표제지

제출문

보고서 초록

요약문

SUMMARY

목차

체중 및 당대사조절 생물지표를 활용한 다원적 기능성 평가시스템 연구 16

제출문 18

보고서 초록 20

요약문 22

SUMMARY 26

CONTENTS 28

목차 30

제1장. 연구개발과제의 개요 32

제2장. 국내외 기술개발 현황 37

제3장. 연구개발수행 내용 및 결과 45

제4장. 목표달성도 및 관련분야에의 기여도 127

제5장. 연구개발결과의 활용계획 134

제6장. 연구개발과정에서 수집한 해외과학기술정보 138

제7장. 참고문헌 140

심혈관기능유지 관련 다원적 생물지표를 활용한 바이오식품의 기능평가 기술연구에 관한 연구 144

제출문 146

보고서 초록 148

요약문 150

SUMMARY 152

CONTENTS 154

목차 156

제1장 연구개발과제의 개요 158

제2장 국내외 기술개발 현황 159

제3장 연구개발 수행 내용 및 결과 162

제4장 목표달성도 및 관련분야에의 기여도 207

제5장 연구개발결과의 활용계획 210

제6장 연구개발과정에서 수집한 해외과학기술정보 211

제7장 참고문헌 213

연구개발 결과 활용계획서 217

[첨부1] 연구개발 결과 및 활용계획서 218

자체평가의견서 222

바이오식품소재의 기능특이적 분자지표 발굴 및 응용연구 226

제출문 228

보고서 초록 230

요약문 232

SUMMARY 236

CONTENTS 240

목차 242

제1장 연구개발과제의 개요 244

제2장 국내외 기술개발 현황 246

제3장 연구개발수행 내용 및 결과 249

제4장 목표달성도 및 관련분야에의 기여도 407

제5장 연구개발결과의 활용계획 413

제6장 연구개발과정에서 수집한 해외과학기술정보 414

제7장 참고문헌 416

연구개발 결과 활용계획서 421

[첨부1] 연구개발 결과 및 활용계획서 422

자체평가의견서 426

바이오식품소재의 효능 평가를 위한 세포주 개발 432

제출문 434

보고서 초록 436

요약문 438

SUMMARY 442

CONTENTS 446

목차 448

제1장 연구개발과제의 개요 450

제1절 연구개발의 목적 450

제2절 연구개발의 필요성 450

제3절 연구개발의 범위 452

제2장 국내·외 기술개발 현황 454

제1절 국내·외 기술개발현황 454

제2절 연구결과가 국내·외 기술개발현황에서 차지하는 위치 456

제3장 연구개발수행 내용 및 결과 457

제1절 신 기능성 유전자 발현 세포주 모델 개발 457

제2절 발현시스템 조절 세포주 구축 465

제3절 다중 유전자 발현 세포주 개발을 위한 유전자 연구 470

위탁과제 연구결과 473

제4절 단백질체 기술 확립 473

제5절 신기능성 바이오마커의 탐색 476

제6절 Leptin 수용체 결여 세포주와 과다 발현 세포주의 효능비교 488

제7절 새로이 개발된 신기능성 biomarker 발현 세포주의 효능 검증 489

제4장 목표달성도 및 관련분야에의 기여도 493

제5장 연구개발결과의 활용계획 497

제6장 연구개발과정에서 수집한 해외과학기술정보 498

제7장 참고문헌 499

연구개발 결과 활용계획서 502

[첨부1] 연구개발 및 활용계획서 503

자체평가의견서 508

바이오식품의 효능 평가를 위한 동물모델 개발 514

제출문 516

보고서 초록 518

요약문 520

SUMMARY 522

CONTENTS(영문목차) 524

목차 526

제1장 연구개발과제의 개요 528

제2장 국내외 기술개발 현황 530

제3장 연구개발수행 내용 및 결과 531

제4장 목표달성도 및 관련분야에의 기여도 571

제5장 연구개발결과의 활용계획 574

제6장 연구개발과정에서 수집한 해외과학기술정보 575

제7장 참고문헌 576

연구개발 결과 활용계획서 578

[첨부1] 연구개발 결과 및 활용계획서 579

자체평가의견서 583

체중 및 당대사조절 생물지표를 활용한 다원적 기능성 평가시스템 연구 16

Table 1. Classification of rodent obesity models (Tschop 등, 2001) 40

Table 2. 비만 관련 유전자들 42

Table 3. Composition of experimental diets 58

Table 4. DNA sequence of primers used in either RT-PCR or real-time PCR 59

Table 5. Body weight gain, food intake and food efficiency ratio of rats fed ND or HFD for 9 weeks 60

Table 6. Visceral adipose tissue weight of rats fed ND or HFD for 9 weeks 61

Table 7. Serum lipid concentrations of rats fed ND of HFD for 9 weeks 63

Table 8. Hepatic lipid concentrations of rats fed ND or HFD for 9 weeks 63

Table 9. Serum GOT and GPT activities of rats fed ND or HFD for 9 weeks 64

Table 10. Hepatic lipogenic enzymes activities of rats fed ND or HFD for 9 weeks 65

