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外科分子生物学 Molecular Biology of Surgery

外科分子生物学 Molecular Biology of Surgery. Bingya Liu, M.D., Ph. D. , Shanghai Institute of Digestive Surgery. History of Surgery. 解剖外科学 Anatomy Surgery 病理外科学 Pathological Surgery 病理生理外科学 Patho-physiological Surg 细胞分子生物学外科学 Cell Molecular Biology of Surgery (CMBS).

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外科分子生物学 Molecular Biology of Surgery

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  1. 外科分子生物学Molecular Biology of Surgery Bingya Liu, M.D., Ph. D. , Shanghai Institute of Digestive Surgery

  2. History of Surgery 解剖外科学 Anatomy Surgery 病理外科学 Pathological Surgery 病理生理外科学 Patho-physiological Surg 细胞分子生物学外科学 Cell Molecular Biology of Surgery (CMBS)

  3. 外科细胞分子学Cell Molecular Surgery 从现代细胞分子生物学角度，以细胞分子生物学技术为手段研究外科疾病的病因、发病机制、诊断、治疗和预防 The science study on the etiology, mechanisms, diagnosis , treatment and prevention of the diseases using cell biology and molecular biology

  4. 问题 • 肿瘤的发病机理是什么? • 什么是基因重组药物? • 什么是基因治疗? • 同样是胃癌病人,为何化疗敏感性差异很大?

  5. 第一节基因的结构与功能Structure & Function of Gene

  6. 概念 基因:编码一条多肽链或一个RNA分子所必需的全部DNA序列。就是一个转录单位. Gene：DNA Fragment for coding a peptide molecule ( a trancript unit ) 外显子exon 内含子intron 增强子enhancer 启动子promoter、沉寂子(silencer) 前导序列5’ untranslated regions 5’UTR 终止序列 3’ untranslated regions 3’UTR

  7. 基因按其功能可分为： 结构基因：可被转录成mRNA，进而翻译成多肽链，构成各种结构蛋白质、酶和激素等。 调控基因:指某些可调节控制结构基因表达的基因 Classification of genes as its function: Structural gene Regular gene

  8. 基因组（genome）：细胞内所有的基因总和 结构基因（structural gene)：与成熟mRNA的5’和3’对应的基因区域 基因表达gene expression：基因产生功能分子的过程 transcript into RNA and/or translate into protein 转录Transcription ：以DNA为模板合成mRNA的过程 翻译Translation：以mRNA为模板合成多肽链的过程

  9. DNA复制Replication DNA replication is semi-conservation and synthesis of DNA strands is semi-discontinuous

  10. Gene Expression The Flow of genetic information from DNA→RNA→Polypeptide(protein) Central Dogma of Molecular Biology

  11. Gene Expression Transcription: RNA synthesis using a DNA-dependent RNA polymerase Translation: RNA directs Polypeptide synthesis

  12. 基因表达调控Regulation of Gene Expression 基因组水平：基因组结构改变 Genome: Genomic DNA structural change 转录水平:转录激活、转录延长 Transcription：Activation and extension 转录后水平:剪接、带帽、加尾 post-transcription:Splicing,Capping Polyadenylation 翻译水平：起始mRNA稳定性调节，蛋白质因子的修饰，反义RNA Translation: mRNA stable, Pro.Factors, Antisense 翻译后水平:修饰、加工、聚合 Post-translation：Modification

  13. DNA的损伤与修复DNA Damage and Repair 基因突变 ：DNA分子的改变 Gene mutation: Any change of DNA molecule. / Heritable change

  14. Classes of Mutation ●Chromosomal Abnormalities: Numerical & structural ● Insertions: including duplication ● Deletions :1 bp to megabases ● Frameshifts:Produced by del, ins,splicing errors ● Base Substitutions:

  15. Single Base Substitutions ● Mis-sense Mutation ● Non-sense ● Splice site mutation

  16. DNA修复系统Repair System 直接修复 Direct repair 切除修复 Excision repair 错配修复 Mismatch repair 重组修复 recombinant repair

  17. 癌基因和抑癌基因Oncogene &Tumor suppessor gene

  18. 癌基因Oncogene Genes sufficient to transform a cell, Genes that encodes a protein able to transform cells in culture or to induce cancer in animals 病毒癌基因 原癌基因：细胞癌基因,细胞原癌基因。 Viral oncogene proto-oncogene, cellular oncogene

  19. Classification of Onco-proteins ● Growth Factors ● Receptors for Growth Factors & Hormones ● Intracellular Signal Transducers ● Nuclear Transcription Factors ● Cell-Cycle Control Protein

