B Y T TRNG I HC DC H NI NNG TH THANH PHNG NH GI TC DNG BO V T BO THN KINH V CI THIN KH NNG HC TP NHN THC CA PHN ON N-BUTANOL CHIT T CY RAU NG BINKHA LUN TT NGHIP DC S H NI - 2014B Y T TRNG I HC DC H NI NNG TH THANH PHNG NH GI TC DNG BO V T BO THN KINH V CI THIN KH NNG HC TP NHN THC CA PHN ON N-BUTANOL CHIT T CY RAU NG BIN KHA LUN TT NGHIP DC S Ngi hng dn: 1. TS. Th Nguyt Qu 2. TS. Phm Th Nguyt Hng Ni thc hin: Phng Dc l Sinh ha, Vin Dc liu _ S 3B, Quang Trung, Qun Hon Kim, H Ni H NI 2014i LI CM N Sau mt thi gian hc tp v nghin cu, em hon thnh kha lun tt nghip ny.Em xin by t li cm n su sc ti:TS. Th Nguyt Qu - ging vin b mn Dc lc - trng i hc Dc H Ni TS. Phm Th Nguyt Hng - Trng phng Dc l Sinh ha Vin Dc liu trung ng - nhng ngi thy trc tip hng dn, du dt v to mi iu kin thun li cho em trong qu trnh nghin cu v thc hin ti. Em cng xin gi li cm n n Tp th cn b phng Dc l Sinh ha -Vin Dc liu trung ng, nhit tnh gip , ch bo thm cho em nhiu kin thc qu bu, to iu kin tt nht cho em trong qu trnh thc hin ti. Em xin cm n ton th cc thy gio, c gio ca Trng i hc Dc H Ni, tn tnh ging dy cho em cng nh cc bn sinh vin ca trng trong sut nm nm hc tp. Cui cng, em xin gi li cm n gia nh, bn b quan tm, gip v to iu kin tt nht em hon thnh kha lun ny. H Ni, ngy 14 thng 5 nm 2014 Sinh vin Nng Th Thanh Phng ii MC LC Danh mc cc t vit tt ..........................................................................................iv Danh mc bng .......................................................................................................... v Danh mc hnh, s ............................................................................................... v T VN ............................................................................................................ 1 CHNG 1. TNG QUAN ..................................................................................... 3 1.1. Mt vi khi nim v bnh Alzheimer v chng sa st tr tu ................... 3 1.1.1. Khi nim .................................................................................................. 3 1.1.2. Phn loi .................................................................................................... 3 1.2. C ch bnh sinh ............................................................................................. 4 1.2.1. Gi thuyt beta Amyloid .......................................................................... 4 1.2.2. Gi thuyt m ri thn kinh (Neurofibrillary Tangles - NFTs)......... 5 1.2.3. Gi thuyt Cholinergic v nhng bt thng v cc cht dn truyn thn kinh khc .................................................................................................... 5 1.2.4. Cc cht gy vim trung gian .................................................................. 6 1.2.5. Cc c ch khc ........................................................................................ 7 1.3. Cc thuc iu tr Alzheimer ......................................................................... 8 1.3.1. Cc nhm thuc iu tr Alzheimer ........................................................ 8 1.3.2. Tacrin ........................................................................................................ 9 1.3.3. Hn ch ca cc thuc iu tr Alzheimer ........................................... 10 1.3.3.1. V hiu qu iu tr ............................................................................ 10 1.3.3.2. V tng tc thuc, tc dng khng mong mun .............................. 11 1.4. Nghin cu thuc iu tr Alzheimer .......................................................... 12 1.4.1. Nghin cu thuc nhm lm gim AP ............................................ 12 1.4.2. Nghin cu thuc tc dng trn cc ch khc ................................ 14 1.5. Mt s m hnh nghin cu thuc tc ng ln nhn thc v cc bnh sa st tr tu ............................................................................................................... 15 1.6. Vi nt v cy Rau ng bin ......................................................................... 17 2.1. Nguyn liu .................................................................................................... 21 iii 2.2. ng vt th nghim ...................................................................................... 21 2.3. Ha cht, trang thit b ................................................................................ 21 2.4. Phng php nghin cu ............................................................................. 22 2.4.1.nhgitcdngbovtbothnkinhcacnphnonn-butanolchittRaungbintrntbovnonguynphtgyc bng beta-amyloid ............................................................................................ 22 2.4.1.1. Phng php tch v nui cy t bo thn kinh v no nguyn pht 22 2.4.1.2. nh gi ctnh camu th trn t bo thn kinh v no nguyn pht .................................................................................................................. 23 2.4.1.3.nhgitcdngbovtbothnkinhcacnphnonn-butanol chit t Rau ng bin trn t bo v no nguyn pht gy c bng A 25-35 ......................................................................................................... 24 2.4.2. nh gi tc dng ci thin kh nng hc tp v ghi nh in vivo...... 26 2.4.2.1. Phng php gy thiu mu no cc b tam thi theo m hnh 2VO + hypo ................................................................................................................. 26 2.4.2.2. Th nghim m l nc Morris ......................................................... 