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Tgp chi C6ng nghe Sinh hoc 11(3): 425-432, 2013 TAO MO HINH RUOI GIAM CHUYEN GEN BIEU HIEN PROTEIN a-SYNUCLEIN NHAM tiNG DUNG TRONG NGHIEN CtrU CO CHE PHAT SINH BENH PARKINSON Nguyen Thi Ttrimg Vy, flung Thi Phircmg Than, Trim Linh Think Triremg Doi hoc Khoa hoc to nhien, Dgi hoc Quac gia Thanh ph a Ho Chi Minh TOM TAT Bing nhieu mo hinh nghien ciru khac nhau, dac bi0t la me hinh d6ng vat chuyen gen, cac nha khoa hoc dang tang co nhieu brat tien nghien ciru b0nh di truyen a ngtrai va co the sinh hoc phin tir cua bOnh Vai nhieu tru diem not trOi, mo hinh ruoi giam Drosophila melanogaster da va dangduqc sir dung nhieu nghien ciru y sinh Nhieu ke't qua nghien ciru benh thoai hoa than kinh, ung thu, tieu &rang, tim mach, tren mo hinh ruoi giant da duqc cong bo ten cac tap chi khoa hoc nhu Nature, roi loan bien Science , Clang gap lan cho cac hieu biet khoa hoc ye ca the b0nh Mic du \fay, tai V* Nam, viec tat) va sir dung cac mo hinh mai giam chuyen gen kha mai, hiu nhu chua cOsphOng thi nghiem nao trien khai Trong bai bao nay, chung trinh bay cac ket qua nghien ciru too mo hinh ruoi giim chuyen gen a-synuclein (SNCA) nham img dung nghien turn ca the benh Parkinson Cac cong b8 tnrac day cho thiy hi0n din cita the viii Lewy, c6 phan chit yen la protein a-synuclein, lam thoai h6a din cac to bao thin kink san sinh dopamine va IA nguyen nhin chit yeu gay 130nh Bang cac ky thuit sinh hoc phin tir thing toi chuyen cDNA gen a-synuclein cua ngtrai vao trang ruoi giim da thy tinh nham sang loc thu nhin cite dang ruoi mang gen SNCA Ch'ing toi di to ding ba dung ruoi mang gen SNCA va hat dang d6t bien thay the acid amin A3OP va A53T Han the nua, chung toi cOngda cong viec sir dung he thong UAS-GAL4 nham bleu hi0n.dinh htrang gen SNCA nao ruoi nham tim hieu ca cite tic dOng cita str Lich to SNCA tren hoot dOng dm 110 than kinh cac nghien ciru sau Tir Wet: Benh Parkinson, a -synuclein, the Lewy, Drosophila melanogaster, he thijng UAS-GAL4 DAT VAN DE Benh Parkinson (PD, Parkinson Disease) la benh thoai h6a thin kinh gay bii sv thieu hitt dopamine nao gitza Tuy tinh hinh benh hien rat pito hien, Ian reng va mang tinh nghiem trong, nhung nhfing nghien ciru ve nguyen nhin giy benh, lieu phap phong benh, cac mo hinh nghien ciru mo ph6ng va trj benh van chua mang Igi nhang dap an mong dqi Nhieu ciu h6i Ian ve benh Parkinson van chua co ciu tra lai Nhang nghien ciru gin day bat diu hiring ve mgc tieu phatshien nhang det bien don co lien quan den sv di truyen cga met so kieu benh PD, dieu cho phep chgng ta hieu sau han ve ca the phan tir cua benh PD thong qua viec thiet lip cac mo hinh nghien ciru sir dung deng vat chuyen gen co kieu hinh benh PD (Feany, Bender, 2000; Bilen, Bonini, 2005; Pendleton et al., 2002) Khong nhang co the dap ung nhu cau nghien ciru cac nguyen lY giy benh a mac sinh hoc phan tir, mo hinh nghien dm cho phep nghien ciru sang loc cac chit hem hoc co host tinh tri benh (Sang, Jackson, 2005) MO hinh chuet chuyen gen da va dang duqc sir dung Ong rai de nghien ciru cac benh tren Tuy nhien, mo hinh thuing ton nhieu chi phi cling nhu can met khoang thin gian kha dal Met cac &di tuang sinh vat co the sir dung lam