1、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據。在本研究中 , 我們發現livin 基因在人類胃癌中的表達并調查了介導的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。方法一mRN及蛋白質livin基因的表達用逆轉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轉染進入SGC-7901細

2、胞。逆轉錄聚合酶鏈 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。細胞生長狀態和5-FU的50%卬制濃度(IC50)和順鉑都由MTTt匕色法來決定。結果一livin mRN和蛋白質的表達檢測40例中有19例有胃癌和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。相關發現在 livin 基因的表達和腫瘤的微小分化和淋巴結轉移一樣 (P < 。 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

3、,能有效地減少 livin 基因的表達 ,抑制基因不少于 70%(P < 。重組的質粒被提取和轉染到胃癌細胞。 G418篩選所得到的穩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 < 。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 <。結論 livin 基因在胃癌中的過分表達與腫瘤分化與淋巴結轉移建立聯系 , 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。

4、 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、缺氧、營養損失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的 4。酶抑制劑 (IAPs), 是一種新型的凋

5、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(TNF- a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構相關的蛋白質構成 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livin11-14。有證據表明 , 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 基因被發現在腫瘤細胞中限制性表達 , 但是不存在或是很少數量存在于 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

6、抑制 livin 基因表達可能會呈現出一個有趣的治療策略。 在目前的研究中 , 我們調查了 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 了胃內視鏡檢查 （患者年齡從 3377歲）。這些病例均來自南京醫科大學第一附屬

7、醫院。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 立即被凍結在液態氮中，儲存在-80C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。逆轉錄聚合酶鏈反應技術程序總共RNA（2毫克）提取冷凍組織反轉錄進行，最后的體積2微升是用100 pmolof oligo（dT）15 和200UM-MLV與逆轉錄酶（promega、美國）,根據制造商的說明。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內到達94C。熱處理在 30s

8、內59C（ livin and b-actin ）擴大到30s 內72C。沒有RNA勺病例作為陰性對照物包含在 RT- PCR中。一系列常用的的 livin 和 B -actin 處理劑如下： livin a / B upstream，5 -TCCACAGTGTGCAGGAGACT-3 ;livin a / B downstream;5 -TCCACAGTGTGCAGGAGAC;TB-3-actin upstream,5 -ACGGCACAAAGACGATGGAC-3 B -actin downstream,5 ' -AGCGCAAGTACTCCGTGTG-產品 的尺寸分別為 livi

9、n a /B 是 312/258 bp ，B -actin 是 501bp。西方吸干技術分析病變同質性與沖力緩沖 50mMTris-HCI （pH , 250 mMNaCl,% NP40和 5mMEGTA 包含50mM氟化鈉，60mMB -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。用考馬斯亮藍微盤比色法測定蛋白質含量。蛋白質樣品電泳10%變性SDS凝膠并轉移到PVDF膜（Roche、美國）。 膜是培養特定的主要的抗體 , 與過氧化物酶繼發性抗體反應 （ 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 ） 。 Al

10、exesis（ 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%CO空氣的條件下的37C濕潤培養

11、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DMS（并且4C儲存。ShRNA勺合成和PGPU / GFP /Neo/livin 質粒的制造通過siRNASequence-Selector軟件設計并合成了 Livin的ShRN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/GFP/Neo上海GenePharm股份有限公司。中國 ）Bbsl and BamH地址產生 pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control 質子。的建立穩定表達 pGPU/GFP/

12、Neo/livin and pGPU/GFP/Neo/Control 轉染實 驗,SGC-7901細胞被鍍成6孔板（3 X 105孔密度）,96 孔板（1 X 104孔密度）和12孔 板X 105孔密度）轉染之前培養24小時。按照制造商的說明這些細胞被調控子用 Li-pofectAMlNE 2000 轉染4毫克/孔 空的 pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin 或pGPU/GFP/Neo/Control 質粒 （生命技術公司、大島嶼,NY）。轉染48小時后，這些細胞被轉移在1:15 （v/v）并用 Geneticin （G418） 1000克/毫升培養

13、4周。穩定轉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。使用計數器 確定細胞數目 （Coulter Electronics, Miami, FL）.。種植一些天數后用平均 SD記錄每孔細胞的數量。測定通過MTT對細胞毒性進行了測量。呈幾何數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

