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์‚ฌ๋žŒ ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์—์„œ ๊ณ ๋†๋„ ํฌ๋„๋‹น ์ฒ˜์น˜ ํ›„ ํ™œ์„ฑ์‚ฐ์†Œ๋ฅผ ํ†ตํ•œ MUC5B ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„

Abstract

Background and Objectives

The effects of hyperglycemia on the mucin secretion in inflammatory respiratory diseases are not clear. Therefore, this study was conducted to characterize the effect of hyperglycemia, and the mechanism involved, on MUC5AC and MUC5B expression in human airway epithelial cells.

Materials and Method

The NCI-H292 cells and the primary cultures of human nasal epithelial cells were exposed to different concentration of glucose (5, 10, 15, 20, 30 mM) for 8 or 24 hours, the effects of high concentration of glucose (20 mM) on MUC5AC and MUC5B expression were determined using reverse transcriptase-polymerase chain reaction (PCR), real-time PCR and enzyme immunoassay. Measurement of reactive oxygen species (ROS) production was performed by flow cytometry. To investigate the role of ROS in high concentration of glucose-induced MUC5B expression, the cells were pretreated with N-acetyl-cysteine (NAC, 50 mM) as a ROS scavenger, or diphenyleneiodonium (DPI, 100 nM) as a nicotinamide adenine dinucleotide phosphate oxidase inhibitor for 1 hour.

Results

In the NCI-H292 cells and the primary cultures of human nasal epithelial cells, High concentration of glucose increased MUC5B expression but did not increase MUC5AC expression (p๏ผœ0.05). ROS production was also increased by high concentration of glucose (20 mM) (p๏ผœ0.05). In addition, high concentration of glucose (20 mM)-induced MUC5B expression and ROS production were significantly attenuated by pretreatment of NAC (50 mM) or DPI (100 nM) (p๏ผœ0.05).

Conclusion

High concentration of glucose induces MUC5B expressions via ROS in human airway epithelial cells.

์„œ ๋ก 

์ •์ƒ ์‚ฌ๋žŒ์˜ ๊ธฐ๋„๋Š” ์™ธ๋ถ€ ํ™˜๊ฒฝ์œผ๋กœ๋ถ€ํ„ฐ ์œ ์ž…๋œ ํก๊ธฐ๋กœ๋ถ€ํ„ฐ ๊ธฐ๋„์ ๋ง‰์„ ๋ณดํ˜ธํ•˜๊ธฐ ์œ„ํ•˜์—ฌ ์ ์•ก ์„ฌ๋ชจ ์šด๋ฐ˜๊ณ„(mucociliary transport system)๊ฐ€ ๋ฐœ๋‹ฌ๋˜์–ด ์žˆ๋‹ค. ๊ธฐ๋„์˜ ์ ์•ก ์„ฌ๋ชจ ์šด๋ฐ˜๊ณ„์—์„œ ์ ์•ก์€ ์ˆ˜๋ถ„๊ณผ ์ด์˜จ, ์ ์•ก๋‹จ๋ฐฑ(mucin)์œผ๋กœ ๊ตฌ์„ฑ๋˜๋Š”๋ฐ, ๊ธ€๋ฆฌ์ฝ”์‹คํ™”๋œ ๊ณ ๋ถ„์ž ๋‹น๋‹จ๋ฐฑ์ธ ์ ์•ก๋‹จ๋ฐฑ์— ์˜ํ•ด ์ ์•ก์˜ ์„ฑ์ƒ์ด ๊ฒฐ์ •๋œ๋‹ค. ์ ์•ก๋‹จ๋ฐฑ์€ ์ ์•ก์œ ์ „์ž์— ์˜ํ•ด ์ƒ์„ฑ์ด ์กฐ์ ˆ๋˜๋ฉฐ, ํ˜„์žฌ๊นŒ์ง€ 20์—ฌ ๊ฐœ๊ฐ€ ๋ฐœ๊ฒฌ๋˜์—ˆ๋‹ค. ํŠนํžˆ ํ˜ธํก๊ธฐ์—์„œ ๋ฐœํ˜„๋˜๋Š” ์ค‘์š”ํ•œ ์ ์•ก์œ ์ „์ž๋Š” MUC2์™€ MUC4, MUC5AC, MUC5B, MUC8๋กœ ์•Œ๋ ค์ ธ ์žˆ๋‹ค[1,2].
MUC5AC ์ ์•ก๋‹จ๋ฐฑ์€ ๋ถ„๋น„์„ ๊ด€๊ณผ ์ ๋ง‰ ์ƒํ”ผ ํ‘œ๋ฉด์˜ ๋ฐฐ์„ธํฌ(goblet cell)์—์„œ ๋ถ„๋น„๋˜๋ฉฐ, MUC5B ์ ์•ก๋‹จ๋ฐฑ์€ ์ ๋ง‰ํ•˜ ๋ถ„๋น„์„ ๊ณผ ๋ณด๋‹ค ์ž‘์€ ๋ฒ”์œ„์˜ ์ ๋ง‰ ์ƒํ”ผ ํ‘œ๋ฉด ๋ฐฐ์„ธํฌ์—์„œ ๋ถ„๋น„๋˜์–ด ์ ค(gel) ํ˜•ํƒœ์˜ ๊ธฐ๋„ ์ ์•ก ํ˜•์„ฑ์— ์žˆ์–ด ์ฃผ์š” ์ž‘์šฉ์„ ํ•œ๋‹ค๊ณ  ์•Œ๋ ค์ ธ ์žˆ์–ด์„œ[2-4], MUC5AC์™€ MUC5B ์ ์•ก์œ ์ „์ž์˜ ๋ฐœํ˜„ ๊ธฐ์ „๊ณผ ๊ทธ ์กฐ์ ˆ ๋ฐฉ๋ฒ•์— ๋Œ€ํ•˜์—ฌ ๊ธฐ์ดˆ์ ์ธ ์—ฐ๊ตฌ๊ฐ€ ํ™œ๋ฐœํ•˜๊ฒŒ ์ด๋ฃจ์–ด์ง€๊ณ  ์žˆ๋‹ค[5-8]. ์ž„์ƒ์ ์œผ๋กœ๋„ ๋งŒ์„ฑ ๊ธฐ๊ด€์ง€์—ผ์ด๋‚˜ ์ฒœ์‹๊ณผ ๊ฐ™์€ ๋งŒ์„ฑ ํ˜ธํก๊ธฐ ์—ผ์ฆ์„ฑ ์งˆํ™˜์—์„œ ์ ๋ง‰ํ•˜ ๋ถ„๋น„์„ ์˜ ๋น„๋Œ€๋‚˜ ๋ฐฐ์„ธํฌ์˜ ์ˆ˜ ๋˜๋Š” ํฌ๊ธฐ์˜ ์ฆ๊ฐ€๊ฐ€ ์ผ์–ด๋‚˜, MUC5AC์™€ MUC5B ์ ์•ก๋‹จ๋ฐฑ์˜ ๊ณผ๋ถ„๋น„๊ฐ€ ๊ด€์ฐฐ๋œ๋‹ค[9]. ์ด๋Ÿฌํ•œ MUC5AC์™€ MUC5B ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์˜ ๋ณ€ํ™”๋กœ ์ ์•ก์˜ ์„ฑ์ƒ์ด ๋ณ€ํ™”ํ•˜๊ฑฐ๋‚˜ ๊ทธ ๋ถ„๋น„๊ฐ€ ๊ณผ๋‹คํ•ด์ ธ ๋ณ‘์˜ ๊ฒฝ๊ณผ๊ฐ€ ์•…ํ™”๋  ์ˆ˜ ์žˆ์–ด, ํ˜ธํก๊ธฐ ์—ผ์ฆ์„ฑ ์งˆํ™˜์„ ๊ฐ€์ง„ ํ™˜์ž์˜ ์น˜๋ฃŒ์— ์žˆ์–ด MUC5AC์™€ MUC5B ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์„ ์กฐ์ ˆํ•˜๋Š” ๊ฒƒ์ด ๋งค์šฐ ์ค‘์š”ํ•˜๋‹ค.
๊ณ ํ˜ˆ๋‹น์ฆ์€ ๋‹น๋‡จ๋ณ‘์˜ ํ•ฉ๋ณ‘์ฆ ๋ฐœ์ƒ์— ์žˆ์–ด ์ฃผ๋œ ์ธ์ž์ด๋ฉฐ ๋ฏธํ† ์ฝ˜๋“œ๋ฆฌ์•„์—์„œ ํ™œ์„ฑ์‚ฐ์†Œ(reactive oxygen species)์˜ ํ˜•์„ฑ์„ ์œ ๋ฐœ์‹œ์ผœ์„œ ์‚ฐํ™” ์ŠคํŠธ๋ ˆ์Šค๋ฅผ ํฌํ•จํ•œ ๋‹ค์–‘ํ•œ ๊ธฐ์ „์„ ํ†ตํ•ด ์ฒด๋‚ด ์—ผ์ฆ๋ฐ˜์‘์„ ์ผ์œผํ‚ค๋ฉฐ, ๊ณ ํ˜ˆ๋‹น์ฆ์— ์˜ํ•ด ์œ ๋„๋œ ์—ผ์ฆ๋ฐ˜์‘์€ ์—ฌ๋Ÿฌ ์กฐ์ง๊ณผ ์žฅ๊ธฐ์— ์˜ํ–ฅ์„ ๋ฏธ์ณ ํ˜ˆ๊ด€์งˆํ™˜, ์‹ ์žฅ์งˆํ™˜, ์‹ ๊ฒฝ์งˆํ™˜, ์‹ฌ๊ทผ์งˆํ™˜ ๋“ฑ ๋‹ค์–‘ํ•œ ์งˆํ™˜์„ ์ผ์œผํ‚ค๋Š” ๊ฒƒ์œผ๋กœ ์•Œ๋ ค์ ธ ์žˆ๋‹ค[10-15]. ๋˜ํ•œ ๋‹น๋‡จ๋ณ‘ ํ™˜์ž์—์„œ๋„ ํ˜ธํก๊ธฐ ์—ผ์ฆ์„ฑ ์งˆํ™˜์˜ ์œ ๋ณ‘๊ธฐ๊ฐ„์ด ๊ธธ์–ด์ง€๊ณ , ๋นˆ๋ฒˆํ•˜๊ฒŒ ์žฌ๋ฐœํ•จ์ด ๋ณด๊ณ ๋˜๊ณ  ์žˆ๋‹ค[16,17]. ํ•˜์ง€๋งŒ ๊ณ ํ˜ˆ๋‹น์ฆ์ด ํ˜ธํก๊ธฐ ์—ผ์ฆ์„ฑ ์งˆํ™˜์— ๋ฏธ์น˜๋Š” ์˜ํ–ฅ ์ค‘ ์ ์•ก๋ถ„๋น„์— ๋Œ€ํ•ด์„œ๋Š” ์•„์ง๊นŒ์ง€ ๋ช…ํ™•ํ•˜๊ฒŒ ์•Œ๋ ค์ ธ ์žˆ์ง€ ์•Š์€ ์‹ค์ •์ด๋‹ค.
์ด์— ํ˜ธํก๊ธฐ ์—ผ์ฆ์„ฑ ์งˆํ™˜์—์„œ ์ค‘์š”ํ•œ ์ ์•ก์ธ MUC5AC์™€ MUC5B ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„๊ณผ ์ ์•ก๋‹จ๋ฐฑ ์ƒ์„ฑ์— ๊ณ ๋†๋„ ํฌ๋„๋‹น์ด ์–ด๋– ํ•œ ์˜ํ–ฅ์„ ๋ฏธ์น˜๋Š”์ง€ ์•Œ์•„๋ณด๊ณ ์ž ์ด ์—ฐ๊ตฌ๋ฅผ ์‹œํ–‰ํ•˜์˜€๋‹ค.

