{"id":4903,"date":"2024-01-15T06:33:58","date_gmt":"2024-01-15T06:33:58","guid":{"rendered":"https:\/\/corp.farlong.com\/protective-effect-of-tisochrysis-lutea-on-dry-eye-syndrome-via-nf-%ce%bab-inhibition\/"},"modified":"2024-01-27T10:30:56","modified_gmt":"2024-01-27T10:30:56","slug":"protective-effect-of-tisochrysis-lutea-on-dry-eye-syndrome-via-nf-%ce%bab-inhibition","status":"publish","type":"post","link":"https:\/\/directcm.com\/vi\/protective-effect-of-tisochrysis-lutea-on-dry-eye-syndrome-via-nf-%ce%bab-inhibition\/","title":{"rendered":"T\u00e1c d\u1ee5ng b\u1ea3o v\u1ec7 c\u1ee7a Tisochrysis lutea \u0111\u1ed1i v\u1edbi h\u1ed9i ch\u1ee9ng kh\u00f4 m\u1eaft th\u00f4ng qua \u1ee9c ch\u1ebf NF-\u03baB"},"content":{"rendered":"
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Nguy\u00ean v\u1eadt li\u1ec7u<\/h3>\n

V\u1eadt li\u1ec7u in vivo\/in vitro<\/h4>\n

T. lutea<\/i> \u0111\u01b0\u1ee3c l\u1ea5y t\u1eeb Microalgae Ask us Corp. (Gangneung, Korea). Scopolamine, fluorescein, hematoxylin v\u00e0 eosin (H&E) \u0111\u01b0\u1ee3c mua t\u1eeb Sigma-Aldrich (St. Louis, MO, USA) \u0111\u1ec3 th\u00ed nghi\u1ec7m tr\u00ean \u0111\u1ed9ng v\u1eadt. Dulbecco's Modified Eagle's Medium (DMEM): F12, dung d\u1ecbch mu\u1ed1i \u0111\u1ec7m ph\u1ed1t ph\u00e1t (DPBS) c\u1ee7a Dulbecco, huy\u1ebft thanh b\u00f2 b\u00e0o thai (FBS) v\u00e0 dung d\u1ecbch trypsin\u2013EDTA 0,25% \u0111\u01b0\u1ee3c l\u1ea5y t\u1eeb Life Technologies (Carlsbad, CA, USA). Dung d\u1ecbch kh\u00e1ng sinh, bao g\u1ed3m 10.000 U\/mL penicillin v\u00e0 10.000 \u03bcg\/mL streptomycin, \u0111\u01b0\u1ee3c mua t\u1eeb Hyclone Laboratories Inc. (South Logan, UT, Hoa K\u1ef3). TNF-\u03b1 t\u00e1i t\u1ed5 h\u1ee3p c\u1ee7a con ng\u01b0\u1eddi \u0111\u01b0\u1ee3c l\u1ea5y t\u1eeb c\u00e1c h\u1ec7 th\u1ed1ng R&D (Minneapolis, MN, USA). C\u00e1c kh\u00e1ng th\u1ec3 ch\u00ednh bao g\u1ed3m kinase \u0111\u01b0\u1ee3c \u0111i\u1ec1u h\u00f2a t\u00edn hi\u1ec7u ngo\u1ea1i b\u00e0o (ERK), kinase ch\u1ed1ng c-jun N-terminal (JNK), anti-p38, anti-\u03b1-tubulin v\u00e0 anti-\u03b2-actin \u0111\u01b0\u1ee3c mua t\u1eeb C\u00f4ng ngh\u1ec7 sinh h\u1ecdc Santa Cruz ( Santa Cruz, CA, Hoa K\u1ef3). C\u00e1c kh\u00e1ng th\u1ec3 ch\u00ednh kh\u00e1c v\u00e0 kh\u00e1ng th\u1ec3 th\u1ee9 c\u1ea5p li\u00ean h\u1ee3p peroxidase (HRP) \u0111\u01b0\u1ee3c l\u1ea5y t\u1eeb Cell Signaling Technology, Inc (Beverly, MA, USA). C\u00e1c h\u00f3a ch\u1ea5t bao g\u1ed3m 3\u20134,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) \u0111\u01b0\u1ee3c mua t\u1eeb Sigma-Aldrich (St. Louis, MO, USA).<\/p>\n

V\u1eadt li\u1ec7u ph\u00e2n t\u00edch h\u00f3a h\u1ecdc<\/h3>\n

PUFA \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh b\u1eb1ng c\u00e1ch s\u1eed d\u1ee5ng dung d\u1ecbch ch\u1ee9a 37 ch\u1ea5t ti\u00eau chu\u1ea9n PUFA \u0111\u01b0\u1ee3c mua t\u1eeb Sigma-Aldrich (St. Louis, MO, USA). Axit valeric \u0111\u01b0\u1ee3c l\u1ea5y t\u1eeb Sigma-Aldrich (St. Louis, MO, USA) \u0111\u1ec3 s\u1eed d\u1ee5ng l\u00e0m ch\u1ea5t chu\u1ea9n n\u1ed9i. C\u00e1c dung m\u00f4i, bao g\u1ed3m dung d\u1ecbch boron triflorua metanol, N<\/i>-hexan, Na2<\/sub>V\u00cc TH\u1ebe4<\/sub>, CH3<\/sub>Cl, MeOH v\u00e0 NaCl, \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u1ec3 chi\u1ebft xu\u1ea5t PUFA t\u1eeb T. lutea<\/i> \u0111\u01b0\u1ee3c mua t\u1eeb Sigma-Aldrich (St. Louis, MO, USA).<\/p>\n

