TAP cm SINH HQC, 2012, 34(4): 515-520
NGHlfeN ClTU TAC DVNG BAO V | TE BAO THAN KINH CUA o C O N O T O X I N MVIIA TAI T 6 H O P d D^NG PROTEIN DUNG HOP v O l THIOREDOXIN
Bdi Thj HuyJn, Dodn Vift Binh*, NguyJn Thj Kim Dung, L£ Thj BIch Thdc, NguySn Bich Nhi, Phan Vfin Chi
Vifn Cdng nghf sinh hpc, *[email protected]
TOM TAT: Omega-conotoxin MVllA Id mflt loai peptide cfl tdc dting khoa chpn Ipc cdc kenh ion Ca^*
tai cac khdp ciia te bdo IhAn kinh vk do dfl, cfl tdc dyng h^in ch^ dflng Ca^* Iran vio trong tfi bao va bdo vf 16 bao ihSn kinh. Bao cio ndy trinh bdy kel qud thir nghifm tdc dyng bSo vf ll bjlo ihSn kinh cua Omega- conotoxin MVIIA lai 16 hpp d d{ing dung hpp vdi ihiorcdoxin (Trx-CTX) do Vifn Cflng nghf sinh hpc chi tao tr6n mo hinh chuflt nhflt thi^ii mau lodn bfl ndo trong 5 phiil vk 10 phut va mfl hinh chuflt nhat ihiiu mdu eye bfl nao. Trx-CTX dupc ticm tinh m^ch mflt Idn vdi li^u 5 hoflc 10 mg/kg trong mo hinh thi§u mau toan bfl nao vd ticm hui lan lieu 5 mg/kg trong infl hinh thilu mdu c^ic bfl nSo. K^t qua cho thdy Trx- CTX cfl tac dyng gidm so te bao CAI ciia viing doi hai mi! bj chit, giam the lich nao bj ton thuong va co tac dyng bdo vf tfi bao thdn kmh trong cac mo hinh tren. Trx-CTX co the dupc sir dyng lam nguyen lifu cho cac cflng trinh nghien cim tiip theo vdi myc dich lam thudc chtta bfnh cho ngudi.
Tif khoa: Bao vf thdn kinh, conotoxin, dflt quy, omega-conotoxin MVIIA, Trx-CTX, MO DAU
Ca^*" cfl vai trd quan trpng trong ho^t dflng ciia tl bdo than kinh. Ca^* tham gia vdo qud trinh ddn truyfn tin hifu thdn kinh, true tifp difu hfla ho^l dflng cua protein mang, ciia cac enzym vd cdc protein tham gia difu hfla qud trinh sao chep trong bifu hifn gen. CJ trang thai hoat dflng binh thudng, hdm lupng Ca^* trong vd ngodi tf bdo thdn kinh dupc giCi d trang thai can bdng bdi cdc cachf rdt tinh vi [16]. Cac co chi nay hoat dpng phu thuflc rdt chat chg vao trang thai ndng lupng cung nhu vao hoat dflng cita tl bdo vd giup cho lupng Ca^* trong te bdo dupc gid dn djnh trong mflt gifli hgn rpng. Tuy nhien, niu lupng Ca^* trong tf bdo vupt qud gioi ban nay thi Ca^*se gdy ra mflt loat cdc phdn dng cfl h?ii cho tl bao vd cd thf lam te bao bj chit [7].
Dot quy Id mpt bfnh hay xdy ra d nhiJng Dgirfli cao mfli ho§c nhOng bfnh nhan bi bfnh tim m?ich vd thudng dl I?i di chdng nguy hifm.
Trong can dflt quy, do mach mdu bj nghSn I^i, man khdng dua dupc din mflt sd viing cua nao Idm cho nhOng vung nao nay khflng dupc cung cdp du 6 xy vd ndng lupng. Sy thilu hut ndng lupng sS Idm cho mdng ll bao bj khu eye vd pha vd su can bdng Ca^*. Ham lupng Ca^* trong tf bao tdng vupt qud gidi h^u vi Idm tl bao thdn kinh bi tdn thuong [7].
