp ，-act

9、in 是501bp。2.3 西方吸干技術(shù)分析病變同質(zhì)性與沖力緩沖50mM Tris-HCl (pH 7.5), 250 mM NaCl,0.1% NP40和5mM EGTA包含50mM氟化鈉，60mM -丙三醇-磷酸鹽,0.5mM釩酸鈉,0.1mM苯甲基磺醯化氟10g/ml亮抑蛋白酶肽。用考馬斯亮藍微盤比色法測定蛋白質(zhì)含量。蛋白質(zhì)樣品電泳10% 變性SDS凝膠并轉(zhuǎn)移到PVDF膜(Roche、美國)。膜是培養(yǎng)特定的主要的抗體,與過氧化物酶繼發(fā)性抗體反應 (細胞信號技術(shù)、美國),最后通過增強化學熒光達到可視化 (細胞信號技術(shù)、美國)。Alexesis(美國) 和散塔克魯茲生物技術(shù)(美國)購買的單克

10、隆抗體livin (1:250) and actin (1:400)24細胞系和細胞培養(yǎng)我們選了一個人類胃腺癌細胞系在這一研究中。SGC-7901(上海細胞研究所,中國上海,)是一種附著中度分化胃腺的人類細胞株。線是胃癌細胞上皮細胞,并成長為附著細胞RPMI 1640 (Hyclone Inc, USA)含10%FCS (Life Technologies, Inc.),每毫升100個單位的青霉素和毫升100微克的鏈霉素(BioWhittaker)。在含有5%CO2空氣的條件下的37濕潤培養(yǎng)器中保存SGC-7901細胞。溶解順鉑和氟尿嘧啶(齊魯制藥廠、中國)在DMSO并且4儲存。2.5。ShR

11、NA的合成和PGPU / GFP /Neo/livin質(zhì)粒的制造通過siRNASequence-Selector軟件設計并合成了Livin的ShRNA序列(上海生物技術(shù)有限責任公司。集團公司、中國)。序列如下(表1),然后被插入pGPU/GFP/Neo(上海GenePharma股份有限公司。中國) BbsI and BamH地址產(chǎn)生pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control質(zhì)子。2.6SGC-7901的建立穩(wěn)定表達pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control轉(zhuǎn)染實驗,SGC-7901細胞被鍍成6孔板(3

12、15;105孔密度), , 96孔板(1 ×104孔密度)和12孔板(1.5 ×105孔密度)轉(zhuǎn)染之前培養(yǎng)24小時。按照制造商的說明這些細胞被調(diào)控子用Li-pofectAMINE 2000轉(zhuǎn)染4毫克/孔空的pGPU/GFP/Neo/vector， pGPU/GFP/Neo/livin或pGPU/GFP/Neo/Control 質(zhì)粒 (生命技術(shù)公司、大島嶼,NY)。轉(zhuǎn)染48小時后,這些細胞被轉(zhuǎn)移在 1:15 (v/v)并用Geneticin (G418) 1000克/毫升培養(yǎng)4周。穩(wěn)定轉(zhuǎn)染的克隆體取出并保存在媒介容器400 g/ml G418用作另外的研究。27依賴貼壁細胞細

13、胞生長的測定親本細胞和細胞穩(wěn)定表達pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control被種到6孔盤子中。每隔一天收集三孔的細胞。使用計數(shù)器確定細胞數(shù)目(Coulter Electronics, Miami, FL).。種植一些天數(shù)后用平均SD記錄每孔細胞的數(shù)量。2.8MTT測定通過MTT對細胞毒性進行了測量。呈幾何數(shù)增長的細胞被鍍在密度為10000細胞/孔的96孔板上作用24小時。接下來這些細胞被以不同濃度的藥物治療48小時。每孔加入100微升MTT溶液 (1毫克/毫升),并且這些細胞放在37下培養(yǎng)四小時。上層清液用異丙

14、醇代替溶解有色甲品。用micro-ELISA測量到吸光率的波長為595nm(ClinBio-128 SLT, ,奧地利)。治療細胞的吸光率相當于計算控制細胞的吸光率并用細胞死亡百分率顯示出來。2.9流式細胞術(shù)細胞被收集并加入冰冷的70%乙醇于PBS緩沖液中儲存在-4攝暖直到使用。懸浮后后，100 ml核糖核酸酶I (1 mg/ml) and 100 ml 的聚酰亞胺(400 g/ml) 37下培養(yǎng)細胞并用流式細胞術(shù)(BD,美國)進行分析。2.10統(tǒng)計分析數(shù)據(jù)的展現(xiàn)要用至少三個不同實驗±SD的方法。實驗結(jié)果用學生的t檢驗來分析和當P < 0.05時被認為是具有統(tǒng)計學顯著