Tuy\u00ean b\u1ed1 \u0111\u1ea1o \u0111\u1ee9c<\/h3>\n

Nghi\u00ean c\u1ee9u n\u00e0y \u0111\u00e3 \u0111\u01b0\u1ee3c ph\u00ea duy\u1ec7t b\u1edfi \u1ee6y ban Ch\u0103m s\u00f3c v\u00e0 S\u1eed d\u1ee5ng \u0110\u1ed9ng v\u1eadt Th\u1ec3 ch\u1ebf (IACUC) c\u1ee7a Vi\u1ec7n Khoa h\u1ecdc v\u00e0 C\u00f4ng ngh\u1ec7 H\u00e0n Qu\u1ed1c (KIST): KIST s\u1ed1 2020-002, Vi\u1ec7n Gangneung v\u00e0 tu\u00e2n theo tuy\u00ean b\u1ed1 c\u1ee7a Hi\u1ec7p h\u1ed9i Nghi\u00ean c\u1ee9u Th\u1ecb gi\u00e1c v\u00e0 Nh\u00e3n khoa v\u1ec1 S\u1eed d\u1ee5ng \u0111\u1ed9ng v\u1eadt trong nghi\u00ean c\u1ee9u nh\u00e3n khoa v\u00e0 th\u1ecb gi\u00e1c. Ngo\u00e0i ra, ch\u00fang t\u00f4i tu\u00e2n theo c\u00e1c khuy\u1ebfn ngh\u1ecb v\u1ec1 Nghi\u00ean c\u1ee9u \u0110\u1ed9ng v\u1eadt: B\u00e1o c\u00e1o Th\u00ed nghi\u1ec7m In Vivo (ARRIVE). T\u1ea5t c\u1ea3 c\u00e1c th\u1ee7 t\u1ee5c \u0111\u01b0\u1ee3c th\u1ef1c hi\u1ec7n tu\u00e2n th\u1ee7 c\u00e1c quy \u0111\u1ecbnh v\u00e0 h\u01b0\u1edbng d\u1eabn hi\u1ec7n h\u00e0nh.<\/p>\n

Th\u00ed nghi\u1ec7m tr\u00ean \u0111\u1ed9ng v\u1eadt v\u00e0 c\u1ea3m \u1ee9ng m\u00f4 h\u00ecnh DES<\/h3>\n