26 2.5. X l s liu ................................................................................................... 29 3.1.Tcdngbovtbothnkinhtrnmhnhgyctbobng protein A25-35 ca cn phn on n-butanol chit t Rau ng bin ......... 30 3.1.1 nh hng ca mu nghin cu i vi kh nng sng st ca t bo thn kinh v no nguyn pht ......................................................................... 30 3.1.2Tcdngbovtbothnkinhvnonguynphtkhitn thng bi protein A25-35 ............................................................................ 31 3.2. Tc dng ci thin kh nng hc v nh in vivo bng th nghim m l nc Morris ca cn phn on n-butanol chit t Rau ng bin ............... 32 CHNG 4. BN LUN ....................................................................................... 36 4.1. V tc dng bo v t bo thn kinh ca phn on butanol chit t Rau ng bin ............................................................................................................... 36 4.2. V tc dng tng kh hc tp v tr nh in vivo ca phn on butanol chit t cy Rau ng bin .................................................................................. 38 KT LUN V KIN NGH ................................................................................ 45 TI LIU THAM KHO ...................................................................................... 46 iv Danh mc cc t vit tt ADAS-CogAlzheimer's Disease Assessment Scale-cognitive (Thang ng gi nhn thc trong bnh Alzheimer) ApoE4Apolipoprotein E4 APPAmyloid precursor protein (protein tin cht amyloid) BuOHn-butnol GSK3bglycogen synthase kinase 3bHDLHigh-density lipoprotein (lipoprotein t trng cao) ICD 10International Classification of Diseases 10th

(Phn loi bnh quc t ln th 10) LDLLow-density lipoprotein (lipoprotein t trng thp) NFTsNeurofibrillary Tangles (m ri thn kinh) NMDAN-methyl-D-aspartateA25-35Amyloid -Protein Fragment 25-35 ( on acid amin -amyloid peptid cha 11 acid amin) AP-amyloid peptidv Danh mc bng STTTrang 1Bng 1.1Phn loi chng sa st tr tu trong bnh Alzheimer4 2Bng 1.2 Cc nhm thuc iu tr triu chng suy gim nhn thc9 3Bng 2.1Phnnhmthkhnngsngstsaugyctbo bng A 25-35 25 4Bng 2.2 Phn nhm th nghim v iu tr27 5Bng 3.1Thigiantimtngtmthybncacclth nghim bi tp nhn thy bn 32 6Bng 3.2Thigiantimtngtmthybncacclth nghim bi tp khng nhn thy bn 33 Danh mc hnh, s STTTrang 1Hnh 1.1 Cu trc cc thnh phn Saponin chnh trong bacosid A18 2Hnh 2.1 Sccbcnhgikhnngsngsttrntbo gy c bng beta-amyloid26 3Hnh 2.2 S qui trnh bi tp trn m hnh m l nc Morris 28 4Hnh 2.3M hnh th nghim m l nc Morris29 5 Hnh 3.1nhhngcaphnonn-butanolRaungbini vi kh nng sng st ca t bo thn kinh v no sau 24 gi nui cy v th thuc 30 6Hnh 3.2 Tcdngbovtnthngtbothnkinhvno nguyn pht gy bi A 25-35 ca cn phn on BuOH chit t Rau ng bin 31 7Hnh 3.3 T l thi gian chut cung phn t ch34 1 T VN Sasttrtulhuqucanhiubnhlthnkinhkhcnhautrong,bnh Alzheimer l nguyn nhn ph bin nht gy nn chng sa st tr tu. Chng sa st trtugyrassuygimnnglcnhnthcvhnhvicabnhnhn,rtngn cucsngbnhnhnkhibnhtintrin.Cnbnhnyvnccoilbnhtui gi, do nguy c sa st tr tu tng ln theo tui. Theo bo co ca T chc y t th gii (WHO) nm 2012, khu vc u Mbnh hay gp nht tui 80-89, chul75-84tuivChuPhihaygpnhttrongtui70-79[101].Tuy nhin,tuimcbnhhinanggimdn[49],[101].Ccnhnghincuc tnh trn ton th gii c gn 7,7 triu ca mi mc mi nm. Chng sa st tr tu cn tng gnh nng cho gia nh bnh nhn, cho h thng chm sc sc khe v cho nn kinh t ca hu ht cc quc gia [101]. Theo hip hi Alzheimer quc t, Alzheimer vchngsasttrtulnguynnhngytvongngth6BcM.Trong nm 2012, nc M cn ti 15,4 triu ngi chm sc dnh cho cc bnh nhn, tr gi hn 216 t USD [10]. Vit Nam hin cha c con s thng k y .Hin cha c phng php no thc s c hiu qu trong iu tr chng sa st tr tunichungvbnhAlzheimerniring.Ccphngphpiutrbngthuc kt hp vi chm sc ch c th lm chm s tin trin ca bnh, lm chm s mt chcnngnhnthc,hnhvicabnhnhn.Hnna,ccthucangcs dng iu tr suy gim nhn thc cn nhiu tc dng khng mong mun v gy nhiuctnhtrnbnhnhn.Ccthucmivangcnghincu,tuy nhin, c ch bnh sinh phc tp cng khin cho vic nghin cu cc phng php iu tr kh khn v km ton din. Hin nay, xu hng nghin cu thuc iu tr suy gim nhn thc t dc liu l mt hng c trin vng. Vit Nam,mt s nghin cu c thc hin nh gi tc dng tng kh nng hc tp v bo v tbothnkinhtdcliunhccsaponintrongSmNgcLinh,HngNhu Ta, C bnh vi, RaungBinhaycngilRausamng,tnkhoahcBacopamonnieri (Linn.) Pennell h Hoa Mm Ch (Scofulariacea) l cy thuc c s dng trong 2 yhcctruynnlmthucbthnkinh,tngcngtrnh,trbnhng kinh [1], [2]. Vit Nam, Rau ng bin mc hoang di, c s dng lm rau n vcngcdngnhmtvthuciutrmtschngbnhtrongyhcdn gian.Vicnghincutcdngtrntrnh,khnnghctpcacyRaung BincnghincutrnthgiivbcucnghincuVitNam [3],[4]tuynhinvnchacyvcslngvchtlngktqu nghincu.Do,chngtithchinti:nhgitcdngbovtbo thn kinh v ci thin kh nng hc tp nhn thc ca phn on n-butanol chit t cy Rau ng bin . Kha lun nm trong ti cp B Y t Nghin cu tc dng cithinkhnnghc,nhvbovtbothnkinhcacyRaungbin (Bacopamonnieri (Linn.)Wettst.)theohnglmthuciutrAlzheimer(Ph duytQs769/Q-BYT,ngy13thng3nm2012,chnghimti:TS. Phm Th Nguyt Hng Vin dc liu trung ng.) Mc tiu nghin cu:- nh gi tc dng bo v t bo thn kinh ca phn on n-butanol chit t cy Raungbintrntbovnonguynphtbgycbngproteinbeta-amyloid. - nh gi tc dng ci thin kh nng hc tp nhn thc trn chut nht trng b gysuygimtrnhdothiumunoccbtmthicaphnonn-butanol chit t cy Rau ng bin. 3 CHNG 1. TNG QUAN 1.1. Mt vi khi nim v bnh Alzheimer v chng sa st tr tu 1.1.1. Khi nim Chngsasttrtu(Dementia)cnhnghalssuygimkhnngnhn thc v gim khnng thc hin thnh