Df chdng Igi tdc hai gay ra do Ca tdng dflt ngflt trong can dflt quy, gdn day, nguoi ta chu y din nhung lo?i chdt co khd ndng ch$n dong Ca^*
tu ben ngodi vdo trong le bdo qua cdc kenh ion vd do dfl cfl thf cd tac dyng bdo vf tf bao than kinh. Nhifu lo^i thuflc ndy dd chdng td co hifu qud trfn dflng vdt thyc nghifm [11, 12,14].
Omega-conotoxin MVIIA Id mflt loai peptide nho dupc tdch chift td bdu dflc ciia loai dc cdi biSn C magus, cd tdc dyng khod chpn Ipc cac kf nil ion Ca^* lai cac khflp (synapse) cua tf bao thdn kinh [2, 8]. Cho d€n nay, dd cfl nhifu cflng trinh nghifin cdu chdng minh mflt sfl lo^u to-conotoxin MVIIA tflng hpp hfla hpc cfl khd ndng bdo vf tl bdo thdn kinh tren mo hinh gay thilu mdu ndo thyc nghifm [4, 13, 15].
Omega-conotoxin MVIIA da dupc tdch ddng, bilu hifn trudc tien d dang protein dung hpp vdi thioredoxin (Trx-CTX), sau dfl dirpc linh chi thdnh cflng (co-CTX) t?i Phdng Hfla sinh protein, Vifn Cong nghf sinh hpc. Omega- conotoxin MVIIA d dgng protein dung hpp cfl thi dupc sdn xudt vdi khfli lupng Ifln va dupc tinh chi theo mflt quy trinh khflng phuc tap [6], vi vdy sg rdt thudn Ipi nfu dupc dung lam thuflc.
Bai bao ndy trinh bay cdc kit qua nghifn cdu khd ndng sd dung Trx-CTX do Vien Cflng nghe sinh hpc tao ra de bdo vf ll bao thdn kinh
Bui Thi Huyen et al.
tren md hinh gay dflt quy thyc nghifm trfn chuflt nhdt,
VAI Ll$lJ VA IMIUONG PHAP INGH|£N CVV Vilt lifu
Trx-CTX chi t^o igi Phflng Hod sinh protein, Vifn Cflng nghf sinh hpc. Ketamin ciia hdng Rliflne Mfrcux, 2.3,5- Triphenyltetrazolium chloride (TTC) ciia Merck, chi khdu Surclon* ciia Suremed ( USP size 6/0). hematoxylin - cosin cua hdng Thenno Shandon, kinh hifn vi BX51 cua hdng Olympus.
Chuflt thi nghifm Id chuflt nhflt trflng dflng Swiss, gidng dye. 2 thdng tufli, trpng lupng trung binh la 40g/con, nufli bflng thirc fln vifn cd thdnh phdn protein 23%, md 5%, carbonhydrate 45-55%, xo 5%.
Phuong phdp
M6 hinh thieu mau toan bp nao Thieu mdu trong 5 phut
Mfl hinh thilu mdu loan bfl ndo dupc thyc hifn theo phuang phdp ciia Valentino et al, (1993) [15]. Chuflt dupc gdy me bdng ketamin vfli lifu 60yg/g, sau dfl dupc cfl djnh ndm ngua trfn bdn md. Dimg dao mfl r?ch mflt dudng d gida cfl dai khoang I -2 cm qua da co chupt rfli bflc tdch cho den khi nhin thay dflng m^ch cdnh hai ben. Dung kim lufln spi chi khdu tdch dflng mach canh hai ben rfli thdt Igi. Sau 5 phiit thi nfli long, rfli thao hfln spi chi ra. Sau dfl khdu Idp da ngodi lai. Chuflt d Ifl thi nghifm lifn sau khi thdo chi thdt dflng mgch cdnh 45 phut, dupc tiem vdo iTnh mach Trx-CTX vfli lieu 5 mg/kg.
Sau 24 gid thi gilt chuflt, mfl Idy ndo, cd djnh nao trong dung djch phflc mfln trung tinh, ddc parafin, cdt Idt vd nhupm tieu ban bflng hemaioxylin-eosin rdi dpc vd chyp mau tieu bdn tren kinh hifn vi.