Table 11. Fasting serum glucose level of rats fed ND or HFD for 9weeks(9wks) 66

Table 12. Classification by biological function of genes with altered expression(≥1.5 fold) in the liver of rats 73

Table 13. Classification by biological function of genes with altered expression(≥1.5 fold) in the epididymal adipose tissue of rats 74

Table 14. Genes up-or down-regulated in the liver of rats fed high fat diet 75

Table 15. Genes up-or down-regulated in the epididymal adipose tiss 79

Table 16. Composition of experimental diets 92

Table 17. DNA sequences of primers used in RT-PCR of real-time PCR analyses 93

Table 18. Body weight gain, food intake and food efficiency ratio of mice fed experimental diets for 12 weeks 95

Table 19. Visceral adipose tissue weights of mice fed experimental diets for 12 weeks 96

Table 20. Serum and hepatic biochemistris of mice fed experimental diets for 12 weeks 97

Table 21. Genes related to obesity up- or down-regulated in the epididymal adipose tissue of mice by the high fat diet as assessed by oligoDNA microarray analyses 102

Table 22. Comfirmation of oligoDNA microarray results by real time RT-PCR analyses of high-fat diet responsive genes in the epididymal adipose tissue 104

Table 23. Composition of the experimental diets 117

Table 24. Body weight gain, visceral fat pad weight, and plasma and hepatic biochemistries of mice fed experimental diets. 118

Table 25. Genes up- or down-regulated in the epididymal adipose tissue of mice fed high fat diet as determined by the oligo DNA microarray analysis. 120

Table 26. HFD-induced changes in the mRNA level of epididymal adipose tissue genes as determined by real time RT-PCR analysis 120

Table 27. Composition of the experimental diets 122

Table 28. Body weight, food intake, and visceral fat-pad weight of rats fed experimental diets 123

Table 29. Plasma and hepatic biochemistry of rats fed experimental diets 123

Table 30. Primer Sequences and PCR Conditions 124

심혈관기능유지 관련 다원적 생물지표를 활용한 바이오식품의 기능평가 기술연구에 관한 연구 144

Table 1-1. Changes of various biomarkers in hamster and mouse 162

Table 1-2. Effect of resveratrol and fibrate on free cholesterol, free fatty acid, HDL cholesterol, Apo A-I and CETP concentration of hamsters fed with high fat diet. 165

Table 1-3. Effect of resveratrol and fibrate in TAS, GOT and GPT concentration of hamsters fed with high fat diet. 165

Table 1-4. Effect of Quercetins on free fatty acid and HDL cholesterol concentration of hamsters fed with high fat diet. 168

Table 1-5. Effect of Quercetins on Apo A-I and CETP concentration of hamsters fed with high fat diet. 168

Table 1-6. Effect of Quercetins on TBARS and total antioxidant status of hamsters fed with high fat diet. 169

Table 1-7. Weight of liver and adipose tissues of mouse fed with experimental diets. 172

Table 1-8. Effect of quercetin and resveratrol on hepatic lipid contents of mouse fed with high fat diet. 175

Table 1-9. Up- or down regulated genes by biological function(＞1.5 fold) 180

Table 1-10. Comparison of mRNA expression between the microarray and the real-time PCR analysis 182

Table 2-1. Effects of resveratol for 20 weeks on the age-dependent plasma lipids concentration change in Apo E-/-(이미지참조) mice 186

Table 2-2. Effects of resveratrol supplement for 20 weeks on the plasma and hepatic lipidsconcentrations in normal diet-fed Apo E-/-(이미지참조) mice 186

Table 2-3. Effects of resveratrol supplement on hepatic HMG-CoA reductase and ACAT activities in Apo E -/-(이미지참조) mice 187

Table 2-4. Effects of resveratrol supplement for 20 weeks on TBARS levels and paraoxonase activity in normal diet-fed Apo E-/-(이미지참조) mice 189

Table 2-5. Effects of resveratrol supplement for 20 weeks on erythrocyte antioxidant enzyme activities in normal diet-fed Apo E-/-(이미지참조) mice 190

Table 2-6/6. Effects of resveratol supplementation on hepatic ad adipocyte fatty acid metabolism in Apo E-/-(이미지참조) mice 191

Table 2-7. Array comparision in the liver of resveratrol supplemented Apo E-/-(이미지참조) mice compare to the control mice 191

Table 2-8. Effects of tannin supplement for 20 weeks on the plasma and hepatic lipids concentrations in normal diet-fed Apo E-/-(이미지참조) mice 194

Table 2-9. Effects of tannin supplementon hepatic HMG-CoA reductase and ACAT activities in Apo E -/-(이미지참조) mice 196

Table 2-10. Effects of tannin supplementation on fatty acid metabolism in Apo E -/-(이미지참조) mice 197

Table 2-11. Effects of resveratrol on serum lipids and lipoprotein cincentrations in rabbits fed a high cholesterol diet 200

