5/7/2018 1 introducing Zebrafish as Animal Model Husnul Khotimah Lab/Dept Farmakologi Fakultas Kedokteran Univ. Brawijaya - Non-human species widely studied to understand human disease. usually because it is easy to maintain and breed in a laboratory setting and has particular experimental advantages. - Model organisms are used when experimentation using humans is unfeasible or unethical. Can you think of a model organism? WHAT IS A MODEL ORGANISM/ANIMAL MODEL ? Types of model organism Genetic model organism Experimental model organism Genomic model organism Good candidates for genetic analysis. Good candidates for developmental biology. Good candidates for genome research. Breed in large numbers. Have short generation times so large scale crosses can be followed over several generations. Produce robust embryos that can be easily manipulated and studied. Easy to manage genomes e.g. small genome size or limited number of repeats. Genome is similar to a human. Humans Animal models Mutant Gene Mutant or missing Protein Mutant Phenotype (disease) Mutant Gene Mutant or missing Protein Mutant Phenotype (disease model) Currently there is no easy way to connect mutant phenotypes to candidate human disease genes
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5/7/2018
1
introducing
Zebrafish as Animal Model
Husnul KhotimahLab/Dept Farmakologi
Fakultas Kedokteran Univ. Brawijaya
- Non-human species widely studied tounderstand human disease. usually because itis easy to maintain and breed in a laboratorysetting and has particular experimentaladvantages.
- Model organisms are used whenexperimentation using humans is unfeasible orunethical.
Can you think of a model organism?
WHAT IS A MODEL ORGANISM/ANIMAL MODEL ?
Types of model organism
Geneticmodel organism
Experimentalmodel organism
Genomicmodel organism
Good candidates forgenetic analysis.
Good candidates fordevelopmental biology.
Good candidates forgenome research.
Breed in large numbers.
Have short generation timesso large scale crosses can befollowed over severalgenerations.
Produce robust embryos thatcan be easily manipulated andstudied.
Easy to manage genomes e.g.small genome size or limitednumber of repeats.
Genome is similar to a human.
Humans Animal models
Mutant Gene
Mutant or missingProtein
Mutant Phenotype(disease)
Mutant Gene
Mutant or missingProtein
Mutant Phenotype(disease model)
Currently there is no easy way to connect mutantphenotypes to candidate human disease genes
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Humans Animal models
Mutant Gene
Mutant or missingProtein
Mutant Phenotype(disease)
Mutant Gene
Mutant or missingProtein
Mutant Phenotype(disease model)
Sequence analysis (BLAST) can connectanimal genes to human genes
Humans Animal models
Mutant Gene
Mutant or missingProtein
Mutant Phenotype(disease)
Mutant Gene
Mutant or missingProtein
Mutant Phenotype(disease model)
Shared ontologies and syntax can connect mutantphenotypes to candidate human disease genes
ZEBRA – FISH ?
WHAT IS ZEBRAFISH ?
Small freshwater fish from South Asia.4 cm long when fully grown.Dark: light cycle 10h : 14h
Life span 3-5 years
Adult : 3 months
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Type of Zf
Transgenic Zebrafish
University of Oregon, USA
GEORGE STREISINGER DEVELOPED ZEBRAFISH ASA MODEL SYSTEM
“FOUNDER FATHER” :DEVELOPMENTAL ANDGENETICS RESEARCH
1970
WHY ZEBRAFISH ?
Optical translucent embryosOptical translucent embryos
High fecundity, Rapid hatching of eggsHigh fecundity, Rapid hatching of eggs
Maintenance cost significantly lowMaintenance cost significantly low
Amenable for molecular and genetic :Whole genome have been identifiedAmenable for molecular and genetic :Whole genome have been identified
Fertilzed and developed out side ofmother’s bodyFertilzed and developed out side ofmother’s body
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Zebrafish development ORTHOLOGUE GENES SHARED (KERSTIN ET AL, 2013)
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75%75-80%
Protein coding 70% related to human, 84%ascociated with human disease (Priyadharsini, et al.,2013).
