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Tap chi Cong nghe Sinh hpc 13(3): 801-810, 2015 NGHIEN CVU THIET LAP QUY TRiNH THU NHAN TE BAO TIEN THAN NQI MO MAU CUONG RON NGLIYI Nguyen Thanh Tam', Phan) Le Biru Traci , VII Bich NgQc', Trjnh Ngoc Le Van', Nguyen Minh Hang', Dining Thi Bach Tuyit2, Phom Van Pink' 'Throng flai hpc Khoa hpc to nhien, Doi hpc Qujc gia Thanh pita lid Chi Minh 2Trwang Doi hpc pham Thanh Ho Chi Minh Ngay nhall bai: 21.3.2015 Ngay titian (tang: 30.7.2015 TOM TAT Hien nay, to bac) tien than dang la tam diem cho sir phit trien lieu phi!, to bio nhim phuc vu cho viec dieu trj cite benh ve thieu mau cue b4 Trong do, to brio den than nei m6 clang vai tr6 quan tong viec tham gia vim qui trinh stia chfra va di tao mach Vi the, cluing toi tien hinh nghien cfru thietl#p quy trinh thu nh#n &mg to bio tir matt cuing rOn ngueri Te bio dun nhin &Arc thu nhon bang phuang phip li tarn ding ty tong Te bio dart nhan sau thu nhin duqc thu nghiem nuoi cay tronif mai tnrerng khac bang phuang phip khic nham khio sat va thiet lop duqc quy trinh nuoi cay toi tru nhit cho (long to bio tien thin nei m6 Te bito duqc dinh gii thong qua tieu chi hinh thii, tang ham dinh va tang sinh cic th6i diem khio sat 7, 14, 21 va 28 sau thu n110 Sau dO, matt to bio Ong vien duqc phan tich cac dtic diem kieu hinh mien djch th8ng qua cic marker be met: CD14, CD34, CD44, CD45, CD73, CD90 va CD133 Ket qui cho ditty mau to bao du qc nuoi cay m8i twang EBM-2 vOi SingleQuots: 12 µg/ml BBE, 10 ng/ml hEGF, I pg/m1 hydrocortison & antibiotics, 5% FBS b6 sung 50 ng/ml VEGF, 50 ng/ml hIGF, 50 ng/ml hEGF bang phucmg phip chnyin djch not to bio sau 24 gi6 nuoi cAy la hieu qui nil& so v6i cac nghiem thirc khao sat khic ea tai Te bio tien thin nei mo thu nh#n duqc bieu hien throng tinh vii cac marker CD34, CD133, CD44 va am tinh vOi CD14, CD45, CD73 vi CD90 Them vao do, cac to bio co kha nang tang sinh nuoi cay in vitro vi hinh thii to bio On dinh theri gian nuoi cAy Tin khOa: mau cuing ron, nuoi cay to btio, quy trinh thu nkm to bcio, d bcio gjc, to boo tin than nen mo DAT VAN DE vira qua, to bao gecitien Han met vai th#p than ter tam diem cila sir phut trien cac hirang lieu phap to bao cho rat nhieu benh, bao gem cac benh ye tim mach (Fan et aL, 2010; Moubarik et al., 2011; Chen et al., 2011) Giai (loon quyet dinh viec sira chi-a cdc mo bi thieu mau cuc be la cal trien he thong phan be mach duqc dieu hoa beri qua trinh hinh mach mau mai co the hoc sir phit trien cita cac mach mau mei Met so nghien ciru tit& day chUng minh sir ten tai cita cac to bao nei mo tufin hoan nhieu benh ve tim mach, cau hoi la tai nhimg to bao (hay tien than duqc Cita cuing) bien hien vai tiro si,r phut trien mach co the tre so sinh TO bao nguyen they hay to brio geetien than nei mo c6 the g6p phan vac sir hinh mach mail mai co the.truerng bang qua trinh hinh mach, dieu du qc cluing minh (Janic et al., 2010; Yoder et al., 2012) Cac cong trinh di chirng h stir hien dien cite cac to bao tien thin Wei me CD34+ mau ngoai vi ngueri, c6 kha nang sat nhOp vac cac vi tri hoot hoa qua trinh hinh mach Sau do, cac nha nghien cuu tim dirqc bang chimg quan tong qua trinh hinh mach mau a tre so sinh IA to bao tien than nei mo vat kha rang "homing" den cac vi tri hinh mach mai co the va biet hoe, cac to bao nei mo truemg Do v#y, to bao tien than nei me duqc xem nhtr ting vien tiem nang cho sir