Table 2-12. Effects of resveratrol on serum apolipoprotein concentrations in rabbits fed a high cholesterol diet 200

Table 2-13. Proportion of the area of aortic plaque 201

Table 2-14. Computer-assisted morphometry of aortic cross sections of rabbits in each group after 8 weeks 202

바이오식품소재의 기능특이적 분자지표 발굴 및 응용연구 226

Table 1. Established animal models of intestinal inflammation 252

Table 2. Histological and inflammatory indices 252

Table 3. Histopathological index in DSS-induced colon tissue. 270

Table 4. Gene functional classification in DSS-induced colon tissue. 275

Table 5. Gene functional classification 294

Table 6. Comparison of gene expression between microarray and real-time PCR 299

Table 7. Gene functional classification. 312

Table 8. Comparison of gene expression between microarray and real-time PCR 316

Table 9. Gene functional classification. 325

Table 10. Comparison of gene expression between microarry and real-time PCR. 329

Table 11. Gene functional classification. 338

Table 12. Comparison of gene expression between microarry and real-time PCR 342

Table 13. Effect of water extracts from PJ on food intake, body weight, and liver weight in rats against ethanol induced hepatic injury in rats 344

Table 14. Effect of water extracts from PJ on biochemical parameters exposed to ethanol in rats 349

Table 15. Differentially expressed genes induced by acute ethanol treatment in rat 353

Table 16. Effect of water extracts from P.J. on food intake, body weight, alcohol intake, and liver weight against chronic ethanol administration in rats. 357

Table 17. Effect of water extracts from PJ on biochemical parameters exposed to chronic ethanol administration in rats. 362

Table 18. Differentially expressed genes induced by chronic ethanol treatment 367

Table 19. Comparison between microarray and real-time PCR data 372

Table 20. The commonly expressed genes against both of acute and chronic ethanol-induced liver damage in rat 374

Table 21. Effect of Pycnogenol on ethanol-induced upregulation of gene expression 385

Table 22. Effect of Pycnogenol on ethanol-induced downregulation of gene expression 386

Table 23. Caryophyllene effect on TNF-α regulation of gene expression 389

Table 24. Caryophyllene effect on TNF-α regulated inflammation-related gene expression 390

Table 25. Caryophyllene effect on TNF-α-regulated antipoptoic gene expression 390

Table 26. Caryophyllene effect on TNF-α-regulated proapoptotic gene expression 391

Table 27. Representative examples 391

Table 28. Identification of target genes associated with anti-inflammatort effect 395

Table 29. Effect of lipoic acid and silymarin on TNF-α-induced gene expression 396

Table 30. Summary of biomarkers for evaluating anti-inflammatory function of biofood 405

Table 31. Summary of biomarkers for evaluating anti-oxidative function of biofood 406

바이오식품소재의 효능 평가를 위한 세포주 개발 432

Table. 1. The proteins were identified by MALDI-TOF & MALDI-TOF/TOF. 474

Table.2. PPAR-γ유전자의 PCR primer sequence 477

Table 3. The results of blast search of differentially expressed genes. 483

Table 4. Up-regulated genes after naphthalene exposure for 24h. 484

Table 5. Down-regulated genes after naphthalene exposure for 24h. 484

Table 6. RhoC유전자의 RT-PCR primer sequence 491

바이오식품의 효능 평가를 위한 동물모델 개발 514

Table 1. Microinjection list 539

Table 2/1. Production of F1 transgenic mice 546

Table 3/2. Production of F2 transgenic mice 546

Table 4/3. Production of F3 transgenic mice 547

Table 5/1. Production of F4 transgenic mice 558

Table 6/2. Production of F5 transgenic mice 559

Table 7/3. Production of F6 transgenic mice 559

Table 8/4. Microinjection list 567

Table 9/5. Production of F1 transgenic mice 569

Table 10/6. Production of F2 transgenic mice 569

체중 및 당대사조절 생물지표를 활용한 다원적 기능성 평가시스템 연구 16

FIGURE 1. Body weight gain of rats fed experimental diets for 9 weeks... 60

FIGURE 2. Viseral fat pads weight of rats fed ND or HFD for 9 weeks.... 61

FIGURE 3. Increased visceral fat cell size in rats fed the high fat diet (HFD) compared to those fed the normal diet(ND).... 62

FIGURE 4. Histological profiles of liver tissues from rats fed the normal diet (ND) or the high fat diet (HFD) for 9 weeks(wks). Images captured at 200x magnification. 64

FIGURE 5. Hepatic lipogenic enzyme activities of rats fed ND or HFD for 9 weeks... 65

FIGURE 6. Serum fasting glucose concentrations of rats fed ND or HFD... 66

FIGURE 7. Serum glucose, insulin, c-peptide, and leptin levels, and insulin resistance index of rats fed the normal diet (ND) or the high fat diet (HFD) for 9 wk.... 67

FIGURE 8. Western blot analysis of adiponectin receptor 2 in liver of rats fed experimental diets for 9 weeks... 68

