KR101557746B1 - Marker Composition for Estimating Exposure of Hydrofluoric Acid and Toxicity - Google Patents

Marker Composition for Estimating Exposure of Hydrofluoric Acid and Toxicity Download PDF

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KR101557746B1
KR101557746B1 KR1020130135523A KR20130135523A KR101557746B1 KR 101557746 B1 KR101557746 B1 KR 101557746B1 KR 1020130135523 A KR1020130135523 A KR 1020130135523A KR 20130135523 A KR20130135523 A KR 20130135523A KR 101557746 B1 KR101557746 B1 KR 101557746B1
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이미영
류아름
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순천향대학교 산학협력단
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Abstract

본 발명은 불산의 노출과 독성 평가용 단백질 검출제제를 포함하는 마커 조성물에 관한 것으로, 더욱 상세하게는 불산을 사람 피부섬유아세포에 24시간 처리한 후 프로테오믹 분석을 진행한 결과 증가되거나, 감소되는 발현 변화를 보이는 총 16개의 단백질 중 어느 하나 이상의 단백질 검출제제를 포함하는 불산의 노출과 독성 평가용 마커 조성물에 관한 것이다.
불산 처리에 의해 발현증가한 5종의 단백질과 발현감소한 11종 단백질들로서, 베타-액틴(beta-actin), 아넥신(annexin) A5, 아넥신(annexin) A4, 프로히비틴(prohibitin) 1, 열충격 단백질(heat shock protein) 27, 콜라겐(collagen), MSN protein, MTHSP75, 스트레스-유도 포스포프로테인 1(stress-induced phosphoprotein 1), 디-3-포스포글리세라이트 디하이드로게네이즈 (PHGDH), EF-Tu, hnRNP 2H9B, 포스포글리세라이트 뮤타아제 1(phosphoglycerate mutase 1), 퍼옥시리독신(peroxiredoxin), 40S 리보솜 단백질(ribosomal protein) S12 또는 HLA-A2 로서, 이들 중 어느 하나 이상의 검출제제를 포함하는 마커 조성물은 불산 노출에 의한 독성 진단을 위한 유용한 바이오마커로서, 사용될 수 있다. The present invention relates to a marker composition comprising a protein detection agent for evaluating exposure and toxicity of hydrofluoric acid. More particularly, the present invention relates to a marker composition comprising a protein detection agent for evaluating exposure and toxicity of hydrofluoric acid. More specifically, The present invention relates to a marker composition for evaluating exposure and toxicity of hydrofluoric acid.
Five proteins with elevated expression by hydrofluoric acid and eleven proteins with reduced expression were identified as beta-actin, annexin A5, annexin A4, prohibitin 1, Proteins such as heat shock protein 27, collagen, MSN protein, MTHSP75, stress-induced phosphoprotein 1, di-3-phosphoglyceride dehydrogenase (PHGDH), EF -Tu, hnRNP 2H9B, phosphoglycerate mutase 1, peroxiredoxin, 40S ribosomal protein S12 or HLA-A2. Can be used as useful biomarkers for toxicity diagnosis by exposure to fluoric acid.

Description

불산의 노출과 독성 평가용 단백질을 검출하는 마커 조성물{Marker Composition for Estimating Exposure of Hydrofluoric Acid and Toxicity}Technical Field [0001] The present invention relates to a marker composition for detecting exposure to toxic chemicals,

본 발명은 불산의 노출과 독성 평가용 단백질 검출제제를 포함하는 마커 조성물에 관한 것으로, 더욱 상세하게는 불산을 사람 피부섬유 아세포에 24시간 처리한 후 프로테오믹 분석을 진행한 결과 증가되거나, 감소되는 발현 변화를 보이는 총 16개의 단백질 중 어느 하나 이상의 검출제제를 포함하여 불산의 노출과 독성을 평가할 수 있는 불산의 노출과 독성 평가용 마커 조성물에 관한 것이다.The present invention relates to a marker composition comprising a protein detection agent for evaluating exposure and toxicity of hydrofluoric acid. More particularly, the present invention relates to a marker composition comprising a protein detection agent for evaluating exposure and toxicity of hydrofluoric acid. More specifically, The present invention also relates to a marker composition for evaluating exposure and toxicity of hydrofluoric acid, which is capable of evaluating exposure and toxicity of hydrofluoric acid, including at least one of 16 proteins exhibiting changes in expression.

불산은 플루오르화수소가 용해된 형태의 물질로 주로 기름 정제, 유기불소화합물의 생산, 보일러의 성능검증, 치과치료, 금속작업, 석유산업, 유리산업에 주로 쓰인다 (Burgher F, 2011a). 불산은 또한 세척제, 냉각제, 의약품, 플라스틱, 형광등, 해충제와 같은 생활용품에도 사용되어 진다 (Franzblau A, 2003).Fluoric acid is a substance in the form of hydrogen fluoride dissolved mainly in oil refining, production of organic fluorine compounds, verification of boiler performance, dental treatment, metal working, oil industry, and glass industry (Burgher F, 2011a). Foshan is also used in household goods such as detergents, coolants, medicines, plastics, fluorescent lights, and insect repellents (Franzblau A, 2003).

불산은 부식성을 띠며 표면, 피부, 눈, 호흡기에 손상을 주는 수소이온(H+)과 국소적, 조직적 세포독성을 가지는 플루오라이드이온(F-)으로 이루어져 있으며, 두 이온의 특성으로 인해 불산에 노출이 되면 조직의 괴사와 심각한 고통이 유발 된다 (Burgher F, 2011b). 불산은 분해가 되었을 때 조직 깊숙이 침투한다. 플루오라이드이온은 칼슘이온, 마그네슘이온과 강하게 반응하여 중화시킨다. 이 반응은 많은 효소 시스템과 같은 세포대사를 방해하여 세포괴사나 죽음에 이르게 하며 저칼슘혈증과 저마그네슘혈증의 원인이 된다 (Bertolini JC, 1991; Kirkpatrik JJ 1995; Ohtani M 2007). FOSHAN is composed of hydrogen ion (H + ), which is corrosive and damages the surface, skin, eyes and respiratory system, and fluoride ion (F - ) which has local and systematic cytotoxicity. Exposure causes tissue necrosis and severe pain (Burgher F, 2011b). When hydrofluoric acid decomposes, it penetrates deeper into the tissue. Fluoride ions react strongly with calcium ions and magnesium ions to neutralize them. This reaction interferes with cell metabolism such as many enzyme systems leading to apoptosis or death and causes hypocalcemia and hypomagnesemia (Bertolini JC, 1991; Kirkpatrik JJ 1995; Ohtani M 2007).

불산의 의한 부상은 주로 플루오라이드와 칼슘사이의 결합으로 인해 칼슘 의존적 프로세스의 붕괴로 일어난다 (Schultz CH, 1989). 불산에 피부가 노출되면 칼슘, 마그네슘과 플루오라이드이온이 결합해 불용성염을 만들고 몸속 양이온을 감소시켜 부정맥을 일으킨다 (Coffey JA, 2007; Wong A, 2012). Inhomogenesis by hydrofluoric acid occurs mainly due to the collapse of calcium-dependent processes due to the bond between fluoride and calcium (Schultz CH, 1989). When exposed to hydrofluoric acid, calcium, magnesium, and fluoride ions combine to form insoluble salts and reduce body cations to produce arrhythmias (Coffey JA, 2007; Wong A, 2012).

