课题规划与实施阶段的文献查阅-.ppt

1、课题规划与实施阶段的文献查阅,苏 川南京医科大学病原生物学系 86862774江苏省现代病原生物学重点实验室 86862773,GR-3,一、科研任务的三个阶段,科研选题阶段课题实施阶段查阅文献草拟研究方案、选择实验方法预试验/方案与方法修正正式实验结果分析与论文撰写阶段,二、课题实施阶段文献查阅的原则,不善于查找文献或未能认真、有效地检索和阅读文献，是造成课题重复和无效劳动的关键因素。合理使用、组合不同的检索词查阅不同种类的期刊、不同种类的文献大量阅读不同质量的文献，比较并批判性地采用,实验方案的选择（选用什么实验方案）：三多一少多看好文章多比较：找出共同点，分析各研究中采用不同方法的可能原

2、因多请教有经验的人少单纯追求方法上的创新最适，并受诸多条件的限制实验方法设计的基本原则：,三、课题实施/方案修正阶段文献阅 读的特点（着眼点）与科研选题阶段文献查阅的不同之处：,对照的设置（实验设计之关键）对照的意义：对照的意义首先在于：通过对照鉴别实验因素与非实验因素的差异。对照的意义其次在于：通过对照消除和减少实验误差。设立对照的要点：对照组与实验组有可比性对照的数量要足够对照处理的均衡性（一致性）对照的形式：自身对照组间对照潜在对照历史对照重复实验重复多样本量有统计学意义不浪费,随机化：单纯随机抽样系统抽样分层抽样整群抽样双步抽样 实验方法细节的确定：不盲目相信文献不完全相信文献多请教有

3、经验的人,阅读文献时要注意实验设计的基本要素所谓实验，就是尽可能地排除外界的许多影响，突出主要因素，并且在能够仔细地观察到各种现象之间相互关系的条件下，使某一事物（或过程）发生或重演。大多数医学研究课题，其目的都是为了阐明某些因素作用于某一研究对象时所产生的效应或影响。因此，因素、对象、效应便成了实验设计中的三个基本组成部分（或三个基本要素）。,四、实验方案设计时的文献阅读须关注的细节,因素：包括内在因素和外加因素A.内在因素：受试对象本身的因素研究人群血脂水平与冠心病发病率的关系时，血脂水平是该项研究中的（内在）因素研究子女高血压发生频率与父母高血压病之间的关系时，要探索和考虑的是遗传因素B

4、.外加因素：处理因素，目的是为了观察受试对象 的反应化学的：药物、毒物、营养物等物理的：按摩、理疗、温度、压力等生物的：病毒、细菌、原虫等,C.对于因素，要注意数量、质量和强度三个方面数量：一个实验中要研究的因素可为一个或多个，如同一种药可分为大中小三个剂量水平质量:因素为一种化学药品：生产厂家、剂型、批号、质量贮存方法等必须一致。因素为一种手术：在实验正式开始前必须先达到较为熟练的程度，否则在实验的前一阶段很不熟练，后一阶段才渐渐稳定下来，必然会对实验结果造成较大的误差。强度：对于外加因素的研究，要注意合理的、持续的强度。如给药方法、用药次数、疗程和间隔时间等，在不同对象及整个研究过程中应保

5、持一致。,对象：受试的人、动物、细胞、器官、组织等受试对象基本条件是否具备：诊断标准等受试对象选择标准必须统一、恰当。受试条件的生物学特性：注意动物的（品系）敏感性及国家标准、细胞的状态等。研究对象的选择：合适、最有利于检验假说人的选择动物的选择细胞、器官、组织等的选择人、动物、细胞等之间的选择,效应：试验结果，其指标的检测要注意指标的性质合适观察指标与检测指标、定量指标与定性指标指标与研究目的的相关性指标的数量合适适量但足够说明问题数量太多也是一种浪费分清主要指标与次要指标尽量采用客观指标：通过仪器或特定程序记录下来的各种数据资料。指标的精密与准确,Targeted Expression o

6、f GFP/TRAIL Fusion Protein from hTERT Promoter Elicits Antitumor Activity without Toxic Effects on Primary Human Hepatocytes,Tongyu Lin,Jian Gu,Lidong Zhang,Xuefeng Huang,L.Clifton Stephens,Steven A.Curley,and Bingliang Fang2Departments of Thoracic and Cardiovascular Surgery T.L.,J.G.,L.Z.,X.H.,B.F.