์žฌ๋ฃŒ ๋ฐ ๋ฐฉ๋ฒ•

์„ธํฌ ๋ฐฐ์–‘ ๋ฐ ์ฒ˜์น˜

์‚ฌ๋žŒ ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์˜ ์ ์•ก์ƒํ”ผ์–‘ ์•” ์„ธํฌ์ฃผ(human pulmonary mucoepidermoid carcinoma cell line)์ธ NCI-H292 ์„ธํฌ(American Type Culture Collection, Manassas, VA, USA)๋ฅผ 6-well plate์— 1ร—106 cells/well์˜ ๋†๋„๋กœ ์ ‘์ข…ํ•œ ํ›„ 2 mM L-glutamine, 100 U/mL penicillin, 100 ฮผg/mL streptomycin๊ณผ 10% Fetal bovine serum(FBS; Hyclone Laboratories, Logan, UT, USA)์ด ํฌํ•จ๋œ RPMI 1640 ๋ฐฐ์ง€(Invitrogen, Carlsbad, CA, USA)๋ฅผ ์ด์šฉํ•˜์—ฌ 95%์˜ ์‚ฐ์†Œ์™€ 5%์˜ ์ด์‚ฐํ™”ํƒ„์†Œ๊ฐ€ ํ˜ผํ•ฉ๋œ ์™„์ „ ์Šต์œค ํ™˜๊ธฐ๋œ ๋ฐฐ์–‘๊ธฐ์—์„œ 37โ„ƒ์˜ ์˜จ๋„๋กœ ๋ฐฐ์–‘ํ•˜์˜€๋‹ค. 70~80% ์ •๋„ ๋ฐฐ์–‘์ด ์ด๋ฃจ์–ด์ง€๋ฉด ์„ธํฌ๋ฅผ 0.5% fetal calf serum์ด ํฌํ•จ๋œ RPMI 1640 ๋ฐฐ์ง€๋กœ ๊ต์ฒดํ•œ ํ›„ 24์‹œ๊ฐ„ ๋™์•ˆ ๋ฐฐ์–‘ํ•˜๊ณ , ๋‹ค์‹œ FBS๊ฐ€ ํฌํ•จ๋˜์ง€ ์•Š์€ RPMI 1640 ๋ฐฐ์ง€๋กœ ์„ธ์ฒ™ํ•˜์—ฌ ์‚ฌ์šฉํ•˜์˜€๋‹ค.
๊ณ ๋†๋„ ํฌ๋„๋‹น์— ์˜ํ•œ MUC5AC์™€ MUC5B ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„๊ณผ MUC5B ๋‹จ๋ฐฑ ์ƒ์„ฑ์„ ์•Œ์•„๋ณด๊ณ ์ž ํฌ๋„๋‹น์„ ์ด์šฉํ•˜์˜€๋‹ค. ๋‹ค์–‘ํ•œ ๋†๋„(5, 10, 15, 20, 30 mM)๋กœ ์„ธํฌ ๋ฐฐ์–‘ ๋ฐฐ์ง€์— ํˆฌ์—ฌํ•˜์˜€์œผ๋ฉฐ, ๋™์ผ ๋†๋„(20 mM)์—์„œ๋Š” ๋…ธ์ถœ ์‹œ๊ฐ„(0, 2, 4, 8, 24, 48์‹œ๊ฐ„)์„ ๋‹ค๋ฅด๊ฒŒ ํ•˜์—ฌ ๊ด€์ฐฐํ•˜์˜€๋‹ค. ๋˜ํ•œ ๊ฐ ํฌ๋„๋‹น ๋†๋„์—์„œ MUC5B ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์— ๊ด€๊ณ„ํ•˜๋Š” ํ™œ์„ฑ์‚ฐ์†Œ ์ƒ์„ฑ์„ ์•Œ์•„๋ณด๊ธฐ ์œ„ํ•˜์—ฌ ํ™œ์„ฑ์‚ฐ์†Œ ์–ต์ œ์ œ์ธ N-acetyl-cysteine(NAC; Sigma-Aldrich, St. Louis, MO, USA)๊ณผ nicotinamide adenine dinucleotide phosphate(NADPH) ์‚ฐํ™”ํšจ์†Œ ์–ต์ œ์ œ์ธ diphenyleneiodonium(DPI; EMD Biosciences, Inc., San Diego, CA, USA)์„ ๊ณ ๋†๋„ ํฌ๋„๋‹น์— ๋…ธ์ถœ๋˜๊ธฐ 1์‹œ๊ฐ„ ์ „์— ์ „์ฒ˜์น˜ํ•˜์˜€๋‹ค. ์‚ฌ๋žŒ์˜ ํ˜ˆ์•ก ๋‚ด ์ƒ๋ฆฌํ•™์  ํฌ๋„๋‹น ๋†๋„์ธ 5 mM ๋†๋„์—์„œ ๋Œ€์กฐ๊ตฐ ์„ธํฌ๋ฅผ ๋…ธ์ถœ์‹œ์ผœ ์‚ฌ์šฉํ•˜์˜€๋‹ค[18].
์‚ฌ๋žŒ ์ฝ”์ ๋ง‰ ์ƒํ”ผ์„ธํฌ๋ฅผ ์–ป๊ธฐ ์œ„ํ•ด์„œ ๊ธฐ์ € ๋‹น๋‡จ๋ณ‘์ด๋‚˜ ์•Œ๋ ˆ๋ฅด๊ธฐ ์งˆํ™˜๊ณผ ๊ทธ ๊ฐ€์กฑ๋ ฅ์ด ์—†๊ณ , ํ”ผ๋ถ€๋‹จ์ž์‹œํ—˜(skin prick test)๊ณผ ๋‹ค์ค‘ ์•Œ๋ ˆ๋ฅด๊ธฐํ•ญ์›๊ฒ€์‚ฌ(multiple allergen simultaneous test)์—์„œ ์Œ์„ฑ๋ฐ˜์‘์ด ๋‚˜์˜จ 10๋ช…์„ ๋Œ€์ƒ์œผ๋กœ ํ•˜์—ฌ ์œต๋น„์ˆ  ์‹œํ–‰์ค‘์— ๋ฐœ์ƒํ•˜๋Š” ๋น„๊ฐ• ๋‚ด ์ ์ถœ๋ฌผ ์ค‘ ์ •์ƒ ํ•˜๋น„๊ฐ‘๊ฐœ ์กฐ์ง์„ ์–ป์—ˆ๋‹ค. ์ผ์ฐจ๋ฐฐ์–‘์„ ํ•˜๊ธฐ ์œ„ํ•ด ํ•˜๋น„๊ฐ‘๊ฐœ ์ ๋ง‰์กฐ์ง์„ phosphate-buffered saline(PBS)์œผ๋กœ ์„ธ์ฒ™ํ•œ ํ›„, 90๋ถ„ ๋™์•ˆ dispase(Boehringer Mannheim Biochemica, Mannheim, Germany)์— ์นจ์ „์‹œ์ผฐ๋‹ค. ์™ธ๊ณผ์šฉ ์ˆ˜์ˆ ์นผ์„ ์‚ฌ์šฉํ•˜์—ฌ ํ•˜๋น„๊ฐ‘๊ฐœ ์ ๋ง‰์˜ ํ‘œ๋ฉด์„ ๋ฒ—๊ฒจ๋‚ด์–ด, 1% PBS๋ฅผ ์ถ”๊ฐ€ํ•œ ํ›„ mesh๋ฅผ ํ†ตํ•ด ์—ฌ๊ณผํ•˜์˜€๋‹ค. ์ด๋Ÿฌํ•œ ๊ณผ์ •์„ ํ†ตํ•ด ์–ป์€ ํ•˜๋น„๊ฐ‘๊ฐœ ์ ๋ง‰์˜ ์ƒํ”ผ์„ธํฌ๋“ค์„ 24-well(2.5ร—105 cells/well) plate์— ์ ‘์ข…ํ•œ ํ›„, EpiLife medium(Cascade Biologics, Portland, OR, USA)๊ณผ keratinocyte growth supplement(5/500 mL of medium, Cascade Biologics, Portland, OR, USA)์—์„œ ๋ฐฐ์–‘ํ•˜์˜€๋‹ค. ์‹คํ—˜์—์„œ ์‚ฌ์šฉํ•œ NCI-H292 ์„ธํฌ์™€ ์ผ์ฐจ ๋ฐฐ์–‘ํ•œ ์‚ฌ๋žŒ ์ฝ”์ ๋ง‰ ์ƒํ”ผ์„ธํฌ์— ๋Œ€ํ•œ ํฌ๋„๋‹น์˜ ์„ธํฌ๋…์„ฑ ์œ ๋ฌด๋Š” 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) ๋ถ„์„(Sigma-Aldrich, St. Louis, MO, USA)๊ณผ ํ˜„๋ฏธ๊ฒฝ์„ ์ด์šฉํ•œ ์„ธํฌ ํ˜•ํƒœ ๋ณ€ํ™” ์œ ๋ฌด๋ฅผ ํ™•์ธํ•˜์—ฌ ๊ฒ€์ฆํ•˜์˜€๋‹ค.
๋ณธ ์—ฐ๊ตฌ๋Š” ๋ณธ์› ์ž„์ƒ ์‹œํ—˜ ์‹ฌ์‚ฌ ์œ„์›ํšŒ(Institutional Review Board: YUH-13-0375-O33)์˜ ์Šน์ธ์„ ๋ฐ›์•„ ์‹œํ–‰ํ•˜์˜€๋‹ค.