cng cc hot ng sinh hot hng ngy [24]. Theo phn loi bnh quc t ln th 10- ICD10, Chng sa st tr tu (F00-F03)lmthichngdobnhcanob,thngctnhchtmntnhhaytin trin, trong c s xo trn ca nhiu chc nng v no cao hn, bao gm c tr nh, t duy, nh hng, tnh ton, nng lc hc tp, ngn ng v phn xt. thc cabnhnhnkhngbmti.Ssuygimnhnthcthngcikmhoc xut hin trc s suy gim kh nng kim sot cm xc, hnh vi x hi, hoc ng ccabnhnhn.HichngsasttrtuhaygpbnhAlzheimer,bnhmch munovtrongccbnhkhclnguynnhnnguynphthocthphtnh hngtibno[102].Trong,bnhAlzheimerlnguynnhnphbinnht, chim khong 50% - 70% s trng hp ri lon chc nng nhn thc [24], [49].1.1.2. Phn loia. Phn loi hi chng sa st tr tu Cc ri lon to thnh chng sa st tr tu c phn loi v xc nh trn c s cc yu t gy bnh khc nhau v nhng con ng sinh l khc nhau. Trong mt stiliu,chngsasttrtucphnloithnhcctyphocccdng[10], [24], [29], [49]. Cc typ ph bin ca chng sa st tr tu gm:Sa st tr tu bnh Alzheimer (Dementia in Alzheimer disease)Sa st tr tu bnh mch mu (Vascular dementia) Sa st tr tu do th Lewy (Lewy body dementia) Sasttrtuthytrn-thidng,gmcbnhPick(Frontotemporal dementia, Picks disease) Sasttrtudoccnguynnhncthongckhcnh:nhimc mntnh(doruhocthuc),thiuvitamin,bnhtmthn,hormonvsuy 4 chc nng cc c quan (suy gip, suy gan, suy thn), chn thng u v tn thng no lan ta, nhim trng mn tnh (c bit l HIV), b. Phn loi sa st tr tu trong bnh Alzeimer BnhAlzheimerthngcphnloitheothiimkhipht[49],[102]. HthngICD10phnloichngsasttrtutrongbnhAlzheimernhsau (bng 1.1): Bng 1.1Phn loi chng sa st tr tu trong bnh Alzheimer F00.0SasttrtutrongbnhAlzheimerxuthinsm:Mttrnhtrong bnhAlzheimerkhiphttrctui65,qutrnhdinbinxuinhanh chng vi nhiu du hiu ri lon chc nng v no cao hn. F00.1Sa st tr tu trong bnh Alzheimer xut hin mun: Mt tr nh trong bnh Alzheimer khi pht sau tui 65, thng t >70 tui, tin trin chm, vi s suy gim tr nh l nt c trng chnh. F00.2 SasttrtutrongbnhAlzheimerkhnginhnhhockiuhn hp F00.9Sa st tr tu trong bnh Alzheimer khng xc nh 1.2. C ch bnh sinhNguyn nhn v cc c ch bnh sinh ca bnh Alzheimer cha c bit chnh xc. Tuy nhin, tui cao v yu t gia nh l nhng nguy c quan trng nht. Cc du hiu tn thng trong bnh Azheimer l mng bm v m ri thn kinh nm khuvcvnovcccutrcthythidngtrunggiancano(entorhinal/ perirhinalcortexvvnghim),vthidngbnvnhnyMaynert[24], [49]. Cng vi nhng tn thng, xy ra s thoi ha t bo thn kinh v cc khp thn kinh v s teo v no [24], [40], [49]. 1.2.1. Gi thuyt beta Amyloid5 -amyloid peptid (AP) l nhng sn phm t nhin ca qu trnh chuyn ha bao gm 36-43 axit amin. A42 t ph bin hn so vi cc AP nhng li d b kt t v hnh thnh mng bm. AP c ngun gc t s phn gii protein tin cht amyloid (amyloidprecursorprotein-APP)tuntbiccenzym:beta-siteamyloid precursorproteincleavingenzyme1(BACE-1)-mt-secretasev-secretase- mtphchpproteinvipresenilin1.Smtcnbnggiasnxut,thanhthi cc peptid lm cho ccAP d tha tch lyv ktt li gyccho tbo. Gi thitnydatrnccnghincuvcchnhthcditruyncabnhAlzheimer (trong c hi chng Down) v cc bng chng cho thy A42 gy c cho cc t bo [24], [40], [49]. 1.2.2. Gi thuyt m ri thn kinh (Neurofibrillary Tangles - NFTs) m ri thn kinh-NFTs thngc tm thytrong cc t boca vng hi mvvnongibnhAlzheimer.NFTschnhthnhtproteintau phosphoryl ha bt thng. Proteintau c vai trn nh cu trc ving, gip h thngvingthchinchcnngnngtbo,chcnngvnchuynvvn ngtbocht.Khisitaubphosphorylhabtthngtimtvtrcth, chngkhngthlinktmtcchhiuquviccvinglmchcnngca chngbginondntigychttbo.MtcaccNFTstngquanvi mc nghim trng ca bnh mt tr nh bi chng l mt du hiu v s cht i cacctbothnkinh.NFTscngctmthytrongbnhsasttrtukhc ngoi bnh Azheimer [24], [40], [49].1.2.3.GithuytCholinergicvnhngbtthngvccchtdntruyn thn kinh khc Tn thng t bo thn kinh c th c nhn thy cng vi cu trc mng bm. Ri lon chc nng v thoi ha t bo lan ta dn n mt lot cc khim khuyt dntruynthnkinh,trongccbtthngtrnhcholinergiclnibtnht. Mthotngcacholinergictngquanvimcnghimtrngcabnh 6 Azheimer. Vo giai on mun ca bnh Azheimer, s lng cc t bo thn kinh cholinergicvslngccththnicotinictrongvnghimvvnogim xung.Ththnicotinictrcsinapcvaitrkimsotvicgiiphngca acetylcholin, cng nh cc cht dn truyn thn kinh quan trng khc cho b nh v tm trng, bao gm glutamat, serotonin v norepinephrin. Gi thuyt cholinergic l giivicmttbocholinergiclnguynnhnsuygimnhnthctrongbnh Azheimer,do,victngchcnnghcholinergiccthcithintriuchng mt tr nh. Ngoi nhng bt thng h cholinergic, cc nghin cu cng cho thy mt s ri lon hot ng nhiu con ng dn truyn thn kinh khc nh t bo thn kinh serotonincahtnhnraphevcctbonoradrenergiccanhnlc(ceruleus locus) b cht, tng hot ng ca monoamin oxidase typ B (monoamin oxidase typ Bctmthychyutrongnovtrongtiucuvchutrchnhimchoqu trnh chuyn ha dopamin), ri lon iu ha glutamat (c coi l mt trong nhng qutrnhtrunggianchnhdnnsuygimtrtutrongnhngchnthngthn kinh sau t qu hoc sau chn thng s no cp tnh) [24], [40], [49]. 1.2.4. Cc cht gy vim trung gian Gi thuyt cho rng, khi AP kt t, cc thc bo tiu thn kinh m (microglia) c kch hot v tng cng phn ng ca cc t bo hnh sao vi cc mng bm. Cc thc bo tiu thn kinh m lm gim AP qua thc bo. Cc t bo hnh sao aAP vo tbocht gin tip qua receptor v vn chuynAP vo tun hon chung, do cng lm gim AP. Tuy nhin, vic kch hot mn tnh cc tiu thn kinh m dn ti gii phng cc cytokin nh interleukin-1(IL-1), interleukin-6 (IL-6) v yu t hoi t u (TNF-), nictric oxid lm tn thng cc t bo thn kinh vthcyqutrnhvimlintc.Trnthct,nhiucytokinvchemokintng ln trong no bnh nhn Azheimer v mt s kiu a hnh gen tin vim c bo co l c lin quan vi bnh Azheimer [24], [40], [49]. 