7、,Veterinary Medicine L.C.S.,and Surgical Oncology S.A.C.,The University of TexasM.D.Anderson Cancer Center,Houston,Texas 77030,CANCER RESEARCH 62,36203625,July 1,2002,Some Abbreviations,TRAIL tumor necrosis factor-related apoptosis-inducing ligandGV16 GAL4/VP16 fusion proteinhTERT human telomerase r

8、everse transcriptaseGT Gal4/TATANHFB normal human fibroblastNHPH normal human primary hepatocyte,Background,TRAIL:Apo2 LA 40 kDa type II transmembrane proteinOne cytokine of the TNF family The highest amino acid homology with FasL(28%)TNF-(23%)lymphotoxin-(23%)lymphotoxin-(22%),The function of TRAIL

9、,Apoptosis:anticancer activitySuppress tumor metastasis Prevent auto-immunity Virus induced diseases,T.M.Baetu,J.Hiscott/Cytokine&Growth Factor Reviews 13(2002)199207,Recent findings:Normal human hepatocytes,brain tissue,and certain epithelial cells are susceptible to recombinant TRAIL proteins The

10、potential toxicity of the TRAIL protein if administered systemicallydirect introduction of the TRAIL gene into cancer cells can elicit apoptosis and suppress tumor growth in vitro and in vivo.nontransduced neighboring cancer cells can be killed by the TRAIL or GFP/TRAIL fusion genes through bystande

11、r effects.,the hTERT promoter target proapoptotic genes to cancersthe GAL4 gene regulatory system augment transgene expression from the hTERT promoter without losing the target specificity.Ad/gTRAIL:express the GFP/TRAIL fusion gene driven by the hTERT promoter via GAL4 gene-regulatory components.,Q

12、uestions,1.If Ad/gTRAIL have toxic effects on normal human hepatocytes or not?11：in vitro 12：in vivoIf Ad/gTRAIL can induce apoptosis in cancer cells or not?21：in vitro 22：in vivo,1.Construction and Characterization of Ad/gTRAIL(Fig 1A),the hTERT promoter a GAL4 gene-regulatory system,Answer,a bicis

13、tronic adenoviral vector,Ad/gTRAIL which expresses the GFP/TRAIL fusion gene from the hTERT promoter via the GAL4 gene-regulatory system.,2.characterize this vectors functionality(Fig 1B),Human colon cancer cell line:DLD-1 Ad/CMV-GFP 50%GFP-positive cells no cell death.Ad/gTRAIL the same level of GF

14、P-positive cells 90%of the Ad/gTRAIL-treated cells were killed 48h after treatment,Question 1:If Ad/gTRAIL have toxic effects on normal human hepatocytes?,Question 1-1:In vitro Transgene Expression and Toxicity of Ad/gTRAIL in Normal Human Primary Hepatocytes(NHPH)or Normal Human Fibroblast(NHFB)Que

15、stion 1-2:In vivo Transgene Expression and Toxicity of Ad/gTRAIL after Systemic Administration,To answer Question 1-1 Transgene Expression and Toxicity of Ad/gTRAIL in NHPHs or NHFBs,NHPHs or NHFBs PBS,Ad/CMV-GFP,Ad/gTRAIL,or Ad/GT-TRAIL+Ad/PGK-GV16 Two days laterAnalyze GFP expressionQuantify apopt

16、otic cells by flow cytometric assay,Cytometric analysis of apoptosis(Fig 2A),GFP-positive cells(NHPHs or NHFBs)Ad/CMV-GFP:50%Ad/gTRAIL:30%apoptotic cells in NHFBs Ad/gTRAIL Ad/GT-TRAIL+Ad/PGK-GV16 background levels of apoptosis,top panel:levels of GFP expression bottom panel:apoptotic cell death,Fig

17、2A,microscopic study(Fig 2B),cell morphology of NHPHs at 48 h after treatment,Fig2B,cell viability assay(Fig 2C),Fig 2C:PBS(),Ad/CMV-GFP(),Ad/gTRAIL(),and Ad/GT-TRAIL+Ad/PGK-GV16(*).,In vitro Ad/gTRAIL have no toxic effects on NHPHs or NHFBs,conclusion 1-1,To answer Question 1-2 Transgene Expression

18、 and Toxicity of Ad/gTRAIL after Systemic Administration(Fig 3),Adult BALB/c mice PBS,Ad/CMV-GFP,Ad/gTRAIL,and Ad/GT-TRAIL+Ad/PGK-GV16 Histopathological changes Liver function test:AST ALTWestern blot analysis of GFP or GFP/TRAIL fusion protein expression,Histopathological Changes,Ad/GT-TRAIL+Ad/PGK

19、-GV16Ad/gTRAILAd/CMV-GFPPBS,liver function test(Fig 3A),PBSAd/CMV-GFP PBS Ad/CMV-GFP Ad/gTRAIL Ad/gTRAIL Ad/GT-TRAIL+Ad/PGK-GV16 Ad/GT-TRAIL+Ad/PGK-GV16,1,DLD-1 cancer cells treated with Ad/gTRAIL 24,liver samples after systemic administration of Ad/GT-TRAIL+Ad/PGK-GV1657,liver samples after systemi

