上海交通大學(xué)醫(yī)學(xué)院2012級研究生文獻導(dǎo)讀如何全面把握科學(xué)文獻的內(nèi)容PPT學(xué)習(xí)課件

1、 Kinds of reading skill p Intensive reading skill （精讀）（精讀） p Extensive reading skill（泛讀）（泛讀） p Skimming reading skill（略讀）（略讀） p Scanning reading skill（掠讀）（掠讀） Intensive reading p 精讀、細讀精讀、細讀 p 完全理解每詞、每句和每段意思完全理解每詞、每句和每段意思 Extensive reading p 泛讀、快讀泛讀、快讀 p 理解段落的大體意思理解段落的大體意思 p 理解關(guān)鍵字、關(guān)鍵句理解關(guān)鍵字、關(guān)鍵句 Skimmi

2、ng reading p 略讀、快讀略讀、快讀 p 尋找文章的關(guān)鍵詞、主題句尋找文章的關(guān)鍵詞、主題句 p 了解文章大體意思了解文章大體意思 p 查找、選取精讀內(nèi)容查找、選取精讀內(nèi)容 Scanning reading p 掠讀、快速閱讀掠讀、快速閱讀 p 查找文章中特定信息查找文章中特定信息 Reading fast & mastering the topic? 把握主題和快速閱讀的可能技巧？把握主題和快速閱讀的可能技巧？ p Title p Abstract p Results: Figures and Tibles p Conclusions Intensive reading skill

3、p Results p Introduction Extensive reading skill p Material and Methods p Reference Scanning reading skill p Discussion p Reference Skimming reading skill Selection and validation of differentially expressed genes in head and neck cancer Paper 1 Selection and validation of differentially expressed g

4、enes in head and neck cancer Title Abstract We applied a robust combinatorial (multi-test) approach to microarray data to identify genes consistently up- or down-regulated in head and neck squamous cell carcinoma (HNSCC). RNA was extracted from 22 paired samples of HNSCC and normal tissue from the s

5、ame donors and hybridized to the Affymetrix U95A chip. Abstract (continued) Forty-two differentially expressed probe sets (representing 38 genes and one expressed sequence tag) satisfied all statistical tests of significance and were selected for further validation. Selected probe sets were validate

6、d by hierarchical clustering, multiple probe set concordance, and target-subunit agreement. In addition, real-time PCR analysis of 8 representative genes performed on both microarray tested and independently obtained samples correlated well with the microarray data. Abstract (continued) The genes id

7、entified and validated by this method were in comparatively good agreement with other rigorous HNSCC microarray studies. We conclude that combinatorial analysis of microarray data is a promising technique for identifying differentially expressed genes with few false positives. Abstract (continued) D

8、ifferentially expressed genes in OSSC detected by microarray step I Testable Hypothesis: Differentially expressed genes can be detected by microarray analysis Normal tissue OSSC tissue GeneChip Human Genome U95A2 12,626 full-length genes. One sample One chip Total 22 pairs Selection of differentiall

9、y expressed genes between Tumor tissues and Normal tissues step II a t-test b Wilcoxon rank-sum c paired t-test d SAM e PPV f MDMR g WEPO Group A Group B Group C Tier 42 A 246 103 2 62 8 93 3 B 55 C 200 七種統(tǒng)計學(xué)方法篩選差異表達基因 Combinatorial approach for analysis and selection Step III Validation of Tier I g

10、enes Hierarchical clustering All genes vs. Tier I genes RT-PCR Chip tested samples Independent (chip un-tested) samples Hierarchical Clustering based on all (12642) probes Hierarchical Clustering based on Tier I (42) probes Selected genes may be the the useful target for molecular diagnosis of OSSC