7 1.2.5. Cc c ch khc Ngy cng c nhiu bng chng v mt mi quan h nhn-qu gia cc bnh tim mch v nhng yu t nguy c tim mch vi t l mc bnh Azheimer. Cc yu t nguy c tim mch cng l nhng yu t nguy c sa st tr tu bao gm: tng huyt p,tnglipoproteincholesterolttrngthp(LDL-cholesterol),gimlipoprotein cholesterol t trng cao (HDL - cholesterol) v bnh tiu ng.Bnh tiu ng c th lmtng nguycmt tr nh thng qua ccyut lin quann"hichngchuynha"(rilonlipidmuvtnghuytp)vthng qua bt thng insulin. Ri lon trong con ng tn hiu insulin (insulin-signaling pathways), c ngoi vi v no, c lin quan n bnh Azheimer. Insulin cng c vai tr trong vic iu chnh s chuyn ha ca AP v protein tau.Bnhmchmunocthlmnngthmssuygimnhnthccabnh Azheimerdoccmchmubtthngcthlmgimcungcpccchtdinh dng cho t bo thn kinh v gim thanh thi ca AP t no b, lm tng tc lng ng amyloid v lm tng c tnh ca amyloid cho t bo thn kinh. Nhng nghin cu tm thy nhiu mi lin h gia cholesterol v s xut hin cabnhAzheimer.ApolipoproteinElmtlipoproteincnhimvvnchuyn cholesterol trong no. ApoE4 c lin quan n tng lng ng ca AP v c cho lngvaitrnhmtchtiuhathcytrongqutrnhbnhsasttrtu mch mu. Cholesterol c vn chuyn bng LDL vo t bo thn kinh v lin kt vi NFTs. Mc cholesterol cao trong t bo thn kinh no c th lm thay i chc nng mng t bo, dn n hnh thnh cc AP v cc mng bm. Nhng gi thuyt khc c xut gii thch bnh Azheimer bnh bao gm soxyha,rilonchcnngtythvnhngnhhngcaestrogenphn sau mn kinh. Ri lon chc nng ca ty th c th lm gin on qu trnh chuyn hannglngtrongtbothnkinh,giiphngccgctdovgymtcn bng oxi ha. Estrogen c cho l c vai tr thc y tng trng t bo thn kinh 8 v ngn chn qu trnh oxi ho nh mt tc nhn chng oxi ha, do s c li cho cctbotipxcviAP.Estrogencthngvaitrtrongvicduytrdn truyn thn kinh qua cholinergic, tng s lng th th NMDA cc vng no lin quannvicghilinhngkcmivngnngatnthngtbo.Thct, estrogenlmtnglulngmuno,tngsdngglucose,lmgimnng ApoEtronghuyttngvlmgimcngthngdolinquannqutrnhgii phng glucocorticoid. Chotinay,chaccchbnhsinhnocchngminhlcchchnh. Nhng tt c cc gi thuyt u dn n mt hu qu: s thoi ha ca t bo thn kinhtrongvngnocaohn;hnhthnhNFTsvmngbm;ssuygimhot ngnghimtrnghcholinergicvsmtnhnthctintrinttchotikhi bnh nhn t vong [24], [40], [49].1.3. Cc thuc iu tr Alzheimer Mc tiu chnh ca vic iu tr trong bnh Azheimer l iu tr triu chng kh khn v nhn thc v bo v cc chc nng ca bnh nhn cng lu cng tt. Mc tiu th cp bao gm iu tr cc di chng tm thn v hnh vi ca bnh. Cc bin phpkhngdngthucchyulcungcpthngtinchongichmscbnh nhn [24], [29], [49]. Ngoi ra, vic phng bnh c thc hin bng cch can thip vo c ch gy bnh quan trng giai on trc khi c triu chng [29]. 1.3.1. Cc nhm thuc iu tr Alzheimer NhngnhmthucchyucsdngtrongiutrbnhAlzheimer nhm can thip vo nhng triu chng suy gim nhn thc (cognitive symptoms) v bo v duy tr chc nng l [24], [49], [70], [8]:Nhm c ch cholinesterase : donepezil, galantamin, rivastigmin Nhm c ch glutamat: memantin Cc thuc s dng vi nhng cch tip cn iu tr khc: liu php estrogel, thuc chng vim, thuc h lipid mu. 9 Bng 1.2 Cc nhm thuc iu tr triu chng suy gim nhn thc (cognitive symptoms) Nhm thuc i dinC ch 1Nhmcch cholinesterase tacrinc ch c hi phc acetylcholinesterase. donepezilc ch c hi phc acetylcholinesterase. rivastigmincchchiphcacetylcholinesterase v butyrylcholinesterase. galantamincchchiphcacetylcholinesterase v iu bin th th nicotinic. 2Tcdngtrn h glutamat memantinikhngcaththN-methyl-D-aspartate (NMDA) Nhngnhmthuccsdngiutrcanthipnhngtriuchng khng thuc nhn thc (non-cognitive symptoms) [24], [49], [70], [85]: Nhmthucchnglonthn(tmthnhngcm):haloperidol,risperidon, quetiapin, olanzapin. Nhm thuc chng trm cm: c ch ti hp thu chn lc serotonin (SSRIs) nh: citalopram, fluoxetin, sertralin..., nhm chng trm cm ba vng, trazodon, venlafaxin Nhm thuc chng co git: carbamazepin, valproic acid ... 1.3.2. Tacrin - IUPAC Name: 1,2,3,4-tetrahydroacridin-9-amine - M ATC: N06DA01 - Cng thc phn th: C13H14N2 - Tnh cht: tacrin l bt trng, d tan trong nc, trong dung dch acid hydrocloric 0.1N,dungdchmacetatpH4,dungdchmphosphat(pH7.0npH7.4), 10 dung dch methanol, ethanol, dimethylsufoxid v propylen glycol, hi tan trong acid linoleic v polyethylen glycol 400 [95]. -Tcdng:Tacrinlthucutinthucnhmcchacetylcholinesterase c cc qun l dc M ph duyt iu tr bnh Alzheimer nm 1993. Tuy nhin, doctnhtrngan,cngvisxuthincanhiuthuccngnhmctc tnh hn, tacrin khng cn c s dng hoc vi mt s hng dn ch dng nh la chn th 2 trong iu tr bnh Alzheimer [72].1.3.3. Hn ch ca cc thuc iu tr Alzheimer 1.3.3.1. V hiu qu iu tr Chng sa st tr tu trong bnh Alzheimer l hu qu ca nhng thoi ha khng hi phc thn kinh no b. Cc phng php iu tr hin ti khng c hiu qu ko dituith,chakhibnhbnhAzheimer,hoctmdnghocongcqu trnhsinhlbnh[49].Hainhmthucchnhangsdnglthuccch cholinesterasevthuctcdngtrnhglutamatdatrnhaigithuytcch bnh sinh ca bnh Alzheimer. Tuy nhin, c ch bnh sinh chnh bnh Alzheimer n nay cn cha c xc nh r rng v hai gi thuyt ny ch l mt phn trong rt nhiu con ng dn n vic t bo thn kinh b ph hy [49]. Nhm thuc c ch cholinesterase ch nh cho trng hp bnh nh n trung bnh, c hiu qu n nhnhnthctmthi,hoclmgimrtnhssuygimnhnthc,20-30% bnhnhniutrgimc7imtrongthangimngginhnthc- ADAS-Cog (tng ng gim c 1 nm suy gim nhn thc), hay ch gim 5-15% so vi gi dc khi theo di t 6 n 12 thng [44], [45], [62]. Memantin c dngchobnhnhngiaiontrungbnhnnng,nhngngikhngdng c thuc c ch cholinesterase [70]. Mts th nghim lm sng cho thy hiu qu ca memantin khi nh gi bng thang ADAS-cog so vi gi dc thi im 12 - 24 tun iu tr, nhng khng cho thy li ch t 24 48 tun [69]. Nhng nhm thuc iu tr triu chng khng thuc nhn thc: cc thuc chng lonthn,thucchngtrmcm,chngcogitchcsdngkhinhngtriu chng