15、性。3結(jié)果3.1。livin在胃腸癌中的表達在目前的研究中,我們第一次驗證了逆轉(zhuǎn)錄聚合酶鏈反應技術(shù)和西方吸干技術(shù)的存在在40胃癌中,13 癌組織和13良性病變胃粘膜損傷。在癌組織和良性病變胃粘膜損傷中, 每個mRNA亞型不可見水平被發(fā)現(xiàn)后,在腫瘤組織中,19/40(47.5%)顯示出mRNA及l(fā)ivina和livin蛋白質(zhì)表達(Figs. 1 and 2). 。livin表達與預后變量相關,如組織學惡性度和淋巴結(jié)轉(zhuǎn)移,但包括年齡、性別、階段和腫瘤細胞浸潤程度(表2)。3.2。穩(wěn)定轉(zhuǎn)染表達pGPU/GFP/Neo/livin與pGPU/GFP/Neo/Control的特征我們建立了SGC-790

16、1 與任一pGPU/GFP/Neo/livin穩(wěn)定轉(zhuǎn)染，pGPU/GFP/Neo /Control 質(zhì)粒，或空pGPU/GFP/Neo/vector (圖3)。用西方吸干技術(shù)和逆轉(zhuǎn)錄聚合酶鏈反應技術(shù)選擇每個轉(zhuǎn)染的克隆并分析決定livin mRNA,和蛋白質(zhì)表達。其他的都被選擇作為擴展和另外的研究。(如圖4及5)顯示， livin mRNA和蛋白質(zhì)的水平在SGC-7901pGPU/GFP/Neo/livin2中轉(zhuǎn)染降低了90%以上。Livin表達抑制在pGPU/GFP/Neo/livin1轉(zhuǎn)染和消極控制中沒有被發(fā)現(xiàn)。所以SGC-7901 pGPU/GFP/Neo/livin2被用來做后續(xù)實驗。3

17、.3穩(wěn)定轉(zhuǎn)染中抑制細胞生長SGC-7901的增長率pGPU / GFP /Neo/ livin2均有顯著的抑制轉(zhuǎn)染。如圖顯示，SGC-7901 Pgpu/GFP/尼歐/ livin2 / GFP細胞數(shù)目有顯著下降轉(zhuǎn)染在72小時到96小時后鍍(P < 0.01),而陰性對照和家長的細胞。3.4。穩(wěn)定的轉(zhuǎn)染容易受細胞凋亡因子刺激我們認為SGC-7901 pGPU/GFP/Neo/livin2 轉(zhuǎn)染和陰性對照細胞同細胞毒順鉑的增長速率明顯抑制，圖表6中顯示SGC-7901 pGPU/GFP/Neo/livin2轉(zhuǎn)染細胞數(shù)量培養(yǎng)后72小時和96小時與消極控制和親本細胞相比明顯降低陰性對

18、照細胞和細胞毒藥物(5 -氟尿嘧啶和順鉑)。pGPU MTT測定表明,SGC-7901 /Neo/ livin2 / GFP更敏感轉(zhuǎn)染順鉑和氟尿嘧啶比消極的控制和親本細胞(Figs. 7A, 6B)。由順鉑和5氟尿嘧啶誘導凋亡細胞數(shù)增加至約2.5 - 3倍的pGPU/GFP/Neo/livin2轉(zhuǎn)相比，其控制細胞（P<0.001;圖7C條。）。此外，經(jīng)歷了穩(wěn)定轉(zhuǎn)染無自發(fā)性凋亡更容易比對照細胞（P<0.05。圖7C條）凋亡刺激。討論在本研究中,我們表明,推薦的新成員: 一個新的IAP家族成員，被認為是不符合非癌胃組織的表達，只有在胃癌患者（47.5）的比例計算，也表明，抑制Livin

19、的表達或功能的原因自發(fā)性細胞凋亡和對SGC - 7901細胞生長的抑制，使細胞更容易凋亡刺激。據(jù)認為，活著有兩個亞型，A和B雖然這兩個亞型在阻止腫瘤壞死因子誘導細胞凋亡參與- A和抗CD95的在體外，他們表現(xiàn)出一些不同的抗凋亡的特性?；钪鴅似乎是在阻斷DNA損傷劑誘導細胞凋亡13超過活著有效。一些組織中Livin分布研究表明，最近都活著升高亞型A和B已發(fā)現(xiàn)在心臟，胎盤，肺，脾，卵巢，而活著balone特別是在已檢測到胎兒組織和dult腎臟和Livin一單是在腦，骨骼肌和外周血淋巴細胞的檢測11-14。此外，雖然Livin的表達是在一個癌細胞的細胞株和腫瘤組織中的一些品種檢測14-18和反活著抗