Reverse transcription-polymerase chain reaction(RT-PCR) ๋ถ„์„

๋ชจ๋“  RNA๋Š” reverse transcription-polymerase chain reaction(PCR) kit(Roche Applied Science, Mannheim, Germany)๋ฅผ ์ด์šฉํ•˜์—ฌ ๋™์ •๋˜์—ˆ๊ณ , GeneAmp RNA PCR cored kit(Roche Applied Science)๋ฅผ ์ด์šฉํ•˜์—ฌ cDNA๋กœ ์—ญ์ „์‚ฌ๋˜์—ˆ๋‹ค. ์‹คํ—˜์— ์‚ฌ์šฉ๋œ primer์˜ ์—ผ๊ธฐ์„œ์—ด์€ MUC5AC ์ ์•ก์œ ์ „์ž์˜ ๊ฒฝ์šฐ sense๋Š” 5โ€™-TCA ACG GAG ACT GCG AGT ACA C-3โ€™, antisense๋Š” 5โ€™-CTT GAT GGC CTT GGA GCA-3โ€™์ด๊ณ , MUC5B ์ ์•ก์œ ์ „์ž์˜ ๊ฒฝ์šฐ sense๋Š” 5โ€™-CAC ATC CAC CCT TCC AAC-3โ€™, antisense๋Š” 5โ€™-GGC TCA TTG TCG TCT CTG-3โ€™์ด๋ฉฐ, ๊ฐ ๋ฐ˜์‘์˜ ๋‚ด๋ถ€ ์–‘์„ฑ ๋Œ€์กฐ๊ตฐ(internal positive control)์€ glyceraldehyde-3-phosphate dehydrogenase(GAPDH)๋ฅผ ์‚ฌ์šฉํ•˜์˜€์œผ๋ฉฐ, ์—ผ๊ธฐ์„œ์—ด์€ sense๋Š” 5โ€™-CCT CCA AGG AGT AAG ACC CC-3โ€™, antisense๋Š” 5โ€™-AGG GGT CTA CAT GGC AAC TG-3์ด๋‹ค.
๊ฐ๊ฐ์˜ ์ค‘ํ•ฉํšจ์†Œ์—ฐ์‡„๋ฐ˜์‘ ๊ณผ์ •์„ ๊ฐ„๋žตํžˆ ์„ค๋ช…ํ•˜๋ฉด, ๋ฐฐ์–‘๋œ ์„ธํฌ๋ฅผ 2% bovine serum albumin์„ ํ•จ์œ ํ•œ PBS๋กœ 3ํšŒ ์„ธ์ฒ™ํ•œ ํ›„ Trizolโ“‡(Molecular Research Center, Cincinnati, OH, USA)์„ ์ด์šฉํ•˜์—ฌ ์ด mRNA๋ฅผ ์ถ”์ถœํ•˜์˜€๋‹ค. MUC5AC mRNA์— ๋Œ€ํ•œ PCR์€ 95โ„ƒ์—์„œ 60์ดˆ๊ฐ„ ๋ณ€์„ฑ๊ณผ์ •๊ณผ 60โ„ƒ์—์„œ 60์ดˆ๊ฐ„ ๊ฒฐํ•ฉ๋ฐ˜์‘, 72โ„ƒ์—์„œ 60์ดˆ๊ฐ„ ์—ฐ์žฅ๋ฐ˜์‘์„ 33ํšŒ ๋ฐ˜๋ณตํ•œ ํ›„ 72โ„ƒ์—์„œ 20๋ถ„๊ฐ„ ์ตœ์ข… ์—ฐ์žฅ๋ฐ˜์‘์„ ์‹œํ–‰ํ•˜์˜€๊ณ , MUC5B mRNA์— ๋Œ€ํ•œ PCR์€ 95โ„ƒ์—์„œ 60์ดˆ๊ฐ„ ๋ณ€์„ฑ๊ณผ์ •๊ณผ 72โ„ƒ์—์„œ 60์ดˆ๊ฐ„ ๊ฒฐํ•ฉ๋ฐ˜์‘, 72โ„ƒ์—์„œ 60์ดˆ๊ฐ„ ์—ฐ์žฅ๋ฐ˜์‘์„ 33ํšŒ ๋ฐ˜๋ณตํ•œ ํ›„ 72โ„ƒ์—์„œ 20๋ถ„๊ฐ„ ์ตœ์ข… ์—ฐ์žฅ๋ฐ˜์‘์„ ์‹œํ–‰ํ•˜์˜€๋‹ค. ์ฆํญ๋œ ์ค‘ํ•ฉํšจ์†Œ์—ฐ์‡„๋ฐ˜์‘์˜ ์‚ฐ๋ฌผ์€ SYBR green์ด ํ•จ์œ ๋œ 1% agarose gel์„ ํ†ตํ•œ ์ „๊ธฐ์˜๋™์„ ์ด์šฉํ•˜์—ฌ ๋ถ„๋ฆฌ ๊ด€์ฐฐํ•˜์˜€๋‹ค. ํ™•์ธ๋œ ๋ (band)์˜ ์„ธ๊ธฐ๋Š” Scion Image software(Scion Corporation, Frederick, MD, USA)๋ฅผ ์ด์šฉํ•˜์—ฌ ๋ฐ˜์ •๋Ÿ‰์ ์œผ๋กœ ๋ถ„์„ํ•˜์˜€๋‹ค. MUC5AC ์ ์•ก์œ ์ „์ž์™€ MUC5B ์ ์•ก์œ ์ „์ž ๊ฐ๊ฐ์˜ ๋ ์˜ ์„ธ๊ธฐ๋Š” GAPDH ๋Œ€์กฐ๊ตฐ์˜ ์„ธ๊ธฐ์™€ ๋น„๊ตํ•˜์—ฌ ์ƒ๋Œ€์ ์ธ ๊ฐ’์œผ๋กœ ๋‚˜ํƒ€๋‚ด์—ˆ๋‹ค.