v tm-thn kinh mc nghim trng, gy kh khn cho chc nng hot 11 ng ca bnh nhn v cc hot ng chm sc cn thit ca ngi chm sc, hoc c th gy nguy himcho bn thn bnh nhn hoc ngi khc. Vic iu tr phi da trn s cn bng gia li ch v nguy c [49], [70], [85]. Ngoi ra, thuc hng thncnctcdngkhngcholinergic,vvychngcthlmbnhtrmtrng hn hoc tc ng n vic s dng thuc c ch cholinesterase [49].1.3.3.2. V tng tc thuc, tc dng khng mong munNhm c ch cholinesterase Tc dng khng mong mun ca nhm c ch cholinesterase thng do cc tc dng kch thch cholinergic qu mc v ph thuc vo liu nh: bun nn, nn, tiuchy,aubng,chngmt,nhcu,titncbt,mhi,nhpchm, chut rt, ngt, mt mi, mt ng, kch ng, trong nn, bun nn, tiu chy c bo co nhiu nht. Tacrin gy nhim c gan sau khong 3 thng 30 - 40% s bnh nhn biu hin bi tng men gan v tr li bnh thng sau khi ngng thuc [44], [49], [72]. Tng tc thuc ca cc thuc trongnhm c ch cholinesterase c th gp nh: donepezil v glantamin chuyn ha qua h Cytocrom P450, emzym CYP3A4, nn chng c nguy c gy tng tc vi cc thuc c ch hoc gy cm ng trn h enzym ny: carbamazepin, dexamethason, phenobarbital, phenytoinand, rifampicin, erythromycin Ngoi ra, cc thuc trong nhm cn c th gy tng tc thuc vi ccnhm:thucchnglonthn,ccazolchngnm,nhmcchththH2, chn knh calci, cc thuc tc dng ln acetylcholin khc, [51] Nhm c ch glutamatTc dng khng mong mun: to bn, l ln, chng mt, au u, o gic, ho v cao huyt p [49], [62]. Thuc c th gy tng tc vi cc nhm thuc: chng iu tr tiu ng, co-trimoxazol, cc cht gy kim ha nc tiu [51]. 12 1.4. Nghin cu thuc iu tr Alzheimer Pht trin thuc mi ch yu theo hai hng. Hng th nht, tp trung vo cc thuc lm gim nng AP no hoc cc dng hot ng ca n. Hng th hai bao gm tt c cc chin lc iu tr khc [49], [81]. 1.4.1. Nghin cu thuc nhm lm gim AP gim nng AP trong no, thuc c th tc dng vo cc qu trnh: gim tng hp AP, gim lng ng v thc y qu trnh thi tr AP. lm gim to thnh AP cc thuc c nghin cu theo hng tc dng c ch -secretase v -secretase. Do -secretase 1 cn c cc c cht khc (bao gm neuregulin -1 tham gia vo qu trnh myelin ha) nn vic c ch enzym ny c th gyctnh.Chonnaychacdliuvhiuqucathuccchca-secretase trn th nghim lm sng [49], [81]. Ccthuccch-secretasecngangcchnghincu.-secretasel phchpenzym,ngoicchtlAPP,-secretasecnthamraphnctmts protein trong mng t bo nh th th Notch, ErbB4, th th p75NTR neurotrophin, N-cadherin,tiunvknhnatri4[81].Viccch-secretasecthcch con ng tn hiu Notch gy ra nhng tc dng ph nghim trngtrn ng tiu ha,tuync,llch,trndangvtthnghimvtrnngi.Semagacestatl cht c ch -secretase u tin qua th nghim lm sng v c chng minh l lm gim nng AP trong huyt tng v gim sn xut AP thn kinh trung ng. Hai th nghim lm sng ngu nhin c i chng pha 3 vi semagacestat dng li sm v mt s tc dng bt li nghim trng, bao gm huyt hc, tiu ha v nhim c da, c cho l do semagacestat c ch ca con ng tn hiu Notch. Mtsthuckhccngnhmnhbegacestat,avagacestatangcnhvgi mc chn lc v an ton so vi hiu qu trn lm sng [23], [39], [42], [43]. Mt sloithuckhngvimkhngsteroid(NSAIDs)ctcngln-secretase nhngkhngcchhonton-secretase,chngctcdnglmgimA40v A42,trongkhilmtngA38.Tarenflurbil(mtngphnquanghc flurbiprofen)cthnghimlmsngpha3nhngkhngctcdnglm 13 chm suy gim nhn thc [36], trong khi lm tng chng mt, thiu mu v nhim trng.Sthtbicatarenflurbilcthdohiulcthpvkhnngthmnhp no km [49], [81]. Hn na, c ch cyclo-oxygenase trong tiu thn kinh m c th dn n s c ch thi trAP [49]. Mt s nghin cu gn y cho thy, cc thucthucnhm1,4-dihydropyridin(DHP)chnknhcanxitypLcnhhng n qu trnh sn xut AP [32], [33]. Nilvadipin v amlodipin cng c tc dng n nh nhn thc mt s bnh nhn tng huyt p c suy gim nhn thc nh, thuc dung np tt v khng gy ra tc dng h huyt p nguy him [38] [52]. Nilvadipin vamlodipinangcnghincutnhngtcdnglmgimsnxutAPt APP in vitro v lm gim tch ly do gim sn xut v tng thi trAP qua hng ro mu no khi iu tr th nghim bng ng ung di hn [33]. Cc thuc lm gim s lng ng ca AP c nghin cu vi c ch c ch cc tiu n v AP tp hp thnh cc oligomer kh tan, hoc kch hot thi tr qua phnngmindch.MtschtcchkttpAPnhTramiprosat,PBT2, Scyllo-inositol ang c nghin cu. Tramiprosat cho thy tc dng gim nng APtrongdchnotynghinlmsngpha2,tuynhinkhngctcdng ci thin tr nh th nghim lm sng pha 3 [34], [49]. PBT2 l mt quinolone 8-hydroxy,thmttquadchnotykhidngbngngung,ctcdngthc ythitrAPoligomervphchikhnngnhnthctrongmhnhbnh Azheimer trn chut [8] [30], tuy nhin trong th nghim lm sng pha 2a gn y, PBT2 lm gim A42 trong dch no ty v ci thin nhn thc, nhng khng thy mitngquancnghagiasgimAPtrongdchnotyvsthayi nhn thc [30]. Scyllo-inositol c th lin kt trc tip vi AP oligomer, thc y phnlycaAPoligomer[11],[31].Trongthnghimlmsng,Scyllo-inositol lm tng khi lng no tht, nhng t ci thin v tnh trng lm sng, th nghim liu cao ca thuc buc phi ngng sm do gy nhiu tc dng ph [81]. Phng phpmin dch c nghin cubao gm vaccin AN-1792 vmin dchthngthngquatruynkhngth,nhmtngthitrAP.VaccinAN-14 1792 l liu php min dch u tin c nghin cu, nghin cu lm sng pha 2 trn ngi ca AN-1792 phi dng li do mt s lng bnh nhn b vim no, sau tin trin thnh vim mng no v khun. Nhng nghin cu di hn v ton din v bnh hc lm sng ca cc bnh nhn iu tr AN-1792 c th cho kt qu khquantrongviccithinsuygimnhnthcvlmgimmngbmno; nhng tim chng AN-1792 khng c nhiu li ch c ngha cho nhng bnh nhn khiphtmun-lhnhthcphbincabnhAzheimer[49][81].Liuphp khngthndngbtucnghincugny,ktquthaithnghim lm sng pha 3 vi bapineuzumab v solaneuzumab khng mang li kt qu mong mun. Nguyn nhn c th l cc cht ny chn sai loi AP ch hoc do thuc s dngqumuntrongqutrnhbnh.Mtskhngthndngmivich nhm l nhiu loi amyloid- khc nhau ang c th nghim [49], [81]. 