송사리에서의 유해성 분자마커 개발과 Micro-capillary Electrophoresis Chip에서의 유해성 분자마커 분리

소개

본 연구의 목적은 유해 환경오염물질인 유기인산계 농약(Diazinon)에 노출된 모델 생물체(송사리)의 비정상적인 행동과 형태적 변화가 일어나는 100ppb의 농도에서 분자생물학적인 방법을 이용하여 transcription 수준에서의 이상발현을 통해 생물지표(biomarker)를 개발하는데 있다. 이러한 이상행동과 형태적 변화와 연계된 차별 발현된 유전자의 발현 분석을 통하여 적절한 생물지표를 동정, 검증하고자 하였다.

또한, 랩온어칩의 플랫폼 기술의 생물학적 환경 모니터링에 응용하기 위해 PDMS 기반의 micro-capillary electrophoresis (MCE) chip을 제작하였다. 이 칩은 Photo공정을 통해 negative 감광제(SU-8)로 mold를 제작한 후 PDMS를 열경화시키고, O2 plasma로 표면처리하여 접합시켰다. Biomarker로써 발현양의 차이가 가장 큰 ribosomal protein S17을 PDMS기반의 MCE chip에서 미세관을 통해 분리 실험을 하였다. 랩온어칩의 다양한 장점과 유용성을 고려할 때 환경 시료의 분석, 환경 독성 분석, 환경 미생물 분석 분야에 칩, 센서 기술이 광범위하게 적용될 수 있어 유해성 생물지표 개발에 랩온어칩의 적용은 환경 모니터링을 위한 의미있는 시도라고 사료된다.

목차

Ⅰ. INTRODUCTION

Ⅱ. THEORETICAL BACKGROUND
2.1 Biomarker
2.2 Ecotoxicogenomics
2.2.1 Historical and current perspectives in fish toxicology
2.3 Model Organism
2.4 Hazardous Toxic Chemicals
2.5 Lab-on-a chip
2.6 Plasma Treatments in Surface Chemistry
2.7 Electrophoresis

Ⅲ. MATERIALS AND METHODS
3.1 Experimental Fish and Chemical
3.2 Total RNA Extraction
3.3 Annealing Control Primer RT-PCR (ACP RT-PCR)
3.4 Cloning and Analysis of cDNA
3.5 Annealing Control Primer RACE PCR for getting Full-length cDNA
3.6 Phylogenetic Analysis
3.7 Real Time qPCR Analysis
3.8 Statistical Analysis
3.9 Fabrication of Micro-Capillary Electrophoresis (MCE) Chip
3.9.1 Mask design for forming pattern of MCE chip
3.9.2 Fabrication process of PDMS-based MCE chip
3.9.3 Bonding of PDMS/glass or PDMS/PDMS
3.9.4 Measurements of Contact Angle
3.10 Separation and Instrumentation
Ⅳ. RESULT AND DISCUSSION
4.1 Differential expressed genes in medaka
4.2 Analysis of Gene Ontology
4.3 Isolation of Full-length cDNA
4.4 Phylogenetic Analysis
4.5 Analysis of Real-Time Quantitative PCR
4.6 Ribosomal Protein Cording Genes and Various DEGs
4.7 Fabrication and Characteristics of PDMS-based MCE chip
4.8 Separation of Ribosomal Protein S17 gene on MCE chip

Ⅴ. CONCLUSION

Ⅵ. ACKNOWLEDGEMENTS

Ⅶ. LITERATURE CITED

본문내용

환경모니터링과 환경질의 개선에 유용한 도구로 활용되는 환경 독성평가는 오늘날 환경문제의 접근에 있어서 많은 발전을 거듭해 왔다. 이전의 전형적 분석방법은 고비용과 낮은 신뢰도 그리고 화학적 분석법의 단점인 독성반응과 생체이용률(bioavailability)의 복합적인 예측의 불가능으로 진보된 분자생물학적 기법의 적용이 점차적으로 필요하게 되었다. 생태학적 유해성은 유해성(hazard) 측면에 있어서 특정한 화학물질과 혼합된 화학물질의 노출(exposure)과 동등하게 적용되어야 하며, 화학물질의 노출과 유해성의 폭넓은 측정은 생물학적 시스템을 이용한 독성평가에 의하여 그 목적을 달성할 수 있다. 이러한 유해성 평가에 있어서 환경오염물질의 영향을 효과적으로 알기 위해 분자마커를 이용한 독성분석 방법이 개발되었다. 그러나 생물모델을 이용한 다양한 환경 유해성 biomarker의 개발이 충분하게 진행되지 않아서, 환경오염물질이 생물체에 미치는 영향을 정확히 평가할 수 있을 다양한 종류의 분자마커 개발 연구가 요구되고 있다.

본 연구의 목적은 유해 환경오염물질인 유기인산계 농약(Diazinon)에 노출된 모델 생물체(송사리)의 비정상적인 행동과 형태적 변화가 일어나는 100ppb의 농도에서 분자생물학적인 방법을 이용하여 transcription 수준에서의 이상발현을 통해 생물지표(biomarker)를 개발하는데 있다. 이러한 이상행동과 형태적 변화와 연계된 차별 발현된 유전자의 발현 분석을 통하여 적절한 생물지표를 동정, 검증하고자 하였다.

우선, 정상송사리(대조군)와 노출된 송사리(실험군) 각각 5개체로부터 total RNA를 추출하였고, reverse transcriptase PCR을 통해 cDNA를 만들어 임의의 primer를 사용해 PCR 하여, 대조군과 실험군의 발현 차이를 agarose gel 상에서 확인하였다. 이 중 발현의 차이가 나는 83개의 유전자를 추출하여 cloning을 한 후, sequencing 분석을 하였다. 추출된 유전자는 NCBI Blaster를 사용해 정리하고, 그 기능은 Gene Ontology DB를 사용해 정리하였다. 이 중 관심있는 5개의 ribosomal protein coding 유전자만을 선택하여 발현양의 차이를 검증하기 위해 real-time PCR 을 사용하였고, 생물지표로써의 가능성을 확인하였다. 더 나은 연구를 위해 5개의 ribosomal protein coding gene의 partial cDNA로부터 RACE PCR 방법을 사용해 Full-length cDNA를 확보하였다.

또한, 랩온어칩의 플랫폼 기술의 생물학적 환경 모니터링에 응용하기 위해 PDMS 기반의 micro-capillary electrophoresis (MCE) chip을 제작하였다. 이 칩은 Photo공정을 통해 negative 감광제(SU-8)로 mold를 제작한 후 PDMS를 열경화시키고, O2 plasma로 표면처리하여 접합시켰다. Biomarker로써 발현양의 차이가 가장 큰 ribosomal protein S17을 PDMS기반의 MCE chip에서 미세관을 통해 분리 실험을 하였다. 랩온어칩의 다양한 장점과 유용성을 고려할 때 환경 시료의 분석, 환경 독성 분석, 환경 미생물 분석 분야에 칩, 센서 기술이 광범위하게 적용될 수 있어 유해성 생물지표 개발에 랩온어칩의 적용은 환경 모니터링을 위한 의미있는 시도라고 사료된다.

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