이번 연구는 2018년 내몽골 자치구 과학기술부 과학기술사업 지원사업의 '몽골 의학의 지역질병에 대한 쿠미스의 치료효과 핵심과제 연구'에 의해 부여되었다.

참고: 이 프로토콜에 사용된 모든 재료, 기기 및 시약과 관련된 자세한 내용은 재료 표를 참조하십시오.
1. 세포 배양
<ol><li>Dulbecco's Modified Eagle Medium(DMEM)(10% 소 태아 혈청[FBS], 100단위/mL 페니실린 및 100μg/mL 스트렙토마이신 포함)을 함유한 배양 플라스크에서 HepG2 세포 배양. 배양 플라스크를 5%CO2 인큐베이터에서 37°C로 유지합니다.</li>
</ol><p c...

대사성 질환 분석을 위한 올레산 유도 HepG2 세포 

<ol>
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MASLD는 알코올 및 기타 확립된 간 손상 물질 이외의 요인으로 인해 간세포에 과도한 세포 내 지방 침착을 특징으로 하는 임상병리학적 증후군이다18. MASLD는 후천성 대사 스트레스 간 손상과 복잡하게 연결되어 있으며, 특히 인슐린 저항성 및 유전적 감수성과 관련이 있습니다. MASLD에 대한 약물을 효과적으로 연구하고 스크리닝하려면 적절한 실험 모델을 선택하는 것이 중요합니?...

대사기능 장애 관련 지방성 간 질환(metabolic dysfunction-associated steatotic liver disease, MASLD)은 단순 지방증, 비알코올성 지방간염(NASH), 간경변증, 간세포 암종 1,2,3,4,5,6 등 다양한 질환을 포함하며, 모두 알코올 섭취 이외의 요인에 기인한다 7 . MASLD는 대사성 간 손상으로 인해 발생하는 가장 흔한 간 질환으로, 전 세계 인구의 거의 4분의 1이 영향을 받습니다 8,9,10,11,12. MASLD의 정확한 발병 기전은 아직 밝혀지지 않았지만 다양한 이론이 그 발병을 설명하려고 시도합니다. 한 가지 지배적인 개념은 고전적인 "2 히트" 이론에서 "다중 히트" 모델1로 출발할 것을 제안합니다. 이러한 가설의 핵심은 인슐린 저항성의 역할이며, 이는 MASLD 발병기전에서 중추적인 역할을 하는 것으로 여겨진다13. 연구에 따르면 간세포의 인슐린 저항성은 유리 지방산 수치를 증가시켜 간 내에 저장된 트리글리세라이드를 형성합니다14,15.
연구원들은 MASLD에서 지방 침착을 시뮬레이션하기 위해 in vivo 및 in vitro 모델을 모두 사용했습니다. 그러나 그 병리기전을 완전히 복제하는 것은 여전히 어려운 과제입니다. 이러한 한계에도 불구하고 이러한 모델은 MASLD에 대한 잠재적인 치료 표적을 연구하는 데 중요한 역할을 했습니다. 그러나 MASLD의 안정적인 모델을 개발하는 것이 중요합니다. 동물 모델은 효과적이지만 시간과 비용이 많이 들기 때문에 체외 세포 모델에 대한 관심이 높아지고 있습니다. 이러한 모델은 종종 올레산(OA) 및 팔미트산과 같은 단일 또는 다중 유리 지방산을 사용하여 식이 유발 MASLD를 재현합니다. 이 중 인간 간모세포종 세포주인 HepG2는 MASLD의 시험관 내 세포 모델을 확립하는 데 자주 사용됩니다.
OA 유도는 HepG2 세포를 자극하여 역사가 잘 확립된 방법인 MASLD와 유사한 지방 침착을 복제합니다. 이 연구의 목적은 0.25mM OA로 처리된 HepG2 세포의 생존율, ORO(Oil Red O) 염색, 지질 함량 및 트리글리세리드(TG) 수준을 입증하는 것이었습니다. 이 실험의 목적은 MAFLD 모델링 연구의 개발을 위한 추가 증거를 제공하는 것이었습니다.

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			<td>0.22 &#181;m filter</td>
			<td>Millex</td>
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			<td>0.25% Trypsin-EDTA (1x) Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
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			<td>0.45 &#181;m filter</td>
			<td>Millex</td>
			<td></td>
			<td></td>
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			<td>2 mL Crygenic Vials</td>
			<td>CORNING</td>
			<td>430659</td>
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			<td>25 cm2 Cell Culture Flask</td>
			<td>CORNING</td>
			<td>430639</td>
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			<td>6-well cell culture plate</td>
			<td>CORNING</td>
			<td>3516</td>
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			<td>96-well cell culture plate</td>
			<td>CORNING</td>
			<td>3599</td>
			<td></td>
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			<td>Blood Count Plate</td>
			<td>Shanghai Jing Jing Biochemical Reagent &#38; Instrument Co.</td>
			<td>02270113</td>
			<td></td>
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			<td>Cell Counting Kit-8 assays</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>CA1210-1000T</td>
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			<td>CO2 incubator</td>
			<td>NUAIRE</td>
			<td>NU-5710E</td>
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			<td>&#160;DMSO Dimethyl sulfoxide&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>D8371</td>
			<td></td>
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			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>8122691</td>
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			<td>Enzyme Labeling Equipment</td>
			<td>Tecan</td>
			<td>Spark</td>
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			<td>Fetal Bovine Serum, Qualified</td>
			<td>Gibco</td>
			<td>10099141</td>
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		<tr>
			<td>HepG2 cells line</td>
			<td>Beijing North China Chuanglian Biotechnology Research Institute (BNCC)</td>
			<td>221031</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Triglyceride (TG) ELISA instruction</td>
			<td>Nanjing Jiacheng Bioengineering Institute</td>
			<td>20170301</td>
			<td></td>
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		<tr>
			<td>Inverted Microscope for Cell Culture</td>
			<td>Leica</td>
			<td>DMi1&#160;</td>
			<td></td>
		</tr>
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			<td>Isopropanol</td>
			<td>Tianjin Zhiyuan Chemical Reagent Co.</td>
			<td>2021030141</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red Stain Kit, For Cultured Cells</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>G1262</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid&#160;</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A502071</td>
			<td></td>
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		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
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		<tr>
			<td>SPSS 24.0</td>
			<td></td>
			<td>Statistics software</td>
			<td></td>
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</table>

