Applied Microscopy

  • 제37권2호
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  • Pages.93-102
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  • 2007
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  • 2287-5123(pISSN)
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  • 2287-4445(eISSN)
한국현미경학회 (Korean Society of Microscopy)
권호 표지

대식세포주 (RAW264.7)에서 박테리아성 지질다당류 (LPS) 자극에 의한 분비백혈구단백분해효소억제제 (SLPI)와 성장인자들의 발현

Expression of Growth Factors and Secretory Leukocyte Protease Inhibitor (SLPI) in RAW264.7 Cells after Lipopolysaccharide (LPS) Stimulation

  • 손욱희 (조선대학교 치과대학 치과보존학교실) ;
  • 최백동 (조선대학교 치과대학 구강조직학교실) ;
  • 정순정 (조선대학교 치과대학 구강조직학교실) ;
  • 왕관림 (조선대학교 치과대학 구강조직학교실) ;
  • 황호길 (조선대학교 치과대학 치과보존학교실) ;
  • 정문진 (조선대학교 치과대학 구강조직학교실)
  • Son, Wook-Hee (Department of Conservative Dentistry, College of Dentistry, Chosun University) ;
  • Choi, Baik-Dong (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Jeong, Soon-Jeong (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Wang, Guan-Lin (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Hwang, Ho-Keel (Department of Conservative Dentistry, College of Dentistry, Chosun University) ;
  • Jeong, Moon-Jin (Department of Conservative Dentistry, College of Dentistry, Chosun University)
  • 발행 : 2007.06.30
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초록

분비백혈구단백분해효소억제제 (SLPI)와 여러 성장인자들은 상처 감염이나 박테리아 침입 시 일어나는 염증반응에 서로 관계가 있지만, 대식세포에서 LPS 자극시 SLPI와 VEGF, bFGF, PDGF 등과 같은 성장 인자들의 발현관계에 대해서는 아직까지 알려진 연구 결과가 없다. 따라서 본 연구는 대식세포 세포주로 알려진 RAW264.7 세포에 SLPI 발현의 적정농도인 LPS에 반응하는 SLPI 및 성장 인자들과 세포외부기질이 발현을 규명하고자 하였다. 역전사효소 중합반응(RT-PCR)과 면역학적 단백질 검출법(Western blotting)은 LPS 처리 후 SLPI와 몇몇 성장인자들 (VEGF, bFGF, PDGF)와 제1형 아교질 mRNA와 SLPI 단백질의 검출을 위해 수행하였다. RAW264.7 세포 주를 mL 당 100 ng의 LPS에 각각 30분, 60분, 90분, 24시간, 48시간동안 노출시켰다. RT-PCR 결과 SLPI mRNA는 시간이 지남에 따라 점점 발현 양이 증가하였고 VEGF와 PDGF mRNA는 초기에 높은 발현 양상을 보였다. 그러나 bFGF와 I형 아교질의 발현은 매우 미약하게 나타났다. SLPI 단백질 발현 역시 mRNA 수준과 마찬가지로 증가하는 양상을 보였는데, 배양액내의 SLPI 단백질양은 전체적으로 감소하는 경향을 보였다. 또한 광학현미경 관찰과 주사전자현미경 관찰결과, LPS가 RAW264.7 세포주의 형태학적인 변화를 일으킴을 확인하였다. 본 결과를 종합하면 SLPI 발현증가의 적정 농도라 생각되는 100ng의 LPS에 의해서 발현되는 VEGF나 PDGF는 SLPI의 발현에 관계가 있는 것으로 생각되지만 추후에 이들 인자들의 단백질이나 유전자 도입을 통하여 발현 관계를 명확히 확인해야 하는 추가실험이 진행되어야 할 것이다.하게 된다.토끼 면역항체를 선모충유충 조직항원에 반응시켰을 때 충체의 표피와 기저층 그리고 EIM 및 stichocyte의 ${\alpha}_0\;{\alpha}_1$ 과립에 황금입자가 표지되었다. 따라서 1일 동안 배설되는 분비배설항원은 선모충 유충의 표피와 stichocyte의 ${\alpha}_0\;{\alpha}_1$ 과립에서 유도되는 반면에 3일 동안 배설되는 분비배설항원은 표피와 stichocyte의 ${\alpha}_0$ 과립에서 유도되고, 선모충유충 감염후 1주, 4주에 실험쥐에서 형성되는 감염항체는 선모충의 표피와 기저층 그리고 EIM에서 분비되는 항원에 의하여 생성된다. 이상의 결과로 선모충의 분비배설항원과 감염항원은 선모충 유충의 표피와 EIM및 stichocyte의 ${\alpha}_0\;{\alpha}_1$ 과립에서 유도되며 이들은 45 kDa 단백을 포함하고 있는 것으로 생각된다.성하고 있는 세포들에는 세포질이 어두운 세포와 밝은 세포가 있었으며, 세포질내에는 전자밀도가 높은 분비과립이 관찰되었다. 전체적인 특징은 눈물샘분비세포 중 장액세포의 것과 비슷하였으나, 과립의 크기는 작았다. 분비관을 구성하는 세포들 사이에도 연접복합체가 매우 잘 발달되어 있었다. 샘포에서 사이관으로 이행되는 곳에서도 샘포세포와 사이관세포 사이에서도 연접복합체가 관찰되었다. 분비관세포의 분비과립 가운데는 중심부분에 전자밀도가 더 높은 중심을 가진 다른 모양의 과립이 관찰되기도 하였다. 의해 사망한 환자는 없었다. 결 론: 자궁경부암 환자에 항암화학요법과 동시에 외부 방사선조사와 고선량률의 강내조사를 시행한 결과 독성이 심하지 않고 국소제어율과 단기 생존율이 양호하여 안전하고 효율적인 치료방법으로 생각된다.

Secretory leukocyte protease inhibitor (SLPI) was known as one of bacterial lipopolysaccharide (LPS)-induced products of macrophage. Macrophages play an important role in the development of inflammatory responses by secreting an array of cytokines and chemokines in a tissue microenvironment. To identify the function and relationship between potent growth factors and SLPI after LPS stimulation, we conducted reverse transcriptase polymerase chain reaction (RT-PCR) and Western blots for the detection of mRNA and protein expression of SLPI and growth factors such as VEGF, PDGF, bFGF after 100 ng LPS stimulation on the RAW264.7 cells. The result of RT-PCR was showed SLPI mRNA expression was increased from 60 min to 48h in RAW 264.7 cells after incubation with LPS. VEGF and PDGF mRNA was expressed highly at initial stage by LPS stimulation. The mRNA of bFGF and type I collagen was very weakly expressed after LPS stimulation. SLPI protein level was increased likely the mRNA levels in RAW 267.7 cells. Additionally, phase contrast and scanning electron microscopic observation demonstrated that the LPS induce the change of morphology of the RAW264.7 cells. From these results, it suggest that expression of SLPI by LPS treatment may associate with VEGF and PDGF expression in RAW264.7 cells.

키워드

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