phut trien cac huerng dieu tri cho benh tim mach a ngtrai (Janic et al., 2010; Asahara et al., 1997; Asahara et al., 1999) Te bao nen than nei mo tir xucmg hoOc mau ngooi vi, d#c biet la fir mau day roil ngueri c6 vai to quan tong qua trinh sinh ljr va sinh 1y benh khic Chung tham gia vao qua trinh tao mach va d Ong mach, chinh vi the, cluing c6 chirc rang quan tong viec sUra chira mach (Hur et al., 2004; Di et al., 2009; Urbich et al., 2005; Rchman et 801 Nguyen Thanh Tam et aL al., 2003; Yoder et al., 2007) Nhiing nghien ciru ten ding vat di khim phi ring sv to mach mai ma thieu mau cvc be c6 the duqc cai thin nha ghep tit), xtrang tr than Chien luqc dieu trj dAy hira hen dva tren nen tang cac bao tien than nal mo co the biet hem cac to bao nei ma truing (Fadini et al., 2012; Hur et al., 2004; Murayama et al., 2002) Trong tucmi lai, de (mg clung lieu phap tri lieu bang cac bio tien than nei m8 (mg vien rang rai hors thi viec xac dinh kha nang bit h6a, marker dinh dau va vai tre sinh ljf tiem nang cite chimg la rat can thiet (Hur et al., 2004; Yoder 2012) Nhieu quy trinh phan lOp te bao tien than nal mo tir cite nguen khac nhu mau ngoai vi, tuSI xuang, mach hay tir cac ma va ca quan khfic da duqc ma to (Uhler et aL, 2007; Loomans et al., 2004; Yang et al., 2011) Tuy nhien, te bao tien than nal m8 tuan hoan mau ngoai vi, mach hay cac ca quan khac chiem tjr le nit thip Cfic nghien ciru truac day cho they, co Ichang 0,05-0,2 te bao/ml mau ngoai via ngtrai truing thanh, co 2-5 bao/ml mau cuOng ron (Ingram et aL, 2004) MOt khfic, viec thu nhen lai phirc tap horn mang tinh xam len cao Do do, viec phan lop loai te bao thuOng it co hieu qua Gan day, mau cluing ron ngoai duqc chirng minh la met ngu6n phong phis de thu nhOn te bao tien than nal mo vai tien nang dieu trj da clang (Lin et aL, 2011; Wu et al., 2007) Vi mei throng nuoi cAy te bao la di em quan tong tronf viec xac dinh dOc tinh cita te bao Hien tai, met so mei truing thong dung viec nuoi cAy tang sinh te bao tien than nei m8 nhu M-199, MCDB 131 be sung cac nhan to tang truing VEGF, bFGF, EGF, R3-IGF-1, hydrocortisone, ascorbic acid va mai truing tang truing nei ma EGM-2 da duqc sir dung (Taniguchi et aL, 2006; Loomans et al., 2004; Yang et al., 2011; Ye et aL, 2008) Mvc tieu cita huang nghien c(ru la thiet lap met dieu kien tot nhAt cho str tang sinh in vitro dm cac te bao tien than nei m6 thu nhOn tir mau cuOng A ron ngueri co kha nang tang sinh thoi gian dai ma kh6ng anh hiring den kha nang tao mach ding nhu cac dOc tinh, chirc nang cita quan the te bao tang sinh D6I TUQNG VA PHUONG PHAP NGHIEN OYU Thu MO miu cuong ron MAu matt cuOng ron ngoai duqc thu nhOn tir Benh vien Hung Vuang — Thanh phO Ho Chi Minh 802 Cfic mkt mau phai d am bao cac tieu chitin: (1) — MAu phai binh nhau, day ron nguyen yen va duqc thu nhOn tir tre sa sinh kb:5e manh, di thing; (2) — MAu mau sach, duqc xet nghiem am tinh vai HIV, HBV, HCV va met se benh truyen nhiem khac; (3) — Mau ducre thu nhOn sau sin phi sinh va duqc sir dung — gib sau Thu nh$n to bio don nhan tir miu cueng ran Te bao dan nhan tir mau cueng ron ngtrai duqc thu nhan bang phuang phap li tam tat gradient nong de Ficoll-Paque Plus , (Amersham Biosciences, Uppsala, Thy Dien) 8ng Ii tam 15 ml (Coming, Hoa Kjr) Mei Ong li tam chira phan lap g6m miu miu cuOng ron QA duqc pha loang vai dung djch PBS va dung djch Ficoll-Paque Plus vai ti lg 2:1 