14、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37E下培養 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。流式細胞術細胞被收集并加入冰冷的70聽醇于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°C 下培養細胞并用流式細胞術（BD,美國）進行分析。統計分析數

15、據的展現要用至少三個不同實驗土 SD的方法。實驗結果用學生的t檢驗來 分析和當 P < 時被認為是具有統計學顯著性。3 結果。 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。在癌組織和良性病 變胃粘膜損傷中,每個mRN亞型不可見水平被發現后，在腫瘤組織中，19/40%）顯 示出 mRNAi livina a 和 livin B 蛋白質表達（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轉移 ， 但包括年齡、性別、階段和腫瘤細 胞

16、浸潤程度 （表2）。穩定轉染表達 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 穩定轉染， pGPU/GFP/Neo /Control 質粒，或空 pGPU/GFP/Neo/vector （ 圖3）。用西方吸干技術和逆轉錄聚 合酶鏈反應技術選擇每個轉染的克隆并分析決定 livin mRNA， 和蛋白質表達。其他的都被選擇作為擴展和另外的研究。（如圖4及5）顯示，livinmRN和蛋白質的水平在SGC-7901pGPU/GFP/Neo/livin2中轉染降低了 90%以上。Liv

17、in表達抑 制在pGPU/GFP/Neo/livi n1轉染和消極控制中沒有被發現。所以 SGC-7901 pGPU/GFP/Neo/livin2 被用來做后續實驗。穩定轉染中抑制細胞生長SGC-7901的增長率pGPU / GFP /Neo/ livin2 均有顯著的抑制轉染。如圖顯 示，SGC-7901 Pgpu/GFP尼歐/ livin2 / GFP細胞數目有顯著下降轉染在 72小時到 96 小時后鍍 （P < ， 而陰性對照和家長的細胞。穩定的轉染容易受細胞凋亡因子刺激我們認為 SGC-7901 pGPU/GFP/Neo/livin2 轉染和陰性對照細胞同細胞毒順 鉑的增長速率明

18、顯抑制， 圖表 6中顯示 SGC-7901pGPU/GFP/Neo/livin2 轉染細胞 數量培養后 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）。pGPLMTT測定表明，SGC-7901/Neo/ livin2 / GFP更敏感轉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增加至約 - 3 倍的 pGPU/GFP/Neo/livin2 轉相 比，其控制細胞（P<圖7C條。）。此外，經歷了穩定轉染無自發性凋亡更容易比 對照細胞（Pv圖7C條）凋亡刺激。討論在本研究中

19、, 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 （）的比例計算， 也表明， 抑制 Livin 的表達或功能的原因自發性細胞凋亡和對 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已發現在心臟， 胎盤，肺，脾，卵巢，而活著 balone 特別是在已檢測到胎兒組織

20、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 抗體在胃癌和肺癌患者血清識別 19,20 ，沒有數據有關亞型中 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（）和 Livin 表達與一些已知的預后因素，如分級，淋巴結轉移，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 。 這些結果表

21、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 具體干預措施的戰略，我們選擇了作為一個分子靶點的Livin基因。的shRNA技術 representiong 一個極其有力的工具，抑制內源性基因表達 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

22、因產物與靶mRN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 是經常更強烈的沉默比硅 livin2 Livin 基因。我們的研究結果還表明，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 - 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 細胞類似的結果報告了 Crnkovic - 梅坦斯 18 ?？傊?，我們的結果表明， Livin 的表達和功能抑制自發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果。由于在胃癌中的表達，但活著的優惠 在正常組織中，這些數據表明，

23、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，如高效輸送到目標細胞的免疫 反應或規避，一般的挑戰。值得注意的是，在最近的研究表明，體內的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。這些數據表明，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

24、-7901 cells by shRNA-mediated silencing of livin geneBackground-Because of increased resistance to apoptosis in tumor cells, inhibition of specific antiapoptotic factors may provide a rational approach for the development of novel therapeutic , a novel inhibitor of apoptosis protein family, has been

25、 found to be expressed in various malignancies and is suggested to have 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

