長庚大學中醫系

潘台龍 老師

email:pan@mail.cgu.edu.tw

後基因世代於中醫藥研究應用與展望

•中藥基因體計畫

•中藥化學研究

•中藥藥理學研究

•中藥複方基礎研究

•中藥資源研究

•中藥與辨正研究

真菌界 Fungi　真菌群 Eumycota　　　　子囊亞菌群 Ascomycetes　　　　　核菌綱 Pyrenomycetes　　　　　　球殼菌目 Sphaeriales　　　　　　　麥角菌科 Clavicipitaceae　　　　　　　　冬蟲夏草屬 Cordycpes

Examples : 冬蟲夏草

分佈區：青海及青康藏高原一帶。

藥效：增強免疫力、防止及致療急性腎臟衰竭暨肝硬化。

品管：冬蟲夏草分類及演化因區域不一而有差異，如何科學化鑑定，成為重要課題之一。

Backgrounds

Japanese society for cordyceps research Cordyceps nutans

Isaria japonica

中藥材成份鑑定 /農藥檢測(LC-NMR-MS)

活性分析(Cell-based HTS)

Previously strategy

•中藥化學研究

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Fibroblast

HeLa

Calu-1

Vero

Wish

K562

Raji

活性分析Cell proliferation and viability assays are of particular importance for routine applications. Tetrazolium salts (e.g. MTT, XTT, WST-1) are especially useful for this type of analysis. These tetrazolium salts are cleaved to formazan by the "succinate-tetrazolium reductase" system (EC 1.3.99.1) which belongs to the respiratory chain of the mitochondria and is active only in metabolically active cells. The figure below shows an example of this type of reaction.

中藥材

DNA 抽取

標的篩選

DNA 擴增

指紋圖譜

成份鑑定 /農藥檢測(LC-NMR-MS)

活性分析(Cell-based HTS)

The post-genomics era

•RFLP : restriction fragment length polymorphism

•PCR-RFLP : Polymerase chain reaction RFLP

•RAPD : Random amplified polymorphic

•AFLP : Amplified Fragment Length Polymorphism

Technologies

Classification and polymorphism o

n Cordycpes sinensis

RFLP is one of the DNA fingerprinting techniques that is used to determine plant strain and purity in nutraceutical and herb production.

RFLP

The Polymerase Chain Reaction is used to amplify a sample of DNA.

B

A

109 bp

H HH H

H HH

Plant A

Plant B

C M A B C D E

35 bp

22 bp

57 bp

17 bp

12 bp

109bp

109bp

PCR-RFLP

The primers used for amplification of the rDNA internal transcribed spacer regions were primer A (forward): 5'-CGTAGGTGAACCTGCGGAAGGATCA-3' (18S rDNA 3' terminal region of eukaryotic organisms) and primer B (reverse): 5'-TTCCCTGTTCACTCGCCGTTACT-3' (position 6080 bp in eukaryotic 25S rDNA)

PCR-DNA sequencing

DNA Sequencing

• Like PCR, it utilizes DNA polymerase and thermal cycling

• Only reads the sequence of 1 strand of DNA using 1 primer

• The CEQ 8000 utilizes the Sanger dideoxy termination method

• Reaction generates a population of dye-terminated DNA fragments

Cycle Sequencing

DTCS

DeoxyNucloetide

DideoxyNucloetide

DTCS Extension and Termination

Extension and Termination

•Simultaneous reactions terminate at different lengths

•Reactions generate multiple fragments of all sizes from 1 to 1000+ bases

Separation and Detection

•Fragments are separated by capillary gel electrophoresis

•Laser-induced fluorescence of dye terminators is sequentially read by the PMT sensor

Sequencing Results

www.ncbi.nlm.nih.gov

Random amplified polymorphic DNA (RAPD), a simple

chain reaction (PCR) amplification of genomic DNA by

a single synthetic oligonucleotide primer, can generate

a complex pattern.

RAPD usually reveals considerable polymorphism in

genomic DNA and genetics marker for estimating

genetic, taxonomic, and phylogenetics relationships of

species.