또한, 플루오라이드는 크렙스 회로와 Na/K+ ATPase 펌프의 활성에 포함되는 세포내 효소를 억제해 에너지 생산에 영향을 줄 뿐만 아니라 세포가 죽음에 이르게 한다(Wong A, 2012), 플루오라이드 음이온은 매우 작아 쉽게 수용액에 잘 확산되며 지질막을 쉽게 통과 한다(Makarovsky I, 2008). 몇몇 연구결과에서는 플루오라이드에 의해 다량의 활성산소가 생산되며 SOD, catalase, glutathione peroxidase와 같은 항산화효소의 활성을 감소시킨다는 것을 밝혔다 (Mittal and Flora, 2006).In addition, fluoride inhibits intracellular enzymes involved in the activity of the Krebs cycle and the Na / K + ATPase pump, affecting energy production as well as causing cell death (Wong A, 2012). Fluoride anions They are very small and easily diffuse into aqueous solutions and pass easily through lipid membranes (Makarovsky I, 2008). Several studies have shown that fluoride produces large amounts of reactive oxygen and reduces the activity of antioxidant enzymes such as SOD, catalase, and glutathione peroxidase (Mittal and Flora, 2006).

불산 노출에 의한 분자적 세포적 독성이 단백질수준에서의 실험결과들이 불산이 가지는 일반적인 독성에 대한 정보에 비해 부족하다. 이에 사람 피부섬유아세포에 불산을 처리하였을 때의 단백질 발현변화를 프로테오믹 기법을 사용하여 연구하였다. 불산에 의해 발현증가하거나 감소된 단백질들은 불산노출에 의한 독성평가용 마이오마커로 사용가능할 것이다Molecular cellular toxicity due to exposure to FOSH The experimental results at the protein level are insufficient compared to information on the general toxicity of FOSHAN. Protein expression of human fibroblasts treated with hydrofluoric acid was studied using proteomic techniques. Proteins expressed or reduced by hydrofluoric acid may be used as myo-markers for assessing toxicity by exposure to hydrofluoric acid

본 발명은 상기 문제점을 해결하기 위하여, 사람 피부섬유아세포에 불산을 처리하였을 때의 단백질 발현변화를 프로테오믹 기법을 사용하여 연구한 결과, 불산에 의해 발현증가하거나 감소된 단백질들을 확인하고 이의 검출제제를 포함하는 불산노출에 의한 독성평가용 마커 조성물를 제공하는 것을 목적으로 한다. In order to solve the above problems, the inventors of the present invention have studied the change of protein expression in human fibroblasts treated with hydrofluoric acid by using proteomic techniques. As a result, It is another object of the present invention to provide a marker composition for evaluating toxicity by exposure to hydrofluoric acid containing a preparation.

상기와 같은 목적을 달성하기 위해, 본 발명은 베타-액틴(beta-actin), 아넥신(annexin) A5, 아넥신(annexin) A4, 프로히비틴(prohibitin) 1, 열충격 단백질(heat shock protein) 27, 콜라겐(collagen), MSN protein, MTHSP75, 스트레스-유도 포스포프로테인 1(stress-induced phosphoprotein 1), 디-3-포스포글리세라이트 디하이드로게네이즈 (PHGDH), EF-Tu, hnRNP 2H9B, 포스포글리세라이트 뮤타아제 1(phosphoglycerate mutase 1), 퍼옥시리독신(peroxiredoxin), 40S 리보솜 단백질(ribosomal protein) S12 또는 HLA-A2 중 어느 하나 이상의 검출제제를 포함하는 것을 특징으로 하는 불산의 노출과 독성 평가용 마커 조성물을 제공한다. In order to accomplish the above object, the present invention provides a pharmaceutical composition comprising beta-actin, annexin A5, annexin A4, prohibitin 1, heat shock protein, 27, collagen, MSN protein, MTHSP75, stress-induced phosphoprotein 1, di-3-phosphoglyceride dehydrogenase (PHGDH), EF-Tu, hnRNP 2H9B, Wherein the detection agent comprises at least one detection agent selected from the group consisting of phosphoglycerate mutase 1, peroxiredoxin, 40S ribosomal protein S12, and HLA-A2. A marker composition for toxicity evaluation is provided.

바람직하게는 불산에 의해 그 발현량이 증가되는, 베타-액틴(beta-actin), 아넥신(annexin) A5, 아넥신(annexin) A4, 프로히비틴(prohibitin) 1, 열충격 단백질(heat shock protein) 27 중 어느 하나 이상을 검출하는 단백질 검출제제를 포함하는 것을 특징으로 하는 불산의 노출과 독성 평가용 마커 조성물을 제공한다. Annexin A5, annexin A4, prohibitin 1, heat shock protein (hereinafter referred to as " heat shock protein "), 27. A marker composition for assessing exposure and toxicity of hydrofluoric acid, comprising:

또한, 바람직하게는 불산에 의해 그 발현량이 감소되는, 콜라겐(collagen), MSN protein, MTHSP75, 스트레스-유도 포스포프로테인 1(stress-induced phosphoprotein 1), 디-3-포스포글리세라이트 디하이드로게네이즈 (PHGDH), EF-Tu, hnRNP 2H9B, 포스포글리세라이트 뮤타아제 1(phosphoglycerate mutase 1), 퍼옥시리독신(peroxiredoxin), 40S 리보솜 단백질(ribosomal protein) S12 또는 HLA-A2 중 어느 하나 이상을 검출하는 단백질 검출제제를 포함하는 것을 특징으로 하는 불산의 노출과 독성 평가용 마커 조성물을 제공한다. Also, it is preferable to use collagen, MSN protein, MTHSP75, stress-induced phosphoprotein 1, di-3-phosphoglyceride dehydrogenase (PHGDH), EF-Tu, hnRNP 2H9B, phosphoglycerate mutase 1, peroxiredoxin, 40S ribosomal protein S12 or HLA-A2 The present invention provides a marker composition for evaluating exposure and toxicity of hydrofluoric acid.

본 발명은 사람 피부섬유아세포에 불산을 처리하였을 때의 단백질 발현변화를 프로테오믹 기법을 사용하여 연구한 결과, 불산에 의해 그 발현량이 증가하거나 감소된 단백질들을 측정하는 검출제제를 포함하는 불산의 노출과 독성 평가할 수 있는 마커 조성물을 제공한다. The present invention relates to a method for detecting the expression of protein in humic skin fibroblasts by treatment with hydrofluoric acid and a method for detecting the amount of protein expressed by hydrofluoric acid, To provide a marker composition that can be evaluated for exposure and toxicity.