Chu\u1ed9t \u0111\u01b0\u1ee3c nh\u1ed1t trong ph\u00f2ng \u0111\u1ed9ng v\u1eadt c\u00f3 m\u00e1y l\u1ea1nh v\u00e0 duy tr\u00ec \u1edf nhi\u1ec7t \u0111\u1ed9 22 \u00b0 C \u00b1 2 \u00b0 C, \u0111\u1ed9 \u1ea9m 50% \u00b1 10% v\u00e0 chu k\u1ef3 sinh h\u1ecdc 12 gi\u1edd s\u00e1ng\/12 gi\u1edd t\u1ed1i. Th\u1ee9c \u0103n v\u00e0 n\u01b0\u1edbc \u0111\u01b0\u1ee3c cung c\u1ea5p t\u1ef1 do. Chu\u1ed9t \u0111\u01b0\u1ee3c l\u00e0m quen v\u1edbi kh\u00ed h\u1eadu trong m\u1ed9t tu\u1ea7n tr\u01b0\u1edbc khi \u0111\u01b0\u1ee3c chia ng\u1eabu nhi\u00ean th\u00e0nh n\u0103m nh\u00f3m g\u1ed3m b\u1ea3y con chu\u1ed9t BALB \/ c \u0111\u1ef1c 6 tu\u1ea7n tu\u1ed5i (Orientbio, Gyunggi-do, H\u00e0n Qu\u1ed1c). DES \u0111\u01b0\u1ee3c g\u00e2y ra th\u1ef1c nghi\u1ec7m tr\u00ean chu\u1ed9t hai l\u1ea7n m\u1ed7i ng\u00e0y th\u00f4ng qua ti\u00eam trong ph\u00fac m\u1ea1c 200 \u03bcL (2,5 mg\/mL) scopolamine pha lo\u00e3ng trong dung d\u1ecbch mu\u1ed1i \u0111\u1ec7m ph\u1ed1t ph\u00e1t (PBS; Sigma-Aldrich, St. Louis, MO, USA). T. lutea<\/i> \u0111\u01b0\u1ee3c d\u00f9ng b\u1eb1ng \u0111\u01b0\u1eddng u\u1ed1ng cho c\u00e1c nh\u00f3m chu\u1ed9t h\u00e0ng ng\u00e0y \u1edf n\u1ed3ng \u0111\u1ed9 0 (ki\u1ec3m so\u00e1t ph\u01b0\u01a1ng ti\u1ec7n), 100, 150 ho\u1eb7c 300 mg\/kg trong 200 \u03bcL n\u01b0\u1edbc c\u1ea5t. Nh\u00f3m \u0111\u1ed1i ch\u1ee9ng nh\u1eadn PBS kh\u00f4ng c\u00f3 scopolamine. Khi T. lutea<\/i> \u0111\u01b0\u1ee3c d\u00f9ng b\u1eb1ng \u0111\u01b0\u1eddng u\u1ed1ng, 200 \u03bcL n\u01b0\u1edbc c\u1ea5t \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng trong nh\u00f3m \u0111\u1ed1i ch\u1ee9ng v\u00e0 nh\u00f3m DES. Vi\u1ec7c s\u1ea3n xu\u1ea5t n\u01b0\u1edbc m\u1eaft \u0111\u01b0\u1ee3c \u0111\u1ecbnh l\u01b0\u1ee3ng sau hai tu\u1ea7n b\u1eb1ng c\u00e1ch s\u1eed d\u1ee5ng que th\u1eed Schirmer ti\u00eau chu\u1ea9n \u0111\u1eb7t \u1edf 1\/3 d\u01b0\u1edbi c\u1ee7a m\u00ed m\u1eaft th\u00e1i d\u01b0\u01a1ng tr\u01b0\u1edbc khi nh\u1eafm m\u1eaft l\u1ea1i trong 1 ph\u00fat. Sau \u0111\u00f3, d\u1ea3i n\u00e0y \u0111\u01b0\u1ee3c l\u1ea5y ra v\u00e0 \u0111o chi\u1ec1u d\u00e0i c\u1ee7a \u0111i\u1ec3m \u01b0\u1edbt t\u00ednh b\u1eb1ng milim\u00e9t \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh gi\u00e1 tr\u1ecb th\u1eed nghi\u1ec7m c\u1ee7a Schirmer. Nhu\u1ed9m b\u1ec1 m\u1eb7t gi\u00e1c m\u1ea1c \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh m\u1ee9c \u0111\u1ed9 thay \u0111\u1ed5i b\u1ec1 m\u1eb7t gi\u00e1c m\u1ea1c. B\u1ec1 m\u1eb7t gi\u00e1c m\u1ea1c \u0111\u01b0\u1ee3c quan s\u00e1t v\u00e0 ghi \u0111i\u1ec3m sau khi ti\u00eam m\u1ed9t gi\u1ecdt fluorescein 3% v\u00e0o k\u1ebft m\u1ea1c b\u00ean d\u01b0\u1edbi. Nhu\u1ed9m gi\u00e1c m\u1ea1c \u0111\u01b0\u1ee3c \u0111\u00e1nh gi\u00e1 m\u1ed9t c\u00e1ch m\u00f9 qu\u00e1ng. Chu\u1ed9t \u0111\u00e3 \u0111\u01b0\u1ee3c ti\u00eau h\u1ee7y do tr\u1eadt kh\u1edbp c\u1ed5 t\u1eed cung. Gi\u00e1c m\u1ea1c \u0111\u01b0\u1ee3c l\u1ea5y ra b\u1eb1ng c\u00e1ch b\u00fang mi chu\u1ed9t v\u00e0 d\u00f9ng k\u1eb9p c\u1eaft d\u00e2y th\u1ea7n kinh th\u1ecb gi\u00e1c ra kh\u1ecfi nh\u00e3n c\u1ea7u v\u00e0 l\u1ea5y gi\u00e1c m\u1ea1c ra. Th\u1ea5u k\u00ednh tinh th\u1ec3 \u0111\u01b0\u1ee3c l\u1ea5y ra t\u1eeb b\u00ean trong gi\u00e1c m\u1ea1c v\u00e0 \u0111\u01b0\u1ee3c b\u1ea3o qu\u1ea3n \u1edf -80 \u00b0 C. Tuy\u1ebfn l\u1ec7 \u0111\u01b0\u1ee3c c\u1eaft b\u1ecf kh\u1ecfi h\u00e0m d\u01b0\u1edbi c\u1ee7a chu\u1ed9t b\u1eb1ng k\u1eb9p v\u00e0 l\u1edbp bi\u1ec3u b\u00ec c\u1ee7a v\u00f9ng k\u00e9o \u0111\u01b0\u1ee3c c\u1eaft \u0111i \u0111\u1ec3 l\u1ed9 n\u1eeda d\u01b0\u1edbi c\u1ee7a khu\u00f4n m\u1eb7t. Tuy\u1ebfn l\u1ec7 g\u1ea7n h\u00e0m d\u01b0\u1edbi \u0111\u01b0\u1ee3c c\u1ed1 \u0111\u1ecbnh v\u00e0 c\u1eaft b\u1ecf sau \u0111\u00f3 b\u1ea3o qu\u1ea3n \u1edf nhi\u1ec7t \u0111\u1ed9 -80\u00b0C, tr\u00e1nh \u00e1nh s\u00e1ng.<\/p>\n

m\u00f4 h\u1ecdc<\/h3>\n

C\u00e1c m\u00f4 bi\u1ec3u b\u00ec gi\u00e1c m\u1ea1c v\u00e0 tuy\u1ebfn l\u1ec7 \u0111\u01b0\u1ee3c thu th\u1eadp v\u00e0 c\u1ed1 \u0111\u1ecbnh trong formaldehyde 10%, sau \u0111\u00f3 \u0111\u01b0\u1ee3c x\u1eed l\u00fd \u0111\u1ec3 nh\u00fang v\u00e0 c\u1eaft parafin. C\u00e1c ph\u1ea7n \u0111\u01b0\u1ee3c nhu\u1ed9m b\u1eb1ng H&E v\u00e0 ki\u1ec3m tra \u1edf \u0111\u1ed9 ph\u00f3ng \u0111\u1ea1i 40 \u00d7 b\u1eb1ng k\u00ednh hi\u1ec3n vi (TE-2000U, Nikon, Tokyo, Nh\u1eadt B\u1ea3n). \u0111\u1ed9 d\u00e0y bi\u1ec3u m\u00f4 gi\u00e1c m\u1ea1c trung t\u00e2m \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh b\u1eb1ng c\u00e1ch chia m\u1ed7i gi\u00e1c m\u1ea1c th\u00e0nh n\u0103m ph\u1ea7n.<\/p>\n