11、Results of Real Time-PCR Ratio grids showing selected and unselected probe sets for multi-probe set genes. Tier I Genes Annotation Up-regulated Down-regulated Structural: Col-1 (1, 2), Col-4 (1, 2), Col-5 (2), FN*, MFAP2 Enzymes: FAPA*, MMP1*, PLAU*, SERPINH2* TAA: OPN (x2)* Other: C5ORF13*, ITAG6,

12、PTHLH*, Est Structural: KRT*(4, 13), PPL*, SCEL* Enzymes: Enzyme: ACPP* (x2), CYP3A5, DUSP5, GPX3*, HPGD, PP11, SPINK5*, TGM3* TAA: CEACAM* (1, 5), LAGY*, EMP1* Other: BLNK, IL1RA*, KIAA0790 MAL, NMU, PITX1*, ZNF185 Discussion Study design and overall strategy The method is with fewer positive false

13、, but some putative genes were missed. Rationale of the approach to selection of differentially expressed genes. Validation of selected genes. Annotation and relevance of selected genes to malignancy. conclusion pCombinatorial analysis of microarray data from 22 paired tumor/normal samples from HNSC

14、C patients yielded 42 probe sets (representing 38 genes plus one EST) which satisfy highly stringent criteria for significant over- or under-expression in HNSCC tissue. pA subset of selected genes was validated by in silico analysis and real-time PCR of both chip- tested and independently obtained s

15、ample pairs to confirm their differential expression in HNSCC versus normal tissues. Conclusion(continued) pSeveral genes identified in the present study were also found to be concordant with genes identified in other HNSCC microarray analyses of similar rigor. pThe strength of validation of these t

16、argets suggests that the intersection of mathematically distinct statistical methods can yield a reliable list of differentially expressed genes containing fewer false positives than a single method. pThis approach may have broad utility in the analysis of gene expression in HNSCC and other malignan

17、cies. MA kuriakoseFangan Chen Zhiyuan Zhang Ping ZhangWantao Chen Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line Paper 2 Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line Title Ab

18、stract Established a drug-resistant cell line to cisplatin, named Tca/cisplatin. Its IC50 was 6.5 times as that of parent cells Step I Tca Tca/cisplatin DNA staining by AO and EB Viable: green, dead: red Viable: attached, dead: disattached A Concentration of cisplatin (M) B Concentration of cisplati

19、n (M) C Tca/cisplatinTca8113 0 M0.8 M3.2 M12.8 M51.2 M Platinum/DNA (pg/ g) Platinum accumulation on DNA in Tca/cisplatin and Tca8113. Tca/cisplatin-0hr G1：56.445.2% G2/M：82.31% S：35.564.74% Tca/cisplatin-6hr G1：55.346.1% G2/M：9.892.91% S：34.773.1% Tca/cisplatin-12hr G1：66.336.1% G2/M：1.670.84% S：33

20、.673.33% Tca/cisplatin-24hr G1：25.656.74% G2/M：0.430.26% S：74.357.9% Tca8113-0hr G1：61.685.3% G2/M：3.471.3% S：34.856.7% Tca8113-6hr G1：23.784.3% G2/M：30.684.8% S：45.547.2% Tca8113-12hr G1：73.396.54% G2/M：1.110.56% S：22.612.2% Tca8113-24hr G1：54.235.6% G2/M：2.870.28% S：42.913.1% Effects of 10M cispla

21、tin on Tca/cisplatin and Tca cell cycle distribution TcaTca/cisplatin Step II 12626 probe sets RT-PCR RT-PCR Western blot Western blot CCND1 CCND3 Pgp GST-Pi 口腔鱗癌耐藥相關(guān)基因的驗證口腔鱗癌耐藥相關(guān)基因的驗證 Step III Cyclin D1 Cyclin D3 Tca/cisplatin TcaTca/cisplatin Tca RT-PCRWestern blot 細胞周期調(diào)控基因細胞周期調(diào)控基因CCND1CCND1和和CCND