불산 처리에 의해 발현량이 증가한 5종의 단백질과 발현량이 감소한 11종 단백질들로서, 베타-액틴(beta-actin), 아넥신(annexin) A5, 아넥신(annexin) A4, 프로히비틴(prohibitin) 1, 열충격 단백질(heat shock protein) 27, 콜라겐(collagen), MSN protein, MTHSP75, 스트레스-유도 포스포프로테인 1(stress-induced phosphoprotein 1), 디-3-포스포글리세라이트 디하이드로게네이즈 (PHGDH), EF-Tu, hnRNP 2H9B, 포스포글리세라이트 뮤타아제 1(phosphoglycerate mutase 1), 퍼옥시리독신(peroxiredoxin), 40S 리보솜 단백질(ribosomal protein) S12 또는 HLA-A2 로서, 이들 중 어느 하나 이상의 단백질을 검출하는 제제를 포함하는 마커 조성물은 불산 노출에 의한 독성 진단을 위한 유용한 마커로서, 사용될 수 있다. Five proteins with increased expression levels by hydrofluoric acid treatment and 11 proteins with decreased expression levels were identified as beta-actin, annexin A5, annexin A4, and prohibitin 1 , Heat shock protein 27, collagen, MSN protein, MTHSP75, stress-induced phosphoprotein 1, di-3-phosphoglyceride dehydrogenase (PHGDH) , EF-Tu, hnRNP 2H9B, phosphoglycerate mutase 1, peroxiredoxin, 40S ribosomal protein S12 or HLA-A2. The marker composition comprising the agent to be detected can be used as a useful marker for toxicity diagnosis by exposure to fluoric acid.

도 1은 그림 1. 불산이 사람 피부섬유아세포의 생존에 미치는 영향.
도 2는 프로테오믹스 기법을 이용한 불산 처리에 따른 사람피부섬유아세포의 단백질발현 변화 분석. (A) 대조군, (B) 불산 처리군.
도 3은 불산 처리에 따른 사람피부섬유아세포의 단백질 발현변화 분석. (A) 대조군 대비 단백질 발현 증가 그룹, (B) 대조군 대비 단백질 발현 감소 그룹.Figure 1 shows the effect of hydrofluoric acid on the survival of human skin fibroblasts.
FIG. 2 is a graph showing changes in protein expression of human skin fibroblasts by hydrofluoric acid treatment using the proteomics technique. (A) control group, (B) hydrofluoric acid treatment group.
FIG. 3 is a graph showing changes in protein expression of human skin fibroblasts by hydrofluoric acid treatment. (A) increased protein expression compared to the control group, and (B) decreased protein expression relative to the control group.

본 발명은 불산의 노출과 독성 평가용 단백질 검출제제를 포함하는 마커 조성물에 관한 것으로, 더욱 상세하게는 불산을 사람 피부섬유아세포에 24시간 처리한 후 프로테오믹 분석을 진행한 결과 증가되거나, 감소되는 발현 변화를 보이는 총 16개의 단백질 중 적어도 하나 이상의 단백질 검출제제를 포함하는 불산의 노출과 독성 평가용 마커 조성물에 관한 것이다.The present invention relates to a marker composition comprising a protein detection agent for evaluating exposure and toxicity of hydrofluoric acid. More particularly, the present invention relates to a marker composition comprising a protein detection agent for evaluating exposure and toxicity of hydrofluoric acid. More specifically, The present invention relates to a marker composition for evaluating exposure and toxicity of hydrofluoric acid, which comprises at least one protein detection agent out of a total of 16 proteins exhibiting a change in expression.

이하, 본 발명을 실시예를 들어 상세히 설명하기로 한다.
Hereinafter, the present invention will be described in detail with reference to examples.

[실시예 1 : 세포배양과 불산 처리][Example 1: Cell culture and treatment with hydrofluoric acid]

사람 피부섬유아세포는 열불활성화된 10% FBS와 1% P/S가 포함된 Dulbecco’s modified essential 배지 (DMEM)를 사용하여 5% CO2와 37℃의 조건에서 배양하였다. 불산의 독성실험을 하기 위해 사람 피부섬유아세포를 96-well plate에서, 프로테오믹스 분석을 위해서는 100 mm 배양접시에서 24시간 배양하였다. 그 후, 세포에 불산을 처리하고 37℃에서 24시간 배양하였다.
Human dermal fibroblasts were cultured in Dulbecco's modified essential medium (DMEM) containing 5% heat-activated 10% FBS and 1% P / S at 5% CO 2 and 37 ° C. Human fibroblasts were cultured in 96-well plates for proteolysis and in 100-mm culture dishes for 24 hours. Then, the cells were treated with hydrofluoric acid and cultured at 37 DEG C for 24 hours.

[실시예 2 : MTT assay][Example 2: MTT assay]

세포 생존률 실험을 위해 MTT assay를 실시하였으며 사람 피부섬유아세포를 96-well plate에 각 well당 3 × 104의 수로 계대배양 하였다. 세포에는 불산 처리 농도를 0 ~ 5 mM 까지 0.5 mM의 간격으로 24시간 동안 처리하였다. 처리 후 PBS로 수세한 후 5 mg/ml의 농도인 MTT 시약을 넣고 37℃에서 3시간 동안 반응시켰다. 반응이 끝나면 MTT 시약을 제거한 후에 DMSO 100 ul을 넣고 상온에서 30분 동안 반응시킨다. 반응이 끝나면 570 nm에서 흡광도를 측정하였다. 각 실험은 세 번 반복실험 하였다.
For cell viability, MTT assay was performed. Human skin fibroblasts were subcultured in a 96-well plate at a rate of 3 × 10 4 cells per well. Cells were treated with 0 to 5 mM HOC treatment at 0.5 mM intervals for 24 h. After treatment, the cells were washed with PBS, and MTT reagent with a concentration of 5 mg / ml was added thereto, followed by reaction at 37 ° C for 3 hours. After the reaction is complete, remove the MTT reagent, add 100 μl of DMSO, and allow to react at room temperature for 30 minutes. After the reaction, absorbance was measured at 570 nm. Each experiment was repeated three times.

[실시예 3 : 시료 준비][Example 3: Sample preparation]

사람 피부섬유아세포를 세포용해 버퍼(lysis buffer, 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 100 mM DTT, protease inhibitor cocktail)에 넣은 후 초음파 분쇄기로 분쇄하였다. 그 후 12,000 × g, 4℃에서 30분 동안 원심분리 하였다. 상등액을 새로운 튜브에 옮겨 브래포드(Bradford)법을 이용하여 단백질 정량을 하였으며 각 시료는 사용 전까지 70℃에 보관하였다.
Human skin fibroblasts were plated in lysis buffer (7 M urea, 2 M thiourea, 4% (w / v) CHAPS, 100 mM DTT, protease inhibitor cocktail) and then disrupted with an ultrasonic grinder. Thereafter, centrifugation was carried out at 12,000 x g, 4 ° C for 30 minutes. The supernatant was transferred to a new tube and protein quantification was performed using the Bradford method. Each sample was stored at 70 ° C until use.