Real-time polymerase chain reaction(real-time PCR) ๋ถ„์„

ํ•ฉ์„ฑ๋œ cDNA 0.5 ฮผL๋ฅผ ๋Œ€์ƒ์œผ๋กœ LC Fast Start DNA Master SYBR Green kit(Roche Applied Science)๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ real-time PCR์„ ์ˆ˜ํ–‰ํ•˜์˜€๋‹ค. Real-time PCR์€ ์ตœ์ข…๋Ÿ‰์ด 10 ฮผL๊ฐ€ ๋˜๊ฒŒ, 2.5 mM์˜ MgCl2์™€ ๊ทธ ์ตœ์ข… ๋†๋„๊ฐ€ 0.5 ฮผM์ด ๋˜๊ฒŒ primer๋ฅผ ํˆฌ์—ฌํ•˜์˜€์œผ๋ฉฐ, 25 ng์˜ RNA 1 ฮผL๋ฅผ ์ด์šฉํ•˜์—ฌ ์‹คํ—˜์„ ์ˆ˜ํ–‰ํ•˜์˜€๋‹ค. ์ •๋Ÿ‰์ ์ธ PCR์€ Light-Cycler(Roche Applied Science)๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ 95โ„ƒ์—์„œ 10์ดˆ๊ฐ„ ๋ณ€์„ฑ(denaturation)๊ณผ์ •์„ ๊ฑฐ์น˜๊ณ  60โ„ƒ์—์„œ 5์ดˆ๊ฐ„ ๊ฒฐํ•ฉ(annealing) ๋ฐ˜์‘์„ ์‹œํ‚จ ํ›„, 72โ„ƒ์—์„œ 10์ดˆ๊ฐ„ ์—ฐ์žฅ(extension) ๋ฐ˜์‘ํ•˜์˜€๊ณ , ์ด๋Ÿฌํ•œ ๊ณผ์ •์„ 45ํšŒ ๋ฐ˜๋ณตํ•˜์˜€๋‹ค. ๊ฐ ์‹คํ—˜์—์„œ ์ฆํญ๋œ cDNA์˜ ์œ ๋ฌด๋ฅผ ํ™•์ธํ•˜๊ธฐ ์œ„ํ•˜์—ฌ GAPDH๋ฅผ ๋Œ€์กฐ ์œ ์ „์ž(internal control)๋กœ ์‚ฌ์šฉํ•˜์˜€๋‹ค.

Enzyme-linked immunosorbent assay(ELISA) ๋ถ„์„

MUC5B ์ ์•ก๋‹จ๋ฐฑ์˜ ํ•จ๋Ÿ‰์„ ์ธก์ •ํ•˜๊ธฐ ์œ„ํ•ด์„œ enzyme-linked immunosorbent assay(ELISA)๋ฒ•์„ ์ด์šฉํ•˜์˜€๋‹ค. ์‹œ๋ฃŒ๋ฅผ ์ฒ˜๋ฆฌํ•œ ๋ฐฐ์–‘๋œ ์„ธํฌ์—์„œ lysis buffer[50 mM trisยทCl(pH 7.5), 1 mM ethylene glycol tetraacetic acid, 1% triton X-100, and 1 mM phenylmethylsulfonyl fluoride]๋กœ ๋‹จ๋ฐฑ์„ ์ถ”์ถœํ•˜์—ฌ ์ •๋Ÿ‰ํ•˜์˜€๋‹ค. ์ถ”์ถœํ•œ ๋‹จ๋ฐฑ 100 ฮผg์„ 96-well plate์— ๋‹ด๊ณ  40โ„ƒ์—์„œ ๊ฑด์กฐ๋  ๋•Œ๊นŒ์ง€ ๋ฐฉ์น˜ํ•œ ํ›„ plate๋ฅผ PBS๋กœ 3ํšŒ ์„ธ์ฒ™ํ•˜์˜€๋‹ค. ๋น„ํŠน์ด์  ๊ฒฐํ•ฉ์„ ๋ฐฉ์ง€ํ•˜๊ธฐ ์œ„ํ•ด 2% bovine serum albumin์œผ๋กœ ์‹ค์˜จ์—์„œ 1์‹œ๊ฐ„ ๋™์•ˆ ์ฐจ๋‹จํ•œ ํ›„ PBS๋กœ 3ํšŒ ์„ธ์ฒ™ํ•œ ๋‹ค์Œ 0.05% Tween 20์„ ํ•จ์œ ํ•œ PBS์— 1:200์œผ๋กœ ํฌ์„๋œ MUC5B ์ ์•ก๋‹จ๋ฐฑ ์ผ์ฐจํ•ญ์ฒด(Santa Cruz Biotechnology, Santa Cruz, CA, USA)๋กœ ๋ฐ˜์‘์‹œ์ผฐ๋‹ค. ๋‹ค์‹œ PBS๋กœ 3ํšŒ ์„ธ์ฒ™ํ•œ ํ›„ HRP-conjugated ์ด์ฐจํ•ญ์ฒด(Santa Cruz Biotechnology)๋ฅผ 0.05% Tween 20์„ ํ•จ์œ ํ•œ PBS์— 1:5000์œผ๋กœ ํฌ์„ํ•˜์—ฌ ๊ฐ well์— ์ฒจ๊ฐ€ํ•˜์˜€๊ณ , 1์‹œ๊ฐ„ ํ›„์— ๊ฐ well์„ PBS๋กœ 3ํšŒ ์„ธ์ฒ™ํ•˜์˜€๋‹ค. 3,3,5,5โ€™-tetramethyl benzidine ์šฉ์•ก์œผ๋กœ ๋ฐœ์ƒ‰ํ•œ ํ›„, 2N-H2SO4๋ฅผ ์ด์šฉํ•˜์—ฌ ์ค‘๋‹จ์‹œ์ผฐ๋‹ค. ELISA reader(EL800โ“‡; BIO-TEK Instruments, Winooski, VT, USA)๋กœ 450 nm์—์„œ ํก๊ด‘๋„๋ฅผ ์ธก์ •ํ•œ ํ›„ ํ‘œ์ค€๊ณก์„ ์„ ์ด์šฉํ•˜์—ฌ ๋‹จ๋ฐฑ์˜ ์–‘์„ ์ •๋Ÿ‰ํ•˜์˜€๋‹ค.

Flow cytometry ๋ถ„์„

์ƒ์„ฑ๋œ ํ™œ์„ฑ์‚ฐ์†Œ์˜ ํ•จ๋Ÿ‰์„ ์ธก์ •ํ•˜๊ธฐ ์œ„ํ•ด์„œ flow cytometry๋ฒ•์„ ์ด์šฉํ•˜์˜€๋‹ค. ๋Œ€์ƒ ์„ธํฌ์— 50 ฮผM์˜ 2,7-dichlorodihydrofluorescein diacetate์™€ aredox-sensitive fluorescent dye๋ฅผ 40๋ถ„ ๋™์•ˆ ์ฒ˜๋ฆฌํ•œ ํ›„ PBS๋กœ 3ํšŒ ์„ธ์ฒ™ํ•˜์˜€์œผ๋ฉฐ, Flow cytometry๋ฅผ ์‹œ์ž‘ํ•˜๊ธฐ ์ „๊นŒ์ง€ ์–ผ์Œ์— ๋‹ด๊ธด 1 mL PBS suspension์—์„œ ์„ธํฌ๋ฅผ ์œ ๋ฆฌํ•˜์˜€๋‹ค. FACSCanto II(BD Bioscience, Rutherford, NJ, USA)์—์„œ 488 nm excitation beam์„ ์ด์šฉํ•˜์—ฌ flow cytometry๋ฅผ ์‹œํ–‰ํ•˜์˜€๋‹ค. ์‹ ํ˜ธ๋Š” 530 nm ํˆฌ๊ณผ ๋ฐด๋“œ๋ฅผ ์ด์šฉํ•˜์—ฌ ์ถ”์ถœํ•˜์˜€์œผ๋ฉฐ, 5000๊ฐœ ์„ธํฌ์—์„œ์˜ ํ‰๊ท  ํ˜•๊ด‘๊ฐ•๋„๋ฅผ ๊ธฐ์ค€์œผ๋กœ ์‚ผ์•˜๋‹ค.

ํ†ต ๊ณ„

ํ†ต๊ณ„ ์ฒ˜๋ฆฌ๋Š” Windows์šฉ SPSS version 10.0(SPSS Inc., Chicago, IL, USA)์„ ์‚ฌ์šฉํ•˜์˜€๋‹ค. ๋ชจ๋“  ์‹คํ—˜์€ p๊ฐ’์ด 0.05๋ฏธ๋งŒ์ธ ๊ฒฝ์šฐ๋ฅผ ์œ ์˜ํ•œ ๊ฒƒ์œผ๋กœ ์ •ํ•˜์—ฌ Student t-test์™€ analysis of variance test, Mann-Whitney U test๋ฅผ ์ด์šฉํ•˜์˜€๋‹ค.