Thieu mdu trong 10 phut
Chuflt dupc gdy mf, mfl nhu trfn, rdi that dflng mach cdnh hai bfn. Sau 10 phut thi ndi long, rfli thdo hdn spi chi ra. Sau dd khdu ldp da ngodi lai. Chuflt d Ifl thi nghifm lien sau khi thao chi thdt dpng m^ch cdnh 45 phut, dupc tiem vao tTnh mgch Trx-CTX vdi lifu 10 mg/kg.
Sau 120 gid thi gilt chu$t, mfl Idy ndo, cdt lat va nhuom tieu ban bdng hematoxylin-eosin rfli
dpc vd chyp mSu tifu bdn trfn kinh hifn vi.
Md hinh thiiu mdu c^c bQ ndo
Hai muai con chuflt dupc chia dfu thdnh 2 16, mdi 16 10 con. M6 hinh thilu mdu eye bfl ndo dupc tiln hdnh theo phuang phdp ciia Pinte ct al. (2011) [10] cfl cdi tiln nhu sau: chuflt dupc guy mf bdng ketamin vdi lifu 60pg/g, sau dfl dupc c6 djnh nflm ngua tren bdn md. Dimg dao md r^ch mflt dudng d gida cd, khodng 1- 2cm qua da cd chuOt rdi bdc tdch cho din khi nhin thiiy dflng m^ch cdnh bfn trdi vd chd ch^c ba chia thdnh hai nhdnh. Dung kim lufln spi chi phSu thu^t (6/0) tdch ddng m^ch canh bfn trdi trong (left internal carotid artery) rdi that chi l^i.
Cd dl niit that nhu vdy rdi khdu Iflp da ngodi l^i. Chuflt d 16 thi nghifm liln sau khi thdo chi that dflng mgch cdnh 30 j>hut, dupc tifm vdo ITnh m^ch Trx-CTX vdi lifu 5 mg/kg vd sau 60 phut dupc tifm nhac l^i vdi lifu nhu tren. Sau 24 gid thi quan sdt, ddnh gid tr^ng thdi than kinh df cho diim theo thang diim ciia Berdeson et al. (1986) [1]. Sau 48 gid gilt chuflt, md Idy ndo, nhupm TTC dl xdc djnh thi tich phdn ndo bj tdn thucmg hojc cd djnh ndo trong dung djch formol trune tinh, due parafin, cdt Idt vd nhupm tieu bdn bdng hematoxylin-eosin rfli dpc va chyp mdu tieu bdn Uf n kinh hien vi.
Ddnh gid muc dg tdn thuong thdn kinh
Mdc dfl tfln thuong thdn kinh cua chupt sau khi md dupc tiln hdnh ddnh gid sau khi mfl 24 gid vd cho diim theo phuang phdp cua Berdeson et al. (1986) [1]. Difrn O: chuflt vfn dflng binh thirdng, khflng cfl bifu hifn tdn thuong thdn kinh; diim 1: chuflt bj cong gdp chdn trudc; difm 2: chuOt bi ddy trdi vf phia tay bj lift; difm 3:
Chudt cfl bifu hifn chgiy vdng trdn.
Do the tich phdn ndo bf l6n thuang Phdn ndo chuflt bj tdn thuang dirpc xdc djnh theo phuang phdp ciJa Pateliya et al. (2008) [9]
cfl cdi tiln nhu sau: ndo chuflt dirpc cdt thanh nhilng Idt cdt cd chilu ddy 1mm rdi nhupm cac Idt cat trong dung djch TTC 1% trong 30 phdt, Ldt cdt se cfl nhdng vCmg bat mdu do khflng dfu nhau: do sdm hojc hdng tdi nh^t. Viing bdt mdu It han chinh Id vung ndo bj tdn thuang. Sau do d$t Idt cdt trfn mflt philn kinh. Trfn bl mflt ldt cat d%\. mOt tdm nylon mdng trong sudt cfl kf .cdc 6 vuong nhd cfl difn tich 1 mm . Dim cac 6
TAP CHi SINH HQC, 2012, 34(4) 515-520 vudng nhfl dl tinh thf tich phdn nao bj tdn
thucmg vd thi tich ctia mdi ldt cat, Cudi cung cflng cac sd Iifu trfn tdt cd cdc ldt cdt I^i df tinh tflng sfl.
