Effect of polymer of lysine on the mucin release from primary cultured hamster tracheal surface epithelial cells

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(1)Journal of Dental Hygiene Science Vol. 2, No. 1, pp. 0~0 (2002).

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(3) Effect of polymer of lysine on the mucin release from primary cultured hamster tracheal surface epithelial cells †. Choong-Jae Lee , Seon Kim and Kyung-Hee Hong Lab of Basic Medical Sciences, Department of Dental Hygiene and Research Institute of Health Sciences, Sahmyook Nursing and Health College, Seoul 130-711, Korea. In the present study, we tried to investigate whether poly-L-lysine(PLL)(MW 78,000 and 9,600) significantly affect mucin release from cultured hamster airway goblet cells. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled with 3H-glucosamine for 24 hr and chased for 30 min in the presence of varying concentrations of PLL to assess the effects on 3H-mucin release. Possible cytotoxicities of PLL were assessed by measuring Lactate Dehydrogenase(LDH) release during treatment. The results were as follows : (1) PLL significantly inhibited mucin release from cultured HTSE cells in a dose-dependent manner; (2) there was no significant release of LDH by treatment of PLL 9,600; (3) however, in the case of treatment of PLL 78,000, there was significant release of LDH during treatment. We conclude that PLL which has molecular weight under 10,000 might inhibit mucin release from airway goblet cells without significant cytotoxicity. This finding suggests that PLL might be used as a tool of research for the hypersecretion of airway mucus.

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(14) oq A vKG ( -./ 012 (w I x y, V z>! {L>, p> |}~K . 3,4). 1). 4). 5-8). 2). †. Corresponding author Tel: 011-9736-9501 Fax: 02-3407-8504 E-mail: [email protected]. 9). .

(15) . Effect of polymer of lysine. ¸3. B¤ ¹K> 4l D8 \], ^_ # ºT 5 e./ ! y# »¼K O½¤ 5 e# ®¾ v¿ RL À/ Á/Â p# ÃX :) oÄ, C ( -B3.. . o. Poly-L-lysine(PLL)(MW 78,000 and 9,600), pronase(Type XIV), insulin, transferrin, epidermal growth factor, hydrocortisone, sodium selenite, testicular hyaluronidase(Type VI-S), LDH assay kit(LD-L 10), retinoic acid, gentamicin, sodium dodecyl sulfate Sigma (St. Louis, Mo., USA) , penicillin-G, streptomycin, Joklik-modified Minimal Essential Medium(S-MEM), Dulbecco's Modified Eagle's Medium (DME), fetal bovine serum(FBS), Medium 199(M199) GIBCO (Grand Island, New York, USA) , Vitrogen 100 Celtrix Lab.(Santa Clara, CA, USA) , reagent grade , .. j. ). j ) ) x ¸. Ú ¨Ñipl BL# -l x Û j' ¸. |} j'f ÜB B©cÝC Þ S ÜB Û j' ¸3.

(16) . «¬t# La E ªJ >'f |}$ß Kim # $ß j' ¸3. 8 \ 10 7Å# Æ8 «¬t Bà³/ _j iá, Êâ LÒ) ãiä µ åâf æç èé(PE tube) ê ¸3. PE tube Û Ca , Mg free Minimum Essential Medium 'iä 0.1% pronase 7 y, 4 C) 16 iÎ Ç¡ $® ¸3. 16iÎ µ, ' ë g Cq y, 10% fetal bovine serum ±W MEM ) 200ìg/ íTE ±./² Cq a 2î ëï3. ëf cell pellets insulin(5 µg/ml), transferrin(5 µg/ml), 9,10). 2+. o.

(17) # $ß B' ¸ A, ªJa # <= 8 ý ªJa(24 well plate, 5ì10 cells/well) 10 µCi/ml # [6- H] glucosamine(39.2 Ci/mmol, New England neuclear) ±W þªJ well 9 200 µl ÿ G y 32 C ) 24iÎ Ç¡ ªJ±./² $j \(metabolic radiolabeling)ú3. 5. Poly-L-lysine(PLL) 24iÎ Ç¡# j> $j \G þ~f µ ªJ (pretreatment sample, B PT / p) Cq ôú3. ªJ a well 9 0.5 ml # Dulbecco's Ca , Mg free PBS G y ë & 2î Ñ±./² ªJa# j oq I 0.1 µM-10 µM# PLL ±W. PBS G y 32 C) 30EÎ ªJ ¸3. 30E# ªJB. Ñ Cq Û, treatment sample(B T sample) / # ¸3. Cqf