๊ฒฐ ๊ณผ

NCI-H292 ์„ธํฌ์—์„œ ๊ณ ๋†๋„ ํฌ๋„๋‹น์ด MUC5AC์™€ MUC5B ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์— ๋ฏธ์น˜๋Š” ์˜ํ–ฅ

๋‹ค์–‘ํ•œ ๋†๋„์˜ ํฌ๋„๋‹น(5, 10, 15, 20, 30 mM)์— 8์‹œ๊ฐ„ ๋…ธ์ถœ์‹œํ‚จ ํ›„ ๋ถ„์„ํ•œ MUC5AC mRNA์™€ MUC5B mRNA์˜ ๋ฐœํ˜„ ์ •๋„๋ฅผ ํ™•์ธํ•ด ๋ณธ ๊ฒฐ๊ณผ, MUC5AC mRNA๋Š” ํฌ๋„๋‹น์˜ ๋†๋„ ๋ณ€ํ™”์— ๋”ฐ๋ผ ๋ฐœํ˜„ ์ •๋„์˜ ์ฐจ์ด๋ฅผ ๋ณด์ด์ง€ ์•Š์€ ๋ฐ˜๋ฉด, MUC5B mRNA์˜ ๊ฒฝ์šฐ ํฌ๋„๋‹น ๋†๋„๊ฐ€ 15 mM ์ด์ƒ์—์„œ ๋ฐœํ˜„์–‘์˜ ์œ ์˜ํ•œ ์ฆ๊ฐ€๋ฅผ ํ™•์ธํ•  ์ˆ˜ ์žˆ์—ˆ๋‹ค(Fig. 1A). ํฌ๋„๋‹น ๋†๋„์˜ ์ฆ๊ฐ€์™€ ๋…ธ์ถœ ์‹œ๊ฐ„์— ๋”ฐ๋ฅธ MUC5B mRNA ๋ฐœํ˜„ ์ •๋„๋ฅผ ์ •๋Ÿ‰์ ์œผ๋กœ ํ™•์ธํ•˜๊ธฐ ์œ„ํ•ด real-time PCR์„ ์ด์šฉํ•˜์—ฌ ๋ถ„์„ํ•˜์˜€๋‹ค. ํฌ๋„๋‹น ๋†๋„์— ๋”ฐ๋ผ MUC5B mRNA ๋ฐœํ˜„์€ ์˜๋ฏธ ์žˆ๊ฒŒ ์ฆ๊ฐ€๋˜์—ˆ์œผ๋ฉฐ, ํฌ๋„๋‹น 20 mM ๋†๋„์—์„œ ์ตœ๋Œ€์น˜๋ฅผ ์ด๋ฃจ๊ณ , 30 mM ๋†๋„์—์„œ๋Š” ๊ฐ์†Œํ•˜๋Š” ์–‘์ƒ์„ ๋ณด์˜€๋‹ค(Fig. 1B). ๋…ธ์ถœ ์‹œ๊ฐ„์— ๋”ฐ๋ฅธ MUC5B mRNA ๋ฐœํ˜„ ์ •๋„๋ฅผ ๋ณด๋Š” ์‹คํ—˜์—์„œ๋Š” 20 mM ํฌ๋„๋‹น์— ๋Œ€ํ•œ ๋…ธ์ถœ ์‹œ๊ฐ„์ด ์ฆ๊ฐ€ํ•จ์— ๋”ฐ๋ผ MUC5B mRNA ๋ฐœํ˜„์ด ์ฆ๊ฐ€ํ•˜์˜€์œผ๋ฉฐ, 8์‹œ๊ฐ„์งธ MUC5B mRNA ๋ฐœํ˜„์ด ์ตœ๋Œ€์น˜๋ฅผ ์ด๋ฃจ๋Š” ์–‘์ƒ์„ ๋ณด์˜€์œผ๋‚˜, ์‹œ๊ฐ„ ์˜์กด์„ฑ์€ ๊ด€์ฐฐ๋˜์ง€ ์•Š์•˜๋‹ค(Fig. 1C). ํฌ๋„๋‹น ๋†๋„์— ๋”ฐ๋ฅธ MUC5B ๋‹น๋‹จ๋ฐฑ ์ƒ์„ฑ๋ณ€ํ™”๋ฅผ ํ™•์ธํ•˜๊ธฐ ์œ„ํ•ด ๋‹ค์–‘ํ•œ ๋†๋„์˜ ํฌ๋„๋‹น(5, 10, 15, 20, 30 mM)์— 24์‹œ๊ฐ„ ๋…ธ์ถœ์‹œํ‚จ ํ›„ ELISA ๊ฒ€์‚ฌ๋ฅผ ์‹œํ–‰ํ•˜์˜€์œผ๋ฉฐ, MUC5B ๋‹น๋‹จ๋ฐฑ์˜ ์ƒ์„ฑ์ด ๋…ธ์ถœ๋œ ํฌ๋„๋‹น์˜ ๋†๋„ ์ฆ๊ฐ€์— ๋”ฐ๋ผ ๋‹น๋‹จ๋ฐฑ์ƒ์„ฑ์ด ์ฆ๊ฐ€๋จ์„ ํ™•์ธํ•  ์ˆ˜ ์žˆ์—ˆ๋‹ค(Fig. 1D). ์ด๋Ÿฌํ•œ ๊ฒฐ๊ณผ๋“ค์€ ์„ ํ–‰์—ฐ๊ตฌ๋ฅผ ํ†ตํ•ด ํ™•์ธ๋œ ๊ฒฐ๊ณผ๋“ค๊ณผ ์œ ์‚ฌํ•œ ๊ฒฝํ–ฅ์„ฑ์„ ๋ณด์˜€๋‹ค[19].

NCI-H292 ์„ธํฌ์—์„œ ๊ณ ๋†๋„ ํฌ๋„๋‹น์ด ํ™œ์„ฑ์‚ฐ์†Œ ์ƒ์„ฑ์— ๋ฏธ์น˜๋Š” ์˜ํ–ฅ

ํฌ๋„๋‹น 20 mM ๋†๋„์— ๋…ธ์ถœ๋œ NCI-H292 ์„ธํฌ์—์„œ ํฌ๋„๋‹น์— ๋…ธ์ถœ๋œ ์‹œ๊ฐ„์ด ์ฆ๊ฐ€ํ•จ์— ๋”ฐ๋ผ control(ํฌ๋„๋‹น 5 mM)๊ณผ ๋น„๊ตํ•˜์—ฌ ํ™œ์„ฑ์‚ฐ์†Œ ์ƒ์„ฑ์ด ์ฆ๊ฐ€ํ•จ์„ ํ™•์ธํ•  ์ˆ˜ ์žˆ์—ˆ๋‹ค(Fig. 2).

๊ณ ๋†๋„ ํฌ๋„๋‹น์— ๋…ธ์ถœ๋œ NCI-H292 ์„ธํฌ์—์„œ ํ™œ์„ฑ์‚ฐ์†Œ์–ต์ œ์ œ์™€ NADPH ์‚ฐํ™”ํšจ์†Œ ์–ต์ œ์ œ๊ฐ€ ํ™œ์„ฑ์‚ฐ์†Œ ์ƒ์„ฑ๊ณผ MUC5B mRNA ๋ฐœํ˜„์— ๋ฏธ์น˜๋Š” ์˜ํ–ฅ

ํ™œ์„ฑ์‚ฐ์†Œ ์–ต์ œ์ œ์ธ NAC์™€ DPI์˜ ํšจ๊ณผ๋ฅผ ํ™•์ธํ•˜๊ธฐ ์œ„ํ•ด ๊ณ ๋†๋„ ํฌ๋„๋‹น(20 mM)์— ๋…ธ์ถœ์‹œํ‚ค๊ธฐ 1์‹œ๊ฐ„ ์ „์— NAC(50 mM)์™€ DPI(100 nM)๋ฅผ ๊ฐ๊ฐ ์ „์ฒ˜์น˜ํ•˜์˜€๋‹ค[20]. ๊ณ ๋†๋„ ํฌ๋„๋‹น์— ๋…ธ์ถœ๋œ NCI-H292 ์„ธํฌ์—์„œ ํ™œ์„ฑ์‚ฐ์†Œ ์–ต์ œ์ œ์ธ NAC(50 mM)์— ์˜ํ•ด ํ™œ์„ฑ์‚ฐ์†Œ ์ƒ์„ฑ์ด ์˜๋ฏธ ์žˆ๊ฒŒ ๊ฐ์†Œ๋˜์—ˆ๊ณ , NADPH ์‚ฐํ™”ํšจ์†Œ ์–ต์ œ์ œ์ธ DPI(100 nM)์— ์˜ํ•ด์„œ๋„ ํ™œ์„ฑ์‚ฐ์†Œ ์ƒ์„ฑ์ด ์˜๋ฏธ์žˆ๊ฒŒ ๊ฐ์†Œ๋˜์—ˆ๋‹ค(Fig. 3).
๊ณ ๋†๋„ ํฌ๋„๋‹น์— ๋…ธ์ถœ๋œ NCI-H292 ์„ธํฌ์—์„œ ํ™œ์„ฑ์‚ฐ์†Œ ์–ต์ œ์ œ์ธ NAC(50 mM)์— ์˜ํ•ด MUC5B mRNA ๋ฐœํ˜„๊ณผ ๋‹น๋‹จ๋ฐฑ ์ƒ์„ฑ์ด ์˜๋ฏธ ์žˆ๊ฒŒ ๊ฐ์†Œ๋˜์—ˆ๊ณ , NADPH ์‚ฐํ™”ํšจ์†Œ ์–ต์ œ์ œ์ธ DPI(100 nM)์— ์˜ํ•ด์„œ๋„ MUC5B mRNA ๋ฐœํ˜„๊ณผ ๋‹น๋‹จ๋ฐฑ ์ƒ์„ฑ์ด ์˜๋ฏธ ์žˆ๊ฒŒ ๊ฐ์†Œ๋˜์—ˆ๋‹ค(Fig. 4).