  20. Viruses associated with human cancers Virus Associated tumors DNA viruses Epstein-Barr Burkitt’s lymphoma Nasopharyngeal cancer Hepatitis B Liver cancer Papilloma virus Benign warts Cervical cancer RNA viruses (HIV-1) Kaposi’s sarcoma (HTLV-1) Adult T-cell leukemia HTLV-2 Hairy cell leukemia HTLV-5 Cutaneous T-cell leukemia

  21. Retroviral oncogenes Oncogene Virus Tumor v-ABL Abelson leukemia virus Leukemia v-ERBA Avian erythroblastosis virus Helps-ERBB v-ERBB Avian erythroblastosis virus Erythroleukemia v-FMS Feline sarcoma virus Sarcoma v-HRAS Rat sarcoma virus (Harvey strain) Sarcoma v-KRAS Rat sarcoma virus (Kirsten strain) Sarcoma v-JUN Avian sarcoma virus Fibrosarcoma v-MYB Avian myeloblastosis virus Myeloblastosis v-MYC Avian myelocytomatosis virus Leukemia v-SIS Simian sarcoma virus Sarcoma v-SRC Rous sarcoma virus Sarcoma

  22. 抑癌基因Tumor suppessor gene 在肿瘤形成中起重要作用的第二类基因是抑癌基因，这类基因是控制异常细胞增殖，其丢失或失活与恶性发展有关。 Genes involved in the control of abnormal cell proliferation and whole loss or inactivation is associated with the development of malignancy

  23. 重组DNA技术Recombinant DNA • 在体外将一些不同物种来源的DNA片段用生物化学方法拼接成一个人造的重组分子 • 通常重组DNA一词指的是将任何细胞来源的DNA片段接在染色体外的能迅速复制的质粒或病毒载体。 • Insert and ligate any DNA fragment into a vector such as plasmid or viral vector, to form a recombinant DNA. • Target genes, vector, ligase

  24. 限制性内切酶Restriction Enzymes • Enzymes which have the activity of restricting (cutting) and /or modification of a specific DNA seq. • Mainly 3 types: • Type I, II, and III

  25. 第二节 基因诊断和治疗Gene Diagnosis and Gene Therapy

  26. 基因诊断Gene Diagnosis ●检测基因的存在状态或缺陷 To detect gene status or defect ●检测基因的表达（转录或翻译） To detect gene expression ●内源性：基因相关疾病 Endogenous gene: ●外源性：外来微生物感染 Exogenous , Pathogens ●常用技术：分子杂交、PCR、基因芯片

  27. 聚合酶链反应Polymerase Chain Reaction, PCR ● 体外基因扩增技术 ● 在模板DNA、引物和4种脱氧核糖核苷酸存在的条件下依赖于DNA聚合酶的DNA合成反应 ●In vitro DNA amplification Template Primers dNTP DNA polymerase Buffer

  28. Polymerase Chain Reaction (PCR) is DNA Replication in a test tube. So what do you need to put in the tube??

  29. Requirements for DNA Synthesis • DNA Polymerase • dNTPs • single-stranded template • primer

  30. Step 1: Initiation -Requires a primer 3’-GATCGATTCATC-5’ 3’-GATCGATTCATC-5’ dNTPs DNA polymerase primer + dNTPs DNA polymerase + NO Reaction

  31. Step 2: Elongation The new strand grows in the 5’ to 3’ direction always!

  32. Step 3: ‘Termination’ 1. Polymerase falls off the template 2. Polymerase runs out of DNA Ouch! Ouch! Ouch!

  33. Now weCyclethe temperature - WHY?? • 95oC - DNA unwinds into 2 single strands: ssDNA template requirement • 50o to 65oC - Primers anneal to single strands: Primer requirement • 72oC - DNA polymerase incorporates dNTPs: Elongation of strand, AKA Extension

  34. Repeat the Cycle • Start with x copies and make x in the first cycle • Start with 2x and make 2x in the second cycle • Start with 4x and make 4x in the third cycle • Make 16,777,216x in the 25th cycle • Make 536,870,912x in the 30th cycle

  35. 分子杂交技术Molecular Hybridization • 序列互补单链的RNA和DNA，或DNA和DNA，或RNA和RNA，根据碱基配对原则借氢链相连而形成杂交分子的过程，为核酸分子杂交。 • DNA-DNA, RNA-DNA, RNA-RNA, and Ag-Ab hybridization • in situ hybridization

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