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College Innovative Experiment

College Innovative Experiment. Studies about the roles of DLC-1 gene in HBV-induced Hepatocellular Carcinoma. Reporter. Cai Jie. Group members. Wang Jimeng, Wu Fei. Prof. Wensheng Sun, Liang Xiaohong. Advisors. CONTENTS. 1. Background. 2. DLC-1. 3. New subject. 4.

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College Innovative Experiment

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  1. College Innovative Experiment Studies about the roles of DLC-1 gene in HBV-induced Hepatocellular Carcinoma Reporter Cai Jie Group members Wang Jimeng, Wu Fei Prof.Wensheng Sun,Liang Xiaohong Advisors

  2. CONTENTS 1 Background 2 DLC-1 3 New subject 4 Experimental Procedure 5 Feasibility analysis 6 Innovation

  3. Results control DLC1 mRNA in BEL7402 cells transfected with or without HBc expression vector The difference of DLC1 expression between HBV-transgenic mice and their normal littermates

  4. Paper 1. A paper has been published on Chin J Curr Adv Gen Surg. 2. Another paper would be submitted to Medical hypothesis soon.

  5. Thank You !

  6. Interest Communicating，consulting Subject Designing Preparing Experiment

  7. Experimental Procedure Tumor-bearing nude mice Ayw subtype HBV Tg mice Experiment in clinical samples Experiment in vitro

  8. Background Infection ofHepatitis B Virus (HBV): the most important reason for Hepatocellular Carcinoma（HCC）in China HCC HBV

  9. Background Multiple genetic alterations cancer Identification ofcancer-related genes

  10. DLC-1 DLC-1 (deleted in liver cancer 1) First cloned in a human hepatocellular carcinoma. DLC-1 Located at human chromo- some 8p21.3-22 A potential tumor suppressor A focal adhesion molecule preventing cell transformation

  11. DLC-1 RhoGAP SAM START

  12. New subject DLC-1 HBc ? DLC-1 HBV

  13. New subject Objectives Toexplore the role of DLC-1 gene in HBV-induced HCC NO.1 Toprovide responsible theory evidence for prevention and gene therapy of HCC NO.2

  14. Experimental Procedure Tumor-bearing nude mice Ayw subtype HBV Tg mice Experiment in clinical samples Experiment in vitro

  15. Detect the effect of HBV on DLC-1 in thses cells 1 2 Detect the effect of DLC-1 to HBV-transfected cells Experiment in vitro Plasmids:pcDNA3-HBV1.1、pcDNA3 Hepatocyte cell lines:HepG2、L02 Method: liposome transfection Procedure

  16. Experiment in vitro B A Detect the effect of HBV on DLC-1 in thses cells HepG2 cells or L02 cells Transfection C pcDNA3-HBV1.1 pcDNA3 No transfection Total protein Total RNA by TRIZOL qRT- PCR Western Blot DLC-1

  17. Experiment in vitro D E F Detect the effect of DLC-1 to HBV-transfected cells HepG2-HBV1.1 or L02-HBV1.1 Transfection DLC-1 plasmid Empty plasmid No Transfection Gather cells at different time(0h,24h,48h,72h,5d,7d) Add in TRAIL or FasL Add in 5mg/L RNaseA to digest Gather the cells after 24h Detect the apoptotic rate with TUNEL Detect the cell cycle with PI-FCM Detect the cell proliferation with MTT

  18. Experiment in vitro Detect the methylation of DLC-1 promotor Use LOH to analysis the DLC-1 Mechanisms HepG2-HBV1.1 L02-HBV1.1

  19. Experiment in clinical Samples: Normal liver HBV-related HCC liver Use qRT-PCR to detect the expression of DLC-1 mRNA Use immunohistochemical method to detect the expression of DLC-1 protein

  20. Experiment on mice HBV Tg mice Neoplasia formation Nude mice Tumor suppression

  21. Experiment on ayw subtype HBV Tg mice BALB/c BALB/c HBV-Tg 6，12，20months Total RNA by TRIZOL Total protein qRT- PCR Western Blot DLC-1

  22. Experiment on tumor-bearing nude mice HepG2.2.15 cell （Stable transfected with HBV） DLC-1 plasmid ？ Neoplasia formation

  23. Feasibility analysis Feasible theoretically, and argument reasonable; Infarctate preliminary work; Strong technolongical develop-ment and innovation capacity.

  24. Innovation DLC-1 gene in HBV with whole expression. Research both in vitro and vivo，more clinical significance.

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