Nhu\u1ed9m DAB \u0111\u1ec3 ph\u00e2n t\u00edch h\u00f3a m\u00f4 mi\u1ec5n d\u1ecbch (IHC)<\/h3>\n

C\u00e1c m\u00f4 t\u1eeb tuy\u1ebfn l\u1ec7 c\u1ee7a chu\u1ed9t BALB\/c \u0111\u01b0\u1ee3c l\u1ea5y v\u00e0 c\u1ed1 \u0111\u1ecbnh b\u1eb1ng formaldehyde 10%. C\u00e1c m\u00f4 tuy\u1ebfn l\u1ec7 c\u1ed1 \u0111\u1ecbnh \u0111\u01b0\u1ee3c nh\u00fang parafin, c\u1eaft nh\u1ecf (5 \u03bcm) v\u00e0 parafin \u0111\u01b0\u1ee3c lo\u1ea1i b\u1ecf ba l\u1ea7n trong 3 ph\u00fat b\u1eb1ng xylene (Junsei, Tokyo, Nh\u1eadt B\u1ea3n). C\u00e1c ph\u1ea7n \u0111\u01b0\u1ee3c hydrat h\u00f3a trong 1 ph\u00fat trong ethanol 100, 95, 70 v\u00e0 50%. Kh\u00e1ng nguy\u00ean tuy\u1ebfn l\u1ec7 \u0111\u01b0\u1ee3c chi\u1ebft xu\u1ea5t b\u1eb1ng l\u00f2 vi s\u00f3ng v\u1edbi \u0111\u1ec7m TRIS\u2013EDTA (pH 9,0). Sau khi b\u00f4i hydro peroxide 3% v\u00e0o c\u00e1c phi\u1ebfn m\u00f4 trong 15 ph\u00fat, c\u00e1c ph\u1ea7n n\u00e0y \u0111\u01b0\u1ee3c ch\u1eb7n b\u1eb1ng 2% BSA trong 1 gi\u1edd \u1edf nhi\u1ec7t \u0111\u1ed9 ph\u00f2ng. C\u00e1c ph\u1ea7n \u0111\u01b0\u1ee3c x\u1eed l\u00fd qua \u0111\u00eam \u1edf 4 \u00b0 C b\u1eb1ng CD45 (Bio-RAD, MCA 1258GT, pha lo\u00e3ng 1:100). Sau khi r\u1eeda b\u1eb1ng PBS, c\u00e1c t\u1ebf b\u00e0o \u0111\u01b0\u1ee3c x\u1eed l\u00fd trong 2 gi\u1edd \u1edf nhi\u1ec7t \u0111\u1ed9 ph\u00f2ng b\u1eb1ng IgG-HRP ch\u1ed1ng th\u1ecf (C\u00f4ng ngh\u1ec7 sinh h\u1ecdc Santa Cruz, Dallas, TX, Hoa K\u1ef3, SC-2357, pha lo\u00e3ng 1:100) v\u00e0 nhu\u1ed9m m\u00e0u trong 1 ph\u00fat v\u1edbi 3, 3\u2032-diaminobenzidine tetrahydrochloride (Ph\u00f2ng th\u00ed nghi\u1ec7m Vector, Burlingame, CA, Hoa K\u1ef3). C\u00e1c phi\u1ebfn k\u00ednh \u0111\u01b0\u1ee3c nhu\u1ed9m trong 30 gi\u00e2y b\u1eb1ng hematoxylin v\u00e0 r\u1eeda s\u1ea1ch b\u1eb1ng n\u01b0\u1edbc m\u00e1y. Sau \u0111\u00f3, c\u00e1c l\u00e1t c\u1eaft n\u00e0y \u0111\u01b0\u1ee3c ph\u1ee7 l\u00ean v\u00e0 g\u1eafn b\u1eb1ng Permount\u2122 Mounting Medium (Thermo Fisher Scientific, Waltham, MA, USA). T\u1ea5t c\u1ea3 c\u00e1c slide \u0111\u01b0\u1ee3c ki\u1ec3m tra v\u00e0 ch\u1ee5p \u1ea3nh b\u1eb1ng k\u00ednh hi\u1ec3n vi \u00e1nh s\u00e1ng (Olympus, CX43, Olympus Optical Co., Tokyo, Nh\u1eadt B\u1ea3n). Ph\u1ea7n m\u1ec1m ImageJ (phi\u00ean b\u1ea3n 1.53, Vi\u1ec7n Y t\u1ebf Qu\u1ed1c gia, Bethesda, MD, Hoa K\u1ef3)48<\/a><\/sup> \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u1ec3 th\u1ef1c hi\u1ec7n ph\u00e2n t\u00edch d\u1eef li\u1ec7u \u0111\u1ecbnh l\u01b0\u1ee3ng.<\/p>\n