va du qc li tam 500 g 25 phut a 18 — 20°C Sau d6 thu nhOn phan clop te bao don nhan nim giira lap Ficoll-Paque Plus va 16p huyet tuang ben ten Khio sit nuoi cAy te bio tien than nei mo ting vien Te bao dan nhan duqc huyen phit mei tramg khao sit: (1)-MT1 (M199 + 20% FBS, 1% antibiotic-mycobiotic, 0,4% ECGS /heparin va 12 µg/ml BBE, 10 ng/ml hEGF, ng/ml bFGF, ng/ml VEGF, ng/ml hIGF); (2)-MT2 (MCDB131 + 20% FBS, 1% antibioticmycobiotic, 0,4% ECGS /heparin va 12 1.1g/m1 BBE, 10 ng/ml hEGF, ng/ml bFGF, ng/ml VEGF, ng/ml h1GF); (3)-MT3 (EGM + SingleQuots: 12 tt g/ml BBE, 10 ng/ml hEGF, µg/ml hydrocortison & antibiotics, 5% FBS (Sigma) + 50 ng/ml VEGF, 50 ng/ml hIGF, 50 ng/ml hEGF) vai phuang phap: (1)-PP1 (Thay mai mei tnr0ng sau 72 ti ia nuoi cAy te bio) vi (2)PP2 (Chuyen djch not te bio sau 24 gia nuoi ay); SO luvng te bao duqc hieu chinh ye mot QC? 106 te bio mat binh nuoi cAy a dieu kien 37°C, 5% CO2 Te bao tien than nal ma (mg vien duqc dinh giit dva vao hinh thai, sv barn dinh va tang sinh thoi gian khao sit 7, 14, 21 va 28 Nu8i cAy tang sinh te blo tien than n6i nto ting viers M8i truing nuoi cAy duqc thay mai sau lchoing — tity theo mat da te bio bim dinh va tang sinh binh nuoi cAy Khi mat da te bac, tien than nei m6 (mg vien binh dot khoinf 80 — 85% , tien hinh cAy chuyen tang sinh Cic te bio sau duqc cAy chuyen — lin se duqc sir dung de xac dinh dOc tinh te bao tien than nei mo Tap chi Cong nghe Sinh hpc 13(3): 801 -810, 2015 Xic dinh d#c tinh to ben tan than nOi mo De khing dinh te bao &rig vien thu nhan duce la te bac tien than niji me, tien hanh xac dinh cac dac diem kieu hinh mien djch cua cac te bao ML te bao dirge tien hanh phan tich kieu hinh be mat bang 1E9 thuat flow cytometry Mat d bao binh nuoi d at 75 — 80% se tien hanh tach bang Trypsin/EDTA 0,25% (COng ty GeneWorld, Thanh phi HO Chi Minh, Viet Nam) Te bao duce it vii Idling the cho tong loci marker toi 30 phut or 4°C Li tam lin d e loaf bo khang the thira bang dung dich FACSFIow Chuyen huyen phu te bao sang eng li tam ml (BD Biosciences, Hoa KS') sir Bang Tdm tat cacti bb trf bao citing d e phan tich kieu hinh va phin tram ducmg tinh, am tinh veri timg marker bang hg th8ng FACSCalibur Cie marker d tax dung phan tich genn CD34, CD44, CD133, CD14, CD45, CD73 va CD90 (Rouhl et al., 2008; Hur et al., 2004; Fadini et al., 2012; Hristov et aL, 2003) X* lir se lieu Tit ca cac s6 lieu thu nhan dime xir ljr bang phin mem Microsoft Excel va Statgraphic 7.0 nhim loci 136 cac gia tn bit thuing day s6 cac Ian lap 101, d6ng that tinh gia tri trung binh, sai s6 va dO tin cay er mire LSD 95% cac nghlOm thiic thi nghiem STT Ton nghiem thin Cach bb hi thi nghhlim Nghiem thirc A MT1 + PP1 Nghiem thirc B MT1 + PP2 Nghiem thin C MT2 + PP1 Nghiem thirc D MT2 + PP2 Nghiem thin E MT3 + PP1 Nghiem thirc F MT3 + PP2 KET QUA VA THAO LUAN Khao sat nuoi cay te bao tien than nOi m8 iing vien Nghiem there A: Luqng te bao Mm dinh thua then, co hinh clang khong d Ong nhit, mOt s6 it trai dai, thon nho dau giong nguyen bao sqi, da so lai co dang bach cau truing thanh, troll, barn chat be mat nuoi cay Qua flied gian khao sat, khong thiy co sg phan chia, tang sinh cua cac bao ma chi they str bong trete clan qua cac lan thay moi truing nen so luqng te bao balm dinh cang giam Nghiem thirc B: Sr thich nghi cua te bao vii moi truing nuoi cay la khong co, bang minh chirng la sir giam din se luvng te bao