m6 hinh nghien ciru berth tren ngtrii la mo hinh ruoi giam Drosophila chuyen gen Day la met mo hinh huu ich cho viec sang loc rung rat cac gen giy benh vii chi phi thip va thii gian ngan mo hinh ruoi giim chuyen gen chung ta co the nghien ciru va mo piling met cfich ro rang mang Itrai cac gen lien quan, dieu Ma gen giy benh Vai cac tru diem nay, mo hinh ruoi giim chuyen gen da va dang duqc irng dung cac nghien ciru benh tren ngtrii (Bilen, Bonini, 2005; Pendleton et al., 2002; Sang, Jackson, 2005) Benh Parkinson thuing duqc biet den IA benh liet rung, day la benh thoai hoi than kinh theo deo tuoi, gay anh hiring den kha nang van &ling, giti thang bang va kiem sok ca the dia benh nhan viot Nam, day la met benh tuang del ph6 bien vii tSt 10 mac Will la 90-100 ngtrii ten 100000 din Sr thoai hela dan cac to bao san sinh dopamine duqc cho la nguyen nhin chinh don den benh Parkinson (Gandhi, Wood, 2005; Moore et al., 2005) Yeu to moi truing va yeu to di tuyen co lien quan chat the tai sir thoai hia 425 Nguyen Thi Ttrong Vy et al Cie nghien cuu cho thiy sir hinh thinh the WU Lewy IA nguyEn nhan chinh dAn dEn cic r8i loan chirc nang ty the dua den stress oxy h6a cOng vai viEc dopamine bi oxy h6a vi tra nen gay d'QC cho to bio (Feany, Bender, 2000) Gia thiet dtra li asynuclein, parkin vi DJ-1 c6 lien quan den qua trinh tich to oh protein de hinh thinh nen the Lewy Trong a-synuclein &rig vai tro trung tam bEnh Parkinson vi day la thinh phan chinh the Lewy (Rosner et al., 2008) Protein synuclein dugc biet den vi cong b6 nim 1988 bed Maroteaux nghien cuu ve loii Torpedo californica, co cic clang chinh IA a-synuclein, (3synuclein, vi y- synuclein, synoretin; dugc tao qui trinh splicing de hinh thinh mRNA tnrang thinh Trong a-synuclein co vai tro quan trong qui trinh diet' haa chu trinh hinh tni tiet tai synap vi dieu hoa tier dopamine (Perez et al., 2002) Cac nghien et:1.0 in vitro cho thay clang monomer khong gap cuOn c6 the tich to thinh cic Bang oligomer nh6, cau tnic co the duce on dinh bai nhang twang tic giong u-sheet vi sau d6 hinh thinh clang co lucmg phan tir cao han, khong tan goi la fibril, day IA clang ,cau trite dugc tim thay pito bien cic the Lewy Ngaii cic nghien cuu cho thay khong chi so tich to cila protein a-synuclein dugc tim they cfic the Lewy dic trung cho bEnh Parkinson ma can co cic dot bien khfic ten gen ‘SNCA h6a cho asynuclein Cac dang bien nhu sau: dang dOt bien diem amino acid alanine bi thay the bai threonine a vi tri protein thir 53 (A53T)hoic proline tai vi tri protein thu 30 (MOP), mOt so it trtrang hqp dang amino acid glutamic bi thay the INA lysine tai vi tri protein thir 46 (G46L), vi dang dOt bien lim gia tang so lacing bin cOa gen SNCA clan tai du thira Itrang a-synuclein (Polymeropoulos et al., 1997) Tuy vEy, ca the dm so tich to a-synuclein mOt vai, tnrang hgp bEnh Parkinson van china dugc hieu mOt cach day du Do de, viEc xay dung m8 hinh nghien dui so tic eking cua gen a-synuclein len hE than kinh co vai fro then chat viEc tim ca the phan tir phit sinh bEnh Parkinson CS nghien cuu nay, chimg tai sir dung ruoi giim de xay dung m8 hinh nghien cuu bEnh Parkinson Trong nhang nim qua, nh6m cic nhi khoa hoc Chau Au vi M9 di xay dirng