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)

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

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 ?

第一节基因的结构与功能

Structure & Function of Gene

概念基因 : 编码一条多肽链或一个 RNA 分子所必需的全部 D

NA 序列。就是一个转录单位 .Gene ： DNA Fragment for coding a peptide molec

ule ( 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

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

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

DNA 复制Replication

DNA replication is semi-conservation and synthesis of DNA strands is semi-discontinuous

Gene ExpressionThe Flow of genetic information from DNA→

RNA→Polypeptide(protein)

Central Dogma of Molecular Biology

Gene Expression

Transcription: RNA synthesis using a

DNA-dependent RNA polymerase

Translation: RNA directs Polypeptide

synthesis

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

基因突变 ： DNA 分子的改变

Gene mutation: Any change of DNA m

olecule. / Heritable change

DNA 的损伤与修复DNA Damage and Repair

Classes of Mutation

●Chromosomal Abnormalities: Numerical

& structural

● Insertions: including duplication

● Deletions :1 bp to megabases

● Frameshifts:Produced by del, ins,splicin

g errors

● Base Substitutions:

Single Base Substitutions

● Mis-sense Mutation

● Non-sense

● Splice site mutation

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

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

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

Classification of Onco-proteins

● Growth Factors

● Receptors for Growth Factors & Hormones

● Intracellular Signal Transducers

● Nuclear Transcription Factors

● Cell-Cycle Control Protein

Virus Associated tumors

DNA virusesEpstein-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

Viruses associated with human cancers

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

Retroviral oncogenes

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

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

限制性内切酶Restriction Enzymes

• Enzymes which have the activity of r

estricting (cutting) and /or modificati

on of a specific DNA seq.

• Mainly 3 types:

• Type I, II, and III

第二节 基因诊断和治疗Gene Diagnosis and Gene The

rapy

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

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

TemplatePrimersdNTPDNA polymeraseBuffer

Polymerase Chain Polymerase Chain Reaction (PCR) is DNA Reaction (PCR) is DNA Replication in a test Replication in a test tube.tube.

Polymerase Chain Polymerase Chain Reaction (PCR) is DNA Reaction (PCR) is DNA Replication in a test Replication in a test tube.tube.

So what do you So what do you need to put in need to put in

the tube??the tube??

So what do you So what do you need to put in need to put in

the tube??the tube??

• DNA Polymerase

• dNTPs

• single-stranded template

• primer

Requirements for Requirements for DNA SynthesisDNA SynthesisRequirements for Requirements for DNA SynthesisDNA Synthesis

Step 1: InitiationStep 1: InitiationStep 1: InitiationStep 1: Initiation

-Requires a primer3’-GATCGATTCATC-5’3’-GATCGATTCATC-5’ 3’-GATCGATTCATC-5’3’-GATCGATTCATC-5’

dNTPs

DNA polymerase++

NO Reaction NO Reaction

dNTPs

DNA polymerase

primer

++

Step 2: Step 2: ElongationElongationStep 2: Step 2: ElongationElongation

The new strand grows in the 5’ to 3’ direction always!

The new strand grows in the 5’ to 3’ direction always!

Step 3: Step 3: ‘Termination’‘Termination’Step 3: Step 3: ‘Termination’‘Termination’

1.1. Polymerase falls off Polymerase falls off the templatethe template

2.2. Polymerase runs Polymerase runs out of DNAout of DNA

Ouch!Ouch!Ouch!Ouch!Ouch!Ouch!Ouch!Ouch!Ouch!Ouch!Ouch!Ouch!

Now weNow we CycleCycle the the temperature - WHY??temperature - WHY??Now weNow we CycleCycle the the temperature - WHY??temperature - WHY??

9595ooC - DNA unwinds into 2 single stranC - DNA unwinds into 2 single strands: ds: ssDNA template requirementssDNA template requirement

5050oo to 65 to 65ooC - Primers anneal to single stC - Primers anneal to single strands: rands: Primer requirementPrimer requirement

7272ooC - DNA polymerase incorporates dC - DNA polymerase incorporates dNTPs: NTPs: Elongation of strand, AKA Elongation of strand, AKA

ExtensionExtension

Repeat the Repeat the CycleCycleRepeat the Repeat the CycleCycle

Start with Start with x x copies and make copies and make xx in in the first cyclethe first cycle

Start with Start with 2x2x and make and make 2x2x in the in the second cyclesecond cycle

Start with Start with 4x4x and make and make 4x4x in the in the third cyclethird cycle

Make Make 16,777,216x16,777,216x in the 25th in the 25th cycle cycle

Make Make 536,870,912x536,870,912x in the 30th in the 30th cyclecycle

分子杂交技术Molecular Hybridization

• 序列互补单链的 RNA 和 DNA ，或 DNA 和 DN

A ，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

Molecular hybridization

• Southern blot: detect DNA using DNA probe, D

NA-DNA Hybridization, Edwin Southern 1975

• Northern Blot: detect RNA using RNA or cDNA

probe, RNA-RNA or DNA-RNA hybridization

• Western Blot: detect Protein using Ab probe

Molecular hybridization

• Prepare DNA or RNA

• Electrophoresis

• Transfer DNA or RNA to membrane

• Hybridization

• Imaging

斑点杂交 Dot Blot

• 直接将变性 DNA 或 RNA 点样于硝酸纤维薄膜上，晾干后与放射性核素标记的探针进行杂交及放射自显影，以观察所要研究的基因或 mRNA 是否存在，并可以比较相对量的高低