PUBICATIONS AND PHYLOGENIC OF ZF (STEWART, ET AL., 2014) ZEBRAFISH AS A MODEL IS USED TO STUDY1. Regeneration of heart, tail, retina2. Developmental study : Stunting, Heavy metal, Hypoxia3. Human pigmentation4. Cancer research5. Neuroscience : Autism, PD, Movement6. Genetic Disorders7. Kidney and Hepar disease8. Vaccine9. Metabolik : DM, Obesity10. Drug Development
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Regenerative Cancer
Zebrafish Development Toxicology Zebrafish Embryo Toxicity Test (ZEFT) :Robust and sensitive model
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High Throughput screening
Kind of Defect on Zebrafish
LPS-induce apoptosisAdministration of Asiaticosideameliorate defect and apoptosis
Centella asiatica Effect on Zebrafish
C
LPS+A2
LPS 5
LPS + A3
Representative Embryo in Acridine Orange Staining at 24 hpf
LPS+A1
0
5
10
15
20
25
30
35
40
C LPS 5 LPS+A1 LPS+A2 LPS+A3
Apoptosis at 24 hpf
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Stunting Growth
Centella asiaticacorrected Stuntinggrowth 99.6%
Author Your Presentation
Design and organize
MOLECULAR MARKERS
Centella asiatica as antioxidant Centalla asiatica as anti-inflammation
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Centalla asiatica as anti-inflammation
Centellaasiatica
for P
D
Effect of Centella asiatica on PD?Improved Locomotor function.
You can name, print, andapply effects to an entiresection easily.
DA level in Zf Brain
31
d
c
b
a
d
Linier Regresion : 1 ug/ml could increased DA up to 5.071 UCA extract increased DA level1. CA has antioxidant property ((Haleagrahara and Ponnusamy, 2010 ;
Chippada and Vangalapati, 2011)2. Decreasing oxidation of DA
IHC -synuclein
-synucleinApprox. 14-16 kDA
R+CA5R C R+CA10 R+CA2.5
R CR+CA10 R+CA5 R+CA5-synuclein aggmore than 250 kDA
WB -synuclein
Regressionequation
R Square
Y = 14.65 – 0.863 X 74.1%
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IHC BDNFC Rotenone
R+P2,5 R+P5
R+P10
Regression equationR
SquareY = 6.05 + 1.36 X 77.8%
Correlations
1.000 .882.882 1.000
. .000.000 .16 1616 16
BDNFDosis pegaganBDNFDosis pegaganBDNFDosis pegagan
Pearson Correlation
Sig. (1-tailed)
N
BDNFDosispegagan
Overexpression of α-synuclein downregulateBDNF (Yuan, et al., 2010)
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I bar = 0.01 mm
0
2
4
6
8
10
12
14
16
18
Kontrol Negatif Kontrol positif P1 (dosis 2.5µg/mL)
P2 (dosis 5 µg/mL) P3 (dosis 10µg/mL)
Kelompok
Rat
a-ra
ta A
po
pto
sis
C Rotenone
R+P2.5 R+P5
R+P10
IHC APOPTOSIS
Correlations
1.000 -.794-.794 1.000
. .000.000 .
16 1616 16
ApoptosisDosis pegaganApoptosisDosis pegaganApoptosisDosis pegagan
Pearson Correlation
Sig. (1-tailed)
N
ApoptosisDosis
pegagan
Regression EquationY = 14.70 – 0.839 X 63.0%
Asiaticoside has antioxidant activity,maintaining the metabolic balance ofDA, and increasing ratio of Bcl-2/Bax(Xu, et al., 2012)
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Docking (Autodock Vina on PyRx 0.8), Molecularinteraction (Ligand Scout V.2.0), Molecularvisualization (chimera 1.8.1)
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Energy binding afinityAsiatic acid to PTP-1B : -6.2Kcal/mol
Effect of Centella asiatica onHypoxic Zebrafish larvae
Effect of Centella asiatica onHeavy metal (Pb, Cd, Al)exposure
On Going Research
Effect of Moringa oleifera onstunting growth
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Disadvantage of Zebrafish model
Difficults for surgery procedureDifficults for surgery procedure
Smaller Genome than humanSmaller Genome than human
Very SensitiveVery Sensitive
Sequence flankingSequence flanking
It’s not just about the visuals, but strengthening
YOUR MESSAGE.
It is a tiny fish with a big splash.
Terima Kasih
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