Nu\u00f4i c\u1ea5y t\u1ebf b\u00e0o ARPE-19<\/h3>\n

C\u00e1c t\u1ebf b\u00e0o ARPE-19 bi\u1ec3u m\u00f4 v\u00f5ng m\u1ea1c \u1edf ng\u01b0\u1eddi (B\u1ed9 s\u01b0u t\u1eadp v\u0103n h\u00f3a ki\u1ec3u M\u1ef9, ATCC, Manassas, VA, Hoa K\u1ef3) \u0111\u01b0\u1ee3c nu\u00f4i c\u1ea5y tr\u00ean m\u00f4i tr\u01b0\u1eddng DMEM \/ F-12 (Gibco, Carlsbad, CA, USA) \u0111\u01b0\u1ee3c b\u1ed5 sung 10% FBS (Ph\u00f2ng th\u00ed nghi\u1ec7m HyClone, Logan, UT , Hoa K\u1ef3) v\u00e0 1% penicillin\/streptomycin (Ph\u00f2ng th\u00ed nghi\u1ec7m HyClone). T\u1ebf b\u00e0o \u0111\u01b0\u1ee3c gieo h\u1ea1t v\u1edbi m\u1eadt \u0111\u1ed9 2\u00d7105<\/sup> m\u1ed7i gi\u1ebfng trong c\u00e1c \u0111\u0129a 6 gi\u1ebfng v\u00e0 nu\u00f4i c\u1ea5y \u0111\u1ec3 t\u1ea1o ra ph\u1ea3n \u1ee9ng vi\u00eam v\u00e0 n\u1ed9i ch\u1ea5t.<\/p>\n

Kh\u1ea3 n\u0103ng di \u0111\u1ed9ng<\/h3>\n

\u1ea2nh h\u01b0\u1edfng c\u1ee7a T. lutea<\/i> v\u1ec1 kh\u1ea3 n\u0103ng s\u1ed1ng c\u1ee7a t\u1ebf b\u00e0o \u0111\u01b0\u1ee3c \u0111\u00e1nh gi\u00e1 b\u1eb1ng x\u00e9t nghi\u1ec7m MTT nh\u01b0 nghi\u00ean c\u1ee9u \u0111\u00e3 m\u00f4 t\u1ea3 tr\u01b0\u1edbc \u0111\u00e2y49<\/a><\/sup> nh\u01b0ng v\u1edbi nh\u1eefng s\u1eeda \u0111\u1ed5i nh\u1ecf. T\u1ebf b\u00e0o \u0111\u01b0\u1ee3c c\u1ea5y v\u00e0o \u0111\u0129a nu\u00f4i c\u1ea5y 96 gi\u1ebfng v\u1edbi m\u1eadt \u0111\u1ed9 1,5 \u00d7 104<\/sup> t\u1ebf b\u00e0o\/gi\u1ebfng v\u00e0 \u1ee7 trong 24 gi\u1edd. C\u00e1c t\u1ebf b\u00e0o sau \u0111\u00f3 \u0111\u01b0\u1ee3c x\u1eed l\u00fd v\u1edbi c\u00e1c n\u1ed3ng \u0111\u1ed9 kh\u00e1c nhau c\u1ee7a T. lutea<\/i> v\u00e0 \u1ee7 th\u00eam 24h n\u1eefa. Ph\u1ea7n n\u1ed5i ph\u00eda tr\u00ean c\u1ee7a m\u1ed7i gi\u1ebfng \u0111\u01b0\u1ee3c thay th\u1ebf b\u1eb1ng m\u00f4i tr\u01b0\u1eddng t\u0103ng tr\u01b0\u1edfng m\u1edbi ch\u1ee9a 0,5 mg\/mL MTT v\u00e0 \u1ee7 trong 1 gi\u1edd. Ch\u1ea5t n\u1ed5i ph\u00eda tr\u00ean \u0111\u01b0\u1ee3c lo\u1ea1i b\u1ecf v\u00e0 c\u1eb7n formazan t\u1ea1o ra \u0111\u01b0\u1ee3c h\u00f2a tan trong 200 \u00b5L dimethyl sulfoxide. \u0110\u1ed9 h\u1ea5p th\u1ee5 \u0111\u01b0\u1ee3c \u0111o \u1edf b\u01b0\u1edbc s\u00f3ng 570 nm. Kh\u1ea3 n\u0103ng t\u1ed3n t\u1ea1i c\u1ee7a t\u1ebf b\u00e0o \u0111\u01b0\u1ee3c t\u00ednh to\u00e1n t\u01b0\u01a1ng \u0111\u1ed1i theo t\u1ef7 l\u1ec7 ph\u1ea7n tr\u0103m so v\u1edbi c\u00e1c nh\u00f3m \u0111\u1ed1i ch\u1ee9ng \u00e2m t\u00ednh.<\/p>\n