์‚ฌ๋žŒ ์ฝ”์ ๋ง‰ ์ƒํ”ผ์„ธํฌ์—์„œ ๊ณ ๋†๋„ ํฌ๋„๋‹น์ด MUC5B mRNA ๋ฐœํ˜„, ๋‹น๋‹จ๋ฐฑ ์ƒ์„ฑ ๋ฐ ํ™œ์„ฑ์‚ฐ์†Œ ์ƒ์„ฑ์— ๋ฏธ์น˜๋Š” ์˜ํ–ฅ

์‚ฌ๋žŒ ์ฝ” ์ ๋ง‰ ์ƒํ”ผ์„ธํฌ์—์„œ๋„ ๋‹ค์–‘ํ•œ ๋†๋„์˜ ํฌ๋„๋‹น(5, 10, 15, 20, 30 mM)์— ๊ฐ๊ฐ 8์‹œ๊ฐ„๊ณผ 24์‹œ๊ฐ„ ๋…ธ์ถœ์‹œํ‚จ ํ›„ ๋ถ„์„ํ•œ MUC5B mRNA ๋ฐœํ˜„ ์ •๋„์™€ ๋‹น๋‹จ๋ฐฑ ์ƒ์„ฑ ์ •๋„๋ฅผ ํ™•์ธํ•ด๋ณธ ๊ฒฐ๊ณผ, ํฌ๋„๋‹น ๋†๋„์˜ ์ฆ๊ฐ€์— ๋”ฐ๋ผ MUC5B mRNA ๋ฐœํ˜„๊ณผ ๋‹น๋‹จ๋ฐฑ ์ƒ์„ฑ์ด ์˜๋ฏธ ์žˆ๊ฒŒ ์ฆ๊ฐ€๋จ์„ ํ™•์ธํ•  ์ˆ˜ ์žˆ์—ˆ๋‹ค(Fig. 5A and B).
๊ณ ๋†๋„ ํฌ๋„๋‹น์— ๋…ธ์ถœ๋œ ์ฝ”์ ๋ง‰ ์ƒํ”ผ์„ธํฌ์—์„œ ํ™œ์„ฑ์‚ฐ์†Œ ์–ต์ œ์ œ์ธ NAC(50 mM)์— ์˜ํ•ด MUC5B mRNA ๋ฐœํ˜„๊ณผ ๋‹น๋‹จ๋ฐฑ ์ƒ์„ฑ์ด ์˜๋ฏธ ์žˆ๊ฒŒ ๊ฐ์†Œ๋˜์—ˆ๊ณ , NADPH ์‚ฐํ™”ํšจ์†Œ ์–ต์ œ์ œ์ธ DPI(100 nM)์— ์˜ํ•ด์„œ๋„ MUC5B mRNA ๋ฐœํ˜„๊ณผ ๋‹น๋‹จ๋ฐฑ ์ƒ์„ฑ์ด ์˜๋ฏธ ์žˆ๊ฒŒ ๊ฐ์†Œ๋˜์—ˆ๋‹ค(Fig. 5C and D).