1.4.2. Nghin cu thuc tc dng trn cc ch khc GithuytmrithnkinhtrongbnhsinhcabnhAzheimerkhinTau protein tr thnh mt ch tc dng trong nghin cu thuc iu tr bnh Azheimer. BngchngthcnghimchothyctngtcgiaAPvTauprotein phosphoryl ha bt thng, theo , nhng mng AP kt t kch thch hot ha cc kinase khc nhau nh cyclin kinase, cyclin dependent kinase 5 (CDK5) v glycogen synthase kinase 3b (GSK3b) [40]. Cc nghin cu gn y hng ti pht trin cc thuc c ch protein Tau-phosphoryl ha v cc cht ngn chn kt t Tau protein thnh m ri v/hoc thc y tho g m ri. GSK3b l enzymchnh tham gia vo qu trnh phosphoryl ha Tau protein [16], [40]. Lithium v valproat c cho l c kh nng c ch GSK3b v gim Tau- phosphoryl ha trn m hnh ng vt [41],[99].Tuynhin,khngcbngchngchngminhcthuctrmcm ni chung c tc dng ln s suy gim nhn thc. Hn na, cc thuc ny cn gy tc dng ph nng n, c bit i vi ngi cao tui [103]. Mt s cht khc cng angcnghincunhmttcnhncchGSK3bnh:JGK-263,glucagon-like peptide-1 [21], [46], [59]. Methylthioninium clorua (MTC, methylene blue), l mt hp cht c trin vng do c tc ng ln s khim khuyt chc nng ca ty 15 th v hot ng trao i cht ca t bo, lm gim hnh thnh cc mng AP v cc mrithnkinh[22],[27],[47],[67].Cncthmnhiuthnghimhnna c thc hin kim chng v pht trin thuc theo hng ny.Gn y, liu php min dch tc ng ln c ch bnh sinh qua con ng Tau proteinhaymrithnkinhcch.Nhngthnghimctinhnhcho thy cc tc nhn min dch ny c tc dng lm gim kt t Tau protein, gim to thnh m ri thn kinh. Nhng kt qu ban u ny hi vng s mang li mt hng phttrinmitrongiutrbnhAzheimervnhngbnhsinhlinquanTau protein [19], [89], [104]. Ccloithucthiazolidinedion,rosiglitazonvpioglitazonlmgimkhng insulinvcngcthctcdngkhngvim,gnycthnghimtrn ngibnhAzheimer,nhnghuhtccthnghimchothykhngcbng chng v hiu qu [49]. Latrepirdine (dimebon) l mt khng histamin khng chn lccthnghimtrongiutrAlzheimernhmttcnhnbovtbo thnkinhtnhngnm2000[15],[57],[100].Mtthnghimpha2chokt qungchkhimanglilichtrongstngcngkhnngnhnthctrn bnh nhn [28], , nhng th nghim lm sng pha 3 ca latrepirdin tht bi (c cngbtrnclinicaltrials.gov),tuynhinccloithucbovtbothnkinh khc vn ang c nghin cu. 1.5. Mt s m hnh nghin cu thuc tc ng ln nhn thc v cc bnh sa st tr tu Nghin cucc tcdng ln nhn thc trn ng vt l phng php phbin nghin cu tc dng dc l ca thuc thuc tc dng ln nhn thc v cc bnh gy sa st tr tu. K thut phn tch hnh vi s dng ng vt nghin cu nhng mhnhbnhngivdoncctcdngcathuccsdngrngri [80]. Trn th gii, ccm hnh nghin cutc dng ca thuc trn kh nng hc tp v tr nh c p dng ph bin da trn kh nng nhn din vt th, nhn din mi, khng gian nh: m cung nc Morris (Morris water maze test), m cung ch T/ch Y ( T/Y Maze Spontaneous Alternation Test),m hnh nhn din i tng 16 (2-objectNovelObjectRecognitionTest,Object-Locationmemorytask),mhnh n trnh th ng (passive avoidence task), [14] [54], [63], [71], [73], [74], [75], [93], [96]. Nhng m hnh ny khng gii thch c c ch tc dng dc l ca thuc th nghim. Tuy nhin, nhng th nghimphn tch hnh vivn l cng c khng th thiu, cng vi nhng thng tin thu c t nghin cu phn t v t bo c a vo thc thnh trong nghin cu hnh vi ng vt mt cch y [80]. Cng vi s pht trin cng ngh sinh hc t bo v cng ngh sinh hc phn t, vic tin hnh cc m hnh nghin cu khc mc t bo hay mc phn t l cn thit xc nh ch tc dng ca thuc trong cc qu trnh bnh sinh c th. cp t bo v di t bo, vic nh gi tc dng dc l ca thuc trn nhng qu trnh bnh sinh gy sa st tr tu c tin hnh. Cc m hnh ch yu nh gi cc yu t c coi l c ng gp cho c ch bnh sinh nh: qu trnh vim, s xy ha, h cholinergic, h glutamat, trn biu hin ca cc th th, trn t bo thn kinh v no ca ng vt th nghim. Qu trnh vim c nh gi bng mc tcngnccyutgyvimnhenzymlipoxygenase(LOX), cyclooxygenase-2 (COX2), cc cytokin tin vim TNF-, IL-6, IL-1 [86], [97], [93].Tcdngtrnhcholinergiccnhgichyutrnkhnngcch enzymcholiesterasetrongno.Tcdngchngoxihacnhgithngqua biuhincaccenzymchngoxiha:catalase(CAT),glutathioneperoxidase (GPX),superoxidedismutase(SOD),vsthayicaccyutgyoxiha cbn(nngmalondialdehyd,ccgcoxiha,nnghydroperoxidv carbonyl protein) [12], [60], [76], [ 93]. nh gi cc tc ng trn th th MNDA, GABA, cng c tin hnh.Mt s tc gi s dng m hnh nui cy t bo thn kinh v no nguyn pht [12], [60], [65], [93]. u im ca cc m hnh t bo l kh nng nh gi trc tip nh hng ca thuc ti t bo ch v ch cn lng nh mu nghin cu. 17 1.6. Vi nt v cy Rau ng bin RaungbinctnkhoahclBacopamonnieri(Linn.)Pennell(tnkhc Herpetismonnieri (L.)H.B.K., Gratiolamonniera L., Septasrepens Lour., Bramia indica Lamk.,Bacopamonnieri (Linn.)Wettst.),hHoammch (Scrophulariaceae).Cymchoangdinhiuninhn,SriLanka,i Loan, Trung Quc, cc nc ng Nam. Cy cng ctmthy Hawaii, FloridavmtstiubangminnamncM.VitNam,cyctngiRau ng bin, hayRau sam trng, Rau sam ng, cyRut g [1]. Cymc ven b rung, m ngp nc nhiu vng trung du v ng bng trn c nc. Cy Rau ngbincsdngnhmtloithcphmctnhmt,litiu.Cycng c dng lm thuc li tiu, cha ho, b thn, dng ngoi p cha au nhc do t thp, kt hp mt s v thuc khc trong bi thuc cha rn cn. n , cy Rau ng bin cng c coi nh cythuc truyn thng v c s dng nhmt v thuc b thn kinh, tng cng tr nh, tr bnh ng kinh [1], [2]. Thnh phn ha hc ca cy Rau ng bin gm cc saponin nhm damaran l bacosid (A, B, C) v bacopasaponin (D, E, F) da trn phn genin bacogenin t A1-A5,ngoiracnchersaponin,monnierin;ccalkaloidBrahmine,herpestin;flavonoid(luteolin-7-glucoside,glucoronyl-7-apigenin,glucoronyl-7-luteonin), phytosterol,luteonin,ThnhphnbacosidAcnhlngbngphng phpscklnghiunngcao(HPLC)kthpviNMR(cnghngtht nhn), khi ph. Bn thnh phn chyu c xc nh bao gm: bacosid A3 (1), bacopasid II (2), jujubogenin isomer ca bacopasaponin C (3) v bacopasaponin