Ph\u00e2n t\u00edch Western blot<\/h3>\n

Ph\u01b0\u01a1ng ph\u00e1p Western blot \u0111\u00e3 \u0111\u01b0\u1ee3c ti\u1ebfn h\u00e0nh \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh c\u00e1c con \u0111\u01b0\u1eddng truy\u1ec1n t\u00edn hi\u1ec7u t\u1ebf b\u00e0o l\u00e0m c\u01a1 s\u1edf cho t\u00e1c d\u1ee5ng ch\u1ed1ng vi\u00eam c\u1ee7a T. lutea<\/i> nh\u01b0 \u0111\u00e3 m\u00f4 t\u1ea3 nghi\u00ean c\u1ee9u tr\u01b0\u1edbc \u0111\u00e2y49<\/a><\/sup>, nh\u01b0ng v\u1edbi nh\u1eefng s\u1eeda \u0111\u1ed5i nh\u1ecf. C\u00e1c t\u1ebf b\u00e0o ARPE-19 h\u1ee3p l\u01b0u b\u1ecb b\u1ecf \u0111\u00f3i trong DMEM: F12 ch\u1ee9a kh\u00e1ng sinh 1% FBS v\u00e0 1% trong 24 gi\u1edd. Sau \u0111\u00f3 t\u1ebf b\u00e0o \u0111\u01b0\u1ee3c x\u1eed l\u00fd b\u1eb1ng T. lutea<\/i> (50 v\u00e0 100 \u03bcg\/mL) trong 2 gi\u1edd v\u00e0 sau \u0111\u00f3 \u0111\u01b0\u1ee3c k\u00edch th\u00edch b\u1eb1ng 20 ng\/mL TNF-\u03b1 trong 15 ph\u00fat. C\u00e1c t\u1ebf b\u00e0o \u0111\u01b0\u1ee3c r\u1eeda ba l\u1ea7n b\u1eb1ng DPBS l\u1ea1nh nh\u01b0 b\u0103ng v\u00e0 sau \u0111\u00f3 \u0111\u01b0\u1ee3c l\u1ecdc trong dung d\u1ecbch \u0111\u1ec7m chi\u1ebft protein (C\u00f4ng ngh\u1ec7 sinh h\u1ecdc iNtRON, Kyunggi-do, H\u00e0n Qu\u1ed1c) \u0111\u01b0\u1ee3c b\u1ed5 sung m\u1ed9t lo\u1ea1i cocktail g\u1ed3m c\u00e1c ch\u1ea5t \u1ee9c ch\u1ebf protease v\u00e0 phosphatase. Ch\u1ea5t n\u1ed5i ph\u00eda tr\u00ean c\u1ee7a lysate thu \u0111\u01b0\u1ee3c b\u1eb1ng c\u00e1ch ly t\u00e2m (14.000 \u00d7g<\/i>, 30 ph\u00fat, 4 \u00b0 C) v\u00e0 n\u1ed3ng \u0111\u1ed9 protein c\u1ee7a ch\u00fang \u0111\u01b0\u1ee3c \u01b0\u1edbc t\u00ednh b\u1eb1ng c\u00e1ch s\u1eed d\u1ee5ng b\u1ed9 x\u00e9t nghi\u1ec7m protein DCTM (Bio-Rad, Hercules, CA, USA). L\u01b0\u1ee3ng b\u1eb1ng nhau (15\u201325 \u03bcg) protein t\u1ebf b\u00e0o \u0111\u01b0\u1ee3c ph\u00e2n t\u00e1ch b\u1eb1ng SDS-PAGE v\u00e0 chuy\u1ec3n v\u00e0o m\u00e0ng PVDF. M\u00e0ng \u0111\u01b0\u1ee3c \u1ee7 v\u1edbi albumin huy\u1ebft thanh b\u00f2 5% ho\u1eb7c s\u1eefa g\u1ea7y, sau \u0111\u00f3 \u0111\u01b0\u1ee3c \u1ee7 v\u1edbi c\u00e1c kh\u00e1ng th\u1ec3 ch\u00ednh pha lo\u00e3ng 1:2000 (ch\u1ed1ng phopho-ERK, ch\u1ed1ng ERK, ch\u1ed1ng phospho-JNK, ch\u1ed1ng JNK, ch\u1ed1ng phospho-p38 , anti-p38, anti-phospho-AKT, anti-AKT, anti-phospho-I\u03baB-\u03b1, anti-I\u03baB-\u03b1, anti-phospho-p65, anti-p65, anti-\u03b1-tubulin v\u00e0 anti-\u03b2-action ) \u1edf 4\u00b0C v\u00e0 12 gi\u1edd. Sau \u0111\u00f3, c\u00e1c m\u00e0ng n\u00e0y \u0111\u01b0\u1ee3c r\u1eeda b\u1eb1ng TBS-T v\u00e0 x\u1eed l\u00fd trong 1 gi\u1edd \u1edf nhi\u1ec7t \u0111\u1ed9 ph\u00f2ng b\u1eb1ng kh\u00e1ng th\u1ec3 th\u1ee9 c\u1ea5p li\u00ean h\u1ee3p HRP ch\u1ed1ng th\u1ecf \u1edf \u0111\u1ed9 pha lo\u00e3ng 1:10.000. C\u00e1c d\u1ea3i protein \u0111\u01b0\u1ee3c ph\u00e1t hi\u1ec7n b\u1eb1ng c\u00e1ch s\u1eed d\u1ee5ng Ch\u1ea5t n\u1ec1n c\u00f3 \u0111\u1ed9 nh\u1ea1y t\u1ed1i \u0111a SuperSignal West Femto (Thermo Fisher Khoa h\u1ecdc) v\u00e0 hi\u1ec3n th\u1ecb trong h\u1ec7 th\u1ed1ng t\u00e0i li\u1ec7u gel iBright CL1000 (Thermo Fisher Khoa h\u1ecdc). M\u1eadt \u0111\u1ed9 c\u1ee7a t\u1eebng \u0111\u1ed1m protein \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh b\u1eb1ng ph\u1ea7n m\u1ec1m Image J (phi\u00ean b\u1ea3n 1.53, NIH)48<\/a><\/sup>.<\/p>\n