in vitro modeling of fat deposition in metabolic dysfunction-associated steatotic liver disease

대사 기능 장애 관련 지방성 간 질환(MASLD)의 유병률은 경제 및 생활 패턴의 변화로 인해 급증하여 심각한 건강 문제로 이어지고 있습니다. 이전 보고서에서는 MASLD에 대한 동물 및 세포 모델의 확립을 연구하여 이들 간의 차이점을 강조했습니다. 이 연구에서는 MASLD에서 지방 축적을 유도하여 세포 모델을 만들었습니다. HepG2 세포를 다양한 농도(0.125mM, 0.25mM, 0.5mM, 1mM)의 불포화 지방산 올레산으로 자극하여 MASLD를 모방했습니다. 모델의 효능은 세포 계수 키트-8 분석, Oil Red O 염색 및 지질 함량 분석을 사용하여 평가되었습니다. 이 연구는 MASLD 세포에 대한 작동이 간편한 세포 모델을 만드는 것을 목표로 했습니다. 세포 계수 키트-8 분석 결과에 따르면 HepG2 세포의 생존은 GI50 이 1.875mM인 올레산 농도에 따라 달라지는 것으로 나타났습니다. 0.5mM 및 1mM 그룹의 세포 생존율은 대조군보다 유의하게 낮았습니다(P < 0.05). 또한 Oil Red O 염색 및 지질 함량 분석은 HepG2 세포에서 다양한 올레산 농도(0.125mM, 0.25mM, 0.5mM, 1mM)에서 지방 침착을 검사했습니다. 0.25mM, 0.5mM, 1mM군의 지질 함량은 대조군보다 유의하게 높았다(P < 0.05). 또한, OA 그룹의 트리글리세라이드 수치는 대조군보다 유의하게 높았습니다(P < 0.05).

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a range of conditions, including simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma1,2,3,4,5,6, all attributed to factors other than alcohol consuption7. MASLD is the most prevalent liver disease caused by metabolic liver injury, affecting nearly one-quarter of the global population8,9,10,11,12. While the precise pathogenesis of MASLD has not yet been elucidated, various theories attempt to explain its development. One prevailing notion suggests a departure from the classic &#34;two-hit&#34; theory towards a &#34;multiple-hit&#34; model1. Central to these hypotheses is the role of insulin resistance, which is believed to be pivotal in MASLD pathogenesis13. Research indicates that insulin resistance in hepatocytes leads to increased levels of free fatty acids, subsequently forming triglycerides stored within the liver14,15.
Researchers have used both in vivo and in vitro models to simulate fat deposition in MASLD; yet fully replicating its pathomechanism remains challenging. Despite this limitation, these models have been instrumental in studying potential therapeutic targets for MASLD. However, the development of a stable model of MASLD is crucial. While animal models are effective, they are time-consuming and expensive, thus highlighting the growing interest in in vitro cellular models. These models often use single or multiple free fatty acids such as oleic acid (OA) and palmitic acid to recreate diet-induced MASLD. Among these, the human hepatoblastoma cell line HepG2 is often used to establish in vitro cellular models of MASLD.
OA induction stimulates HepG2 cells to replicate fatty deposition akin to MASLD, a method with a well-established history. The aim of this study was to demonstrate the viability, Oil Red O (ORO) staining, lipid content, and triglyceride (TG) level of HepG2 cells treated with 0.25 mM OA. The objective of this experiment was to provide further evidence for the development of MAFLD modeling studies.

저자 스포트라이트: 질병 메커니즘 및 Koumiss의 지질 저하 효과를 조사하기 위한 MASLD 세포 모델 확립

대사 기능 장애 관련 지방성 간 질환에서 지방 침착의 시험관 내 모델링

세포 생존율에 대한 올레산의 효과 HepG2 세포는 다양한 농도의 OA(0mM, 0.125mM, 0.25mM, 0.5mM, 1mM)에 노출되어 0mM에 비해 0.125mM, 0.25mM, 0.5mM, 1mM에서 세포 생존율이 감소했습니다. 통계적 유의성은 0.5mM(P < 0.05) 및 1mM(P < 0.05)에서 관찰되었습니다. CCK-8 키트로 평가한 OA가 세포 생존율에 미치는 영향은 그림 2에 나와 있습니다. OA-처리된 HepG2 세포...

이 논문에서는 대사 기능 장애 관련 지방성 간 질환의 모델로 올레산 유도 HepG2 세포를 사용하는 방법에 대해 설명합니다.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

in-vitro-modeling-fat-deposition-metabolic-dysfunction-associated

대사 기능 장애 관련 지방성 간 질환에서 지방 침착의 시험관 내 모델링

Abstract