C (4) [1], [2], [26], [58]. Cu trc cc thnh phn Bacosia A c th hin hnh 1.1 [26] 18 Hnh 1.1 Cu trc cc thnh phn Saponin chnh trong bacosid AChotinay,nhiunghincuVitNamvtrnthgiicthchin nhmkimchngvnghincucthhnvnhngtcdngdcltnhng thnhphntrongdchchitcacyraungbin.Tcdngtrntrnhvnhn thccacyraungbincnghincucmtsktqunghincu trn ng vt th nghim [3], [4], [54], [63], [71], [93], [96] v trn ngi [66], [82] [83], [90], [91], [92], [94]. NhngnghincutrnngvtchothydchchitRaungbinlmgim thi gian tr ti ch, gim chiu di ng i th nghim m l Morris [54], [71], [93]; lm tng kh nng phn bit cc i tng m hnh nhn din i tng mi [74], [75]; lm tng t l thay i la chn lun phin m cung ch Y, ch T [96] 19 [63];gimssailm(bias)mhnhmhnhntrnhthng[96],trn chutbgymttrnh.Trnngi,nhiuthnghimlmsng(trongc nhngthnghimlmsngngunhincichngquymln(vitrmbnh nhn/ngi tnh nguyn khe mnh), thi gian di (t 6, 8, 12 tun) [18], [82], [83], [92], [94]), cho thy s ci thin c nghakhi nh gi bngnhng bi kim tra nh:thigianhonthnhanhimv(multitaskingframework),nhndinmu sc, theo di trc quan [83], trn cc thang Wechsler Memory Scale sa i, Mini-MentalStateExamination[82],hthngnhgitrnmytnhCDR(CDR system) [90], [94] so vi nhm chng.C ch tc dng ca cc thnh phn dch chit Rau ng bin mc t bo v phntvnangcnghincu.Nhngnghincutrnchutsuygimnhn thcchothydchchitRaungbinctcdnglnhthngglutamatvh cholinergicnh:gimhotngcaccemzymacetylcholinesterasetrongttc ccvngno[88];lmgimsthiuhtvesicularglutamatetransportertype1 (VGLUT1)hihim1(CA1)v2/3(CA2/3)mhnhchutsuygimnhn thc do tm thn phn lit [74], [75], tc dng bo v thn kinh trong s thay i s gn th th glutamat v lm thay i ng k biu hin gen ca NMDA R1 v gen mgluR8trongvnghippocampuschutngkinh[53],[73].Mtsnghincu cho thy saponin Rau ng bin v bacosid A ngn nga s xut hin ca cn ng kinhdolmgimssuyyuhotngcahGABAergic,suygimngc hctpvtrnh[48],[63],[64].Khinghincuvtcdngchngoxihaca Rau ng bin, kt qu cho thy c s suy gim ng k cc yu t oxy ha c bn (nngmalondialdehyd,ccgcoxiha,hydroperoxidvcarbonylsprotein) trong c t bo cht v ty th ca t bo tt c cc vng no, i km vi tng cng glutathion b thiu ht, tng nng thiol v tng hot ng ca cc enzym chng oxyha(CAT,GPX,SOD)trnchutkhemnh(sovithucchng)vtrn chutphinhimvinhiutcnhnoxihakhcnhau(sovinhmkhngphi nhim) [12], [50], [76], [87], [88], [93]. Cy Rau ng bin cn c nghin cu tc dng chng vim thng qua kh nng c ch LOX v COX-2, gim gii phng cc 20 cytokintinvimnhIL-6,IL-1,TNF-vnitrittrongcctbonnhntrn mhnhchutbgyvim[86],[97],[98].Ngoira,nhiuthnghimc thchinnhgicctcdngkhccathucnhtcdngchngtrmcm thng qua nh hng ln h 5-HT(2C) [55], [56], tc dng h ng huyt [35]21 CHNG 2. NGUYN LIU, THIT B, NI DUNG V PHNG PHP NGHIN CU 2.1. Nguyn liu -Muth:cnphnonn-butanoltdchchitncRaungbinBacopa monieri(Linn.)PennellthuthpNghAn,doVindcliutrungngcung cp. 2.2. ng vt th nghim - Chut nht trng cung cp bi Vin v sinh dch t Trung ng.+Chutdngchothnghimnhgikhnnghctpcamunghincu: cnuinnhtnht1tun,tntrnglng23-25gtrckhitinhnh phu thut. + Chut dng cho thnghim nh gi tc dng camunghin cu ln t bo thn kinh nguyn pht: chut nht trng mang thai khong 18-19 ngy tui. 2.3. Ha cht, trang thit b - Thuc chun: Tacrine do hng Sigma cung cp. - H thng m l nc Morris bao gm:+ B bi: l mt b hnh tr, ng knh 1.1m v cao 30 cm. Lng nc trong b duy tr mc 15cm, nhit khong 200C trong sut qu trnh th nghim. M lcttrongphngkn,ctsnnhnghnhnhnhhngkhnggian, nhng hnh nh ny khng c thay i trong sut qu trnh th nghim. Mt bn hnh tr c ng knh 7,5 cm, trong sut, t trong mt gc ca m l. +Cameracktnivimytnhvphnmmphntchktqudac thitkdatrnphinbnPanLab(TyBanNha)docngtyphttrindchv cng ngh An Nhn cung cp. 22 - B ha cht tch v nui cy t bo (Hng Gibco, M) - Ha cht khc: MTT [3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide]; beta-amyloid25-35protein;trypanblue;dimethylsulfoxid(DMSO)ttiuchun th nghim. 2.4. Phng php nghin cu 2.4.1.nhgitcdngbovtbothnkinhcacnphnonn-butanolchittRaungbintrntbovnonguynphtgycbng beta-amyloid 2.4.1.1. Phng php tch v nui cy t bo thn kinh v no nguyn pht Nguyntc [17], [60], [61]: Vnotchtmtcthsng(thnglchut),xlbngenzymvbin phpchcnhmphnctccmlinktthuccctbothnkinhvmt lngnhtbomringl.Bsungchtdittbommkhnglmnh hng cc t bo thn kinh. Nui cy trong mi trng v iu kin thch hp. Quy trnh tch v nui cy t bo thn kinh v no phi thai chut nguyn pht gm cc bc c bn sau: - Git chut m mang thai khong 18-19 ngy tui. - M tch vng v no, t vo mi trng nui cy (mi trng Neuronbasal b sung12%huytthanhnga,0,6%glucose,200mML-glutaminv1g/ml minomycin (c gi l mi trng Glu (+)) - Bc tch mng v no v cc mch mu bm trn b mt. - Ct nh m v no, sau vi 2 ml trypsin 0,05 % ( 37C trong 15 pht) phn tch cc m v no, ly tm thu c nhng ht nh. 23 - vi enzym deoxyribonuclease (100 U/ml DNase I trong 2 ml m phosphat). phn ct acid deoxyribonucleic ngoi t bo. - Ly mt t huyn dch t bo, nhum bng tripan blue v m trn kinh hin vi xc nh mt t bo v kh nng sng st ca t bo. - T bo c phn tn li trong mi trng Glu (+) v chia vo 96 ging (ging c trng poly-D-lysin) v nui cy 37C,trong mi trng khng kh c cha 10% kh CO2. - Thay mi trng Glu (+) bng mi thng Glu (+) khng c huyt thanh nga, c b sung thm B-27 ( hn ch s pht trin ca t bo thn kinh m) sau khi cy 2 ngy/ ln. 2.4.1.2. nh gi c tnh ca mu th trn t bo thn kinh v no nguyn pht Quy trnh tin hnh: -Cnphnonn-butanolRaungbincphatrongdimethylsulfoxid (DMSO). -tbothnkinhvnonguynphtctchvnuicy(trn)vi muthsaochonngmuthcuicngtrongccgingnuicytboln ltl50,100,200,300,400g/ml,tiptcnuicytbo37C,trongmi trng khngkh c cha 10% CO2. Minng mu th c nh gi trong 3 ging, th nghim c lm lp li 2 ln. - Sau 24 gi, nh lng s lng t bo sng st bng k thut nh lng MTT. Nguyn tc k thut nh lng MTT [60]: MTT l mt mui tetrazolium ha tan trong nc c mu vng nht. Trong t bo sng, enzym hydrogenase ty th s phn gii vng tetratropium ca MTT thnh tinh th formazal mu tm. Ha tan tinh th bng dung mi thch hp. nh lng lng cht phn ng bng o hp th 24 nh sng bc sng 540nm. hp th ca dung dch l mt hm chuyn i t l thun vi lng t bo sng. Tin hnh nh lng MTT trn cc ging t bo th c tnh ca cn phn on n-butanol Rau ng bin: +Bsung20lMTT(nng5mg/ml,trongmmuinatriphosphate)vo mi ging t bo. + Tip tc trong t nui cy duy tr nhit 37C, khng kh c cha 10% CO2