bam dinh va kh8ng co diu hieu tang sinh qua tiled gian nuoi cay Sau khoang thii gian khao sat 28 ngay, cac bao barn dinh nghiem thirc tiep tic giam va bong tr6c din qua cac Ian thay moi truing tiep theo Tiep tic tien hanh thay moi truing cho mL te bao nghiem thirc den thir 40, phin km te bao mat di, lei rat it te bao bitm dinh va van khong co dau hien efia sir tang sinh Nghiem thirc C: S6 te bao barn dinh co giam di, cac te bao phan nhanh, cac to bao dang trung mo truck d6 kh6ng nhung so to bao co clang inch cau van kha nhieu, gay anh hiring den sirc song va tang sinh cua to bao tien than nOi mo iing vien Den thin 28 thl co so tang ye s6 lucmg te bao bam dinh, nhien so luqng te bac dang bach cau van rat nhieu va rat kh6 loci be sau 11111-mg lan thay moi truing tie? theo Ben canh d6, sv bien doi kieu hinh ciia cac te bao (la xay ra, cac to bao quan tam keo dai tau, dOng theri sau thu diem 28 khao sat thi sv tang sinh cua cac te bac la rat yeti, hau nhu chi ding yen mOt eh& Nghiem thirc D: Te bao bam dinh a thin co nhieu hinh ding khac nhau, khong (long nhat, mOt s6 trai dai, thon hai diu, da s6 la clic to bac dang trim gi6ug bach cau So luqng to bao giam din theo thu gian khao sat nhien ca hai dang te bao vim ton tai binh nuoi cay Den thin 28, hiu het cac to bao barn dinh truck d6 bon§ toe va mat di qua trinh thay mei truing, cac te bao Mtn dinh lai rat it va co hinh danf bit djnh, mang te bao loi lam, thieu sire sting Ket qua co the chirng minh cho so khong thich nghi cua te bao vii moi truing nuoi cay va d ac biet la sv bam dinh dui to ,bio Moi truing nuoi cay di kh6ng cam img dirge te bao barn 803 NguyIn Thanh Tam et al dinh tang sinh ma ngtrqc lai to bao chit di qua clic thori diem khao sat Nghiem thug E: Hinh clang to bao er, nghiem thirc tuang dui dung nhat, dai da se to bio c6 hinh clang trai dai‘ nhieu ggc canh gi8ng to bao trung me, met se rat it clic to bao c6 clang vien soi giong to bao nei,m8 va lac dic vai to bao tin than bitch cau bam rat chat vacs be mat binh nuoi cAy Sau thay moi truang, se luqng to bao co giam SO luong te bao &Slut:nig ti bin 600 529.32 120 Nghlipn thirc A 102.1 97.6 di nhung cac to bao yin giu nguyen hinh clang ban dAu Tuy nhien den khao sat tit* 21 va 28, cac to‘ bao chuyen clang sang met hinh thai kirk, met so kgo dai dAu, dAu 1ai hoi dgt met se kgo dai han (dai gap mai dang lai) Tuy nhien, ca hai clang to bao tren deu kitting thay bieu hien gas sv tang sinh luqng mac du ba bao van sang bang, kh8ng c6 bieu hien hoai 100 500 • 80 400 • 60 • Trung binhs6 tc Wan barn dinh 40 Nghi.m thirc B — 427.5 380.88 350.42— 300 • Thing binh so to bio him dinh 200 21.36 20 100 0• gian kliao sat S6 linang to bao 300 263.64 S6 hams te bin 250 210.14 Nghiem thaw C 250 200 Nghiem thin D 197.96 177.96 200 150 138.94 150 • Truing binh him dint, 99.22 100 brio • Trung binh so Id boo him dinh 101.66 100 50 50 28.9 Thrri gian khan sat Thin gian khao sin ngity 14 21 ngisy 28 ugly 14 ngiy 21 28 So Ittvng to bao sd Itroug t• bin 300 Nghiem thirc E 250 ThOi gian khao sit 14 21 ngity 28 ngay 14 ngity 21 28 ngity 232.66 546 84 Nghiem thirc F 600 178.42 183.18 200 700 500 167.98 400 150 • Trung binh so Id bao bin, dinh 300 223.7 • Trung binh so td bac, him dinh 100 200 50 100 52.18 77.62 Thoi gian khan sat 14 ngtiy 21 28 !Tay Thtri gian khan sit nghy 14 ngity 21 28 Hlnh Trung binh so te bac) barn dinh qua thtri gian khao sat el