m8 hinh ruoi de.nghien tiro bEnh Parkinson nhu m8 hinh ruoi chuyen gen bieu hiEn vuot mire gen a-synuclein, DJ1, parkin (Feany, Bender, 2000) Mic du k9 thuit, mo hinh ruoi gam chuyen gene a-synuclein mang tinh ke 426 thira cic thinh tkIll di cong b6, nhung tai ViEt Nam day li nghien ciru dau tien htrang tai viEc sir dung m8 hinh ruoi giam de nghien cuu bEnh Parkinson Ket qui nghien cuu cling tier' de cho viEc thiet ke cic mo hinh ruoi giam chuyen gen de sang icc cfic chat chiet xuat to nhien dE tri bEnh Parkinson tai phang thi nghiEm cua cluing VOL VAT LIEU VA PHYONG PHAP \It liEu Vector tai to hop pBlue/SNCA (ViEn Khoa hoc Cong nghe Kyoto, Nhit Bin) mang cDNA dm gen SNCA ma h6a cho protein a-synuclein Vector pUAST (ViEn Khoa hoc C8ng nghE Kyoto, Nhit Ban) mang gen kiting khing sinh amp' giup cho viEc sang 19c cic the bien nap mang vector tai to hg, Wing MCS, viing,chira nhat mOt vi tri nhin biet cic enzyme cat giai han can thiet cho Bong h6a gen mac tieu vio vector Ngoii ra, ten vector can c6 cic yEu to cho phep then gen vio nhiem sic the ruoi giant nhu: nhan to chuyen gen P, gen chi thi w cho phep khoi phut tinh trang mat de a ruoi giam dOt bien gen mit trang, trinh to UAS (Upstream Activation Sequence) Chung Escherichia coli DH5a [FendA1 hsdR17 (rklmk") supE44 thik-recAl gyrA96 A/acUl 69 (T80 lacZ AM15)] (BO mon C8ng nghe sinh h9c phan tir va mai truang, Twang Dai hoc Khoa hoc To nhien, Dai hoc Quoc Gia Thinh pito H8 Chi Minh) dung clang h6a gen Cfic.doni ruoi giant Drosophila melanogaster: dung mei giam dung chuyen gen A2-3, mat tang; dang ruoi mang balancer P ( w; sp/cyoGFP; pre/TM6B); dang P{+; Elav-GAL4/cyo; +) (8765) bieu hiEn tai mo nio c6 kieu hinh canh cong; clang P{+; +; Ddc-GAL4} (7009) chira promoter cfia gen DOPA decarboxylase ginp bleu hiEn tai cic to bio than kinh sin sinh dopamine; dung ruoi hoang dai dai chUng CantonS +; +} vi dOng bieu hiEn 11galactosidase P{+;+;UAS-LacZ} (Ngfin hang rail giam Bloomington, my) Pinning pilaf) Tgo (long E colt DH5a mang vector tdi to hgp pUASTISNCA cDNA SNCA thu dugc tir vector pBlue/SNCA bing phin Ung PCR vai enzyme Pfu polymerase (Fermentas, Singapore) dirge cat bang Xhol va BglIl Tgp chi Cling nghe Sinh hgc 11(3): 425-432, 2013 (Fermentas, Singapore) va not vao vector pUAST de tao vector tai to, hqp pUAST/SNCA Vector tai to hqp duqc ban nap vao E coli DH5a bang Ma bien nap va sang 19c tren mai trutmg LB-Amp100 Khuins lac m9c tren moi truimg LB-Amp dtrqc sang 19c bang phuang phap PCR khuin lac vai cap mei teen plasmid bao quanh vi tri ellen gen wizl/wiz2 Sau do, nheing khuAn lac cho san pham PCR co kich thuac twang img vai kich thu6c.dq thu nhan plasmid bang doin se duce nuoi cay phucmg phap SDS-kiem Vector thu duac tit cac khuan lac tren duqc PCR viii cap mai dac hieu gen va kiem tra bang enzyme cat giai han XhoI va Bg111 Trinh to gen muc tieu tren vector tai to hop duqc giai trinh to bang phucmg phi') Sanger va phan tich bang phan mem Jellyfish Phwong phdp Igo m6 hinh ruoi gam chuyin gen Vi tiem Thu trimg ru6i cua clang ruoi A2-3 mat trang mai duqc thy tinh giai doan 0-3 gibs va dux hien vi tiem vector tai to hqp pUAST/SNCA vat) phan duoi cua trimg b nhiet de 18°C Trimg mai sau vi tiem duce nuoi