(18) sample l íTE Û sW a Ú j oq y, 50 µl# L ÜC³v³ 8 (LDH activity assay) j' § % µ, $j8 <= ±F , ?\ -70 C ) Ç . ¸3 . 6. !" #$% Hyaluronidase # E\ ., Sepharose CL-4B column./st exclude yEF# glycoconjugate <=./ # ¸3 . Cqf PT, T sample 0.1 M citric acid ²) pH 5.0./ y 100 unit /ml# testicular hyaluronidase(Type VI-S) G 37 C) 16iÎ Ç¡ incubation ¸3. IncubationB digestion mixture 0.2 M NaOH/ iÐy IG 0.1%G I SDS ' G 2EÎ ! 3w 12,000ìg) 5EÎ íT Eï3. PT# » íTE µ# L 50 µl 150 µl# running buffer(50 mM NaAc, 0.1% SDS, pH 7.2), 2 µl# 10% SDS, 3 µl# 5 M NaCl Gïy, T# » L 100 µl 100 µl# running buffer, 2 µl# 10% SDS, 3 µl # 5 M NaCl G ù ðñ ¸3. Bm of loading sample l running buffer/ f Sepharose CL4B column(1ì50 cm) >' ¸3. Column 0.336 ml/min # W./ &Çúy, E # 'F 0.42 ml/ ¸3. 6 ml 'F# vial Cqf n E 3.2 ml# scintillation cocktail G y Ô ! Liquid Scintillation Counter (LKB Wallac) B', $jF ¸3. " E void volume peak# 4E # $jF "ñ sample # <=# J./ # ¸3. n culture well Î ( C ( <= ¹ W¿# [BI 4 § Û, T Î Ç¡ Wf <=# J PT Î Ç¡ Wf <=# J. / #% %\ Y¿ EY\C(secretory index)/ #. y, $K p%$# EY\C â& ¢>./ 10). Kim. ¨©ªJ La § 87Å# Æ8 Golden Syrian «¬t g in vivo |} § 6~87Å# Æ8 S.D. rat Ç jÈ g CÉ"! Ê+) ÄË ÌÍ3. ÇB |}| I µ 2~3¨Î |}| e» ÏiÐy, ÏÎ ¨ÑLÒ , Ó ÇD j' ¸3. eG Ôy, 12iÎ °Õ 7G W\, | B 25 C Ö/ W\ e») j¾ _C W/B ×ØiÙ3. 2. . 3.. o. 4.. 1.. . ú., 1~3¨Î ªJf µ 37 C) 32 C Ó./ [» Û ªJ ¸3. ªJ û" ªJ Ãiµ o 1, 3, 5, 7¨ nn iü ¸3.. 2+. o. epidermal growth factor(12.5 ng/ml), hydrocortisone(0.1 µM), sodium selenite(0.01 µM), retinoic acid(0.1 µM), fetal bovine serum(5%, v/v,), Penicillin G(100 U/ml), streptomycin(100 µg /ml), Gentamicin(50 µg/ml) Dulbecco's Modified Eagle's Medium(DME) M199 1:1 . Vitrogen 100 collagen 24 well plate 104 cells/ gel(0.15 ml/cm2 ) . , , cm2 , 37oC 95% , 5% CO2. B ±Wf K # ðñ ªJ Vò iÙ3 B' Û óYf ôõÂ B Iaf a # öI/ EaiÙ3 a ÷IG øEù W\ ±W. aªJ) ªJ Ä. 5. 3. o. 2+. 2+. o. o. 10). 10). o.

(19) . Journal of Dental Hygiene Science Vol. 2, No. 1, pp. 0~0 (2002). Yû±./², <=W ® p# vK 'c y. ¸3.. HTSE &' ()* +, -./ 0/(LDH) 12 #$ LDH 8 commercial kit(Sigma, LD-L 10) B ' ¸3. 50 µl# ªJ L assay kit# reaction mixture ðñ y 340 nm )# »i> (I cG® UVspectrophotometer / , LDH 8I ¢ ¸3. 8. 34

(20) ~K mean)S.E.M../ ef µ, p% $# ® $ ®# ;E¿/ Ú*3. ¢% unpaired Student's t-test / ¸., p<0.05 ! » ¢>./ W#8B ( -./ + ¸3. 7. PLL.