RAPD

Primer 1 Primer 3

Each primer had the following sequence: 18S F: 5' CAA CCT GGT TGA TCC TGC CAG T 3 ' and 18S R: 5 ' CTG ATC CTT CTG CAG GTT CAC CTA C 3 '.

Ban II Dde I

AFLP: Amplified Fragment Length Polymorphism

Identification and Typing - strain leveluseful for both identification (species) and typing (strain) level

Taxonomy - confirms prior classifications

CATCTGACGCATGGTTAACATCTGACGCATGGTTAAGNNN

NNNTTACTCAGGACTCATTACTCAGGACTCAT

NNNAATGAGTCCTGAGTAGCGAGTCCTGAGTAGCAGAG

CTCGTAGACTGCGTACCCTCGTAGACTGCGTACCAATTCNNN

CATCTGACGCATGGTTAACATCTGACGCATGGTTAAGNNN NNNAATGAGTCCTGAGTAGCGAGTCCTGAGTAGCAGAG

CTCGTAGACTGCGTACCCTCGTAGACTGCGTACCAATTCNNN NNNTTACTCAGGACTCATTACTCAGGACTCAT

CTCGTAGACTGCGTACCCTCGTAGACTGCGTACC CATCTGACGCATGGTTAACATCTGACGCATGGTTAA

TACTCAGGACTCATTACTCAGGACTCAT GAGTCCTGAGTAGCAGGAGTCCTGAGTAGCAG

1. DNA Extraction

AATTCNNN GNNN

NNNTNNNAAT

2. Digestion with EcoRI (E) and MseI(M)

M ME

EE M

Collection of 3 Types of Fragments:

3. Ligation of Sequence Specific Adaptors

4. Amplification:

GACTGCGTACCAATTCACAATGAGTCCTGAGTAG

•Pre-Selective

•Selective

AFLP -How It Works:

GACTGCGTACCAATTCACTTGCAATGAGTCCTGAGTAG

AFLP Fragment Data from CEQ 2000 XL

中藥材

DNA 抽取

標的篩選

DNA 擴增

指紋圖譜

目的基因選擇

目的基因片段擴增

DNA 訂序

特異性 PCR 擴增

限制媒反應

指紋圖譜

成份鑑定 /農藥檢測(LC-NMR-MS)

活性分析(Cell-based HTS)

What are the alternatives to approaches involving whole genome sequencing?

Given the size and complexity of the plant genomes, and the extent of repetitive elements within them, the assembly of a large plant genome after shotgun sequencing would require extensive computing capacity, certainly exceeding that needed to assemble the human genome.

•Searching for transcribed mRNAs provides a much more economic way to gain insight into the gene repository of individual species.

•ESTs are created by partially sequencing randomly chosen gene transcripts that have been converted into cDNA.

•Large-scale EST sequencing provides a cost-effective way

to obtain highly valuable information on the transcribed

genes of individual species. In addition, EST clones can be

used to design expression analysis experiments.

•ESTs provide a high throughput means for identifying

gene transcripts and monitoring complex gene expression

patterns.

•Intrinsic problems with EST analysis include the inherent

unreliable quality of the sequence, the overrepresentation

of highly expressed genes and the incomplete, partial

nature of the sequences.

Summary of cDNA cloning and expressed sequence tag (EST) sequencing.

TRENDS in Plant Science Vol.8 No.7 July 2003

Functional Classification of the Bupleurum root ESTs

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Cellular location molecular function Biological process

Biosynthesis of Beta-amyrin and cycloartenol

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六淫 (風寒暑濕燥火 )

氣象因素

生物性致病因素

人 (各型體質 ) 病症

同氣相求病理

人類各種體質要素及其相互關係模型

DNA

體細胞、組織、器官、系統功能、結構、代謝

反應

氣、血、津、液、五臟、六腑

阴阳

目標確認 目標公證 引導確認 引導最佳化

生物晶片尋找相關之基因

活性藥效分析

活性藥效篩選

最佳效率和毒性輕微

草藥

What is Single Nucleotide Polymorphism ?

