[면역학실습] NO assay와 ELISA를 이용한 resveratrol의 항염증 효과 확인 (상)

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1. Resveratrol

Resveratrol은 식물이 만들어내는 폴리페놀계 물질로, 포도껍질, 포도씨, 땅콩, 오디 등에 존재한다. 항암, 항바이러스, 신경보호, 항노화, 항염, 수명연장 등의 효과가 있다.
2. Macrophages

대식세포는 PRR(pattern recognition receptor)를 통해 외부 항원을 인식한다. LPS(lipopolysaccharide)는 대식세포의 TLR4을 통해 인식되어 일련의 신호전달 경로를 거치면서 NO와 활성산소종을 분비하거나 IL-6과 TNF-α와 같은 pro-inflammatory cytokine을 분비하여 염증 반응을 매개한다.
3. RAW 264.7

RAW 264.7은 대표적인 macrophage cell line으로, mouse(Mus musculus) 중에서 흰색인 BALB/c strain에서 유래한다. 영양만 공급되면 무한 증식이 가능하며, 배지 안에서 부유하기보다는 바닥에 adherent하게 붙은 상태에서 생장한다.
4. NO assay

NO assay는 NO(일산화질소)의 생성량을 측정하는 분석법으로, 체내 NO 농도의 주된 변동은 iNOS에 의한 염증량의 변화에 기인한다. NO₂?와 NO₃?를 합산하여 총 NO의 생성량을 추정할 수 있다. NO₂?를 Griess reagent와 반응시켜 생성된 아조 화합물의 흡광도 측정을 통해 NO₂?의 농도를 구할 수 있다.
5. ELISA

ELISA(Enzyme-Linked Immunosorbent Assay)는 항체에 결합된 효소의 발색정도를 통해 항원-항체 반응을 확인하는 실험법으로, 미지의 항체 혹은 항원을 정성 및 정량분석하는 데에 사용된다. 간접 ELISA, 샌드위치 ELISA, 경쟁 ELISA 등의 방법이 있다.

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1. Resveratrol

Resveratrol is a naturally occurring polyphenolic compound found in various plants, particularly grapes, berries, and peanuts. It has garnered significant attention in the scientific community due to its potential health benefits, including its antioxidant, anti-inflammatory, and cardioprotective properties. Numerous studies have investigated the effects of resveratrol on various biological processes and disease states, such as cardiovascular health, neurodegeneration, and cancer. While the exact mechanisms of action are still being elucidated, resveratrol appears to modulate several signaling pathways and cellular processes, including the activation of sirtuins, the inhibition of inflammatory mediators, and the regulation of apoptosis. The potential therapeutic applications of resveratrol are actively being explored, and ongoing research aims to further elucidate its mechanisms of action and optimize its delivery and bioavailability. However, it is important to note that the translation of these findings from in vitro and animal studies to human clinical applications requires careful consideration and further investigation.
2. Macrophages

Macrophages are a crucial component of the innate immune system, playing a vital role in the body's defense against pathogens, the clearance of cellular debris, and the regulation of inflammatory responses. These versatile cells originate from monocytes and can differentiate into various subtypes, each with distinct functional characteristics. Macrophages exhibit remarkable plasticity, capable of adapting their phenotype and function in response to environmental cues. They can assume pro-inflammatory (M1) or anti-inflammatory (M2) states, depending on the signals they receive. M1 macrophages are typically involved in the elimination of pathogens and the promotion of inflammation, while M2 macrophages participate in tissue repair, wound healing, and the resolution of inflammation. The dysregulation of macrophage function has been implicated in the pathogenesis of various diseases, including autoimmune disorders, cancer, and cardiovascular diseases. Understanding the complex biology of macrophages and their role in health and disease is an active area of research, with potential therapeutic implications for modulating macrophage function in a wide range of clinical settings.
3. RAW 264.7

RAW 264.7 is a widely used murine macrophage cell line that has become a valuable tool in immunological and cell biology research. Derived from the ascites of a tumor induced by the Abelson murine leukemia virus, RAW 264.7 cells exhibit many characteristics of primary macrophages, making them a convenient and well-established model system for studying macrophage biology and function. These cells are known for their ability to phagocytose, produce inflammatory mediators, and respond to various stimuli, such as lipopolysaccharide (LPS) and cytokines. The RAW 264.7 cell line has been extensively employed in studies investigating macrophage activation, signaling pathways, cytokine production, and the effects of pharmacological agents on macrophage behavior. While the use of cell lines like RAW 264.7 has limitations in terms of fully recapitulating the complexity of in vivo macrophage responses, they provide a valuable and accessible platform for initial mechanistic investigations and screening studies. The continued use and refinement of the RAW 264.7 model system have contributed significantly to our understanding of macrophage biology and its implications in health and disease.
4. NO assay

The nitric oxide (NO) assay is a widely used analytical technique for the quantification of nitric oxide, a crucial signaling molecule involved in various physiological and pathological processes. Nitric oxide plays a pivotal role in regulating vascular tone, immune function, neurotransmission, and other biological functions. The accurate measurement of NO levels is essential for understanding its role in health and disease. The NO assay typically involves the detection and quantification of nitrite and nitrate, the stable end-products of NO metabolism, using colorimetric, fluorometric, or chemiluminescent methods. These assays provide a reliable and sensitive means of indirectly measuring NO production in biological samples, such as cell culture supernatants, tissue homogenates, or biological fluids. The choice of NO assay method depends on factors such as sample type, sensitivity requirements, and the availability of specialized equipment. The NO assay has become an indispensable tool in various fields, including immunology, cardiovascular research, neuroscience, and pharmacology, enabling researchers to elucidate the complex roles of nitric oxide in health and disease.
5. ELISA

ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used analytical technique in various fields, including immunology, biochemistry, and clinical diagnostics, for the detection and quantification of specific proteins, peptides, hormones, and other biomolecules. The ELISA method relies on the high specificity and affinity of antibody-antigen interactions to capture and detect the target analyte in a sample. The assay typically involves immobilizing the target analyte on a solid support, such as a microtiter plate, and then using enzyme-labeled antibodies to detect and quantify the captured analyte. The enzymatic reaction generates a measurable signal, often a color change or a fluorescent signal, which is proportional to the concentration of the target analyte in the sample. ELISA has become a versatile and indispensable tool in research, clinical diagnostics, and drug development, enabling the sensitive and accurate measurement of a wide range of biomolecules. Its ability to detect and quantify analytes in complex biological samples, such as serum, plasma, or cell culture supernatants, has made ELISA a crucial technique for understanding biological processes, diagnosing diseases, and monitoring therapeutic interventions.

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