tren moi truang dinh threw 25°C thu ca the Fo Sang 19c ruoi mang vector tai to hQp cla sat nhap vao b0 gen Ruei Fo duqc thu nh'gn va lai vbi ruoi det ban mat trang W118 nham Oat hien the he ruoi lai F mang gen awe tieu co kieu hinh mat do gen chi thi w+qui dinh Cfic clang ruoi chuyen gen sau ductc lai vai ruoi mang balancer de xfic dinh vi stri clan gen tren nhiem sac the ruoi va lam thufin nham thu cac clang citing hqp tir gen muc tieu SNCA Sq hien dien oh gen SNCA ruoi chuyen gen &lac kiem tra bang, phucmg phap PCR yeti moi dac hieu gen va mil bat tren nhiem sac the, bao quanh gen muc tieu SNCA Kiem tra sr, bieu hiin act protein a-synuclein cua Mc &mg mai chuyin gen tgi ma nao Cfic clang ruoi giAm chuyin gen a-synuclein da duce tao va kiem tra duac tien hanh thu cac ruoi cfii trinh va lai vai clang bieu hien protein GAL4 (sir dung clang Elav-GAL4/Cyo) nham kiem tra sq bieu hien dm protein a-synuclein tai m8 nao ElavGAL4/Cyo la clang ruoi mang promoter chuyen bat cho mo nao, do lai dung rail vai cac deng UAS-SNCA thi chi nhitng to bao than kinh mai bieu hien protein a-synuclein The he lai Fl duqc chQn loc dip vao kieu hinh cac balancer quy dinh, vi vgy cac ca the mei lai Fl mane UAS-SNCA va Elav-GAL4 duqc hp chQn co kieu hinh canh thang Protein tong tir cac mau dau ruoi duce thu nhan va tien hanh dien di phan tich bang SDS-PAGE va kiem chirng bang Western blot vsfti khang the dac hieu khfing a-synuclein Deng ruoi Ddc-GAL4 kitting mang balancer va la clang mang promoter chuyen bat gifip bieu hien tai cac to bac, than kinh san sinh dopamine Cfic the he ruoi lai F dime sir dung khao sat str bieu hien protein a-synuclein tai cac m8 than kinh chuyen biet san sinh dopamine bang phuang pita!) hem m8 man dich huStnh quang vai khang the dac hieu khfing asynclein hoa m6 mien dich huynh quang Nhu (1 nun unoh istochemistry) Ruoi lai Fl duqc 25 tuei duce chQn dimg de Lich nao va nhuOm hoa m8 mien dich huStnh quang Nao ruoi duqc tech dung dich PBS va co dinh mo bang dung dich paraformaldehyde 4% pha PBS nhiet dO phong 30 phut Sau (16 rira bang dung (Lich PBS-0.3% TritonX100 (v/v) va khoa mitt mo bang dung dich huyet de (Normal goat serum) 10% PBS-0.15% TritonX100 (v/v) nhiet de phong 30 phut MAu m8 nao duqc it yeti kiting the so cap khfing a-synuclein b 4°C qua dem Sau matt m8 duqc rira 4-5 lan bang dung dich PBS-0.3% TritonX100 va duqc u vai khang the thir cap phat huknh quang FITC 484nm b nhiet de ?hong 2-3 gi0 Mau nao duce rira sach va Lich nao co dinh len lame va quan sat clued kinh hien vi hujrnh quang Hinh inh &Km phan tich bang chtrcmg trinh ImageJ, thong ke bang kiem dinh t-test cita Microsoft Excel KET QUA VA THAO LOAN That ke vector mang va Men MO gene SNCA ruoi gigm Chung Vol tan hanh thu nhgn cDNA gen SNCA bang phan Ong PCR tir khu8n mau plasmid pBlue/SNCA va clan vao vector pUAST bang phin Ong not nha enzyme T4 DNA ligase Vector tai to hqp pUAST/SNCA sau duac sang loc va kiem tra bang phuang phfip PCR vai mei (lac hieu va phuang phap cat giai han Ket qui kiem tra vector tai to hqp cho they vector mang gen SNCA the hien tren san pham PCR kich thuac 435bp ttrcmg Ong vai kich thuac cDNA gen muc tieu (dit lieu thqc nghiem 427 Nguyen Thi Tuang Vy et al kitting