•Many of differences among people have a genetic basis - alterations in the DNA that change the way important proteins are made.•Sometimes the alterations involve a single base pair (the smallest building block of DNA) and are shared by many people. Such single base pair differences are called "single nucleotide polymorphisms", or SNPs. However, the majority of the SNPs do not produce physical changes in people with affected DNA. •Estimate ~ 15M SNPs in total throughout human genome (one SNP every 200 bases).

Genetics of Drug Efficacy and ToxicityPharmacogenomics

Application – High Through-put Screening

•Functional genomics approaches are powerful tools

to accelerate comprehensive investigations of cellular

metabolism in specialized tissues or whole organisms.

Genomics Transcriptomics Proteomics

Biological Knowledgein Herbal plant

+ + +

Metabolomics

•生物晶片尋找草藥反應標的基因 /蛋白

•DNA 微陣列技術協助尋找草藥於組織作用標的，標的確認可能真實地參與疾病的病理生理機制。

•現在一般充份接受單一基因體能在不同的生物學的情況之下性質上地而且數量地引起不同的蛋白質體，這些包括例如轉譯後的蛋白質分解處理和例如磷酸和乙醯化的後平移的修正的機制。

•於對中藥材料及制成品的定性與定量分析，於藥物鑑定、質量保證方面具有極為重要的應用價值，還可用於尋找、發現中藥新基因。

Microarray & DNA Chips

Focus on Technology

Principle: HYBRIDIZATIONSame idea, better use

[Bardeen & Shockley] Transistors to Computer Chips

[Edwin Southern] Southern Blots to DNA ChipsOne to One Correspondence:Clone to Hybridization Signal

History of ScienceOther Landmark One to Ones

1941: Beadle & TatumOne gene, one enzyme

1964: Charles YanofskyDNA sequence colinearwith Protein sequence

Microarray & DNA Chips

Focus on Technology

Ifyou getthis,thenyouhavegot it.

That’sall

there’sto it.

Really!

Ifyou getthis,thenyouhavegot it.

That’sall

there’sto it.

Really!

Microarray & DNA Chips

Focus on Technology

What are the steps?

[1] Choose cell population[or sample for diagnosis]

[2] RNA extraction, purify

[3] Fluorescent label cDNA

[4] Hybridize with PROBE onMicroarray or DNA Chip

[5] Scan

[6] Interpret image

Microarray & DNA Chips

Focus on Technology

Are Microarrays andDNA Chips the same ?

They share the samescientific principle

May be used with similar TARGETS

DIFFER in construction and type of PROBENomenclature

•Cell matrix protein•Inflammation•MMP•Metabolism•Transcription factor•Oncogene

Gene expression profile before and after- treatment of B.kaol

Same Genome

The Challenge of Proteomics

Complex Proteome(s)

•Multiple Proteins for Each Gene

•Varied and Fragile Nature of Protein

•Quantitative and Qualitative Changes of the Proteome

•Structural and Functional Proteomics Studies

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Mass (m/z)

Proteomics: Experimental Approach2-D Electrophoresis

Image analysis and entry into database

Excise spot and In-gel digestion

Extract peptides and Mass analyze

Database search

IEF Machine

Amersham Pharmacia Bio-Rad

IPG strip rehydration

Strip holder on the IPGphor platform

Principle of 2-D Electrophoresis

1 First dimension

– denaturing isoelectric focusing

– separation according to pI

Principle of 2-D Electrophoresis1 First dimension

– denaturing isoelectric focusing

– separation according to pI

2 Second dimension

– SDS electrophoresis

– separation according to MW

The 2-D electrophoresis gel resolves thousands of protein spots

Protein Dyes Stain Advantages Disadvantages

CoomassieBlue

Simple methodologyMS compatibleEasy to image

Very insensitive

Bio-SafeColloidalCoomassieStain

Simple methodologyMS compatibleMore sensitiveEasy to image

Less sensitive thansilver

Silver stain Very sensitiveEasy to image

Complex procedureIncompatible with MSNot quantitative

Sliver stainPlus

Very sensitiveEasy to imageMore MS compatible

than standard silverstaining

Complex procedureNot very MS

compatibleNot quantitative

Ruby stain Very sensitiveMS compatibleQuantitative over 3

orders of magnitudeSimple methodology

Difficult to image

•Powerful capability for the creation of databases

and composite images

•Sophisticated query capability to find unique or

differentially expressed spot

Image Analysis

N C H

Total spots: 682 spots

Matched spots: 501 spots (73.46%)