Nghien ciru to chirc hgc mo ndo Nao cua chuflt tai phdn lung ciia viing hdi ma (dorsal hippocampus ) dupc cdt thanh cdc ldt day 7 ^m dl nhuflm HE vd quan sdt hinh thdi cdc tl bdo thdn kinh CAI (hinh I).
Hmh 1. Hinh dnh nhupm HE cua lat cat vung lung ddi hdi md vdi tl bdo C A1 (hinh chir nhdl).
Dp phdng dai 40 lan KET QUA VA THAO LU^N
Tac dyng cua Tra-CTX trong truong hpp thieu mdu todn bp nao
Thieu mdu trong 5 phut
Ket qud nghien cihi tfl chdc hpc cua mdu
ndo chuflt cho thdy, ndo chuflt bj tliilu mdu trong 5 phut cfl nliifu ll bao CA 1 ciia dfli hdi md cfl bifu hifn bj mdt mdng ll bdo, nguyfn sinh chdt tang bat mdu dfl bj vfln lai, ho$c chi cfln Id nhOng manh vyn (hinh 2A), chung tfl nhOng tl bdo ndy dd bi thuang tfln, thdm chi bj chft. Trong khi dfl, chuflt bj thilu mdu ndo nhimg dupc tifm Trx-CTX cfl da sd ll bdo thdn kinh cfl hinh dnh tl bao binh thudng, chi cd rdt it tl bao cfl bilu hifn bi tdn ihirang (hinh 2B).
ThiC'u mdu trong 10phut
Ndo cliufll bi ihilu mdu liong 10 phiil cfl bicu hifn bj ifln thucmg njng han chupt bj thilu mdu trong 5 phiit. Tuy nhien, ndo chupt dupc ticm Trx-CTX (hinh 3D) vdn cd il tl bdo bi tdn thuong han ndo chuflt khflng duac tiem (hinh 3C).
Tdc dyng ciia Trx-CTX trong trudng hpp thilu mdu eye bfl ndo
Ddnh gid mire dg ton thuang thdn kinh Ket qud xdc djnh muc dp tfln thuong nao theo thang diim ciia Berdeson et al. (1986) [1]
cho thdy chuflt d 16 ddi chung khflng dupc tiem Trx-CTX cfl mdc do nao bi ton thuong ndng ban (nhilu con bi cong gap c6 chan Uudc, bi lift nhe chan trudc phdi vd cfl xu hudng chay vdng iron) vd do do co sfl difm cao ban so vdi chuflt fl Ifl thi nghifm dupc tiem Trx-CTX (da sfl chuflt chi bi cong nhe cfl chan trudc phai, chay deu hai ben) (bdng 1).
Bdng I. Kft qua xdc dinh mdc dp tfln thuang cua nao va do thf lich phdn nao bi tfln thucmg Chi lieu
Muc dfl tfln thuang ciia nao (difm) The tich ndo bi ton thuang (mm^)
Lfl dfli chdng ) Lfl thi nghiem 2,38 ± 0,32
22,00 ±2,70
0,93 ±0,13 11,44±1,17 Do the tich ndo bj ton thuang
Thi tich nao bj tfln thuang ciia chuflt d lfl ddi chiing do dupc Id 22,00 mm', nhieu han han so vdi 16 thi nghifm la 11,44 mm'.
To chdc hpc md ndo
Ndo chudt cua 16 dfli chdng khflng dupc tiem Trx-CTX bi tfln thuang nhifu ban Ifl thi nghifm dupc tifm Trx-CTX cung thf hifn rfl trfn hinh dnh td chdc hpc ciia mo nao (hinh 4).
