THP-1 Cell과 HUVEC을 이용한 Co-Culture Model System에서 최종당화산물에 의한 Cytokines와 RAGE 발현

국문 초록 (Abstract)

Glyceraldehyde를 이용하여 제조된 AGEs 화합물(glycer-AGEs)을 단구세포인 THP-1, 혈관내피세포인 HUVEC 및 이 두 세포가 동시에 배양된 system에서 100 ㎍/mL로 처리후 24시간까지 처리시간을 달리하여 처리하였다. 배양시간동안 각 세포와 배양액을 회수하여 TNF-α와 IL-1β의 발현을 mRNA 수준에서 조사하였다. 그 결과, THP-1의 경우 배양 2시간에서 TNF-α나 IL-1β의 mRNA 발현이 대조구에 비해 증가되었으나 혈관내피세포인 HUVEC의 경우에는 24시간 배양하는 동안 유의적인 차이가 없었다. 그러나 두세포를 동시에 배양한 system에서 혈관내피세포인 HUVEC의 경우에는 배양 4시간에서 대조구보다 TNF-α의 발현은 4.4배, IL-1β의 경우 5.5배 정도 증가되는 것을 확인할 수 있었다. TNF-α와 IL-1β의 배지 내에서의 농도를 측정해 본 결과, THP-1만 배양한 경우 대조구의 배지 내 TNF-α 함량이 배양 6시간에 98.2 pg/mL로 대조구 53.8 pg/mL보다 증가하였으며 HUVEC의 경우 배양 8시간에 93.3 pg/mL로 증가하였다. 그러나 co-culture의 경우 배양 4시간부터 증가하여 배양 8시간에 199.2 pg/mL로 증가하였다. RAGE는 TNF-α와 IL-1β의 발현 pattern과 다르게 단독 및 coculture에서 배양 16시간에 대조구에 비해 각각 1.6배, 24시간에 1.9배 증가하였다. 따라서 본 연구 결과 최종당화산물에 의해 혈관내피세포의 기능상실의 연구에 있어 coculture 조건이 유용하며, 특히 mRNA 수준에서는 4시간에, protein 수준에서는 8시간에 효능을 측정하면 유효성 있는 연구 성과를 얻을 있을 것으로 예측할 수 있었다.

다국어 초록 (Multilingual Abstract)

Although monoculture methods have been remarkably useful due to their simplicity, they have serious limitation because of the different types of cells communication with each other in many physiological situations. We demonstrated levels of markers of endothelial dysfunction such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) as well as stimulation of receptor of advanced glycation endproducts (AGEs) on monoand co-culture system such as only monocyte (THP-1) cultivation system, only endothelial cell (HUVEC) cultivation system, and co-cultivation system of THP-1 and HUVEC. The mRNA levels of TNF-α and IL-1β on HUVEC increased by the co-culture with monocyte after 4 hr at 100 ㎍/mL glyceraldehyde-AGE. The secreted protein contents into medium of TNF-α and IL-1β increased after 8 hr approximately 2～2.5 times compared to mono-cultivation. In contrast, the mRNA level of receptor of AGE (RAGE) was relatively insensitive on the co-culture system. The mediators by which monocytes activate endothelial cell have not been fully elucidated. In this study we confirmed production of soluble cytokines such as TNF-α and IL-1β by monocytes. Use of monocyte conditioned medium, which contains both cytokines, can activate endothelial cell.

목차 (Table of Contents)

• Abstract
• 서론
• 재료 및 방법
• 결과 및 고찰
• 요약
• 감사의 글
• 문헌

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