Proteomics TA Programme

• Proteomics TA is based on fluorescent 2D DIGE technology and is an integral part of the overall Proteomics strategy

– Initial programme gives access to fluorescent 2D DIGE

technology, Cyanine dyes (CyDye™) Cy 2, Cy 3, Cy 5, matching hardware, software and consumables

Fluorescence Difference Gel Electrophoresis

Model System Using Cy3 and Cy5

Ref. Journal of Bacteriology Dec 1997, p.7595-7599

Cy3 Cy5

Protein Identification Methods in Proteomics III

•Protein identification by MALDI-MS peptide mass fingerprinting (ESI and TOF)

•Piptide sequencing by MALDI-MS-PSD analysis

•Miscellaneous techniques for protein identification using MALDI-MS data

•Protein and peptide preparation for MALDI-TOF-MS

In-gel DigestionIn-gel Digestion

Excise WashDry

Rehydrate,add protease

Incubate

Amino acid sequencingPeptide mappingProtein identification.....

ExtractFractionate

(Reference: Hellman et al. Analytical Biochemistry: (1995) 224, 451-455)

K

K

K

K R

R

R

RR

K

RR

K

K

R K R

R

2. Target is introduced into high vacuum of MS

4. Ions are accelerated by an electrical field to thesame kinetic energy, and they drift (or fly ) down afield free flight tube where they are separated inspace.

Flight tube

1. Sample is mixed with matrix & dried on target

High vacuum

Time

High voltage

•

Pulsed laser

3. Sample spot is irradiated with laser ,desorbing ions into the gas phase and startingthe clock measuring the time of flight.

20 - 30 kV

6. A data system controls all instrument parameters,acquires the signal vs. time, and permits dataprocessing.

5. Ions strike thedetector atdifferent times ,depending on themass to chargeratio of the ion.

MALDI : MALDI : MMatrix AAssisted LLaser DDesorption IIonizationTOF : TTime OOf FFlight MMass SSpectrometry

Reflectron Spectra from a Trypsin in-gel Digestion

A 2D-gel from CHO hamster cell extract was coomassie stainedand a spot digested using trypsin in-gel digestion.

Database Searching

Database Searching: Hit List

Hep 3B Mahlavu

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Effect of Bupleurum extracts on hepatoma cell viability

IC50 12.5g/ml IC50 37.5g/ml

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48 hr

Morphological analysis of Hep3B cells- Extracts + Extracts

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Expression Profile of Hep3B between without or with Bupleurum extracts

TABLE 1 List of identified protein spots

Protein nameSwiss-Prot

Accession numberIdentified by

Fructose-bisphosphate aldolase A MGlyceraldehyde 3-phosphate dehydrogenase MPyruvate kinase MThioredoxin peroxidase MRab11 GTP-binding protein MEnolase-α MRim MCyclophilin MCalreticulin MHSP60 MHSP27 M

Vimentin MTriosephosphate isomerase MProtein disulfide isomerase M-antitrypsin MProteasome MAldose A M

2D gel electrophoresis has been the principal tool for t

he separation and analysis of multiple proteins. Howe

ver, it is labor intensive, require large quantities of sta

rting material, lacks interlab reproducibility, and is not

practical for clinical application.

Ciphergen Biosystems, Inc

ProteinChip technology coupled with SELDI-TOF-MS

(surface-enhanced laser desorption/ionization time o

f flight mass spectrometry) to facilitate protein profilin

g of complex biological mixture.

Surface of ProteinChip

Sample Fractionation of ProteinChip

Advantage

It is much faster, has a high-throughput capability, r

equires orders magnitude lower amounts of the prot

ein samples, has a sensitivity for detecting proteins

in the picomole to attamole range, can effectively re

solve low mass proteins (2,000 to 20,000 Da), and i

s directly applicable for clinical assay development

Functional Genomics Approach In Molecular Medicine