T\u1ed5ng s\u1ed1 chi\u1ebft xu\u1ea5t PUFA t\u1eeb T. lutea<\/i><\/h3>\n

T\u1ed5ng s\u1ed1 PUFA \u0111\u01b0\u1ee3c chi\u1ebft xu\u1ea5t t\u1eeb T. lutea<\/i> s\u1eed d\u1ee5ng k\u1ef9 thu\u1eadt Folch50<\/a><\/sup>. T\u00f3m l\u1ea1i, 100 mg m\u1eabu \u0111\u01b0\u1ee3c cho v\u00e0o l\u1ecd ch\u1ee9a 5 mL CH3<\/sub>Dung d\u1ecbch Cl:MeOH (2:1, v\/v) v\u00e0 si\u00eau \u00e2m trong 15 ph\u00fat. Vi\u1ec7c \u0111\u00ecnh ch\u1ec9 \u0111\u01b0\u1ee3c l\u00e0m r\u00f5 b\u1eb1ng c\u00e1ch ly t\u00e2m \u1edf t\u1ed1c \u0111\u1ed9 10.000 v\u00f2ng \/ ph\u00fat trong 5 ph\u00fat. Na2<\/sub>V\u00cc TH\u1ebe4<\/sub> \u0111\u01b0\u1ee3c th\u00eam v\u00e0o ph\u1ea7n n\u1ed5i ph\u00eda tr\u00ean, sau \u0111\u00f3 \u0111\u01b0\u1ee3c l\u1ecdc \u0111\u1ec3 lo\u1ea1i b\u1ecf l\u01b0\u1ee3ng n\u01b0\u1edbc c\u00f2n l\u1ea1i. B\u01b0\u1edbc n\u00e0y \u0111\u01b0\u1ee3c l\u1eb7p l\u1ea1i hai l\u1ea7n n\u1eefa. Dung d\u1ecbch n\u00e0y sau \u0111\u00f3 \u0111\u01b0\u1ee3c c\u00f4 \u0111\u1eb7c trong kh\u00ed nit\u01a1 \u0111\u1ec3 thu \u0111\u01b0\u1ee3c lipit t\u1ea3o tinh khi\u1ebft.<\/p>\n

Methyl h\u00f3a axit b\u00e9o<\/h3>\n

Qu\u00e1 tr\u00ecnh methyl h\u00f3a axit b\u00e9o \u0111\u01b0\u1ee3c th\u1ef1c hi\u1ec7n \u0111\u1ec3 chu\u1ea9n b\u1ecb m\u1eabu lipid cho ph\u00e2n t\u00edch s\u1eafc k\u00fd kh\u00ed (GC). V\u1eadt li\u1ec7u \u0111\u00e3 chi\u1ebft \u0111\u01b0\u1ee3c b\u1ed5 sung v\u00e0o l\u1ecd 10 mL, sau \u0111\u00f3 l\u00e0 1 mL h\u1ed7n h\u1ee3p NaOH:MeOH 0,5 N. Dung d\u1ecbch n\u00e0y ch\u1ee9a 400 \u03bcg\/mL s\u1ee3i b\u00ean trong (axit valeric). H\u1ed7n h\u1ee3p \u0111\u01b0\u1ee3c l\u1eafc trong 30 gi\u00e2y v\u00e0 sau \u0111\u00f3 \u0111\u01b0\u1ee3c \u1ee7 trong 20 ph\u00fat \u1edf 80 \u00b0 C. H\u1ed7n h\u1ee3p n\u00e0y sau \u0111\u00f3 \u0111\u01b0\u1ee3c l\u00e0m l\u1ea1nh \u1edf nhi\u1ec7t \u0111\u1ed9 ph\u00f2ng trong 5 ph\u00fat, sau \u0111\u00f3 th\u00eam 2 mL boron triflorua:MeOH v\u00e0o, sau \u0111\u00f3 khu\u1ea5y trong 30 gi\u00e2y. H\u1ed7n h\u1ee3p n\u00e0y \u0111\u01b0\u1ee3c \u1ee7 trong 20 ph\u00fat \u1edf 80 \u00b0 C sau \u0111\u00f3 l\u00e0m l\u1ea1nh trong 10 ph\u00fat. M\u1ed9t th\u1ec3 t\u00edch 2 mL NaCl si\u00eau b\u00e3o h\u00f2a \u0111\u00e3 \u0111\u01b0\u1ee3c th\u00eam v\u00e0o \u0111\u1ec3 t\u1ea1o ra s\u1ef1 t\u00e1ch pha v\u00e0 thu th\u1eadp axit b\u00e9o. Ti\u1ebfp theo, 1,5 mL N<\/i>-hexane \u0111\u01b0\u1ee3c th\u00eam v\u00e0o v\u00e0 h\u1ed7n h\u1ee3p \u0111\u01b0\u1ee3c khu\u1ea5y tr\u1ed9n trong 30 gi\u00e2y tr\u01b0\u1edbc khi l\u00e0m ngu\u1ed9i \u1edf nhi\u1ec7t \u0111\u1ed9 ph\u00f2ng trong 20 ph\u00fat. C\u00e1c l\u1edbp \u0111\u01b0\u1ee3c t\u00e1ch ra v\u00e0 ch\u1ea5t l\u1ecfng n\u1ed5i ph\u00eda tr\u00ean \u0111\u01b0\u1ee3c lo\u1ea1i b\u1ecf kh\u1ecfi h\u1ed7n h\u1ee3p v\u00e0 n\u01b0\u1edbc \u0111\u01b0\u1ee3c lo\u1ea1i b\u1ecf ho\u00e0n to\u00e0n b\u1eb1ng dung d\u1ecbch Na b\u00e3o h\u00f2a.2<\/sub>V\u00cc TH\u1ebe4<\/sub> l\u1ecdc.<\/p>\n