[실시예 4 : 이차원 전기영동][Example 4: Two-dimensional electrophoresis]

전체 세포 용해물을 rehydration solution (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 100 mM DTT, 0.5% ampholytes (pH 3-10 NL), BPB)에 700 ug을 넣고 최종 부피가 250 ul가 되도록 한다. Immobiline DryStrips (13 cm, pH 3-10 NL, Amersham Biosciences, Uppsala, Sweden)에 시료를 넣은 후 pI값 별로 단백질을 1차원 전기영동 하였다. 그 후, 각 샘플을 평형화를 1차, 2차에 나누어 각각 15분씩 진행하였다. 분자량에 따라 분리하는 2차원 전기영동은 SDS-PAGE를 사용하였으며 15 mm 두께의 12.5% 폴리아크릴아마이드 젤을 사용하였다. 25℃에서 시료가 젤에 들어갈 때까지 20 mA로 전기영동 하였으며 시료가 젤 안으로 들어가면 60 mA로 전기영동 하였다.
700 ug was added to the whole cell lysate in a rehydration solution (7 M urea, 2 M thiourea, 4% (w / v) CHAPS, 100 mM DTT, 0.5% ampholytes (pH 3-10 NL) 250 < / RTI > Immobiline DryStrips (13 cm, pH 3-10 NL, Amersham Biosciences, Uppsala, Sweden) were loaded with samples and one-dimensional electrophoresis of proteins by pI value. Thereafter, each sample was subjected to equilibration by primary and secondary for 15 minutes each. SDS-PAGE was used for two-dimensional electrophoresis according to the molecular weight, and a 12.5% polyacrylamide gel having a thickness of 15 mm was used. At 25 ° C, the sample was electrophoresed at 20 mA until it entered the gel, and when the sample entered the gel, the sample was electrophoresed at 60 mA.

[실시예 5 : Coomassie brilliant blue 염색과 이미지분석][Example 5: Coomassie brilliant blue staining and image analysis]

전기영동이 끝난 젤은 고정액을 사용하여 젤 안의 단백질을 고정시켰다. 그 후 Coomassie brilliant blue 시약을 사용해 염색한 후 5% 아세트산(acetic acid) 용액으로 탈색하였다. Power Look 1100 스캐너를 사용해 젤을 스캔한 후 ImageMaster 2D Platinum 6.0 프로그램을 이용해 젤의 단백질 스팟을 찾고 분석하였다.
After the electrophoresis, gel was immobilized using fixer. It was then stained with Coomassie brilliant blue reagent and decolorized with 5% acetic acid solution. After scanning the gel using the Power Look 1100 scanner, the protein spot of the gel was detected and analyzed using the ImageMaster 2D Platinum 6.0 program.

[실시예 6 : 단백질 동정][Example 6: protein identification]

단백질 스팟을 25 mM ammonium bicarbonate 용액 (pH 8.2)과 50% 아세토니트릴(acetonitrile)로 두 번 수세한 후, 100% 아세토니트릴(acetonitrile)로 건조시켰다. 펩타이드는 트립신 (Promega, Madison, WI)을 사용하여 분해하였으며, α-cyano-4-hydroxycinnamic acid를 녹인 50% acetonitrile/0.1% TFA와 섞었다. 각각의 펩타이드 용액의 염기를 제거한 후 MALDI plate에 점적하였다. 말디토프 질량분석기술(MALDI-TOF-MS)를 사용해 펩타이드를 분석한 후 분석결과는 MASCOT 소프트웨어를 사용하여 단백질동정을 하였다.
Protein spots were washed twice with 25 mM ammonium bicarbonate solution (pH 8.2) and 50% acetonitrile, and then dried with 100% acetonitrile. Peptides were digested with trypsin (Promega, Madison, WI) and mixed with 50% acetonitrile / 0.1% TFA in which α-cyano-4-hydroxycinnamic acid was dissolved. After removing the base of each peptide solution, the solution was applied to a MALDI plate. Peptides were analyzed using Maltitol mass spectrometry (MALDI-TOF-MS) and the results were analyzed using MASCOT software.

< 결과 및 고찰 ><Results and Discussion>

[실시예 7 : 사람 피부 섬유아세포에서의 불산 독성관찰][Example 7: Observation of toxicity to hydrofluoric acid in human skin fibroblasts]

MTT assay를 통해 사람 피부섬유아세포에서 불산 세포독성을 관찰하였다. 그 결과 불산의 농도가 증가함에 따라 세포의 생존율이 감소함을 보였으며 불산 농도 2 mM을 24시간 처리하였을 때 약 20%의 세포독성을 보였으며 이 농도조건에서 단백체 분석을 실시하였다(도 1).
MTT assay was used to observe the cytotoxicity of Fc in human skin fibroblasts. As a result, the survival rate of the cells decreased with the increase of the concentration of hydrofluoric acid, and the cytotoxicity of about 20% was observed when the hydrofluoric acid concentration was 2 mM for 24 hours (FIG. 1) .

[실시예 8 : 단백질체 분석][Example 8: Proteome analysis]

불산을 사람 피부섬유아세포에 24시간 처리한 후 프로테오믹 분석을 진행한 결과 총 16개의 단백질이 발현변화를 보였다(도 2). 16개의 단백질 중 5개의 단백질은 불산을 처리한 실험군에서 증가하였으며 11개의 단백질은 발현이 감소함을 보였다(도 3). After hydrofluoric acid was treated with human fibroblasts for 24 hours, proteolytic analysis was carried out. As a result, a total of 16 proteins showed an expression change (FIG. 2). Of the 16 proteins, 5 proteins were increased in the hydrofluoric acid treated group and 11 proteins were decreased in expression (Fig. 3).

발현증가 단백질은 베타-액틴(beta-actin, 스팟 7번), 아넥신(annexin) A5 (스팟 9번), 아넥신(annexin) A4 (스팟 10번), 프로히비틴(prohibitin) 1 (스팟 11번), 열충격 단백질(heat shock protein) 27 (스팟 14번)이었다, The expression-increasing proteins are beta-actin (spot 7), annexin A5 (spot 9), annexin A4 (spot 10), prohibitin 1 11), heat shock protein 27 (spot 14)

발현감소 단백질은 콜라겐(collagen, 스팟 1번), MSN protein (스팟 2번), MTHSP75 (스팟 3번), 스트레스-유도 포스포프로테인 1(stress-induced phosphoprotein 1, 스팟 4번), 디-3-포스포글리세라이트 디하이드로게네이즈 (PHGDH), EF-Tu, hnRNP 2H9B, 포스포글리세라이트 뮤타아제 1(phosphoglycerate mutase 1), 퍼옥시리독신(peroxiredoxin), 40S 리보솜 단백질(ribosomal protein) S12 (스팟 15번), HLA-A2 (스팟 16번)으로 동정되었다 (표 1).
Expression-decreasing proteins include collagen (spot 1), MSN protein (spot 2), MTHSP75 (spot 3), stress-induced phosphoprotein 1 (spot 4) (PHGDH), EF-Tu, hnRNP 2H9B, phosphoglycerate mutase 1, peroxiredoxin, 40S ribosomal protein S12 (phosphoglycerate mutase 1) Spot 15) and HLA-A2 (spot 16) (Table 1).