20、c administration of Ad/gTRAIL89,liver samples after systemic administration of Ad/CMV-GFP,Fig 3,In vivoAd/gTRAIL doesnt show more toxiceffects.,conclusion 1-2,Treatment of NHPHs with Ad/gTRAIL resulted in no detectable transgene expression and in minimal toxicity In vivo study on mice showed no tran

21、sgene expression from Ad/gTRAIL in liver after system administration of a high dose of Ad/gTRAIL,conclusion 1-1+1-2,4.Question 2:If Ad/gTRAIL can induce apoptosis in cancer cells?,Question 2-1:In vitro Transgene Expression and Apoptosis Induction by Ad/gTRAIL in Cancer Cells in VitroQuestion 2-2:In

22、vivo Suppression of Tumor Growth by d/gTRAIL in Vivo,To answer Question 2-1(Fig 4)Transgene Expression and Apoptosis Induction by Ad/gTRAIL in Cancer Cells in Vitro(Fig 4A),human lung cancer cell lines(A549 and H460)human colon cancer cell lines(DLD-1 and Lovo)Ad/gTRAILAd/CMV-GFPAd/GT-TRAIL+Ad/PGK-G

23、V16,Two days later,cells were harvested and analyzed for GFP expression and apoptosis by FACS.Ad/CMV-GFP or Ad/gTRAIL:similar levels of GFP-positive cells(7090%)Ad/gTRAIL dramatically increase the number of apoptotic cells.,top panel:Levels of GFP expression bottom panel:apoptotic cell death,Fig 4A,

24、Ad/CMV-GFP high levels of transgene expression background levels of apoptosisAd/GT-TRAIL+Ad/PGK-GV16 background levels of GFP-positive cells high levels of apoptosis in cancer cells Ad/gTRAIL high levels of transgene expression high levels of apoptosis in cancer cellscell killing effects XTT assay,c

25、ell viability assay(Fig4B)PBS(),Ad/CMV-GFP(),Ad/gTRAIL(),and Ad/GT-TRAIL+Ad/PGK-GV16(*).,Fig 4B,In vitro Ad/gTRAIL induces apoptosis in cancer cells,conclusion 2-1,To answer Question 2-2(Fig 5)Suppression of Tumor Growth by Ad/gTRAIL in Vivo,Ad/GT-TRAIL+Ad/PGK-GV16 Ad/gTRAIL Ad/CMV-GFP PBSAd/gTRAIL

26、survival advantage,(1)Intralesional administration(Fig 5A),(2)cumulative proportion survival curve of animals bearing DLD-1 tumors(Fig 5B),(3)Posttreatment histochemical examination,Ad/gTRAIL or Ad/GT-TRAIL+Ad/PGK-GV16 dramatically increased apoptosisAd/CMV-GFP or PBS background apoptosis,Fig 5C:,In

27、 vivo Ad/gTRAIL induces apoptosis in xenograft model.,conclusion 2-2,Ad/gTRAIL Elicit apoptosis in malignant cells Suppress tumor growth in vivo,conclusion 2-1+2-2,1.If Ad/gTRAIL can induce apoptosis in cancer cells or not?Ad/gTRAIL can induce apoptosis in cancer cells both in vitro and in vivo.2.If

28、 Ad/gTRAIL have toxic effects on primary human hepatocytes or not?Ad/gTRAIL have no toxic effects on normal human hepatocytes both in vitro and in vivo.,answers,本文的优缺点,优点小有创新实验设计周到对照设置正确实验方法采用合理结果阐述恰当推理合适,缺点仅停留于现象观察而丝毫未及本质（为什么。）研究专题的局限研究结果与意义缺乏普遍适用性新意不足,谢谢！,怎样才是一篇好文章,一、刊登在好杂志上（第一印象）杂志/文章分类按性质分综述类通讯类

29、临床类科研类。按专业类别分公共（通用）类专业类,二、科学意义评价高,影响因子高（多为公共类期刊）当年影响因子多年平均影响因子Journal Impact factor在本专业地位重要（多为专业类期刊）,三、对科研的贡献大,影响因子高的公共类期刊新（发现、理论）从现象到本质实验设计无缺陷从多个角度证明同一个道理影响因子虽不高的专业类期刊在本专业的研究尚属前沿有创新但不大实验设计缺陷少其研究结果的通用性不大,如何来鉴赏一篇好文章,一、标题、作者、单位二、研究背景本研究的意义本研究的国内外研究背景/现状本课题组的研究基础目前尚存在的问题,三、方法、结果与分析实验设计无缺陷用最恰当的方法解决问题！实验结果的合理分析四、讨论对单个实验结果作恰如其份的评价结合研究现状对整个研究的意义进行阐述五、参考文献 时效性、权威性,