FIGURE 9. Quality check results images of ND and HFD group... 69

FIGURE 10. Quality check results images of ND and HFD group.... 69

FIGURE 11. Array images showing different levels of epididymal fat gene expression of normal diet and high fat diet treated rats. 70

FIGURE 12. Array images showing different levels of hepatic gene expression of normal diet and high fat diet treated rats. 70

FIGURE 13. MA-plots represent genes activated and repressed by high fat diet in the epididymal fat tissue of diet-induced obesity rat.... 71

FIGURE 14. MA-plots represent genes activated and repressed by high fat diet in the liver of diet-induced obesity rat.... 71

FIGURE 15. Flow chart of total account of hepatic tissue and epididymal adipose tissue genes analyzed by cDNA microarray analysis 72

FIGURE 16. Expression of energy expenditure-related genes quantified by quantitative real time RT-PCR.... 80

FIGURE 17. Expression of lipid metabolism-related genes quantified by quantitative real time RT-PCR.... 80

FIGURE 18. Expression of adipocyte differentiation-related genes quantified by quantitative real time RT-PCR.... 81

FIGURE 19. Expression of insulin resistance-related genes quantified by quantitative real time RT-PCR.... 81

FIGURE 20. Expression of β-oxidation-related genes quantified by quantitative real time RT-PCR.... 82

FIGURE 21. Body weight gain of mice fed experimental diets for 12 weeks... 95

FIGURE 22. Visceral fat pads weight of mice fed experimental diets for 12 weeks... 96

FIGURE 23. Histological images of epididymal adipose tissue in mice fed experimental diets for 12 weeks.... 97

FIGURE 24. Serum insulin, leptin and adiponectin levels of mice fed the normal diet group(ND), the high fat diet group(HFD) or the high diet + Garcinia canbogia extract group(HFD+G) for 12 weeks... 98

FIGURE 25. Histological profiles of liver tissues from mice fed experiment diet for 12 weeks... 99

FIGURE 26. Quality check of RNA samples prepared from the epididymal adipose tissue of mice... 99

FIGURE 27. An oligoDNA microarray image (A) and MA-plots (B) of the mouse epididymal adipose tissue genes regulated by the high-fat diet 100

FIGURE 28. Flow chart of total account of epididymal adipose tissue genes regulated by the high-fat diet as analyzed by oligoDNA microarray analysis 100

FIGURE 29. Classification by the biological function of genes with altered expression(≥2 fold) by the high-fat diet in the epididymal adipose tissue of mice:... 101

FIGURE 30. Model for major signaling pathways involved in the translocation of the GLUT4 from the cytosol to the plasma membrane in the adipose tissue.... 105

FIGURE 31. Western blot analysis of the whole-tissue extract of epididymal adipose tissue... 106

FIGURE 32. Western blot analysis of the plasma-tissue extract of epididymal adipose tissue... 107

FIGURE 33. Body weight of mice fed experimental diets... 117

FIGURE 34. Western blot analysis of epididymal adipose tissue for Toll-like receptor/NFkB signaling pathway.... 119

FIGURE 35. Western blot analysis of epididymal adipose tissue for adipogenesis.... 121

FIGURE 36. Body weight gain or rats fed experimental diets... 122

FIGURE 37. Gene expressions determined by quantitative RT-PCR analyses in the epididymal fat tissues from rats fed experimental diets.... 125

FIGURE 38. Protein levels of phosphorylated AMPK and ACC determined by Western blot.... 126

FIGURE 39/38. Toll-like receptors such as TLR4 are important for mediating out innate immune response to bacterial pathogenes and also triggers inflammatory processes upon exposure to certain dietary fats (Tschop M, Thomas G., Nature medicine, 12: 1359-1361, 2006). 135

FIGURE 40/39. TLR4 mediated FFA-induced activation of inflammatin and metabolic signalling in insulin resistance (kim, Cell metabol, 4: 417-419, 2006). 135

심혈관기능유지 관련 다원적 생물지표를 활용한 바이오식품의 기능평가 기술연구에 관한 연구 144

Fig. 1-1. Effect of resveratrol and fibrate on serum total cholesterol and triglyceride concentration of hamsters fed with high-fat, high-cholesterol diet.... 163

Fig. 1-2. Effect of resveratrol on serum Apo B and serum Lp(a) concentration of hamsters fed with high fat diet 164

Fig. 1-3. Representative ORO staining of liver tissue on experimental hamsters. 166

Fig. 1-4. Effect of quercetins on serum total cholesterol and triglyceride concentration of hamsters fed with high fat diet.... 167

Fig. 1-5. Effect of quercetins on serum free cholesterol concentration and atherogenic index (AI) of hamsters fed with high fat diet. 167

Fig. 1-6. Effect of Quercetins on serum Apo B and Lp(a) concentration of hamsters fed with high fat diet. 169

Fig. 1-7. Effect of quercetins on hepatic total lipid and cholesterol contents of hamsters fed with high fat diet. 170