< 표 1 > 불산에 의해 발현이 증가하거나 감소한 피부섬유아세포 단백질<Table 1> Dermal fibroblast protein increased or decreased expression by hydrofluoric acid

Figure 112013101958978-pat00001

Figure 112013101958978-pat00001

불산에 의해 감소한 스팟 1은 콜라겐(collagen)으로 동정되었는데, 이 단백질은 피부, 뼈, 연골, 힘줄과 같은 다양한 조직을 유지하는데 중요하다.(Tillet E, 1994; Freise C, 2009). 콜라겐(collagen)은 세포외기질(ECM) 단백질로 세 개의 나선형의 도메인으로 이루어져 있다. 그 중 Collagen VI는 미세섬유의 주요 구성성분이며 α1, α2, α3으로 이루어져 있다. Collagen VI를 암호화하는 유전자에 돌연변이가 일어나면 베쓰렘 근질환, 근이영양증과 같은 상염색체 우성질병이 일어난다 (Lampe AK, 2005). Collagen VI는 서로 다른 시스템에서 세포의 증식과 세포자살을 조절한다 (Cheng IH, 2011). 배아발생, 상처 치유, 조직의 섬유화가 일어나는 동안 collagen VI에 의한 세포자살억제가 일어나며 간엽세포의 증식이 시작된다 (Ruhl M, 1999). Spot 1, reduced by hydrofluoric acid, has been identified as collagen, which is important for maintaining various tissues such as skin, bone, cartilage, and tendons (Tillet E, 1994; Freise C, 2009). Collagen is an extracellular matrix (ECM) protein composed of three helical domains. Among them, Collagen VI is a major component of fine fibers and consists of α1, α2, α3. A mutation in the gene encoding Collagen VI results in autosomal dominant diseases such as Bethlehem myopathy and muscular dystrophy (Lampe AK, 2005). Collagen VI regulates cell proliferation and apoptosis in different systems (Cheng IH, 2011). During embryogenesis, wound healing, and fibrosis of the tissue, cell proliferation is initiated by collagen VI and cell proliferation begins (Ruhl M, 1999).

불산에 의해 증가한 아넥신(annexin) A5 (스팟 9번)는 아넥신 그룹(annexin group)단백질의 하나로 포스파티딜세린(phosphatidylserine, PS), 포스파티딜에탄올아민(phosphatidylethanolamine, PE)과 같은 칼슘 의존적 인지질 결합 단백질이다. 세포자살, 동맥색전증 혈전증, 면역 염증반응, 미세입자형성 억제, 세포표면의 수용체 감소와 같은 다양한 생화학적 세포 공정에 중요한 역할을 한다.Annexin A5 (spot 9), increased by Foshan, is one of the annexin group proteins and is a calcium-dependent phospholipid binding protein such as phosphatidylserine (PS), phosphatidylethanolamine (PE) . It plays an important role in various biochemical cell processes such as cell suicide, arterial embolism thrombosis, immunoinflammatory response, inhibition of fine particle formation, reduction of cell surface receptors.

불산에 의해 증가한 스팟 10은 아넥신(annexin) A4 (ANXA4)로 동정되었는데, 이 단백질은 주로 상피세포에 많으며 세포자살, 세포주기, 항응고와 같은 다양한 생물학적 프로세스에 관련이 있다 (Lin, 2012). 신장암에서 주로 증가하며 in vitro 상에서 MCF7 세포에 annxin A4가 과발현되었을 경우 종양세포의 전이가 촉진되었다. 이에 따라 annexin A4가 종양의 전이에 영향을 줄 것으로 보인다 (Zimmermann U, 2004; Mussunoor S, 2008). ANXA4는 세포 성장이 일어나도록 하며 ANXA4에의한 시그널은 발암과정과 관련이 있다 (Lin, 2012). ANXA4는 세포의 배출현상과 관련이 있는 LAMP2와 RHAMM의 발현을 증가시키고, RHAMM은 AKT를 유도하는 RAS와 PI3K를 활성화 시킨다 (Hall CL, 1995). 결과적으로 ANX4는 AKT, CDK1활성과 PBK 유전자의 발현을 증가시키고 p21의 발현을 감소시켜 세포의 과증식이 일어나도록 한다 (Lin 2012).Spot 10, which is increased by hydrofluoric acid, has been identified as annexin A4 (ANXA4), which is predominantly in epithelial cells and is involved in a variety of biological processes such as apoptosis, cell cycle and anticoagulation (Lin, 2012) . The expression of annxin A4 in MCF7 cells overexpressed tumor cells in the kidney. Thus, annexin A4 appears to influence tumor metastasis (Zimmermann U, 2004; Mussunoor S, 2008). ANXA4 allows cell growth to occur and signals by ANXA4 are associated with the carcinogenesis process (Lin, 2012). ANXA4 increases the expression of LAMP2 and RHAMM, which are associated with cell excretion, and RHAMM activates RAS and PI3K, which induce AKT (Hall CL, 1995). As a result, ANX4 increases AKT, CDK1 activity and PBK gene expression and reduces p21 expression, leading to cell hyperplasia (Lin 2012).

열충격 단백질 패밀리(Heat shock protein family)는 small HSPs (15-30 kDa), HSP60, HSP70, HSP90, HSP100을 포함한다. HSPs는 악성종양으로의 발달시 유전자 발현 억제 해지가 되어 암세포에서 발현이 증가 된다 (Calderwood SK, 2006). HSPs 발현 증가는 암세포의 축적을 야기하고 세포자살을 억제한다 (Calderwood SK, 2006). The heat shock protein family includes small HSPs (15-30 kDa), HSP60, HSP70, HSP90 and HSP100. HSPs are inhibited in gene expression when they develop into malignant tumors and their expression is increased in cancer cells (Calderwood SK, 2006). Increased expression of HSPs causes accumulation of cancer cells and inhibits apoptosis (Calderwood SK, 2006).

불산에 의해 증가한 HSP27 (스팟 14번)은 HSPs의 일종으로 α-crystallin 단백질과 관련있는 저분자 단백질이며, HSP70은 ATP의존적 샤페론의 일부로 새로운 폴리펩타이드의 합성에 관여한다 (Beckmann RP, 1990; Kiriyama MT, 2001). HSP27은 F-actin, Bid, ROS에 의한 미토콘드리아의 cytochrome c 방출을 억제할 뿐 아니라 세포질 내 cytochrome c 농도를 낮추어 세포자살이 일어나는 것을 억제한다 (Beere HM. 2004; Garrido C, 2006). HSP27, a kind of HSPs, is a low molecular protein associated with the α-crystallin protein, and HSP70 is involved in the synthesis of new polypeptides as part of the ATP-dependent chaperone (Beckmann RP, 1990; Kiriyama MT, 2001). HSP27 inhibits mitochondrial cytochrome c release by F-actin, Bid, and ROS as well as inhibits apoptosis by lowering cytochrome c levels in the cytoplasm (Beere HM. 2004; Garrido C, 2006).