20、體在胃癌和肺癌患者血清識別19,20，沒有數(shù)據(jù)有關亞型中Livin的表達在胃癌腫瘤組織。我們的第一次研究表明，活著亞型A和B幾乎都在胃癌組織（47.5）和Livin表達與一些已知的預后因素，如分級，淋巴結(jié)轉(zhuǎn)移，相關的比例計算。從文獻資料表明，這兩個活著亞型參與了阻止細胞凋亡，并可能給活著的過度表達與細胞的強烈抵抗化療誘導細胞凋亡。胃癌一般具有高度抗癌癥放化療和中抗凋亡21。這些結(jié)果表明，Livin的高表達可能對某些癌癥患者和胃癌患者預后化療的責任。與促進腫瘤細胞的凋亡抵抗可能提供一個合理干預策略在癌癥治療的基礎上發(fā)展新的特定因素干擾22,23。由于Livin的表達可能有助于腫瘤細胞和腫瘤的特異

21、性表達及其在細胞凋亡的抗性表型可以讓活著的一個有趣的腫瘤治療靶點的具體干預措施的戰(zhàn)略，我們選擇了作為一個分子靶點的Livin基因。的shRNA技術(shù)representiong一個極其有力的工具，抑制內(nèi)源性基因表達24,25作出抑制Livin基因，并試圖糾正胃癌細胞凋亡的不足。作者：沉默的shRNA功效的tageted基因的表達是不同的，與半的生活和豐富的基因產(chǎn)物與靶mRNA作為24-27，以及無障礙的關系。在這項研究中，我們觀察到硅livin1是經(jīng)常更強烈的沉默比硅livin2 Livin基因。我們的研究結(jié)果還表明，沉默Livin基因的表達可能存在強烈的增加或幾個凋亡的代理人在場下的SGC -

22、7901細胞凋亡反應，抑制細胞的生長，這表明，與美好生活的干擾導致了對凋亡刺激的敏感性。對HeLa細胞類似的結(jié)果報告了Crnkovic -梅坦斯18?？傊?，我們的結(jié)果表明，Livin的表達和功能抑制自發(fā)性細胞凋亡和抑制細胞生長的體外敏感性增強化療藥物的結(jié)果。由于在胃癌中的表達，但活著的優(yōu)惠在正常組織中，這些數(shù)據(jù)表明，針對活著途徑單獨或與細胞毒性藥物可能在胃癌的治療作用。盡管他們的治療潛力，主要技術(shù)障礙仍有待克服，才能申請成為毒品的shRNA。在治療方面，將不得不滿足基因治療的辦法，如高效輸送到目標細胞的免疫反應或規(guī)避，一般的挑戰(zhàn)。值得注意的是，在最近的研究表明，體內(nèi)的shRNA可以直接應用到出

23、生后小鼠臟器highpressure尾靜脈注射，導致靶基因特異性抑制28-30。這些數(shù)據(jù)表明，一個活躍的shRNA通過血液的直接應用是主要可行的。英文翻譯：對照版Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin gene Background-Because of increased resistance to apoptosis in tumor cells, inhibition of specific

24、 antiapoptotic factors may provide a rational approach for the development of novel therapeutic strategies.Livin, a novel inhibitor of apoptosis protein family, has been found to be expressed in various malignancies and is suggested to have poorly prognostic significance. However, no data are availa

25、ble concerning the significance of livin in gastric cancer. In this study, we detected the expression of livin in human gastric carcinoma and investigated the apoptotic susceptibility of SGC-7901 cell by shRNAmediated silencing of the livin gene. Methods-The mRNA and protein expression of livin were

26、 analyzed by RT-PCR and western blot assay.The relationship between livin expression and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. Then it was tr

27、ansfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot assay were used to validate gene-silencing efficiency of livin in SGC-7901 cells. Stable clones were obtained by G418 screening. The cell apoptosis was assessed by flow cytometry (FCM). Cell growth state and 50 % inhibition

28、concentration (IC50) of 5-FU and cisplatin was determined by MTT method. Results-The expression of livin mRNA and protein were detected in 19 of 40 gastric carcinoma cases (47.5%) and SGC-7901 cells. No expression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positiv