26、susceptibility of SGC-7901 cell by shRNAmediated silencing of the livin gene.Methods-The mRNA and protein expression of livin were analyzed by RT-PCR and western blot relationship between livin expression and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expre

27、ssion vector specific to livin was constructed by generecombination, 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-7901cells. Stable clones were obtai

28、ned by G418 screening. The cell apoptosis was assessed by flow 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 %) and SGC-

29、7901cells. No expression of livin was detected in tumor 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 < . Four small interfering RNA eukaryotic expression vector specif

30、ic to livin were constructed by gene recombination. And one of them can efficiently decrease the expression of livin, the inhibition of the gene was not less than 70% (P < . The recombinated plasmids were extracted and transfected gastric cancer cells. The stable clones were obtained by G418 scre

31、ening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the gastric cancer cells was significantlylower than the control groups(P < . The study also showedthat IC50 of 5-Fu and cisplatin on gastric cancer cells treated by shRNA was decreased and the cell

32、s were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) (P < .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 c

33、ancer. ShRNA can inhibit livin expression in SGC-7901 cells and induce cell 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 disease are diagnosed in advanced stages, a

34、nd lose the chance of surgical eradication. Despite muchprogress in chemotherapy, 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, inhi

35、bited process of cell 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 resistanc

36、e is considered to 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 apop

37、tosis induced by a variety of stimuli 5,6, including viral infection, chemotherapeutic drugs, staurosporin, growth factor withdrawal, and by components of the tumornecrosis factor-a (TNF-a)/Fas apoptotic signaling pathways 7C9. TheIAPs consists of a group of structurally related proteins with antiap

38、optotic properties 10, and may play a substantial role in preventing tumor cell from apoptosis, and has becomethe focus of research in recent years. A novel memberof 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 h

39、as been shown that over -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 normal adult tissues 11 C15, and may contribute to tumorig

40、enesis by allowing malignant cell to avoid apoptotic cell 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 expressi

41、on and clinical pathologic parameters was analyzed. Furthermore, we explored the feasibility of shRNAin inhibiting livin gene expression and the apoptotic susceptibility of gastric cancer cell by shRNA-mediated silencing of the livin gene.2. Patients and methods. Patients and tumor samplesForty samp

42、les of gastric 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

43、 examination (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 speci

44、mens were immediately frozen in liquid nitrogen after surgery and stored at -80°C unt il use. Informedconsent was obtained from all patients. 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-

45、MLV reverse transcriptase (promega, USA), according to the manufacturer'sguidelines.Aliquots corresponding to ml cDNAwere then amplified in PCRbuffer 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 prof

46、ile 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 in eluded in each RTCPCR as a n egative con trol.Seque nces of livi n and&act in primers used are as follows:livi na/bupstream,50-TCCACAGTGTGC

47、AGGAG3A0C;iTv-ina/B downstream,50-ACGGCACAAAGACGATGGAC-30;b-acti nu pstream,50-AGCGCAAGTACTCCGTG0fQB-actin downstream, size of the amplified products were312/258 bp for livina/b and 501 bp for b-actin respectively. Western Blot AnalysisTissues were homogenized with lysis buffer 50 mM Tris-HCl(pH , 2

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

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

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

51、 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 DMSOand stored at 4 8C. . ShRNA synthesis and construction of PGPU/GFP/Neo/livin plasmidsShRNA

53、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 pGPU

54、/GFP/Neo/Control plasmids,respectively. . 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-well4plates (3? a 105cells/well),96- well plates (1 x 10 cells/well) and 12-well5plates x 10

55、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 Technologies, Inc., Grand Island,NY) according to the manufacturer? - s instructions. Forty-eight hours a

56、fter transfection, thecells were passaged at 1:15 (v/v) and cultured in mediumsupplemented withGeneticin (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. Assay of anchorage-dependent cell growthParent cel

57、ls 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 Electronics, Miami, FL). The number of cells

58、per well is reported as the average SDat the indicated number of days after plating. 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 dr

59、ugs for 48 h. One hundred microliters of MTT stock solution (1 mg/ml) were addedto each well, and the cells were further in cubated at 37°C for 4 h. T hesupernatant was replaced with isopropyl alcohol to dissolve formazan production.The absorbance at wavelength 595 nm was measured with amicro-ELISA reade