불산에 의해 감소한 미토콘드리아(mitochodrial) HSP75 (mtHSP75; 스팟 3번)는 HSP70의 일종으로 미토콘드리아 샤페론의 일종이다 (Jaattela M, 1999; Voos W, 1999). HSP27과 HSP70은 강력한 샤페론으로 미토콘드리아에 의한 세포자살을 억제한다 (Garrido C, 2006). Mitochondrial HSP75 (mtHSP75; Spot 3), a type of HSP70, is a type of mitochondrial chaperone (Jaattela M, 1999; Voos W, 1999). HSP27 and HSP70 are potent chaperones that inhibit mitochondrial apoptosis (Garrido C, 2006).

불산에 의해 증가한 프로히비틴(prohibitin, PHB; 스팟 11번)는 진핵세포에 높게 보존된 단백질로 PHB1과 PHB2로 나뉜다. PHB는 세포주기조절, 세포자살, 미토콘드리아 호흡사슬 효소의 집결, 노화와 관련한 기능을 한다 (Goates, 2001). PHB1은 주로 미토콘드리아 내막, 세포막, 핵막에 위치한다 (Mishra S, 2006; Schleicher M, 2008). 내피세포에서 PHB1 억제시 미토콘드리아의 ROS 생성의 증가와 혈관형성억제가 일어나며 이를 통해 PHB1이 미토콘드리아의 기능과 ROS에 의한 노화 예방, 혈관형성에 중요한 역할을 함을 알 수 있다 (Schleicher M, 2008). 암세포에서 prohibitin의 억제는 미토콘드리아 네트워크를 무너뜨리고 세포 부착에 문제를 일으킨다 (Sievers C, 2010).Prohibitin (PHB: Spot # 11), which is increased by hydrofluoric acid, is a highly conserved protein in eukaryotic cells and is divided into PHB1 and PHB2. PHB has functions related to cell cycle regulation, cell suicide, mitochondrial respiratory chain enzyme aggregation, and aging (Goates, 2001). PHB1 is mainly located in mitochondrial inner membrane, cell membrane, and nuclear membrane (Mishra S, 2006; Schleicher M, 2008). Inhibition of PHB1 in endothelial cells leads to an increase in mitochondrial ROS production and inhibition of angiogenesis, indicating that PHB1 plays an important role in mitochondrial function, ROS-induced senescence, and angiogenesis (Schleicher M, 2008). Inhibition of prohibitin in cancer cells destroys the mitochondrial network and causes problems with cell attachment (Sievers C, 2010).

불산에 의해 감소한 항산화효소인 퍼옥시리독신(peroxiredoxin) (Prx; 스팟 13번)는 thioredoxin으로 전자를 생성하여 하이드로퍼옥시드(hydroperoxide)와 퍼옥시니트라이트(peroxynitrite)를 줄인다 (Rhee SG, 2005). Prx는 6개의 동형단백질(isoform)을 가지며 산화적 손상을 막는다 (Seo MS, 2000; Rhee SG, 2001). Prx family는 2-cys Prx 단백질 (Prx I ~ IV), atypical 2-Cys (1-Cys Prx, Prx V & VI)로 나뉜다 (Rhee SG, 2005). 2-Cys Prx 단백질은 아미노기와 카르복실기 말단에 둘다 시스테인 잔기를 가지며 1-Cys Prx 단백질은 아미노기 말단에만 시스테인 잔기를 가진다 (Chae HZ, 1994; Kang SW, 1998; Rhee SG, 2001). Prx VI는 과산화수소에 의한 산화적 스트레스로부터 세포를 보호하고 Prx VI 과산화시 세포주기를 멈추거나 세포자살을 유도하며 iPLA2의 활성을 높인다 (Kim SY, 2008).Peroxiredoxin (Prx; spot # 13), an antioxidant enzyme reduced by hydrofluoric acid, produces electrons with thioredoxin to reduce hydroperoxides and peroxynitrite (Rhee SG, 2005 ). Prx has six isoforms and prevents oxidative damage (Seo MS, 2000; Rhee SG, 2001). The Prx family is divided into 2-cys Prx proteins (Prx I to IV), atypical 2-Cys (1-Cys Prx, Prx V & VI) (Rhee SG, 2005). The 2-Cys Prx protein has both cysteine residues at the amino and carboxyl ends, and the 1-Cys Prx protein has only the cysteine residue at the amino terminal end (Chae HZ, 1994; Kang SW, 1998; Rhee SG, 2001). Prx VI protects cells from oxidative stress caused by hydrogen peroxide and inhibits cell cycle or induces apoptosis and increases activity of iPLA2 during Prx VI peroxidation (Kim SY, 2008).

ERM (ezrin, radixin, moesin) family는 세포막에 위치하며 막결합 도메인, α-나선 도메인, 액틴(actin) 결합 도메인으로 구성되어있다 (Tsukita S, 1997). 세 개의 도메인은 세포와 세포, 세포와 기질 사이의 부착, 액틴 필라멘트의 조절, 세포막의 상호작용에 중요한 역할을 한다 (Takeuchi K, 1994; Ichikawa T, 1998). The ERM (ezrin, radixin, moesin) family is located in the cell membrane and consists of the membrane-binding domain, α-helical domain, and actin binding domain (Tsukita S, 1997). Three domains play an important role in cell and cell, adhesion between cell and substrate, regulation of actin filaments, and cell membrane interactions (Takeuchi K, 1994; Ichikawa T, 1998).

Rho는 작은 GTP 결합 단백질로 membrane-actin-filament cross-linkers를 활성화시켜 ERM을 억제한다 (Tsukita S, 1997). Rho inhibits ERM by activating membrane-actin-filament cross-linkers with a small GTP-binding protein (Tsukita S, 1997).

불산에 의해 감소한 모에신(moesin) (스팟 2번)은 T림프구에서 intercellular adhesion molecule-3 (ICAM-3)과 면역침전을 하여 RANTES를 자극한다 (Serrador, 1997). 모에신(Moesin)은 ICAM-3과 CD44와 결합해 막 수용체와 세포골격사이에 링크를 형성하고 세포 이동시 형태변화를 조절한다 (Serrador, 1997).Moesin (Spot 2), reduced by Foshan, stimulates RANTES by immunoprecipitation with intercellular adhesion molecule-3 (ICAM-3) in T lymphocytes (Serrador, 1997). Moesin binds to ICAM-3 and CD44 to form a link between the membrane receptor and the cytoskeleton and regulates morphological changes during cell migration (Serrador, 1997).

스팟 6은 불산에 의해 감소했으며, 신장인자(elongation factor) Tu (EF-Tu)로 동정되었다. 원핵생물의 신장 요소 중 하나로 리보솜의 A-site에 aminoacyl-tRNA (aa-tRNA)가 결합하는 단백질 합성과정에 참여한다 (Ravel JM, 1969; Andersen GR, 2000). EF-Tu는 진화과정에서 높게 보존되었으며 진핵생물에서의 상동단백질인 EF-1a는 구조가 거의 동일하다 (Andersen GR, 2000). 원핵생물 신장회로는 동물의 미토콘드리아에서 일어난다 (Schwarzbach CJ, 1991). 미토콘드리아 EF-Tu (EF-Tumt)는 단백질 열응집 방지, GTP 의존적 환경에서의 단백질 재접힘 강화, 스트레스환경에서의 폴리펩타이드 합성과 같은 샤페론 활성을 보인다 (Suzuki H, 2007).Spot 6 was reduced by hydrofluoric acid and identified as an elongation factor Tu (EF-Tu). (Ravel JM, 1969; Andersen GR, 2000), which binds aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes as one of the elongation factors of prokaryotes. EF-Tu was highly conserved during evolution and EF-1a, a homologous protein in eukaryotes, is almost identical in structure (Andersen GR, 2000). Prokaryotic elongation circuits occur in animal mitochondria (Schwarzbach CJ, 1991). Mitochondrial EF-Tu (EF-Tumt) exhibits chaperone activity such as protein heat aggregation prevention, protein refolding in a GTP-dependent environment, and polypeptide synthesis in a stressed environment (Suzuki H, 2007).