trinh bay tai day) D6ng thtri ket qui xir ljr yeti enzyme cat ihi han va ket qui kiem tra trinh tir gen ding cho thay gen SNCA duqc chen vio vector co trinh ttr giOng 100% vei trinh ti,r cDNA cua SNCA ten ngan hang gen Nhu vay, chang toi da tao clang tong vector tai to hqp mang gen SNCA Ben canh do, cic ket qui &mg hoi wan tu tren hai (long gen SNCA det bien ding cho thay chung Col da thinh ding viec thu nifin hai vector mang clang On bien A3OP_SNCA vi A53T SNCA Cfic vector sau d6 dtrqc tich chiet tit ti bao E coil di vi tiem vio ru6i Sang loc tool mang vector tai vio bQ gen a hop (la sat nhap Sau vi tiem, nhO vio yeti to chuyen vi transposon P tren vector pUAST (Bachmann, Knust, 2008), gen SNCA duqc sat nhip vector, tai to hqp met cfich ndu nhien vio be gen ruoi giam Cic &mg ruoi chuyen gen c6 kieu hinh mat duqc lua ch9n clua vio gen chi thi Chang UM tien hinh lam thufin cic clang ruoi chuyens gen thu nhan duqc vi sir ding phep lai voi ruoi mang balancer de, xic dinh vi tri gen chen tren nhiem sac the rail giam, ket qui phan tich cho thay cluing toi thu nhan dtrqc 03 (long ruoi chuyen gen nhu trinh bay tren bang Sau lim thuAn va xic, dinh nhiem sic the mang gen mac tieu, chfing toi tiep tic tien hinh kiem tra phan tich cic dOng ruoi Tnrac tien, chang toi den hinh kiem tra su hien dien cita phirc hqp gen UAS—SNCA (hoac UAS-A3OPSNCA; UASA53T SNCA) ca the ruoi chuyen gen bing phucmg phip PCR vai cap moi dac hieu bao quanh yang gen mac Lieu (Hinh 1A) Theo ly thuyet, dm% tic Ong cua y'eu to chuyen vi gen P, vector pUAST/SNCA &qv dua vio phoi ruoi, vai boat dOng ciut transposase, phirc hqp UAS-SNCA nem giera cic,trinhtv IR cita yeu to P se duvc chen vac) nhiem sac the, ctia ruoi giam Trong thiet ke thi nghiem chuyen gen dia chimg toi, enzyme transposase chi hien dien nhAt the he P (phoi ruoi) chuyen gen Cic the he sau do, enzyme transposase hoin toin, kh8ng hien dien, vay, sir chen gen vac) nhiem sac the la su chen gen On dinh Chang Col ,thu nhan DNA be gen dm rail chuyen gen du tuyen vi sir dang lim khuon phan irng PCR kiem tra yeti moi dac hieu cho phirc hqp gen mac tieu Ket qui phin.ting PCR vai khuon li DNA be gen dm ru6i chuyen gen cho vach co kich thudfc nhu du doin img vai cap moi so sinh yeti vach chimg dtrcmg (Hinh 1B, gieng 2, 3) nhu sau: vach c6 kich thtrac 435bp img vai cap moi dac hieu cho gen SNCA (Hinh 1B, gieng 9, 11, 13), vach co kich thy& 585bp img v1i cap mei dac hieu bao quanh viing ngoii gen wizl/wiz2 (Hinh 1B, gieng 8, 10, 12) DOng thidong ruoi doi chtng CantonS kh8ng c6 xuat hien vach gen ten hinh PCR vai ca cap moi ten (Hinh 1B, gieng 6, 7) Mar vay, chang toi da tao (long thinh Ong dong ruoi giam chuyen gen mang gen a-synuclein vai vi tri chen gen tren nhiem sac the nhu sau: UASWT_SNCA (nhiem sic the II); UAS-A3OP_SNCA (nhiem sic the II); UAS-A53T_SNCA (nhiem sic the X) (Bing 1) • Cic.clong ruoi tie!) tic duqc kiem tra tang bieu hien protein a-synuclein mo nao bang phuong phip Western blot vi nhuOm mien dich huSmh quang vai khing the doc hieu khing asynuclein Bang Cac clang ru6i chuy4n gen bi6u hiOn gen a-synuclein STT Ten clang ru6i chuyen gen Phirc him gen Dro-AlphaSyn WT UAS-SNCA Dro-Syn A3OP UAS-A3OP_SNCA Dro-Syn A53T UAS-A53T_SNCA