๊ณ  ์ฐฐ

๊ณ ํ˜ˆ๋‹น์ฆ์€ ํ˜ธํก๊ธฐ ์—ผ์ฆ์„ฑ ์งˆํ™˜์˜ ์ดํ™˜๊ณผ ์•…ํ™”, ์žฆ์€ ์žฌ๋ฐœ์— ์žˆ์–ด ๊ด€๊ณ„ํ•˜์—ฌ ํ์„ฌ์œ ์ฆ๊ณผ ํ๋ถ€์ข…, ํ—ˆํŒŒ๊ฝˆ๋ฆฌ์˜ ํŒŒ๊ดด ๋ฐ ํ™•์žฅ ๋“ฑ์˜ ๊ตฌ์กฐ์ ์ธ ๋ณ€ํ™”๋ฅผ ์ผ์œผ์ผœ ํ๊ธฐ๋Šฅ ์ €ํ•˜๋ฅผ ์œ ๋ฐœํ•˜๊ณ , ํ˜ธํก๊ธฐ ๊ตญ์†Œ ์—ผ์ฆ๋ฐ˜์‘์„ ์œ ๋ฐœํ•˜์—ฌ ๊ฐ์—ผ์„ฑ, ์—ผ์ฆ์„ฑ ํ˜ธํก๊ธฐ ์งˆํ™˜์˜ ์•…ํ™”์— ์˜ํ–ฅ์„ ์ฃผ๋Š” ๊ฒƒ์œผ๋กœ ์•Œ๋ ค์ ธ ์žˆ๋‹ค[21-23]. ๋˜ํ•œ, ํ˜ธํก๊ธฐ๋‚ด ํฌ๋„๋‹น ๋†๋„์˜ ์ฆ๊ฐ€๋Š” ์„ธ๊ท ์ฆ์‹์— ํ•„์š”ํ•œ ์˜์–‘๋ถ„์„ ๊ณต๊ธ‰ํ•จ์œผ๋กœ์„œ ์„ธ๊ท ์„ฑ ํ˜ธํก๊ธฐ ์งˆํ™˜์˜ ๋ฐœ์ƒ์— ์ค‘์š”ํ•œ ์œ„ํ—˜์š”์ธ์œผ๋กœ ์•Œ๋ ค์ ธ ์žˆ๋‹ค[24]. ์ •์ƒ์ธ์˜ ๊ฒฝ์šฐ ํ˜ธํก๊ธฐ ๋‚ด๊ฐ•์— ์œ ์ง€๋˜๋Š” ํฌ๋„๋‹น์˜ ๋†๋„๋Š” ํ˜ˆ์žฅ ๋†๋„ ๋น„ํ•ด 12๋ฐฐ ์ •๋„ ๋‚ฎ๊ฒŒ ์œ ์ง€๋˜๋Š” ๊ฒƒ์œผ๋กœ ์•Œ๋ ค์ ธ ์žˆ์œผ๋ฉฐ, ์—ฌ๊ธฐ์—๋Š” ๋Šฅ๋™์  ํฌ๋„๋‹น ์šด์†ก์ฒด์ธ glucose transporter 1, 2, 10 ๋“ฑ์ด ๊ด€์—ฌํ•˜๋Š” ๊ฒƒ์œผ๋กœ ์•Œ๋ ค์ ธ ์žˆ๋‹ค. ์ด๋Ÿฌํ•œ ๋Šฅ๋™์ ์ธ ํ˜ธํก๊ธฐ ๋‚ด์˜ ์ €ํฌ๋„๋‹น ์ƒํƒœ๋Š” ํ˜ธํก๊ธฐ ๊ฐ์—ผ์˜ ์œ„ํ—˜์„ฑ์„ ๋‚ฎ์ถฐ ํ˜ธํก๊ธฐ ํ•ญ์ƒ์„ฑ ์œ ์ง€์— ์ค‘์š”ํ•œ ์ƒ๋ฆฌ์  ํ™˜๊ฒฝ์„ ์ œ๊ณตํ•˜๊ฒŒ ๋œ๋‹ค[24]. ๊ทธ๋Ÿฌ๋‚˜ Philips ๋“ฑ๊ณผ Wood ๋“ฑ์˜ ์—ฐ๊ตฌ ๋“ฑ์„ ํ†ตํ•ด ํ™•์ธ๋˜์—ˆ๋“ฏ์ด ์ „์‹ ์ ์ธ ๊ณ ํ˜ˆ๋‹น์ฆ์ƒํƒœ๋Š” ์ƒํ•˜๋ถ€ ํ˜ธํก๊ธฐ ๋‚ด๊ฐ•์˜ ํฌ๋„๋‹น ๋†๋„์˜ ์ฆ๊ฐ€๋ฅผ ๋ฐœ์ƒ์‹œํ‚ค๋ฉฐ, ๋˜ํ•œ ๊ณ ํ˜ˆ๋‹น์ฆ์œผ๋กœ ์ธํ•ด ์œ ๋ฐœ๋˜๋Š” ์—ผ์ฆ๋ฐ˜์‘์œผ๋กœ ์ธํ•ด ํ˜ธํก๊ธฐ ํ˜ˆ๊ด€๋‚ดํ”ผ์„ธํฌ ๋ฐ ์ƒํ”ผ์„ธํฌ์˜ ์†์ƒ์ด ๋ฐœ์ƒํ•˜์—ฌ ํ˜ธํก๊ธฐ๋‚ด ํ˜ˆ์žฅ ๋ˆ„์ถœ์ด ๋ฐœ์ƒํ•˜๊ณ  ์ด๋Š” ํ˜ธํก๊ธฐ ๋‚ด๊ฐ•์— ์ง€์†์ ์ธ ํฌ๋„๋‹น ๋†๋„์˜ ์ƒ์Šน์„ ๋ฐœ์ƒ์‹œํ‚จ๋‹ค[24-26]. ์ด๋Ÿฌํ•œ ํ˜ธํก๊ธฐ ๋‚ด๊ฐ•์˜ ํฌ๋„๋‹น ๋†๋„์˜ ์ฆ๊ฐ€๋Š” ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ, ํ˜ˆ๊ด€๋‚ดํ”ผ์„ธํฌ, ๋ฉด์—ญ์„ธํฌ, ๊ธฐ๋„ํ‰ํ™œ๊ทผ์„ธํฌ ๋“ฑ์— ์ง์ ‘์ ์ธ ์˜ํ–ฅ์„ ์ฃผ๋ฉฐ ๋‹ค์–‘ํ•œ ๊ธฐ์ „์„ ํ†ตํ•ด ๊ฐ์—ผ์„ฑ, ์—ผ์ฆ์„ฑ ํ˜ธํก๊ธฐ ์งˆํ™˜์˜ ๋ฐœ์ƒ๊ณผ ์•…ํ™”์˜ ์œ„ํ—˜์š”์ธ์ด ๋œ๋‹ค[16].
๋Œ€ํ‘œ์ ์ธ ํ˜ธํก๊ธฐ ์—ผ์ฆ์„ฑ ์งˆํ™˜์—๋Š” ์ฒœ์‹๊ณผ ๋‚ญ์„ฑ ํ์„ฌ์œ ์ฆ, ๋งŒ์„ฑ ํ์‡„์„ฑ ํ์งˆํ™˜, ๊ธฐ๊ด€์ง€ ํ™•์žฅ์ฆ ๋“ฑ์ด ์žˆ๋‹ค. ์ด๋“ค ์งˆํ™˜์—์„œ๋Š” ์—ผ์ฆ์„ฑ ๋ฐ˜์‘์œผ๋กœ ์ ๋ง‰ํ•˜ ๋ถ„๋น„์„ ์˜ ๋น„๋Œ€๋‚˜ ๋ฐฐ์„ธํฌ์˜ ์ˆ˜ ๋˜๋Š” ํฌ๊ธฐ์˜ ์ฆ๊ฐ€๊ฐ€ ์ผ์–ด๋‚˜์„œ MUC2์™€ MUC4, MUC5AC, MUC5B, MUC8 ์ ์•ก์œ ์ „์ž์˜ ๋ฐœํ˜„์ด ์ฆ๊ฐ€ํ•œ๋‹ค[1,2]. ์ด ์ค‘ MUC5B ์ ์•ก์œ ์ „์ž๋Š” ํ˜ธํก๊ธฐ ์—ผ์ฆ์„ฑ ์งˆํ™˜์˜ ์ฃผ์š” ๋ถ„๋น„ํ˜• ์ ์•ก์œ ์ „์ž๋กœ, ์ฒœ์‹ ํ™˜์ž์—์„œ ํก์ž…๋œ ์™ธ๋ถ€ ๋ฌผ์งˆ์ด๋‚˜ ์‚ฐํ™” ์ŠคํŠธ๋ ˆ์Šค, interleukin(IL)-6, IL-8, IL-13, IL-17 ๋“ฑ์— ์˜ํ•ด ๋ฐœํ˜„์ด ์ด‰์ง„๋œ๋‹ค[11,27,28]. ๋‚ญ์„ฑ ํ์„ฌ์œ ์ฆ์ด๋‚˜ ๊ฐ„์งˆ์„ฑ ํ์งˆํ™˜ ํ™˜์ž์—์„œ ํ™œ์„ฑ์‚ฐ์†Œ๋‚˜ ์‚ฌ์ดํ† ์นด์ธ ๋“ฑ์˜ ์—ผ์ฆ์„ฑ ๋งค๊ฐœ ๋ฌผ์งˆ์˜ ์ž‘์šฉ์— ์˜ํ•ด MUC5B ์ ์•ก์œ ์ „์ž์˜ ๋ฐœํ˜„์ด ์ฆ๊ฐ€๋˜๊ณ [3,28], ๋งŒ์„ฑ ํ์‡„์„ฑ ํ์งˆํ™˜ ํ™˜์ž์—์„œ IL-9๊ณผ IL-13, tumor necrosis factor์˜ ํ™œ์„ฑํ™”๋‚˜ ์‚ฐํ™” ์ŠคํŠธ๋ ˆ์Šค๊ฐ€ MUC5B ์ ์•ก์œ ์ „์ž๋ฅผ ๊ณผ๋ฐœํ˜„์‹œํ‚จ๋‹ค[27,29-32]. ์ด์— ๊ณ ํ˜ˆ๋‹น์ฆ์ด ํ˜ธํก๊ธฐ์—์„œ ์—ผ์ฆ๋ฐ˜์‘์„ ์ผ์œผํ‚ค๋ฏ€๋กœ ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์—์„œ ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„ ์กฐ์ ˆ์— ๊ด€์—ฌํ•  ๊ฒƒ์œผ๋กœ ์ถ”์ •๋˜์–ด, ๋ณธ ์—ฐ๊ตฌ๋ฅผ ์‹œํ–‰ํ•œ ๊ฒฐ๊ณผ ๊ณ ๋†๋„์˜ ํฌ๋„๋‹น์ด ์ธ์ฒด ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์—์„œ ์ฃผ์š” ๋ถ„๋น„ํ˜• ์ ์•ก์œ ์ „์ž์ธ MUC5B ์ ์•ก์œ ์ „์ž์˜ ๋ฐœํ˜„์„ ์ฆ๊ฐ€์‹œํ‚ด์„ ์•Œ ์ˆ˜ ์žˆ์—ˆ๋‹ค.
MUC5B ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์— ๊ด€๊ณ„ํ•˜๋Š” ์—ฌ๋Ÿฌ ์—ผ์ฆ ๋งค๊ฐœ๋ฌผ์งˆ๋“ค ์ค‘ ํ™œ์„ฑ์‚ฐ์†Œ๋Š” ์ „์ž ์ „๋‹ฌ๊ณ„ ํ˜น์€ ์—๋„ˆ์ง€๋Œ€์‚ฌ ๊ณผ์ • ์ค‘์— ์‚ฐ์†Œ๊ฐ€ ๋ถˆ์™„์ „ํ•˜๊ฒŒ ํ™˜์›๋˜๊ฑฐ๋‚˜, ํŽฉํ‹ฐ๋“œ ์„ฑ์žฅ์ธ์ž์™€ ์‚ฌ์ดํ† ์นด์ธ, ํšจ์†Œ ๋“ฑ์˜ ๋‹ค์–‘ํ•œ ์‹ ํ˜ธ ์ „๋‹ฌ๊ณ„์˜ ์ž๊ทน์— ์˜ํ•ด ๋ฐœ์ƒํ•˜๋Š” ํ™”ํ•™์  ๋ฐ˜์‘์„ฑ์ด ๋†’์€ ๋ถ„์ž๋กœ, ๊ณผ๋Ÿ‰ ์ƒ์„ฑ๋  ์‹œ ์„ธํฌ ๊ตฌ์กฐ์˜ ์†์ƒ์„ ์•ผ๊ธฐํ•˜์—ฌ ์‚ฐํ™” ์ŠคํŠธ๋ ˆ์Šค๋ฅผ ์กฐ์žฅํ•œ๋‹ค[28,29]. ์ฆ‰, ํ™œ์„ฑ์‚ฐ์†Œ๋Š” DNA์™€ ๋‹จ๋ฐฑ์งˆ, ์ง€์งˆ ๋“ฑ์„ ์‚ฐํ™”์‹œํ‚ด์œผ๋กœ์จ ์ง์ ‘์ ์œผ๋กœ ์„ธํฌ๋ฅผ ์†์ƒ์‹œํ‚ฌ ๋ฟ๋งŒ ์•„๋‹ˆ๋ผ ์ŠคํŠธ๋ ˆ์Šค์— ๋ฏผ๊ฐํ•œ ์„ธํฌ ๋‚ด์˜ ๋‹ค์–‘ํ•œ ์‹ ํ˜ธ ์ „๋‹ฌ์ฒด๊ณ„๋ฅผ ํ™œ์„ฑํ™”ํ•จ์œผ๋กœ์จ ๊ฐ„์ ‘์ ์œผ๋กœ ์„ธํฌ์— ์†์ƒ์„ ์ผ์œผ์ผœ์„œ[27], ์„ธํฌ ๋‚ด์˜ ์ ์ ˆํ•œ ๋ฐฉ์–ด ๊ธฐ์ „์— ๋ฌธ์ œ๊ฐ€ ์ƒ๊ธธ ๊ฒฝ์šฐ, ๋‹น๋‡จ๋ณ‘์„ ํฌํ•จํ•˜์—ฌ, ๋‹ค์–‘ํ•œ ์—ผ์ฆ์„ฑ ์งˆํ™˜, ๋…ธํ™” ๊ด€๋ จ ๋ณ€์„ฑ, ์ข…์–‘์˜ ์ƒ์„ฑ ๋“ฑ์˜ ๋ณ‘์ธ์œผ๋กœ ๊ด€์—ฌํ•  ๊ฒƒ์œผ๋กœ ์ƒ๊ฐ๋œ๋‹ค. ์ด์— ๊ณ ๋†๋„ ํฌ๋„๋‹น์ด ์‚ฌ๋žŒ ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์—์„œ ํ™œ์„ฑ์‚ฐ์†Œ๋ฅผ ์ƒ์„ฑํ•˜์—ฌ ์ ์•ก๋ถ„๋น„์— ๊ด€์—ฌํ•  ๊ฒƒ์œผ๋กœ ์ถ”์ •ํ•˜์˜€์œผ๋ฉฐ, ๋‘ ๊ฐ€์ง€์˜ ๋Œ€ํ‘œ์ ์ธ ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์ฃผ(NCI-H292 ์„ธํฌ์™€ ์‚ฌ๋žŒ ์ฝ” ์ ๋ง‰ ์ƒํ”ผ์„ธํฌ)๋ฅผ ์ด์šฉํ•˜์—ฌ ์ƒ๊ธฐ๋„ ๋ฐ ํ•˜๊ธฐ๋„ ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์—์„œ ๊ณ ๋†๋„ ํฌ๋„๋‹น์ด ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์— ๋ฏธ์น˜๋Š” ํšจ๊ณผ์™€ ์—ฐ๊ด€ ๊ธฐ์ „์œผ๋กœ ํ™œ์„ฑ์‚ฐ์†Œ ๋ฐœ์ƒ์˜ ์—ญํ• ์— ๋Œ€ํ•ด ํ™•์ธํ•ด ๋ณด๊ณ ์ž ํ•˜์˜€๋‹ค. ๋ณธ ์‹คํ—˜์˜ ๊ฒฐ๊ณผ๋ฅผ ํ†ตํ•ด ์ €์ž๋“ค์€ ๊ณ ๋†๋„ ํฌ๋„๋‹น์ด ์„œ๋กœ ๋‹ค๋ฅธ ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์—์„œ ํ™œ์„ฑ์‚ฐ์†Œ ์ƒ์„ฑ์„ ์œ ๋ฐœ์‹œํ‚ค๋ฉฐ, ํ™œ์„ฑ์‚ฐ์†Œ ์–ต์ œ์ œ์™€ NADPH ์‚ฐํ™”ํšจ์†Œ ์–ต์ œ์ œ๊ฐ€ ํ™œ์„ฑ์‚ฐ์†Œ ์ƒ์„ฑ์กฐ์ ˆ์„ ํ†ตํ•ด MUC5B์˜ ๊ณผ๋ฐœํ˜„์„ ๊ฐ์†Œ์‹œํ‚ด์„ ํ™•์ธํ•˜์˜€๋‹ค. ์ด๋Š” ์ž„์ƒ์ ์œผ๋กœ ๋ฐ€์ ‘ํ•œ ์—ฐ๊ด€์„ฑ์ด ์žˆ๋Š” ๊ฒƒ์œผ๋กœ ์•Œ๋ ค์ง„ ๊ณ ํ˜ˆ๋‹น์ฆ๊ณผ ํ˜ธํก๊ธฐ ์งˆํ™˜์˜ ์•…ํ™”์™€ ๊ด€๋ จํ•˜์—ฌ ๊ณ ๋†๋„ ํฌ๋„๋‹น ๋…ธ์ถœ์— ๋”ฐ๋ฅธ ์ ์•ก๊ณผ ๋ถ„๋น„๊ฐ€ ํ•˜๋‚˜์˜ ์ด์œ ๊ฐ€ ๋  ์ˆ˜ ์žˆ์Œ์„ ํ™•์ธํ•œ ์—ฐ๊ตฌ์˜€๋‹ค. ํ•˜์ง€๋งŒ ๊ณ ๋†๋„ ํฌ๋„๋‹น์ด ์‚ฌ๋žŒ ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์—์„œ ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์— ๋ฏธ์น˜๋Š” ์˜ํ–ฅ์„ ๋ณด๋‹ค ์ž์„ธํ•˜๊ฒŒ ์ดํ•ดํ•˜๊ธฐ ์œ„ํ•ด์„œ๋Š” ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์— ๊ด€๊ณ„๋˜๋Š” ์‹ ํ˜ธ ์ „๋‹ฌ ์ฒด๊ณ„์— ๋Œ€ํ•œ ์—ฐ๊ตฌ์™€ ๋”๋ถˆ์–ด ์‹คํ—˜ ๋™๋ฌผ๊ณผ ์ธ์ฒด๋ฅผ ์ด์šฉํ•˜์—ฌ ํฌ๋„๋‹น ํ˜ˆ์ค‘ ๋†๋„์— ๋”ฐ๋ฅธ ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์— ๋Œ€ํ•œ ์ถ”๊ฐ€์ ์ธ ์—ฐ๊ตฌ๊ฐ€ ๋ฐ˜๋“œ์‹œ ํ•„์š”ํ•  ๊ฒƒ์œผ๋กœ ์ƒ๊ฐ๋œ๋‹ค.
๊ฒฐ๋ก ์ ์œผ๋กœ ๋ณธ ์—ฐ๊ตฌ์—์„œ ์‚ฌ๋žŒ ํ˜ธํก๊ธฐ ์ƒํ”ผ์„ธํฌ์—์„œ ๊ณ ๋†๋„ ํฌ๋„๋‹น์ด ํ™œ์„ฑ์‚ฐ์†Œ๋ฅผ ํ†ตํ•ด MUC5B ์ ์•ก์œ ์ „์ž ๋ฐœํ˜„์„ ์œ ๋„ํ•œ๋‹ค๋Š” ๊ฒƒ์„ ์•Œ ์ˆ˜ ์žˆ์—ˆ์œผ๋ฉฐ, ์ด๋Š” ์‚ฌ๋žŒ ํ˜ธํก๊ธฐ์—์„œ ๊ณ ํ˜ˆ๋‹น์ฆ์ด ์ ์•ก ๋ถ„๋น„์— ๋ฏธ์น˜๋Š” ์˜ํ–ฅ์— ๋Œ€ํ•œ ๊ธฐ์ดˆ์ ์ธ ์ž๋ฃŒ๋ฅผ ์ œ๊ณตํ• ๋ฟ๋งŒ ์•„๋‹ˆ๋ผ ๋‹น๋‡จ๋ณ‘์„ ๋™๋ฐ˜ํ•œ ํ˜ธํก๊ธฐ ์งˆํ™˜์—์„œ ์ ์•ก ๋ถ„๋น„ ์กฐ์ ˆ ์น˜๋ฃŒ์ œ ๊ฐœ๋ฐœ์— ๋งŽ์€ ๋„์›€์„ ์ œ๊ณตํ•  ๊ฒƒ์œผ๋กœ ์ƒ๊ฐ๋œ๋‹ค.