불산에 의해 감소한 스트레스-유도 포스포프로테인(stress-induced phosphoprotein) 1 (STIP1; 스팟 4번)은 HSP70과 HSP90의 결합체로 Hop 이라 한다 (Odunuga OO, 2004). STIP1은 세포질에서 HSP90과 HSP70-타겟 단백질의 결합 어답터로 작용하며 최근 연구에서는 HSPs의 샤페론 활동을 조절하는 역할을 함이 밝혀졌다 (Odunuga OO, 2004; Wang TH, 2010). STIP1은 HSP70, HSP90, HSP90 타겟 단백질을 결합하는 단백질로 세포 주기 조절과 세포자살시 발현이 증가한다 (Kamel A, 2003; Pearl, 2005; Kin S, 2010). Stress-induced phosphoprotein 1 (STIP1; Spot No. 4), reduced by hydrofluoric acid, is a combination of HSP70 and HSP90 and is called Hop (Odunuga OO, 2004). STIP1 acts as a binding adapter for HSP90 and HSP70-target proteins in the cytoplasm, and in recent studies it has been shown to play a role in regulating chaperone activity of HSPs (Odunuga OO, 2004; Wang TH, 2010). STIP1 is a protein that binds HSP70, HSP90, and HSP90 target proteins and increases cell cycle regulation and expression during apoptosis (Kamel A, 2003; Pearl, 2005; Kin S, 2010).

불산에 의해 감소한 D-3-phosphoglycerate dehydrogenase (PHGDH; 스팟 5번)는 3-phosphoglycerate가 3-phosphohydroxypyuvate로의 변환을 촉매한다 (Mullarky E, 2011). 어떤 암세포는 많은 양의 glycolytic carbon을 PHGDH를 통해 serine과 glycine 대사로 돌린다 (Locasale JW, 2011). 세포증식이 약화된 세포에서 PHGDH 발현이 감소한다 (Locasale JW, 2011).D-3-phosphoglycerate dehydrogenase (PHGDH; spot number 5) reduced by hydrofluoric acid catalyzes the conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate (Mullarky E, 2011). Some cancer cells convert large amounts of glycolytic carbon into serine and glycine metabolites via PHGDH (Locasale JW, 2011). PHGDH expression is decreased in cells with weakened cell proliferation (Locasale JW, 2011).

불산에 의해 감소한 heterogeneous nuclear ribonucleoprotein (hnRNP) 2H9 (스팟 8번)는 heterogeneous nuclear RNA (hnRNA)에 결합하여 RNA polymerase II에 의해 번역되게 하는 hnRNP 단백질의 아종이다 (Dreyfuss G, 1993; Honore B, 2000). hnRNP 2H9 유전자는 6가지의 전사체(2H9, 2H9A, 2H9B, 2H9C, 2H9D, 2H9E)를 가진다 (Honore B, 2000). hnRNP는 pre-mRNA 처리, 수송과 hnRNA와 다른 핵 구조와의 결합에 참여한다 (Dreyfuss G. 1993; Weighardt F, 1996; Honore B, 2000). hnRNP 2H9는 스플라이싱 과정에 참여하며 일시적으로 hnRNP 복합체에서 떨어져 초기 heat shock-induced splicing을 억제한다 (Mahe C, 1997). 또한, 스플라이싱 조절에 개입하여 유전자 발현에 영향을 주고 hnRNP M의 생리학적 과정에 영향을 준다 (Mahe D, 2000).(HnRNP) 2H9 (Spot 8) is a subspecies of the hnRNP protein that binds to heterogeneous nuclear RNA (hnRNA) and is translated by RNA polymerase II (Dreyfuss G, 1993; Honore B, 2000) . The hnRNP 2H9 gene has six transcripts (2H9, 2H9A, 2H9B, 2H9C, 2H9D, 2H9E) (Honore B, 2000). hnRNP participates in pre-mRNA treatment, transport and binding of hnRNA to other nuclear structures (Dreyfuss G. 1993; Weighardt F, 1996; Honore B, 2000). hnRNP 2H9 participates in the splicing process and temporarily inhibits the initial heat shock-induced splicing away from the hnRNP complex (Mahe C, 1997). It also intervenes in splicing control, influencing gene expression and affecting the physiological processes of hnRNP M (Mahe D, 2000).

불산에 의해 감소한 포스포글리세라이트 뮤타아제(phosphoglycerate mutase) (PGM; 스팟 12번)은 해당과정의 중요 효소로 2.3-bisphoshphoglycerate를 매개로 3-phosphoglycerate (3PG)에서 2-phosphoglycerate (2PG)로의 전환을 촉진한다. PGM은 근육 유래의 m-type과 뇌 유래의 b-type으로 나뉘며 MM, BB, MB로 동형이량체 혹은 이형 이량체로 구성된다. PGM-B는 산소가 적은 상태에서의 해당작용을 조절하며 세포가 저산소증에 적응할 수 있도록 한다(Takahashi Y, 1998). PGM1은 세포증식이 활발한 세포나 암세포에서 과발현되며, 이런 특징으로 바이오 마커로 사용된다 (Ren F, 2010).Phosphoglycerate mutase (PGM; spot number 12) reduced by 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) via 2.3-bisphoshphoglycerate Promote. PGM is divided into m-type derived from muscle and b-type derived from brain, and composed of homodimer or heterodimer as MM, BB, MB. PGM-B regulates the action of oxygen at low levels and allows cells to adapt to hypoxia (Takahashi Y, 1998). PGM1 is overexpressed in cell proliferation-activated cells and cancer cells, and is used as a biomarker with this characteristic (Ren F, 2010).

불산에 의해 감소한 40S 리보솜 단백질(ribosomal protein) S12 (RPS12; 스팟 15번)는 번역과 연관된 단백질로 단백질 합성에 참여하는 리보솜의 30S 서브유닛(subunit)의 기능 중심에 위치한다 (Coukell, 1969; Cheng Z, 2010). 진행중인 암세포에서 RPS12 유전자의 발현이 증가한다 (Cheng Q, 2002a). 자궁경부암 환자의 정상 자궁경부 편평상피 조직에서의 RPS12 유전자 발현은 약하며 초기 자궁경부암 진단마커로서 유용하게 쓰인다(Cheng Q, 2002a; Cheng Q, 2002b, Deng SS, 2006).The 40S ribosomal protein S12 (RPS12; spot 15), which is reduced by hydrofluoric acid, is a protein associated with translation and is located in the functional center of the 30S subunit of ribosomes involved in protein synthesis (Coukell, 1969; Cheng Z, 2010). Expression of the RPS12 gene is increased in ongoing cancer cells (Cheng Q, 2002a). The expression of RPS12 gene in normal cervical squamous epithelium of cervical cancer patients is weak and is useful as an early cervical cancer diagnostic marker (Cheng Q, 2002b, 2002b, Deng SS, 2006).