Fig. 1-8. Effect of quercetins on hepatic triglyceride contents of hamsters fed with high fat diet. 170

Fig. 1-9. Representative ORO staining of liver tissue on experimental hamsters. 171

Fig. 1-10. Changes of body weights of the experimental animals for 8weeks. 172

Fig. 1-11. Effect of quercetin and resveratrol on serum lipids concentration and atherogenic index of mouse fed with high fat diet.... 173

Fig. 1-12. Effect of quercetin and resveratrol on Apo A-I and Apo B concentration of mouse fed with high fat diet. 174

Fig. 1-13. Effect of resveratrol on total antioxidant status and TBARS of hamsters fed with high fat diet. 174

Fig. 1-14. Effect of quercetin and resveratrol on activities of GOT and GPT in mouse fed with high fat diet. 175

Fig. 1-15. Representative ORO staining of liver tissue on experimental mouse 176

Fig. 1-16. Representative HE staining of epididymal fat on experimental mouse 176

Fig. 1-17. Gene expression change by high fat control group compared with normal diet group 177

Fig. 1-18. Gene expression change by quercetin group and resveratrol group compared with high fat control group 178

Fig. 1-19. Ontology profile changes of gene expression by high fat control group compared with normal diet group 178

Fig. 1-20. Ontology profile changes of gene expression by quercetin diet compared with high fat control group 179

Fig. 1-21. Ontology profile changes of gene expression by resveratrol diet compared with high fat control group 179

Fig. 1-22. mRNA expression by real-time PCR in the liver of mouse fed with experimental diet. 183

Fig. 1-23. Lipid metabolism related gene protein expression 184

Fig. 1-24. Dietary quercetin and resveratrol alter lipid metabolism in liver of mouse fed with high fat diet. 184

Fig. 2-1. Morphology of aortic sinus from Apo E-/-(이미지참조) mice given clofibrate(CF) and resveratrol(RV).... 188

Fig. 2-2. Effects of resveratrol supplementation on mRNA expression of PPARα and SREBP-1c in Apo E -/-(이미지참조) mice.... 192

Fig. 2-3. Effects of tannin supplementation on mRNA expression of HMGR and PAP in Apo E-/-(이미지참조) mice.... 192

Fig. 2-4. Hepatic protein expression in Apo E -/-(이미지참조) mice fed normal diets supplemented resveratrol.... 193

Fig. 2-5. Cryosection of aortic sinus from Apo E-/-(이미지참조) mice given clofibrate (CF) and tannin (T).... 195

Fig. 2-6. Effects of tannin supplementation on hepatic antioxidative enzyme activities in Apo E -/-(이미지참조) mice.... 196

Fig. 2-7. Effects of tannin supplementation of mRNA expression of HMGR, ACAT, ACC and PAP in Apo E -/-(이미지참조) mice.... 198

Fig. 2-8. Hepatic protein expression in Apo E -/-(이미지참조) mice fed normal diets supplemented tannin.... 199

Fig. 2-9. Effect of resveratrol on aortic surface plaque by Sudan red IV staining... 201

Fig. 2-10. Representative H&E stained sections from aortic arch of experimental rabbits of each group.... 201

Fig. 2-11. Representative ORO staining of aorta tissue on experimental rabbit.... 202

Fig. 2-12. Representative western blot analysis of aorta tissue on experimental rabbit.... 203

Fig. 2-13. mRNA expression of ICAM-1, VCAM-1, p-Selection and MCP-1 in artery of experimental rabbits.... 204

바이오식품소재의 기능특이적 분자지표 발굴 및 응용연구 226

Fig 1. Microarray work-flow. 255

Fig 2. Total RNA extraction procedure. 256

Fig 3. Example of RNA quality check. 257

Fig 4. Image analysis. 257

Fig 5. Data normalization using scatter plot method. 258

Fig 6. Example of hierachical clustering plot. 258

Fig 7. Example of biological cassification using DAVID. 259

Fig 8. Principle ad procedure of real-time RT PCR. 261

Fig 9. Histological sections of colonic mucosa stained with hematoxylin and eosin.... 270

Fig 10. Hierarchical cluster plot image.... 272

Fig 11. Microarray process flowchart. 273

Fig 12. Gene ontology classification in DSS-induced mice colon. 274

Fig 13. Comparison of gene expression between microarray and real-time PCR.... 280

Fig 14. Protein expressions affected by DSS exposure in mice.... 281

Fig 15. The variations of body weight and colon length in DSS-induced colitis model.... 283

Fig 16. MPO activity and presence of diarrhea and bloody stool.... 284

Fig 17. Histological sections of colonic mucosa stained with hematoxylin and eosin.... 285

Fig 18. Edema and inflammation score in colonic tissue.... 286

Fig 19. Scatter matrix plots represent the correlation of microarray samples.... 288