29、e correlation was found between livin expression and poor differentiation of tumors as well as lymph node metastases (P < 0.05). Four small interfering RNA eukaryotic expressionvector specific to livin were constructed by gene recombination. And one of them can efficiently decrease the expression

30、 of livin, the inhibition of the gene was not less than 70% (P < 0.01). The recombinated plasmids were extracted and transfected gastric cancer cells. The stable clones were obtained by G418 screening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the

31、 gastric cancer cells was significantly lower than the control groups(P < 0.05). The study also showed that IC50 of 5-Fu and cisplatin on gastric cancer cells treated by shRNA was decreased and the cells were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) (P < 0.01). Conclusions

32、-C Livin is overexpressed in gastric carcinoma with a relationship to tumor differentiation and lymph node metastases, which is suggested to be one of the molecular prognostic factors for some cases of gastric cancer. ShRNA can inhibit livin expression in SGC-7901 cells and induce cell apoptosis.Liv

33、in may serve as a new target for apoptosis-inducing therapy of gastric cancer.1. Introduction Gastric cancer is one of the most common malignancies in the world. Most patients with this disease are diagnosed in advanced stages, and lose the chance of surgical eradication. Despite much progress in ch

34、emotherapy, the overall survival of the patients with gastric cancer in advanced stage is still poor. Resistance of cancer cells to chemoagents may contribute to failure of the treatment. Among the reasons of drug resistance, inhibited process of cell apoptosis may play an important role.Cancer cell

35、s are often characterized by increased resistance to apoptosis 1, which mediates their increased resistance to various stimuli of cell apoptosis, such as DNA damage, hypoxia, nutrient-deprivation 2,3. Moreover, apoptosis resistance is considered to be a major cause of therapeutic failure for tumors

36、in clinical practice, since many chemo- and/or radiotherapeutic agents function through the induction of apoptotic tumor death 4.Inhibitor of apoptosis protein (IAPs) is a novel family of intracellular proteins which suppress apoptosis induced by a variety of stimuli 5,6, including viral infection,

37、chemotherapeutic drugs, staurosporin, growth factor withdrawal, and by components of the tumor necrosis factor-a (TNF-a)/Fas apoptotic signaling pathways 7¨C9. The IAPs consists of a group of structurally related proteins with antiapoptotic properties 10, and may play a substantial role in prev

38、enting tumor cell from apoptosis, and has become the focus of research in recent years. A novel member of this family is ML-IAP/livin/KIAP/BIRC7 (in the following termed livin) which has two isoforms, livin a and livin b 11¨C14. It has been shown that over-expression of the livin can block apop

39、tosis induced by a variety of proapoptotic stimuli 12. Interestingly, livin gene has been found to be restrictively expressed in tumor cells, but not, or to lesser amounts in most normal adult tissues 11¨C15, and may contribute to tumorigenesis by allowing malignant cell to avoid apoptotic cell

40、 death. So inhibition of livin expression may represent an interesting therapeutic strategy. In the present study, we investigated the expression of livin in gastric cancinomas and their adjacent tissues. The relationship between livin expression and clinical pathologic parameters was analyzed. Furt

41、hermore, we explored the feasibility of shRNA in inhibiting livin gene expression and the apoptotic susceptibility of gastric cancer cell by shRNA-mediated silencing of the livin gene.2. Patients and methods2.1. Patients and tumor samples Forty samples of gastric carcinoma and 13 samples of paracanc

42、erous tissues were collected from the patients who received gastrectomy (age of patients ranging from 29-77 years). Thirteen samples of benign gastric lesion (chronic superficial gastritis) were gained from the patients undergoing gastric endoscopic examination (age of patients ranging from 33-77 ye

43、ars). These samples were collected from patients admitted to the First Affiliated Hospital of Nanjing Medical University. The patients with gastric cancer were diagnosed as being in stage I to IV based on TNM classification (UICC, 2002). Tumor specimens were immediately frozen in liquid nitrogen aft

44、er surgery and stored at -80 until use. Informed consent was obtained from all patients.2.2. RT-PCR procedureTotal RNA (2 mg) extracted from frozen tissues was reverse transcribed in a final volume of 25 ml with 100 pmol of oligo(dT)15 and 200U M-MLV reverse transcriptase (promega, USA), according t

45、o the manufacturers guidelines. Aliquots corresponding to 2.5 ml cDNA were then amplified in PCR buffer containing 25pmol/ml each primer and 1 U Taq polymerase in a final volume of 50 ml. Each amplification was performed for 35 cycles, one cycle profile consisted of denaturationat 94 8C for 30 s, an