불산에 의해 감소한 휴먼 류코사이트 안티젠(human leukocyte antigen) (HLA)-A2 (스팟 16번)는 HLA A 항원형 그룹으로 HLA는 세포 표면에서 T 세포가 항원으로 인식하도록 해준다. 흑색종과 같은 암세포에서 발현이 줄어들며 이는 CTL에 의한 인식을 낮추어 암세포에 대한 면역반응 낮추어 악성종양으로의 발달을 가능하게 한다 (Pandolfi F, 1991). HLA-A2 발현 감소와 급성백혈병이 재발은 관련이 있다 (Masuda K, 2007).Human leukocyte antigen (HLA) -A2 (spot 16), reduced by Foshan, is an HLA A antigenic group, allowing HLA to recognize T cells as antigen on the cell surface. Expression is reduced in cancer cells such as melanoma, which reduces CTL-mediated cognition, thereby lowering the immune response to cancer cells and enabling development to malignant tumors (Pandolfi F, 1991). Reduced expression of HLA-A2 is associated with recurrence of acute leukemia (Masuda K, 2007).

불산에 의해 베타-액틴(beta-actin) (스팟 7번)도 발현 감소를 보였다. 이 단백질은 구조 단백질로 비근세포의 구조와 모양을 유지시키며 신호전달분자로 쓰일 뿐만 아니라, 웨스턴 반응의 내부 대조군으로 널리 사용되어 왔다. Beta-actin (Spot No. 7) also decreased expression by Foshan. This protein is a structural protein that maintains the structure and shape of non-myocytes and is used as a signaling molecule and has been widely used as an internal control for Western reactions.

그러나, 최근 연구결과에서 내피세포와 혈소판에서 니트릭 옥사이드 합성효소(nitric oxide synthase) type 2 (NOS-3)의 활성에 직접적으로 관련이 있을 것으로 제안되었으며, 본 연구에서는 불산에 의해 발현이 증가됨이 확인되었다. However, recent studies have suggested that nitric oxide synthase type 2 (NOS-3) may be directly related to the activity of endothelial cells and platelets. In this study, .

따라서, 불산 처리에 의해 발현이 증가한 5종의 단백질과 발현이 감소한 11종 단백질 들은 불산 노출에 의한 독성 진단을 위한 바이오 마커로 사용가능할 것으로 보인다. Therefore, five proteins with increased expression by hydrofluoric acid treatment and 11 proteins with decreased expression appear to be available as biomarkers for toxicity diagnosis by exposure to fluoric acid.

상기에 제시된 실시예는 예시적인 것으로 이 분야에서 통상의 지식을 가지는 자는 본 발명의 기술적 사상을 벗어나지 않는 범위에서 제시된 실시예에 대한 다양한 변형 및 수정 발명을 만들 수 있을 것이다. 이러한 변형 및 수정 발명에 의하여 본 발명의 범위는 제한되지 않는다.The above-described embodiments are illustrative and those skilled in the art will be able to make various modifications and alterations to the disclosed embodiments without departing from the spirit of the present invention. The scope of the present invention is not limited by these variations and modifications.

Claims (3)

베타-액틴(beta-actin), 아넥신(annexin) A5, 아넥신(annexin) A4, 프로히비틴(prohibitin) 1, 열충격 단백질(heat shock protein) 27, 콜라겐(collagen), MSN protein, MTHSP75, 스트레스-유도 포스포프로테인 1(stress-induced phosphoprotein 1), 디-3-포스포글리세라이트 디하이드로게네이즈 (PHGDH), EF-Tu, hnRNP 2H9B, 포스포글리세라이트 뮤타아제 1(phosphoglycerate mutase 1), 퍼옥시리독신(peroxiredoxin), 40S 리보솜 단백질(ribosomal protein) S12 또는 HLA-A2 중 어느 하나 이상의 단백질 검출제제를 포함하는 것을 특징으로 하는 불산의 노출과 독성 평가용 마커 조성물. Annexin A5, annexin A4, prohibitin 1, heat shock protein 27, collagen, MSN protein, MTHSP75, Stress-induced phosphoprotein 1, di-3-phosphoglyceride dehydrogenase (PHGDH), EF-Tu, hnRNP 2H9B, phosphoglycerate mutase 1, , Peroxiredoxin, 40S ribosomal protein S12, or HLA-A2. A marker composition for evaluating exposure and toxicity of hydrofluoric acid. 제1항에 있어서,
불산 노출에 의해 발현량이 증가되는, 베타-액틴(beta-actin), 아넥신(annexin) A5, 아넥신(annexin) A4, 프로히비틴(prohibitin) 1, 열충격 단백질(heat shock protein) 27 중 어느 하나 이상의 단백질 검출제제를 포함하는 것을 특징으로 하는 불산의 노출과 독성 평가용 마커 조성물. The method according to claim 1,
Annexin A5, annexin A4, prohibitin 1 and heat shock protein 27, which are increased in expression level by exposure to hydrofluoric acid, such as beta-actin, annexin A5, annexin A4, A marker composition for assessing exposure and toxicity of hydrofluoric acid, comprising at least one protein detection agent. 제1항에 있어서,
불산 노출에 의해 발현량이 감소되는, 콜라겐(collagen), MSN protein, MTHSP75, 스트레스-유도 포스포프로테인 1(stress-induced phosphoprotein 1), 디-3-포스포글리세라이트 디하이드로게네이즈 (PHGDH), EF-Tu, hnRNP 2H9B, 포스포글리세라이트 뮤타아제 1(phosphoglycerate mutase 1), 퍼옥시리독신(peroxiredoxin), 40S 리보솜 단백질(ribosomal protein) S12 또는 HLA-A2 중 어느 하나 이상의 단백질 검출제제를 포함하는 것을 특징으로 하는 불산의 노출과 독성 평가용 마커 조성물.The method according to claim 1,
MSN protein, MTHSP75, stress-induced phosphoprotein 1, di-3-phosphoglyceride dehydrogenase (PHGDH), and di- A protein detection agent of any one or more of EF-Tu, hnRNP 2H9B, phosphoglycerate mutase 1, peroxiredoxin, 40S ribosomal protein S12 or HLA-A2 Wherein the marking composition is used for evaluating exposure and toxicity of hydrofluoric acid.
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US20090305276A1 (en) 2008-06-04 2009-12-10 Mckim James M Method for Predicting Skin Sensitizing Activity of Compounds
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US20090305276A1 (en) 2008-06-04 2009-12-10 Mckim James M Method for Predicting Skin Sensitizing Activity of Compounds
WO2010031799A1 (en) 2008-09-16 2010-03-25 Ait Austrian Institute Of Technology Gmbh Method for identifying irritating and allergenic substances

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