Fig 20. Hierarchical clustering images for normalized signal values.... 289

Fig 21. Schematic diagram of microarray. 290

Fig 22. Gene ontology classification in DSS-induced mice colon. 291

Fig 23. K-means clustering pattern analysis.... 293

Fig 24. Inflammatory gene expression assayed by western blot. 301

Fig 25. IL-6 mRNA and protein expression.... 302

Fig 26. The variations of body weight and colon length in DSS-induced colitis model.... 304

Fig 27. MPO activity and presence of diarrhea and bloody stool.... 305

Fig 28. Histological sections of colonic mucosa stained with hematoxylin and eosin.... 306

Fig 29. Histological score in colonic tissue.... 307

Fig 30. Gene ontology classification in DSS-induced mice colon. 309

Fig 31. K-means clustering pattern analysis.... 310

Fig 32. The variations of body weight and colon length in DSS-induced colitis model.... 318

Fig 33. MPO activity and presence of diarrhea and bloody stool.... 319

Fig 34. Histological sections of colonic mucosa stained with hematoxylin and eosin.... 320

Fig 35. Histological score in colonic tissue.... 321

Fig 36. Hierarchical clustering. 323

Fig 37. K-menas clustering pattern analysis. 324

Fig 38. The variations of body weight and colon length in DSS-induced colitis model.... 331

Fig 39. MPO activity and presence of diarrhea and bloody stool.... 332

Fig 40. Histolofical sections of colonic mucosa stained with hematoxylin and eosin.... 333

Fig 41. Histological score in colonic tissue.... 334

Fig 42. Hierarchical clustering. 336

Fig 43. K-means clustering pattern analysis. 337

Fig 44. Effect of water extracts from PJ on GOT and GPT activities in rats exposed to ethanol induced hepatic injury in rats. 345

Fig 45. Effect of water extracts from PJ on antioxidant enzyme activities and MDA levels wxposed to ethanol in rats. 347

Fig 46. Effect of water extracts from PJ on TG levels exposed to ethanol in rats. 348

Fig 47. Diagrammatic representation of the genes tested in acute ethanol-induced exposed to acute ethanol. 351

Fig 48. Classified differentially expressed genes induced by acute ethanol treatment in rat. 352

Fig 49. Effect of water extracts from PJ on GOT and GPT activities exposed to chronic ethanol administration in rats. 358

Fig 50. Effect of water extracts from PJ on antioxidant enzyme activities, GSH, MDA levels against chronic ethanol administration in rats. 359

Fig 51. Effect of water extracts from PJ on Cholesterol and TG levels against chronic ethanol administration injury in rats. 361

Fig 52. Effect of water extracts from PJ on ethanol metabolic enzyme activities exposed to chronic ethanol administ in rats. 363

Fig 53. Diagrammatic representation of the genes tested in chronic ethanol-induced hepatic injury in rat 365

Fig 54. Differentially expressed genes induced by chronic ethanol treatment in rat. 366

Fig 55. Effect of pycnogenol on GOT, GPT, ALP and LDH activities against ethanol-induced oxidative stress rat model 376

Fig 56. Effect of pycnogenol on Catalase, MDA, GSH and GPx against chronic ethanol-induced hepatic injury in rats. 378

Fig 57. Effect of pinexol on alcohol metabolizing enzymes against ethanol-induced hepatic injury in rats. 380

Fig 58/55. Hierarchical cluster plot 382

Fig 59/56. Diagrammatic representation of the genes tested in ethanol-induced oxidative stress rat model 383

Fig 60/57. Differentially expressed gene number. 384

Fig 61. cDNA microarray analysis of anti-inflammatory effects of caryophyllene using TNF-α-treated HT-29 human colon cells. 388

Fig 62. Clustering : Identification of 9 groups affected by TNF-α and caryophyllene 389

Fig 63. Validation of gene expression by semi-quantitative RT-PCR 392

Fig 64. Validation of gene expression by Western bolt 392

Fig 65. Validation of gene expression by Western and Northern blot 393

Fig 66. Identification of 15 gene groups affected by TNF-α and lipoic acid(A) and TNF-α and silymarin(B) 394

Fig 67. Comparison of anti-inflammatory effect of lipoic acid and silymarin using TNF-α-activated colonic inflammation signaling 395

Fig 68. Effect of Sacchromyces boulardii(SBS) on the TNF-α inflammation signal pathway in HT-29 397

Fig 69. Effect of Sacchromyces boulardii (SBS) on the histologic features of TNBS-induced colitis (A) and the expression of proinflammatory mediaters in colon tissue (B) 397

Fig 70. α-lipoic acid suppresses human colon cancer cell growth by inducing apoptosis and G₁ arrest of the cell cycle 398

Fig 71. Suppression of mRNA expression of RPS6K4(ribosomal protein p90S6K, a novel inhibitor of p53 by α-lipoic acid (ALA) (semi-quantitative RT-PCR) 399

Fig 72. Enhancement of p53 protein stability by α-lipoic acid... 399

Fig 73. α-lipoic acid enhances tumor cell reponse to apoptic stresses 400

Fig 74. Activation of RPS6K4 mRNA expression through NF-kB signaling and ALA inhibition of NF-kB in human colon cancer cells 401