Nao chuflt ciia 16 ddi chung cfl nhilu tl bao
CAI bi chft (hinh 4E), trong khi da sfl le bao ciia ndo chupt Ifl thi nghiem dfu cfl hinh dnh binh thudng, kliflng bj thucmg tdn (hinh 4F).
THAO LUAN
Dpi quy Id mpt bfnh nguy hiem, tliuong xdy ra do dflng m^ch canh hay nhanh Ifn ndo cua nfl bi thdt lai hay bj tdc hoan todn. Trong trudng hpp nay, lupn§ mau Ifn nao bi gidm manh, lam cho nao bi thifu fl xy, Khi dfl se xdy ra mot loat cdc phdn dng nhu tdng cuong dudng phdn hilm klii
Bui Thi Huyen el al dan din t;in kifl ATP lam cho mdng te bdo bi
phdn eye, lc bdo tdng cudng lich IQy glutamale vd sy tich liiy chdt iiiiy Idm iDng d^t ngdl lupng Ca"' di vdo trong lc bdo. Ca'' s5 ho(il hda cdc
enzym protease, phospholipase vd nhiing enzym ndy kit hpp vdi nhilu chdt khdc sf phd huy mdng tl bdo, cdc vi spi, ty thi ciia tl bdo than kinh vd cudi cdng Id 1dm chit tl bdo [16],
///;)/) .'. Anil nhiioni HE lc bao CAI nao chuol ihiOii niiiii IIKUI ho lulo irong III phut C niiini (i(ii durnii | mu. icii clii lc h;io hi Ion ihu-onyl. [), C hiioi Dom \v\-i VX Do |ihong d.ii 4(in \kn.
Hinh 4_ Anh nhuflm HE le bao CAI ndo chuflt thdt dflng m^ch cdnh trdi trong vTnh vifn.
E, Chupt doi chdng ( miii ten chi le bao bi ifln thuang), F. Chuflt tiem Trx-CTX . Dfl phflng dai 400 Idn.
Viing nSo thudng hay bi tfln thuang nhilu nhdl do thifu mdu nao Id viing doi hdi md, ddc biet la cac tf bdo CAI ciia nfl. Ddy cung Id vung CO vai trd rat quan trpng ddi vdi tri nhd cua con ngudi, Chinh vi vdy, cdc cdng trinh nghien cdu ve l6n thuang nao do dpi quy thuong l^p trung nghien cuu ve viing ndy.
Trong thi nghifm, chung tfli da sd dyng cdc mfl hinh gdy thieu mdu nao khac nhau: mfl hinh
gay thieu mdu ndo toan bfl vd mfl hinh thieu mdu ndo cue bp. Trong mfl hinh thifu mau nao todn bfl, thdi gian thieu mau nao ngan, trong 5 phiit vd trong 10 phut. Sau do thdi gian nufli chupt tiep theo ciing khdc nhau, tuong dng Id 24 gid va 120 gid. Ket qud trong ca 2 mfl hinh diu cho thay nao bj ton thuang vdi muc dp khac nhau: chupt thifu mdu trong 10 phiit va gift mfl sau 120 gid cd sfl lupng te bdo nao chet nhieu
TAP CHI SINH HQC, 2012,34(4): 515-520 hem chupt thilu mdu nflo trong 5 phut vd gilt
md sau 24 gid. Dilu dfl cho thdy, mdc dfl tin diuang cua ndo phy thuflc kh6ng nhOng vao thdi gian ndo bj thilu mdu md cfln vao thdi gian xdy ra sau dfl. Kit qua ndy phu hpp vdi kit qud thi nghifm cua Colboume et al. (1999) [5]. Tuy nhifn, cd 2 mfl hinh thi nghifm dfu cho thdy, chupt dupc tiem Trx-CTX cfl ndo it bj tfln thirong han chuflt khflng dupe tifm, chdng td Trx-CTX dd chgn dupc phdn ndo dflng Ca^* trdn vdo qud mdc trong tl bdo thdn kinh klii ndo bi thilu rndu vd do dfl dd cfl tdc dyng bdo vf cdc ic bdo thdn kinh.