22、3CCND3的表達的表達 4 proteins examined by IHC in clinical OSSC samples (n:50) The Tca/cisplatin and Tca8113 cell lines are useful models for identifying candidate genes responsible for the mechanism of cisplatin-resistance. Conclusion Sixty-three genes related to cisplatin-resistance were identified. Amon

23、g these, decreases in cell cycle arrest genes and increase in oncogenes, cell cycle regulation gene and genes involved in metabolism and synthesis led to the cell cycle acceleration, increased proliferation rate and resistance to cisplatin-induced apoptosis in Tca/cisplatin cells. p CCND1 and CCND3

24、seemed to be closely involved in cisplatin resistance. Conclusion(continued) pThe data from this study provide useful clues to screen candidate targets for early diagnosis and intervention in cisplatin-resistance. Inhibition of cyclin D1 expression by cyclin D1 shRNAs in human oral squamous cell car

25、cinoma cells is associated with increased cisplatin chemosensitivity Paper 3 Inhibition of cyclin D1 expression by cyclin D1 shRNAs in human oral squamous cell carcinoma cells is associated with increased cisplatin chemosensitivity Title Cycling D1 is a well-known cell cycle regulator. Recently,its

26、prosurvival function has been revealed in several tumors. Because increasing expression of cyclin D1 is a common event in OSCC and has been correlated with cisplatin resistance, we investigated if cyclin D1 inhibition could increase cisplatin chemosensitivity of OSCC. Five cyclin D1 shRNAs were prep

27、ared and 3 were selected for subsequent experiments. IC50 values for cisplatin were determined by an MTT assay. Cisplatin-induced apoptosis and cell cycle block were investigated. Abstract A tumor transplantation model was generated to examine the cisplatin sensitivity of Tca/cisplatin after in vivo

28、 cyclin D1 silencing. The most effective shRNA resulted in 84.51% knockdown of the cyclin D1 protein level. After the transfection with the 2 most effective shRNAs, the cisplatin IC50 decreased from 5.88 ug/ml to 1.36 ug/ml and 2.47 ug/ml, overexpression of cyclin D1 rendered OSCC cells more resista

29、nt to cisplatin treatment (IC50 increased 1 time) Abstract(continued) This decreasing IC50 was correlated with the down-regulation of cisplatin-induced NF-kB activity in cyclin D1 knockdown cells, and was independent of CDK4 function. In vivo, tumor transplantation models also confirmed a cisplatin-

30、 sensitizing effect of cyclin D1 shRNA in OSCC. A TUNEL assay validated the increase in apoptosis as induced by cisplatin in cyclin D1 knockdown cells. Cyclin D1 may be an important target for future therapy in patients with OSCC. Abstract(continued) FIGURE 1 Cisplatin IC50 and cyclin D1 expression

31、of OSCC cell lines. FIGURE 2 Cyclin D1 shRNA decreased the expression level of cyclin D1 in Tca/cisplatin cells. FIGURE 3 Cyclin D1 shRNA affected cell cycle distribution, enhanced apoptosis and sensitivity to cisplatin in Tca/cisplatin cells. FIGURE 4 Tumor transplanted model and in vivo chemothera

32、py. FIGURE 5 Basal NF-kB activity and cisplatin-induced NF-kB activity. FIGURE 6 CDK4 depletion failed to increase chemosensitivity to cisplatin. In summary, our data show that overexpression of cyclin D1 in OSCC confers resistance to the cytotoxic effects of cisplatin, whereas reduction of cyclin D

33、1 expression results in increased sensitivity to cisplatin partly due to enhanced apoptosis and reduced NF-kB activity. Considering the dual roles of cyclin D1 in promoting cell proliferation and inhibiting cisplatin- induced apoptosis, we propose that cyclin D1 may be an important target for future therapy in patients with OSCC. Celebration of National Day Tel: 23271699-5210(O) Fax: 53591389 p Results p Introduction Extensive reading skill Selection and validation of differentially expressed genes in head and neck cancer Paper 1 Selection and validation