46、nealing at 59 8C (livin and b-actin) for 30 s and extension at 72 8C for 30 s. A sample without RNA was included in each RT¨CPCR as a negative control. Sequences of livin and -actin primers used are as follows:livina/b up stream,50-TCCACAGTGTGCAGGAGACT-30;livina/downstream,50-ACGGCACAAAGACGATGG

47、AC-30;b-actinupstream,50-AGCGCAAGTACTCCGTGTG-30;-actin downstream, 50-AAGCAATGCTATCACCTCCC-30.The size of the amplified products were312/258 bp for livina/b and 501 bp for b-actin respectively.2.3. Western Blot Analysis Tissues were homogenized with lysis buffer 50 mM Tris-HCl(pH 7.5), 250 mM NaCl,

48、0.1% NP40, and 5 mM EGTA containing 50 mM sodium fluoride, 60 mM b-glycerol-phosphate, 0.5 mM sodium vanadate, 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin. The total protein concentrationwas determined using Coomassie Brilliant Blue. Protein samples were electrop

49、horesed in a 10% denaturing SDS gel and transferred to PVDF membrane (Roche, USA). The membranes were incubated with specific primary antibodies, reacted with a peroxidase-conjugated secondary antibody (Cell signaling technology,USA), and finally visualized by enhanced chemiluminescence (Cell signal

50、ing technology, USA). Monoclonal antibodies recognizing livin (1:250) and actin (1:400) were purchased from Alexesis Inc. (USA) and Santa Cruz Biotechnology (USA).2.4. Cell lines and cell culture We selected a human gastric adenocarcinoma cell lines for thisstudy. SGC-7901 (Shanghai Institute of Cel

51、l Research, Shanghai,China) is an adherent, moderately differentiated, human gastric adenocarcinoma cell line. The cell lines are gastric cancer epithelial cells and grow as adherent cells in RPMI 1640 (Hyclone Inc, USA)containing 10% FCS (Life Technologies, Inc.), 100 units/ml penicillin,and 100 mg

52、/ml streptomycin (BioWhittaker). SGC-7901 cells were maintainedat37 8Cina humidified incubatorwithanatmosphere of 5% CO2. Cisplatin and 5-fluorouracil (Qilu pharmaceutical factory,China) were solublized in DMSO and stored at 4 8C.2.5. ShRNA synthesis and construction of PGPU/GFP/Neo/livin plasmidsSh

53、RNA sequences of livin were designed by software of siRNA Sequence-Selector and synthesized (Shanghai Biotech, Ltd.Corp., China). The sequences as following (Table 1)then were inserted into BbsI and BamH sites of the pGPU/GFP/Neo(Shanghai GenePharma Co. Ltd China) to generate pGPU/GFP/Neo/livin and

54、pGPU/GFP/Neo/Control plasmids,respectively.2.6. Establishment of SGC-7901 stable transfectants expressing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control For transfection experiments, SGC-7901 cells were plated into 6-well plates (3¡Á105 cells/well), 96-well plates (1×104 cells/well) and

55、12-well plates (1.5×105 cells/well) for 24 h before transfection The cells were transfected with 4 mg/well of empty pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control plasmid using Li-pofectAMINE 2000 (Life Technologies, Inc., Grand Island,NY) according to the manufacturer¡

56、75;s instructions. Forty-eight hours after transfection, the cells were passaged at 1:15 (v/v) and cultured in mediumsupplemented with Geneticin (G418) at 1000 g/ml for 4 weeks. Stably transfected clones were picked and maintained in medium containing 400 g/ml G418 for additional studies.2.7. Assay

57、of anchorage-dependent cell growth Parent cells and cells stably expressing empty pGPU/GFP/Neo vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control were seeded into 6-well plates. Cells from triplicate wells were collected every other day. Cell numbers were determined using a Coulter counter (Coulter

58、Electronics, Miami, FL). The number of cells per well is reported as the average SD at the indicated number of days after plating.2.8. MTT assay Cytotoxicity was measured by MTT assay. Cells growing exponentially were plated onto 96-well plates at a density of 10000 cells/well for 24 h. The cells we

59、re then treated with different concentrations of drugs for 48 h. One hundred microliters of MTT stock solution (1 mg/ml) were added to each well, and the cells were further incubated at 37 for 4 h. The supernatant was replaced with isopropyl alcohol to dissolve formazan production. The absorbance at wavelength 595 nm was measured with a micro-ELISA reader (ClinBio-128, SLT, Austria). The ratio of th