Tdc dyng bdo vf tl bdo thdn kinh ciia Trx- CTX mpt Idn nfta dupc klidng djnh vd dupc thi hifn rfl trong mfl hinh thilu mdu ndo eye bfl.
Cdc kit i^ua do till tich ndo bj phd huy, xdc djnh mdc dfl tfln thuong nSo vd kit qud nghifn cdu id chuc hpc dfiu cho thdy ndo chuflt dupc tifm Trx-CTX ft bj tfln thuang han. Nhu vdy, Trx- CTX khflng nhOng ehi cd tdc dyng dfli vdi trudng hpp ndo bi thilu mdu thodng qua md con cfl tdc dyng khi mflt phdn cua ndo bi thifu mdu trong mflt thdi gian ddi. NIiQng kit qud nay rdt cfl -^ nghla trong vifc tim ra mflt loai thudc mdi dilu tri cho bfnh dflt quy d ngudi.
Trong mflt cflng trinh nghifn cdu khdc [6], chung tfli cung dd xdc dinh dupc Trx-CTX co ho?t tinh gidm dau rdt tflt. Trx-CTX cfl thf dupc sdn xudt vdi khfli lupng Ifln vd dupc tinh chf theo mflt quy trinh khflng phdc tap. Vi vdy, cfl the sd dyng Trx-CTX Idm nguyen lifu cho cac cflng trinh nghifn cdu tilp theo vdi muc dich Idm thudc chda bfnh cho ngudi.
Ktl LUAN
Omega-conotoxin MVIIA d dang protein dung hpp do Vifn Cflng n ^ f sinh hpc tao ra cd tac dyng bdo vf tl bao than kinh tren mfl hinh chuflt nhdt bi thilu mdu ndo todn bfl vd mfl hinh thifu mdu ndo cue bfl.
L&i cdm ffn: Cdng trinh dupc tai trp bfli kinh phi ciia dl tdi cdp Vifn Khoa hpc vd Cflng nghf Vift Nam (2011-2012, md sd: VAST03.02/11-I2 TAILIEU THAMKHAO
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L., 2010. Drugs fromslugs-Past, present and future perspectives of co-conotoxin research.
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RESEARCH ON NEUROPROTECTIVE EFFECTS O F A FUSION PROTEIN OF (O-CONOTOXIN MVIIA AND THIOREDOXIN
Bui Thi Huyen, Doan Vict Binh, Nguyen Thi Kim Dung, LeThi Bich Thao, >gu>en Bich Nhi, Phan Van Chi
Instimte of Biotechnology, VAST SUMMARY
Ischemic stroke is one of the most dangerous human diseases. Blood supply to the brain is decreased initiating the ischemic cascade with the consequence of a Ca^* influx into nerve-cells. This Ca^* influx can than cause neuronal injury or dcaih of cells. Omega-conotoxin MVIIA (o-CTX) is a neurotoxin isolated from the venom of Cone snail Conus magus. It is a potent selective blocker of the N-lype voltage-sensitive calcium channel in neurons and therefore has the potential to promote neuroprotcclion through inhibition of Ca^*
influx into nerve-cells, (O-CTX has been cloned, expressed in die fusion form with thioredoxin (Trx-CTX ) in E, coli in the institute of biotechnology, Hanoi. In this paper we present our sludy on ncuroprotectiv activity of Trx-CTX in mouse model of global cerebral ischemia and focal brain ischemia. The experiments were carried out in male mice of Swiss race weighing 40 g, Trx-CTX were injected i.v. in the model of global cerebral ischemia, one time with a dose of 5 or 10 mg/kg and in die odier model two times, each with a dose of 5 mg/kg. The results showed that in global cerebral ischemia Trx-CTX has decreased neuronal injury and death of CAI cells of dorsal hippogampus. In other model Trx-CTX also decreased infarction size of die ischemic brain. Trx-CTX proof to possess neuroprotective effect and could be used in further researchs for medicinal purpose.
Keywords: conotoxin, ischemia, ncuroprotectiv, omega-conotoxin MVIIA, Trx-CTX.
Ngdy nhdn bdi: 16-4-2012