(21). ]8 O ±W±./² cationicity Ú l I La/st# <=W cGiÐ »R Ú, -./ -% £ A, BW ¤ lB 5)# K.Ñ ¨.Ðq , La/# 0K I cGiÐ # &' Ú ?@Bú3. ¤, ¨©ªJ «¬t La(HTSE)), EPO MBP # ]8, JB8 !8 G <=W 1o 3 4y , §) 2Ëf |}~Kl 3q -K L Ñf 4y¸3. B¤ |}~K ¢f ¥ x)I, PLL # ]8 O# ñ"G ¨©ªJ «¬t La L./ in vitro )# <=W 1o. -./ Ú43. ¨©ªJf HTSE a PLL 78,000 1 µM I) <=W 85% GF 1o ./ Ú43(Fig. 1). 5 PLL 9,600 10 µM I) <=W 80% GF 1o -./ Ú43(Fig. 2). B ¤ ~K lysine B arginine ]8 O# ñ "G I# La6./st# <=W 1o±./² ` a8 bWc, ^_ ) I

(22) K3EY VL# ´/ &', G68 oi -B 7 C (3. 5 , I La N OP ³ Z ÛÚ#

(23) E Y La# 6 x (%) # xC:./) B'8 G68I oi -B 7 C (3. , 58x W9# !8 ]8 :; ¨s !"# 5¢ :8 Ú -./ 4y% £ A, x">./ I L a# s, ; B 4y% (3 . B¤ 4yl <8 ^_em) = 58x c3c(hypereosinophilia) B , 58x# I B ^_# cL XV * +, , -B {L> > 3q |}~Kl B3 . ?), ¥ x)I <=W 1o &' XV. PLLB Ç¨ ªJa a:8 XV, G6 8 'c 4y ¸3. a/B @L a L6 Am f3. a v³! LDH W Z W a Ö e»./ ! a/# @L # a Ö/ W8 C (., ?) PLL # XV. Effect of PLL 78,000 on mucin release from cultured HTSE cells. Confluent HTSE cells were metabolically radiolabeled with 3Hglucosamine for 24 hrs and chased for 30 min in the presence of 0.01 - 1 µM of PLL and the amount of 3H-mucins in the spent media was measured. Each bar represents a mean S.E.M. from 4 culture wells. *: significantly different from control(p<0.05).. . 5,8,11,12). 6,13). 14). Effect of PLL 9,600 on mucin release from cultured HTSE cells. Confluent HTSE cells were metabolically radiolabeled with 3Hglucosamine for 24 hrs and chased for 30 min in the presence of 0.1 - 10 µM of PLL and the amount of 3H-mucins in the spent media was measured. Each bar represents a mean S.E.M. from 4 culture wells. *: significantly different from control(p<0.05).. . 3,15,16). 3,4,11,12). 8 C ( a:8# \/ BC8 C (3. Fig. 3 Ú ~K) 7 C (DB PLL 9,600 $K Yûi LDH W W#8 (m cGiÐ\ Íw 0 C (3. ¤, Fig. 4) 7 C (DB, PLL 78,000# » yI) L9F# LDHG Wúw 0 C (y, B ¤ ~K EF 9,600# PLL# »D :8B XV \ ¡ ¦§) <=W 1o8B E m XV 8 G68 oi ~KB3. 3C# xl # ,.

(24) . Effect of polymer of lysine. . ¥ x) EF 78,000K 9,600# poly-L-lysine(PLL) B ¨©ªJf «¬t# La/st# <=W %L QR ® \ 'c y ¸3. þù 3 M ªJa H-glucosamine ±W þ ªJ NG. y 24iÎ ªJ±./² ªJa # <= j> $j \ þ~ µ 3J I# PLL 30EÎ % y, W $j8 <=# ±F ¸3. PLL # a: 8 XVÛs 'c § Û PLL % µ ªJa/st W% ªJ Lactate Dehydrogenase(LDH) # 8 ¸3. |} ~K, PLL EF 78,000 g 9,600# ô Äù 'F #>./ <=W 1o ¸3. ¤ a:8# \! LDH W QR PLL 9,600# » W#8B ú., PLL 78,000# » V W cG 4¸3. B¤ ~K PLL# », E FB 10,000 B # ¦§) a:8 XV \ .)I <=W B>./ 1o, G68 oi y (., Ç i PLLB I

(25) K3EY VL x± (%) W' |}C:./ B'8 G68I oi y ( -B3. 3. Effect of PLL 78,000 on LDH release from cultured HTSE cells. Confluent HTSE cells were treated with varying concentrations of PLL for 30 min and supernatants were collected for LDH activity assay at the end of the treatment. Each bar represents a mean S.E.M. from 4 culture wells. *: significantly different from control(p<0.05).. . . Effect of PLL 9,600 on LDH release from cultured HTSE cells. Confluent HTSE cells were treated with varying concentrations of PLL for 30 min and supernatants were collected for LDH activity assay at the end of the treatment. Each bar represents a mean S.E.M. from 4 culture wells.. . ]8 JB8 l# EF ? M> 8B 3Fm Ú 3 4y !, C (., ¥ x j' f ô Î, <=W 1o 8 ©BG . a :8 XV) V ©BG BW ) S xG BG% H*G (3. Çi, -G>! x~K G Iãf µ |ù 2Ë, C ( @oB, B¤ 4l PLLK d ]8 O# ñ"G I

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