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 livin 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 livin 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及西方 吸干化驗進行了分析。小干擾RNA真

2、核表達載體具體到livin基因采用基因重組、 測序核酸。然后用 Lipofectamin2000 轉(zhuǎn)染進入 SGC-7901 細胞。逆轉(zhuǎn)錄聚合酶鏈 反應技術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)果一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 livin 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 liv

3、in 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 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

4、一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。 ShRNA 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

5、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。酶抑制劑（lAPs）,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-a（TNFa凋亡信號通路” Fas信號通路7 - 9。 lAPs是由一組有凋亡特性的結(jié)構相關的蛋白質(zhì)構成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 livin £1-

6、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

7、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）。手術之后腫瘤標本 就立即被凍結(jié)在液態(tài)氮中，儲存在-80°C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）1

8、5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 曲線在30 s內(nèi)到達94C。熱處理在 30s內(nèi)59C（ livin and b-actin）擴大到30s內(nèi) 72C。沒有RNA的病例作為陰性對照物包含在 RT -PCR中。一系列常用的的livin和 伕actin處理劑如下：livin a B upstream 5'-TCCACAGTGTGCAGGAGACT-3-TCCACAGTGTGCAGGA

9、GACT-3-ACGGCACAAAGACGATGGAC-3-AGCGCAAGTACTCCGTGTG-3產(chǎn)品的尺寸分別為;livin a/Bdownstream;5;B-actin upstream,5B-actindownstream,5 a是B312/258 bp ,livin3-actin 是 501bp。2.3 西方吸干技術分析病變同質(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 B-丙三醇-磷酸鹽,0.5mM釩酸鈉,0.1mM苯 甲基磺醯化氟10卩g/ml亮抑蛋白酶肽。用考馬斯

10、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、 美國）,最后通過增強化學熒光達到可視化（細胞信號技術、美國）。Alexesis（美國）和散塔克魯茲生物技術 （美國）購買的單克隆抗體 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 （Hyc

11、lone Inc, USA）含 10%FCS （Life Technologies, Inc.）, 每毫升 1 00個單位的青霉素和毫升 1 00微克的鏈霉素 （BioWhittaker） 。在含有5%CO2空氣的條件下的37C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4C儲存。2.5。ShRNA的合成和PGPU / GFP /Neo/livin質(zhì)粒的制造通過siRNASequence-Selecto軟件設計并合成了 Livin的ShRNA序列（上海生物 技術有 限責任公司。 集團公司、 中 國 ）。 序列 如下（表1）,然后被插入 pGPU/

12、GFP/Neo（上海GenePharm股份有限公司。中國）BbsI and BamH地址產(chǎn)生 pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Co ntro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為05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,

13、pGPU/GFP/Neo/livi n 或 pGPU/GFP/Neo/C on trol 質(zhì)粒 （生命技術公司、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

14、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37C下培養(yǎng) 四小時。上層清液用異丙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

15、胞術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C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卩g/ml） 37下 培養(yǎng)細胞并用流式細胞術（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和西方吸干技 術的存在在 40 胃癌中 ,13 癌組織和 13 良性病變胃粘膜損傷。在癌組織

16、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 livina 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-7901 與任一 pGPU/GFP/Neo/livin 穩(wěn)定轉(zhuǎn)染，pGPU/GFP/Neo /Control 質(zhì)粒，或空

17、 pGPU/GFP/Neo/vector （圖3）。用西方吸干技術和逆轉(zhuǎn)錄聚合 酶鏈反應技術選擇每個轉(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/livi n1轉(zhuǎn)染和消極控制中沒有被發(fā)現(xiàn)。所以 SGC-7901 pGPU/GFP/Neo/livi n2被用來做后續(xù)實驗。3.3穩(wěn)定轉(zhuǎn)染中抑制細胞生長SGC-7901 的增長率 pGPU / GFP /Neo/ l

18、ivin2 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照細胞 和細胞毒藥物（5 -氟尿嘧啶和順鉑）。pGPU MTT測定表明,SGC

19、-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（PV0.001;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 的表達或功能的原因自發(fā)性細胞凋亡和對 SGC - 79

20、01 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。活著 b 似乎是在阻斷 DNA 損傷劑誘導細胞凋亡 1 3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