• Spot DNA or RNA onto membrane and

detect it using appropriate probe

基因芯片基因芯片

多点多点 Dot blotDot blot

Southern blotSouthern blot Northern blotNorthern blot

Dot blotDot blot

每个细胞均会有一整套基因组 并非全部表达何种基因表达就决定了细胞的命运一种细胞之所以区别于其他细胞就是由于其基因表达是特异的

肿瘤细胞基因表达产物与正常细胞的差异有两方面：量：表达水平的高低或表达与不表达质：由于基因突变造成基因产物具有质的差异

把每个细胞全部基因表达以及每个基因的量绘制其基因表达谱，就可建立该细胞的“指纹（ fingerprint ）”，

对比正常细胞的基因表达谱和肿瘤细胞的基因表达谱对差异显著的基因进行更细致的研究，阐明其是否是在癌症中起关键作用的基因，或能否作为早期诊断的指标。

Normal→pre-Cancer→ Cancer in situ →Metastasis

Many Years

差异表达基因研究方法Strategies for Identification differential

expression genes

• 差异显示（ Differential Display,DD-PCR)• 代表性差异分析（ RDA ）• 差减杂交（ Substract Hybridization)• 抑制性差减杂交（ Suppressive Substract Hybridization,

SSH)• 交互减数差异显示（ reciprocal subtraction differential di

splay, RSDD ） • 基因表达系列分析（ Serial Analysis of Gene Expression, S

AGE ） ☆ cDNA 微阵列 ( Micro-array, Gene Chip)• 蛋白质双相电泳 (Two-dimensional gel electrophoresis,

2-DE)

Functional Proteomics

• During human development, cell express different proteins

• Normal and cancer cells express different proteins

• Cell treated with and without drug express different proteins

• Post-translational, and covalent and non-covalent associations etc

Challenges: Genomics vs. Proteomics

Static Very dymanic

Can be am plified Can not be amplified

Little complexity Post translationally

(single component) modified, very complex

Good solubility Veriable solubility

DNA Protein

Why Proteomics?

• Protein expression levels are not predictable from mRNA expression levels.

• Proteins are uniquely modified and processed in ways which are not apparent from the Gene sequence.

• Proteomes are dynamic and reflect the state of Biological Systems.

• Proteomics: Quantitative Protein-Level Measurements of Gene Expression to Characterize Biological Processes,

e.g. Growth, Development, Response, Disease.

• It is more than just a Protein equivalent to DNA Databases - it is the concept of MOLECULAR REGULATION as a systematic science.

Identification markers by Proteomics

1120

16731685

1657

1543

1359

2584

25732570

1328

1050

1055

2551

904

2542

2555

915

2530

512514

230

154 155

232

436453464

522591

601

636675 693735

750 770

784812813817 845846 852 855858

916

10021043

1098

11841189

76.0

66.2

43.0

36.0

31.0

21.5

17.5

Mr

kDa

4.5 5.1 5.5 5.9 6.6 7.0 8.5pI

1256

12871298 1303

13251347

1390

139714101425

1446

1480

1521

15401554

1566 15681589

1606

1635

1665

1732

648

837866892

894 938976

1019 102510321071 1072

10901107 1122

1237

1314 1317

1365 1406

15051529

1647

1688

17001705

Sample Prep

• 2-D Gel Electrophoresis

• Stain and/or Blot

• Image Analysis

• Mass Spectrometry

• Bioinformatics

2-D PAGE For Global Protein Monitoring

BLOT

Stain/Blot Image/DatabaseOrganTissueCells

2-D Array ofseparatedpolypeptidechains.

IPG

Sample Prep

FractionationSolubilization

SDS PAGE

2-D PAGE For Global Protein Monitoring

SDS PAGE

OOOOOOOOOOOOOOOOOOOOOOOOO

Spot Cutter

MALDI- TOF MSNano ESI-MS/MS

Mass Spec

2-D Array

Protein ID

PDQuest/ Melanie Database

BLOT

Global Database Search

BLOT

GEL

DigestProteinLynx

Strategies for Protein characterization

Protein Identification

2-D gel electrophoresis

MALDI-TOF MS Nanoflow ESI-MS/MS

AA sequence

De novo sequencing

cDNA clone

“in-gel” tryptic digestion

BLAST

dbEST -searchBioinformatics

PMF

TAG search

SAGE

Serial A

nalysis o

f Gen

e Exp

ression

基因治疗 Gene therapy

• 基因治疗就是用正常或野生型基因校正或置换致病基因的一种治疗方法

• Any procedure intended to treat or alleviate disease by genetically modifying the cells of a patients