Fig 75. Apoptosis-sensitizing effect of α-lipoic acid is highly dependent on p53 activation via RPS6K4 downstream 401

Fig 76. Schematic representation of p53-activating function of α-lipoic acid through RPS6K4 suppression via NF-kB inhibition 402

바이오식품소재의 효능 평가를 위한 세포주 개발 432

Fig. 1. The cell death (a) and DNA fragmentation (b) by Naphthalene treatment.... 458

Fig. 2. (a) Quantitative real time PCR result after treated 293T cells with Naphthalene for 24h. Reduced COXIII expression by naphthalene treatment (b) The PCR product of COXIII gene. (c) The vector of pCMV-Tag 2 (d)The COXIII gene is introduced into pCMV-Tag 2 vector. 458

Fig. 3. (a) Quantitative real time PCR result after treated 293T cells with Naphthalene for 24h. Reduced Cullin1 expression by naphthalene treatment (b) The PCR product of Culin1 gene. (c) The vector of pLNCX 2 (d) The Cullin1 gene is introduced into pLNCX 2 vector. 458

Fig. 4. The cell line that constitutively overexpresses Cullin1. 459

Fig. 5. The mechanism of RNA interference(RNAi) and sippression of Leptin receptor expression.... 460

Fig. 6. The change of RhoC and Osteopontin in osteoclastogenesis for 6days(a), the clonong of RhoC and Osteopontin genes(b). 462

Fig. 7. The overexpression of RhoC in stable HEK293 cell lines.... 462

Fig. 8. The RT- PCR for candidates.... 464

Fig. 9. The cloning of GSTPi and HNRPH1 genes in pCMV-Tag 2B vector. 464

Fig. 10. Transfection and overexpression of HNRPH1 and GSTPi in 293T cells.... 464

Fig. 11. Nucleotide sequence of adiponectin gene (a) and PCR product from genomic DNA in mature 3T3-L1(b). 465

Fig. 12. The cell lines that constitutively over-expresses adiponectin proteins(a) and the expression of adiponcetin mRNA by natural compounds in adiponcetiin overexpressed cell line(b). 466

Fig. 13. pGL3-Basic vector circle map(a), DNA sequence(b) and PCR product(c) believed to be promoter position. 467

Fig. 14. Comparison of luciferase activites expressed in HEK 293 transfected with pGL3-Basic and pGL3-leptin receptor promoter plasmids. 467

Fig. 15. Luciferase activities expressed in HEK 293 transfected with 14 clones which have fragments by serial deletion. 468

Fig. 16. Luciferase activities expressed in HEK 293 transfected with PPRE. 469

Fig. 17. GeneFishing™ DEG screening result after induction by leptin in HEK293-OB-Rb. 470

Fig. 18. The immunoprecipitated proteins by RhoC-Ab in RAW 264.7 cell(a). The idintified proteins by Maldi-TOF(b). 472

Fig. 19. The immunoprecipitated proteins by RhoC-Ab and then immunoblotting bt β-actin and RhoC antibody in RAW 264.7 cell. 472

Fig. 20. The mRNA expression of Synaptotagmin 13(a) and β-Actin(b) during osteoclast differentiation.... 472

Fig. 1. 2D Gel Elextrophoresis result of 293T cells treated with Aroclor 1254 for 12h. 473

Fig. 2. The RT- PCR for candidates.... 474

Fig. 3. The PCR product of GST1 gene. 474

Fig. 4. The protein level of GSTpi and p-JNK by western blotting after treatment with Aroclor 1254. 475

Fig. 5. The identification of GSTpi activity by western blotting after treatment with Aroclor1254. 475

Fig. 6. The overview of Gene Fishing™ DEG (Differentially Expressed Gene) Assay. 475

Fig. 7. GeneFishing™ DEG screening result after TRANCE/M-CSF induced osteoclast formation in murine RAW 264.7 cell.... 476

Fig. 8. The rate of the mRNA expression of RhoC in RAW 264.7 cells after differentiation during 6 days by Real-Time PCR.... 477

Fig. 9. vector map과 PCR을 수행하여 얻은 PPAR-γ product 478

Fig. 10. Doenjang's water solube fraction suppresses M-CSF/ TRANCE-induced osteoclast formation in primary bone marrow macrophage cells.... 479

Fig. 11. The mRNA expression of osteoclast differentiation markers.... 479

Fig. 12. Effect of the Doenjang's water soluble fraction (1 mg/ml) on the proliferation of MC3T3-E1 cells.... 480

Fig. 13. Alkine phosphatase staining of MC3T3-E1 cells.... 481

Fig. 14. The alkaline phosphatase activity of the MC3Ts-E1 cells.... 481

Fig. 15. Von Kossa staining of MC3T3-E1 cells.... 481

Fig. 16. The results of differentially expressed gene screening.... 482

Fig. 17. The changed creld2 gene expression of the Doenjang treatment in MC3Ts-E1 cells during 6 day differentiation.... 483