21、和反活著抗體 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 L

22、ivin 的表達可能有助于腫瘤細胞和腫瘤的 特異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representiong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 Liv

23、in 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 - 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

24、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 geneBackground-Because of increased res

25、istance to apoptosis in tumor cells, inhibition of specific 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

26、poorly prognostic significance. However, no data are available 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

27、gene.Methods-The mRNA and protein expression of livin were 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 recombi

28、nation, and the nucleic acid was sequenced. Then it was transfected 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 assessedby flo

29、w cytometry (FCM). Cell growth state and 50 % inhibition 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

30、 adjacent tissues and benign gastric lesion. The positive 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 expression vector specific to livin were constructed by gene recombination

31、. And one of them can efficiently decrease the expression 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

32、livin gene was silenced, the reproductive activity of the 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 proapoptoti

33、c stimuli (5-Fu and cisplatin) (P < 0.01).Conclusions-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 ex

34、pression in SGC-7901 cells and induce cell apoptosis.Livin may serve as a new target for apoptosis-inducing therapy of gastric cancer.1. IntroductionGastric cancer is one of the most common malignancies in the world. Most patients with this diseaseare diagnosed in advanced stages, and lose the chanc

35、e of surgical eradication. Despite much progress in chemotherapy,the overall survival of the patients with gastric cancer in advanced stage is still poor. Resistanceof cancer cells to chemoagents may contribute to failure of the treatment. Among the reasons of drug resistance, inhibited process of c

36、ell apoptosis may play an important role.Cancer cells 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

37、 be a major cause of therapeutic failure for tumors 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

38、 variety of stimuli 5,6, including viral infection, 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 propert

39、ies 10, and may play a substantial role in preventing 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.lt has been shown that ov

40、er-expression of the livin can block apoptosis 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 no rmal adult tissues 11 C15, an dmay con tribute to tumorige nesis by allowing

41、 malignant cell to avoid apoptotic cell death. So inhibition of livin expression may represent an interesting therapeutic strategy.ln the present study, we investigated the expression of livin in gastric cancinomas and their adjacent tissues. The relationship between livin expression and clinical pa

42、thologic parameters was analyzed. Furthermore, 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 samplesForty samples of gastric

43、 carcinoma and 13 samples of paracancerous 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 (

44、age of patients ranging from 33-77 years). 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 imme

45、diately frozen in liquid n itroge n after surgery and stored at -80 °C un til use. In formed consent was obta ined 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

46、 reverse transcriptase (promega, USA), according to the manufacturer's 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

47、profile consisted of denaturationat 94 8C for 30 s, annealing 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 RTCPCR as a negative control.Sequences of livin and 3-actin primers used are as follows:livina/b up stream,50-TCCACAGTGTGCAGG

48、AGACT- 30;livina/ B downstreamijCGGCACA AAGACGATGGAC-30;b-actinupstream,50-AGCGCAAGTACTCCGTGTG- 30; -acti n 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 AnalysisTissues were homogenized wit

49、h lysis buffer 50 mM Tris-HCl(pH 7.5), 250 mM NaCl, 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 concentration was determined using Co

50、omassie Brilliant Blue. Protein samples were electrophoresed 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

51、visualized by enhanced chemiluminescence (Cell signaling 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 cultureWe selected a human gastric adenocarcinoma cell line

52、s for thisstudy. SGC-7901 (Shanghai Institute of Cell 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 Tec

53、hnologies, Inc.), 100 units/ml penicillin,and 100 mg/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 synthes

54、is and construction of PGPU/GFP/Neo/livin plasmidsShRNA 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 GenePharm

55、a Co. Ltd China) to generate pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control plasmids,respectively.2.6. Establishment of SGC-7901 stable transfectants expressing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/ControlFor transfection experiments, SGC-7901 cells were plated into 6-well plates (3?a 105 cells/well), 9

56、6-well plates (1 X04 cells/well) and 12-well plates (1.5 105 cells/well) for 24 h before transfectionThe 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 Tech no logies, In c., Grand Isla nd,NY) acc

57、ord ing to the manu facturer?ls 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 add

58、itional studies.2.7. Assay of anchorage-dependent cell growthParent 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

59、 Coulter counter (Coulter 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 assayCytotoxicity 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 were then treated with different concentrations of drugs for 48