ACKNOWLEDGMENTS

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016R1A2B1012606).

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Fig.ย 1.
Effects of high concentration of glucose on expression of mucin genes in human NCI-H292 cells. Results of RT-PCR showed that high concentration of glucose significantly increased MUC5B mRNA expression, but did not increase MUC5AC mRNA expression (A). Effects of high concentration of glucose on MUC5B expression in human NCI-H292 cells. Results of real-time PCR showed that high concentration of glucose significantly increased MUC5B mRNA expression (B) and MUC5B mRNA expression was significantly increased in all duration of time and peaked at 8 hours by treatment of 20 mM glucose in human NCI-H292 cells (C). And results of ELISA showed that high concentration of glucose increased MUC5B protein production and peaked at 20 mM glucose in human NCI-H292 cells (D). The images are representative of three separate experiments performed in triplicated. Bars indicate the meanยฑS.D. of three independent experiments performed in triplicate. *p<0.05 compared with 5 mM glucose, โ€ p<0.05 compared with zero value. RT-PCR: reverse transcription-polymerase chain reaction, PCR: polymerase chain reaction, ELISA: enzyme-linked immunosorbent assay, GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
kjorl-hns-2016-17601f1.tif
Fig.ย 2.
Effects of treatment duration of high concentration of glucose on ROS production in human NCI-H292 cells. Results of flow cytometry showed that ROS production was significantly increased at all times after exposure of glucose (20 mM) in human NCI-H292 cells. The images are representative of three separate experiments performed in triplicated. Bars indicate the meanยฑS.D. of three independent experiments performed in triplicate. *p<0.05 compared with zero value. ROS: reactive oxygen species, DCFDA: dichlorodihydrofluorescein diacetate.
kjorl-hns-2016-17601f2.tif
Fig.ย 3.
Effects of ROS inhibitor and NADPH oxidase inhibitor on ROS production in human NCI-H292 cells. Results of flow cytometry showed that treatment of NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) significantly blocked ROS production in human NCI-H292 cells. The images are representative of three separate experiments performed in triplicated. Bars indicate the meanยฑS.D. of three independent experiments performed in triplicate. *p<0.05 compared with 5 mM glucose, โ€ p<0.05 compared with 20 mM glucose only. ROS: reactive oxygen species, DCFDA: dichlorodihydrofluorescein diacetate, NAC: N-acetyl-cystein, DPI: diphenyleneiodonium, NADPH: nicotinamide adenine dinucleotide phosphate.
kjorl-hns-2016-17601f3.tif
Fig.ย 4.
Effects of ROS inhibitor and NADPH oxidase inhibitor on MUC5B expression on human NCI-H292 cells. Results of RT-PCR and ELISA showed that treatment of NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) significantly blocked MUC5B expression in human NCI-H292 cells (A and B). The images are representative of three separate experiments performed in triplicated. Bars indicate the meanยฑS.D. of three independent experiments performed in triplicate. *p<0.05 compared with 5 mM glucose, โ€ p<0.05 compared with 20 mM glucose only. ROS: reactive oxygen species, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, NAC: N-acetyl-cystein, DPI: diphenyleneiodonium, NADPH: nicotinamide adenine dinucleotide phosphate, RT-PCR: reverse transcription-polymerase chain reaction, ELISA: enzyme-linked immunosorbent assay.
kjorl-hns-2016-17601f4.tif
Fig.ย 5.
Effects of high concentration of glucose on MUC5B expression in the primary cultures of human nasal epithelial cells. Results of RT-PCR and ELISA showed that high concentration of glucose significantly increased MUC5B expression in the primary cultures of human nasal epithelial cells (A and B). Results of RT-PCR and ELISA showed that treatment of NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) significantly blocked MUC5B expression in the primary cultures of human nasal epithelial cells (C and D). The images are representative of three separate experiments performed in triplicated. Bars indicate the meanยฑS.D. of three independent experiments performed in triplicate. *p<0.05 compared with 5 mM glucose, โ€ p<0.05 compared with 20 mM glucose only. ROS: reactive oxygen species, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, NAC: N-acetyl-cystein, DPI: diphenyleneiodonium, NADPH: nicotinamide adenine dinucleotide phosphate, RT-PCR: reverse transcription-polymerase chain reaction, ELISA: enzyme-linked immunosorbent assay.
kjorl-hns-2016-17601f5.tif
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