Fig. 18. The GeneFishing™ DEG screening results after treated 293T cells with the naphthalene for 24h. The 16 spots were identified. 484

Fig. 19. The changed genes expression by the naphthalene exposure for 24h by RT- PCR for candidates:... 485

Fig. 20. Quantitative real time PCR.... 485

Fig. 21. The characterization of cell death by western blotting.... 486

Fig. 22. The characterization of cell cycle by western blotting.... 486

Fig. 23. Transfection and overexpression of Cullin1 and HNRPH1 in 293T cells.... 487

Fig. 24. RT-PCR of OB-Rb overexpressing stable cell line. 488

Fig. 25. Realtime-PCR of OB-Rb overexpressing stable cell line. 488

Fig. 26. Suppression of Leptin receptor expression in 3T3-L1:... 489

Fig. 27. The overexpression of Leptin receptor in 3T3-L1 and HEK293 cells.... 489

Fig. 28. The selection of osteoclastogenesis-positive and negative effect materials by TRAP staining assay. 490

Fig. 29. Osteoclastogenesis의 (a)촉진효능이 있는 것과 (b)억제효능이 있는 천연물질을 Rho C stable-HEK 293 cell 처리한 후 western blot 수행. 490

Fig. 30. Osteoclastogenesis의 (a)촉진효능이 있는 것과 (b)억제효능이 있는 천연물질을 Rho C stable-HEK 293 cell 처리한 후 RT-PCR 수행. 491

Fig. 31. Cullin 과발현 세포주에 naphthalene을 처리하여 western blot 방법으로 세포주로서의 유효성확인. 492

바이오식품의 효능 평가를 위한 동물모델 개발 514

Fig. 1. Construction (A) and agarose gel electrophoresis (B) of the UCP2 1.8kb(-1800/+30)/pGL3-basic plasmid.... 536

Fig. 2. Effects of EGCG on the mRNA level of UCP2 in 3T3-L1 cells (A) and HepG2 cells(B).... 537

Fig. 3. Effect of EFCG diets on the UCP2 mRNA expression in epidydimal adipose tissue (A) and liver (B) of rats fed high-fat diets.... 537

Fig. 4. Effects of EGCG on the UCP3(-1800/+30bp) promoter activity in 3T3L1 cells (A) and HepG2 cells(B).... 538

Fig. 5. Establishment of optimal condition for detecting of transgene expression.... 539

Fig. 6. PCR results of F0 transgenic mice 540

Fig. 1. Results of PCR amplification of genomic DNA isolated from F1 transgenic mice tail 546

Fig. 2. Results of PCR amplification of genomic DNA isolated from F2 transgenic mice tail 547

Fig. 3. Results of PCR amplification of genomic DNA isolated from F3 transgenic mice tail 547

Fig. 4. Body weight gain during high fat diet-feeding in UCP2 transgenic mice.... 548

Fig. 5. Effect of EGCG on the body weight gain in UCP2 transgenic mice.... 549

Fig. 6. Effect of EGCG on the epudydimal adipose tissue weight (g/100g of body weight).... 549

Fig. 7. Luciferase activity in the epidydimal adipose tissue UCP2-Luc transgenic mice.... 550

Fig. 8. Construction (A) and agarose gel electrophoresis (B) of the UC3 1.8kb/pGL3-basic 4.8kb plasmid.... 551

Fig. 9. Effects of EPA on the mRNA level of UCP3 in C2C12 cells.... 552

Fig. 10. Effect of garlic on the UC3 mRNA expression in dpididymal adipose tissue of mice fed highfat diets.... 552

Fig. 11. Effects of EPA on the UC3(-1790/+52 bp) promoter activity in C2C12 cells.... 553

Fig. 1. Results of PCR amplification of genomic DNA isolated from F4 transgenic mice tail 558

Fig. 2. Results of PCR amplification of genomic DNA isolated from F5 transgenic mice tail 559

Fig. 3. Results of PCR amplification of genomic DNA isolated from F6 transgenic mice tail 560

Fig. 4. Body weight gain (A) and energy intake (B) in UCP2-Luc transgenic mice fed experimental diets.... 561

Fig. 5. Plasma triflyceride (A), total cholesterol (B) and leptin (C) in UCP2-Luc transgenic mice fed experimental diets.... 562

Fig. 6. Luciferase activity in various tissues of UCP2-Luc transgenic mice fed experimental diets.... 563

Fig. 7. Body weight gain (A) and energy intake (B) in UCP2-Luc transgenic mice fed experimental diets.... 564

Fig. 8. Plasma triglyceride (A), total cholesterol (B) and leptin (C) in UCP2-Luc transgenic mice fed experimental diets.... 565

Fig. 9. Luciferase activity in various tissues of UCP2-Luc transgenic mice fed experimental diets.... 565

Fig. 10. PCR results of F0 transgenic mice 568

Fig. 11. Results of PCR amplification of genomic DNA isolated from F1 transgenic mice tail 569

Fig. 12. Results of PCR amplification of genomic DNA isolated from F2 transgenic mice tail 570