Bien hiOn protein a-synuclein m0 nao De nghien ciru chirc nang cua a-synuclein ten mo hinh ruoi giAm chuyen gen, cluing toi sir dung he thong UAS-GAL4 nham bieu hien vuqt mirc protein a-synuclein tai vi tri cic mo can khio sat He thong Om phAn, UAS (upstream activation 428 NST nhen gen chen Dec diem Deng hap Deng hap X Deng hap sequence) loan trinh tv duvc thiet ke tnrot gen mac Lieu, vi GAL4 dtrqc thiet ke nim sau promoter bieu hien dinh huong mo Benh Parkinson nhu dtrqc biet la benh thoii hem thin kinh, d6 chang toi ch9n dong ruoi Elav-GAL4 giap bleu hien protein chuyen biet tai m8 nao Khi lai cic (long ruoi UASSNCA yeti clang Elav-GAL4, b the he F1, GAL4 - Tap chi Cong nghe Sinh hoc 11(3): 425 432, 2013 dugc tong hcrp se gin len trinh to UAS IA gen mix tieu dual su kiem soat cua UAS se dugc phien ma giap bleu hien dinh huang protein a-synuclein tai m6 nao ruoi (Hinh 2A) D'ong thei dua vao kieu hinh cua F1, chang toi chqn lai có kieu hinh canh thing se mang phirc hqp caa UAS-SNCA va Elav-GAL4 Ket qua dien di SDS-PAGE mau dau ruoi truans (Hinh 2B) cho they lai Fl,tir dong ruoi d8i dugc chuyen cDNA SNCA chirng CantonS (Hinh 2B, gieng 2) kh6ng the bieu hien protein tuang ang ci cac meu protein ter cac lai Fl (Hinh 2B, gieng 3, 4, 5) dm cac dong ruoi chuyen gen muc tieu cho they có bieu hien protein muc tieu co kich thuac khofing 18kDa so vai thang protein chuan D6ng thei ket qua Western blot vai khang the dac hieu khang a-synuclein Minh 2B, gieng 3, 4, 5) cho they cac d6ng ruoi lai deu bieu hien protein a-synuclein Nhu Vey, protein a-synuclein clang hoang dui va dung det hien A30P, A53T deu dugc bieu hien dinh huang ding tai m6 nao cua ce the mei lai Fl Mat Ichic, chUng toi danh gia su bleu hien asynuclein va cac dung det hien tren m6 nao ruoi tai cac to bio than kinh san sinh dopamine, dua tren ca the tong hqp dopamine (Yu et al., 2005), chimg toi bieu hien dinh huang a-synuclein trong, to bao than dieu hoa cua kinh san sinh dopamine duoi promoter Ddc (DOPA decarboxylase), met promoter dieu hoa tong hcrp DOPA decarboxylase - enzyme co vai tre ,con dueng sinh tong hqp dopamine Chung toi thiet lap cac dong mei chuyen gen mang phac hqp *en Ddc-GAL4>UAS-SNCA va thu nhan mo nao ruoi chuy'en gen, nhuem boa mo mien dich hujrnh quang vai khang the khang a-synuclein Ket qua kiem tra bieu hien protein a-synuclein to bac) than kinh san sinh dopamine (Hinh A) cho they met so vang tren m8 nao a ruoi mang gen bieu hien asynuclein clang khong det bien va dung det bien A30P, A53T cho tin bleu huSfnh quango cac cum to bao, sinh dopamine (yang dugc khoanh trim) doi chieu tren m6 hinh cac yang san sinh dopamine dugc ding be beri White ye (long tic gia nem 2010 (Hinh A3) Cac dong mei d8i chimg Ddc-GAL4 va DdcGAL4>UAS-LacZ khong cho tin hieu lai vai khang the khang ha-synuclein (Hinh Al, A2) Ket qua nhu'em ma dugc xac dinh ctrimg de tin hieu bang chucmg trinh Image) va th8ng ke the hien qua bieu d8 hinh B cho they mew de tin hieu huynh quang thu dugc ter cac (long ruoi co bleu hien protein a-synuclein cao hen nhieu 6? 4-5 Ian so vai cac dong doi chfrng va coy nghia thong ke phan tich bang kiem dinh t-test vai cac gia tri p
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