• 转移 (transfer)• 转导 (transduction)• 转染 (transfection)• 转化 (transformation)

Gene Therapy

• 23 国家 批准基因治疗临床方案 987 个• 美国 66 ％• 欧洲 27 ％

• 肿瘤 656 个 66.5 ％• 单基因遗传病 9.4 ％• 心血管病 8.1 ％• 传染病 6.6 ％

药物名 单位 进展情况

重组人 p53腺病毒注射液 深圳市赛百诺基因技术有限公司 已上市

基因工程腺病毒注射液 H101 上海三维生物技术公司 III期临床

溶瘤性重组腺病毒注射液 上海三维生物技术公司 I期临床

重组腺病毒–胸苷激酶基因制剂 上海市肿瘤研究所 I期临床

重组 AAV-2 人凝血因子 IX注射液 上海复旦大学 I期临床

本元正阳基因技术股份有限公司

重组人白介素 2腺病毒抗癌注射液 成都基因治疗工程技术研究中心 I期临床

血管内皮生长因子基因 北京大学医学部心血管研究所 特批临床

(肢体动脉梗塞 )

细胞因子激活的淋巴细胞 ( 白血病 ) 北京大学人民医院 特批临床

治疗人脑恶性胶质瘤用重组 I 型 天津医科大学总医院 特批临床

单纯疱疹病毒载体

白细胞介素 -2 基因胃癌瘤苗 上海第二医科大学 特批临床

中国基因治疗药物研发状况

药物名 单位 进展情况

细胞融合肝癌疫苗 第二军医大学 完成临床前研究

树突状细胞治疗恶性肿瘤 第二军医大学 完成临床前研究

重组人内皮抑素腺病毒注射液 中山大学肿瘤防治中心生物治疗中心 完成临床前研究

重组腺病毒 -肝细胞生长因子注射液 军事医学科学院 完成临床前研究

糖尿病基因治疗注射液 武汉同济医院基因治疗中心 完成临床前研究

重组逆转录病毒载体 -胸苷激酶 首都医科大学 完成临床前研究基因制剂

重组人 p16逆转录病毒制剂 首都医科大学 完成临床前研究

重组超氧化物歧化酶和血红素加氧 首都医科大学 完成临床前研究

酶 -1逆转录病毒制剂

肝癌靶向的非病毒载体 p21 和 GM- 上海市肿瘤研究所 完成临床前研究

CSF 基因制剂

癌症抑制素基因药物 解放军 302医院基因治疗研究中心 临床前研究

异种血管内皮细胞基因疫苗 四川大学肿瘤生物治疗中心 临床前研究

Methods of gene therapy

• Expression cloning of normal gene products

• Production of genetically engineered Abs

• Production of genetically engineered vaccines

Major disease classes include

• Infectious diseases

• Cancers

• Inherited disorders

• Immune system disorders

Strategies of gene therapy

In vivo gene therapythe genetic material may be transferred directly into cells within a patient

Ex vivo gene therapycells may be removed from the patient and the genetic material inserted into them in vitro, prior to transplanting the modified cell

In vivo and Ex vivo gene therapy

General gene therapy strategies

• Gene augmentation therapy (GAT)

• Targeted killing of specific cells

• Targeted mutation correction

• Targeted inhibition of gene expression

血友病 haemophilia, Factor VIII Gene

自杀基因 suicide gene, TK/ ganciclovir

CEA, MAGE-3 IL-2, GM-CSF

AntiSense, Ribozyme, RNAi

基因治疗的基本程序 Procedure●目的基因的准备（如 TK 、 IL-2 、 p53 ）

Prepare target gene

●受体细胞（靶细胞）的培养Prepare receipt cell

●载体的选择及克隆Prepare a vector, and insert the target gene into the vector

●将目的基因导入靶细胞transfect

●转导细胞的选择和鉴定Selection and identification

●基因治疗的安全性监测及疗效评价Evaluation

TIMP-2 cDNApLXSNpLNCX

pL(TIMP-2)SNpLNC(TIMP-2)

PA317

Virus stock

NIH3T3

virus titer

LAK

PCR : env ,TIMP-2

Northern blot mRNA

SDS-PAGE

Reveres zymography

Treatment test

Transfect

Infect

HpaI

Experimental Procedure

LTR TIMP-2 SV neo LTR

EcoRI XhoI BamHI

ψ+

pA

LTR LTRψ

+

neo CMV TIMP-2

pA

L(TIMP-2)SN

LCN(TIMP-2)

Recombinant TIMP-2 expression plasmids

HindIII ClaI

Application

● Molecular Pathology of Single-Gene Disorders

● Common Polygenic Diseases

● Cancer

● Diagnosis

● Treatment

● Drug discovery

Critical Thinking

Mechanisms of cancer development

Further ReadingHuman Molecular Genetics, 2nd ed.Tom Stachan, and Andrew P. Read

John Wiley & Sons PTE LTD

Molecular Cell Biology, 3rd Ed.Harvey Lodish, et al

Scientific American Books

Gene VIII