X\u00e1c \u0111\u1ecbnh axit b\u00e9o<\/h3>\n

C\u00e1c metyl este c\u1ee7a axit b\u00e9o (FAME) \u0111\u00e3 \u0111\u01b0\u1ee3c ki\u1ec3m tra b\u1eb1ng GC \u0111\u01b0\u1ee3c trang b\u1ecb m\u00e1y d\u00f2 ion h\u00f3a ng\u1ecdn l\u1eeda (FID; Agilent 7890A, Agilent Technologies, Wilmington, DE, USA). C\u00e1c m\u1eabu (1 \u00b5L) \u0111\u01b0\u1ee3c b\u01a1m v\u00e0o c\u1ed9t HP-88 c\u1ee7a GC (J&W 112-88A7, Agilent Technologies, Wilmington, DE, USA) (100 m \u00d7 250 \u03bcm ID, m\u00e0ng 0,2 \u03bcm). Ph\u00e2n t\u00edch GC \u0111\u01b0\u1ee3c th\u1ef1c hi\u1ec7n nh\u01b0 sau: ban \u0111\u1ea7u c\u00e1c m\u1eabu \u0111\u01b0\u1ee3c gi\u1eef \u1edf 140\u00b0C trong 5 ph\u00fat, sau \u0111\u00f3 nhi\u1ec7t \u0111\u1ed9 \u0111\u01b0\u1ee3c t\u0103ng l\u00ean \u1edf 4\u00b0C\/ph\u00fat \u0111\u1ebfn 240\u00b0C trong 15 ph\u00fat. T\u1ed1c \u0111\u1ed9 d\u00f2ng c\u1ee7a c\u1ed9t \u0111\u01b0\u1ee3c c\u1ed1 \u0111\u1ecbnh \u1edf m\u1ee9c 1 mL\/ph\u00fat. Kh\u00ed nit\u01a1 \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng l\u00e0m kh\u00ed mang cho ph\u00e2n t\u00edch, FID \u0111\u01b0\u1ee3c \u0111\u1eb7t \u1edf 280\u00b0C v\u00e0 t\u1ef7 l\u1ec7 ph\u00e2n chia l\u00e0 30:1. FAME \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh b\u1eb1ng c\u00e1ch so s\u00e1nh ch\u00fang v\u1edbi c\u00e1c ti\u00eau chu\u1ea9n \u0111\u00e3 bi\u1ebft. M\u1ed7i m\u1eabu \u0111\u01b0\u1ee3c \u0111o b\u1eb1ng ph\u00e2n t\u00edch GC ba l\u1ea7n.<\/p>\n

Ph\u00e2n t\u00edch th\u1ed1ng k\u00ea<\/h3>\n

T\u1ea5t c\u1ea3 d\u1eef li\u1ec7u \u0111\u01b0\u1ee3c ph\u00e2n t\u00edch th\u1ed1ng k\u00ea b\u1eb1ng c\u00e1ch s\u1eed d\u1ee5ng ANOVA m\u1ed9t chi\u1ec1u v\u1edbi ph\u00e2n t\u00edch h\u1eadu k\u1ef3 c\u1ee7a Tukey b\u1eb1ng SPSS phi\u00ean b\u1ea3n 18.0 (IBM Co., Armonk, NY, USA). \u00cdt nh\u1ea5t ba l\u1ea7n l\u1eb7p l\u1ea1i \u0111\u1ed9c l\u1eadp \u0111\u00e3 \u0111\u01b0\u1ee3c th\u1ef1c hi\u1ec7n cho m\u1ed7i th\u00ed nghi\u1ec7m.<\/p>\n<\/div>\n

[qu\u1ea3ng c\u00e1o_2]<\/p>\n

Li\u00ean k\u1ebft ngu\u1ed3n <\/a><\/p>","protected":false},"excerpt":{"rendered":"

The prevalence of dry eye syndrome (DES) has increased worldwide, affecting around 50% of all adults1. DES is a multifactorial ocular surface disease characterized by tear film instability and hyperosmolarity, ocular surface inflammation and damage, neurosensory abnormalities<\/p>","protected":false},"author":1,"featured_media":5363,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"default","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"default","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"set","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"ast-content-background-meta":{"desktop":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"footnotes":""},"categories":[9],"tags":[],"class_list":["post-4903","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-supplements"],"_links":{"self":[{"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/posts\/4903","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/comments?post=4903"}],"version-history":[{"count":1,"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/posts\/4903\/revisions"}],"predecessor-version":[{"id":5393,"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/posts\/4903\/revisions\/5393"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/media\/5363"}],"wp:attachment":[{"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/media?parent=4903"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/categories?post=4903"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/directcm.com\/vi\/wp-json\/wp\/v2\/tags?post=4903"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}