+ Sau 2 gi, ht loi b mi trng, thm vo 50l DMSO vo mi ging ha tan tinh th. o hp th nh sang bc sng 550nm bng my ELISA. Ch tiu nh gi: T l t bo sng st sau nui cy 24 gi. - Da vo kt qu nh lng t bo sng st, chn nng mu th thch hp tip tc nh gi tc dng bo v t bo gy c bng beta-amyloid. 2.4.1.3. nh gi tc dng bo v t bo thn kinh ca cn phn on n-butanol chit t Rau ng bin trn t bo v no nguyn pht gy c bng A 25-35 Nguyntcchung:Bsungmuthnngkhcnhauvomitrng nui cy t bo thn kinh nguyn pht trc khi gy c bng beta amyloid (A 25-35). Sau , tin hnh nh lng s lng t bo sng st bng k thut nh lng MTT.Quy trnh tin hnh:- Th nghim c tin hnh trn t bo v no nguyn pht sau khi tch v nui cy7ngy.Ccgingtbocchiathnh3nhm,minhm3ging(bng 2.1): 25 Bng 2.1 Phn nhm th kh nng sng st sau gy c t bo bi A25-35 Nhm Th thuc Chng sinh l DMSO 0.01%Chng bnh lDMSO 0.01% + A 25-35 Th Cn BuOH Rau ng bin 100g/ml+ A 25-35 - Chun b beta amiloid 25-35 (A 25-35) kt tp: chun b dung dch A 25-35 trong nc v khun nng 1mM v bo qun - 200C. Trc khi gy c t bo, A 25-35 c 37C trong 48 gi to AP kt tp. - Cn phn on n-butanol chit t Rau ng bin c ha tan trong DMSO v b sung vo cc ging nui cy t bo n nng c chn sau khi nh gi c tnh (mc 2.4.1.2).- Sau 15 pht, b sung A 25-35 trc tip vo mi trng nui cy ca l chng bnh l v l th thuc t c nng cui cng l 10 M v trong t nui cy m bo nhit 37C v khng kh c cha 10% CO2. - 48 gi sau khi thm A 25-35, tin hnh nh lng t bo sng st cc l th nghim bng nh lng MTT. +Bsung20lMTT(nng5mg/ml,trongmmuinatriphosphate)vo mi ging t bo. + Tip tc trong t nui cy duy tr nhit 37C, khng kh c cha 10% CO2 trong 2 gi + Ht loi b mi trng, thm vo 50l DMSO vo mi ging ha tan tinh th. o hp th bng o quang bc sng 550nm bng my ELISA. Th nghim lp li 3 ln trong cng iu kin nghin cu. Ch tiu nh gi: T l t bo sng st ca nhm th so vi nhm chng. 26 Hnh 2.1. Cc bc nh gi kh nng sng st trn t bo gy c bng A 25-35 2.4.2. nh gi tc dng ci thin kh nng hc tp v ghi nh in vivo2.4.2.1.Phngphpgythiumunoccbtamthitheomhnh2VO+ hypo - Chut nht trng sau khi nui n nh trong phng th nghim 5 ngy c chia lm 2 nhm: nhm th v nhm chng sinh l. - Gy m chut bng pentobarbital (liu 50mg/kg) tim phc mc. - Sau khi chut b gy m, bc l 2 ng mch cnh. + i vi nhm th: kp ng mch cnh bng 2 kp ng mch trong 30 pht, ng thi gy h huyt p bng cch rt 0.2ml mu ui chut trong thi gian kp ng mch + i vi nhm chng sinh l: Chut c m bc l 2 ng mch nhng khng kp ng mch v khng rt mu ui. - Chut c hai nhm c m 370C cho n khi tnh dy. 2.4.2.2. Th nghim m l nc Morris Nguyn tc [6], [9], [20], [79]:M l Morris (Morris water maze) l m hnh nh gi kh nng hc tp khng gianchongvtgmnhmnhhngtvtrbtuxungquanhchuvica 27 mt khu vc bi xc nh v tr mt bn (platform) trnhkhi ngp nc da trn cc du hiu gi ngoi vi. Th thuc:Chut c chia lm 5 l th nghim v cho ung mu th nh sau (bng 2.2):Bng 2.2 Phn l th nghim v iu tr th nghim nh gi kh nng hc tp v ghi nh in vivo LnMu th/liu dngCh iu tr Chng sinh l 8 Ungncmuisinh l 0.9% - 1 gi trc khi phu thut -1gitrckhitinhnh th nghim hng ngy Chng bnh l 9 Th liu thp 8 Ungcnphnon BuOH liu0.3g dl/kg Th liu cao 8 Ungcnphnon BuOH liu0.6g dl/kg Tacrin 8 TimphcmcTacrin liu 2.5mg/kg -30phttrckhiphu thut -30phttrckhitin hnh th nghim hng ngy 2 ngy sau khi tin hnh phu thut gy thiu mu cc b tm thi, chut bt u ctinhnhnhgihnhvibngthnghimmlncMorris.Tngthi gian chut phi hc l 8 ngy. Cc bi tp c th v thi gian nh sau: - Bi tp nhn thy bn : 2 ngy sau phu thut, chut c hc bi u tin l bitpnhnthybn(visibletrial).Bnctcaohnmtnc1cm. Mi chut c bi tm thy bn 1 ln. Nu trong 60 giy, chut tm thy bn , chut s c li trong 10 giy. Nu trong 60 giy chut khng tm thy bn 28 ,chutschngdntmnbnvlitrong15giy.Sauchut c lau kh, si m v a v chung. -Bitpkhngnhnthybn:tngyth3tingyth8,chutctp nh khng gian vi bi tp khng nhn thy bn (training trial), khi bn c t di mt nc 1 cm. Mi ngy chut c tp 3 ln, mi ln 3 gc cn li trong m l nc. Nu 60s chut khng tm thy bn , chut s c hng dn cchtmbnvlibn20giy.Khongcchgiacclntpl1pht, trong thi gian ny chut c lau kh v si m. Ton b qu trnh s c ghi li bng camera, thi gian tim tng chut c th tm thy bn s c tnh ton. S liu mi ngy c tnh ton bng gi tr trung bnh thi gian 3 ln tp i vi mi chut. - Bi tp khng c bn : ngy th9 sau khi gy mt tr nh, chut s c tp th li tr nh (probe trial). Trong bi tp ny, bn c b ra ngoi, chut c bi trong m l mt ln duy nht trong 60 giy. Chut s nh li v tr ca bn v cxuhngbiluhntigccamltbnnhngbitptrc. Camera s phn tch v ghi li thi gian chut bi mi gc ca m l. Hnh 2.2 S qui trnh bi tp trn m hnh m l nc Morris 29 Hnh 2.3M hnh th nghim m l nc Morris Ch tiu nh gi: So snh thi gian ca l chut ung mu th vi l chng nh gi tc dng ca mu th. + Thi gian tim tng tm bn trong sut qu trnh tp luyn+ T l thi gian chut gc phn t ca m l cha bn khi khng c bn 2.5. X l s liu S liu c c x l bi phnmm excel 2010, biu din di dng trung bnh sai s chun. Kim nghim gi thit thng k s dng t-test, so snh hai gi tr trung bnh theo cp. S khc bit c ngha thng k khi p