nghiarn thirc Nghiem that F: Tai thai diem ngay, cac to bao bam dinh c6 clang vien soi nho va rat it hay kh8ng c6 cac to bao trai dai, den thai diem 14 ngay, cac to bao • ttep tug bam dinh nhung van each xa ve khoing 804 cach, dac diem cling Ong he gia thuyet cac to biro la limg tiep tvc barn dinh de tang ve so luqng ma kh6ng phai stir tang sinh cila to bao barn dinh tnrac d6 Den thei diem 21 ngay, cac to bao van gift Tap chi Cong nghi Sinh hoc 13(3): 801-810, 2015 nguyen hinh ding ban diu va bit d Au xuAt hien star lien kit gitra cic bao voi (riming cich gifta cic to bao cling thu hero lii),,có nhikng ctun Ho nho (khoing - to bar)) xuat hien Ket qui la met mirth cluing cho s,r tang sinh cita to bao tii filed diem khao sat 21 ngay thfr 28, cic cqm to bao ce chieu Wang tang ve kith thtrac NIA so luang, deng thai quan han het la xuAt hien hien Juqng hop (long dm to bao bim dinh Dieu met Ian nth kiting dinh sq tang ve so Itrqng to bao bim dinh to bao tang sinh da thich nghi yen mei truing nu6i cay Nuoi cay to bio tien thin nOi mo ling vier' mai trtrarng vii phtrang phip khio sit Ket qua a bang cho thAy ring, trung binh se to bar) barn dinh nghiem thirc A, B, D va E giim din theo cic thai diem Bart sat, do, nghiem thirc A va D co so luang bao Mtn dinh a khio sit thir 281i rat thip Te bio a nghiem thirc kh8ng cho thay bieu hien dui sq tang sinh va sau then pan khio sat 28 ngay, cic to bio bim dinh din mat di lin thay mei trterng va hoan town kh6ng cho thAy to bao barn dinh cang nhu tang sinh khoang then gian sau khao sit qua cic Trong Q6, bio bim dinh a nghiem thirc C giam qua th6i diem khao sat ban diu (7, 14 va 21 ngay) nhtrng ce dau hieu tang se ltrqng t>r tiled diem 21 d On 28 ngiy, cho thAy bieu hien sir thich nghi cua to bao vii mei twang nu& cay la co nhtrng hinh ding to bao nghiem thirc trait, bim chit voi be mat nuoi cay (da s6 la hinh clang dm bao tien than cic dong INckcau) kitting phu hop vai hinh thii duqc mieu ti ve to bao tien than !lei m6 la ding vien sei, trop, khong co hai d Au Ice° dai (Deschaseaux et al 2007; He et al., 2005; Hirschi et al., 2008; Hur et al., 2004; Lin et al., 2011; Wu et al., 2007) Tii thai diem khao sit ngay, s6 luqng to bao birn dinh a nghiem thirc la cao duqc giii thich rang, phtrang phip thay mai mei truing sau 72 gia nu8i city to bio la khong phi' hop vi da tao dieu kien cho nhieu loci to bio bim dinh va cinh tranh yen to bao mong muon Bang Trung binh tit bac) barn dinh et nghiarn thtic Trung binh s6 ti bho barn dinh NghiO'm thin Hinh thai ti bao (diritn hinh) nghy 14 21 nghy 28 A 102,1 97,6 62,92 21,36 B 529,32 427,5 380,88 350,12 tEthu duc, nhd C 263,64 138,94 99,22 177,96 Tram barn chat 210.14 197,56 101,66 28,9 Da hinh thai 232.66 178,42 183,18 167,98 Dal, g6c cgnh 52,18 77,62 223,7 546,84 Sei, d6ng nhit E Iron, bam chat S6 luvng t6 bao 600 529,32 546,84 -4- NghiOm thirc A -M-NghtOm thirc B 500 NghlOm thuc C 400 -m-NghiOm thin D *-NghiOm thin E 300 12 63,64 332,66 200 210,14 - NghiOm thin F 100 102,1 ThOi gian kNo sat Hinh So sanh trung binh to bao barn dinh 6' nghiOrn thin SO5 Nguyen Thanh Tim et al nghiem thic F, m#c du bao barn dinh la khong cao a thin diem nhung di cho thAy su tang manh ve so luqng to bao bam dinh qua thai diem khao sat sau de va su tang duce cho la su phin chia dui to bao barn dinh co mau khan sat Dieu cho to nh(in djnh ring, m6i truing nuoi city di gall) bac) cam ung va thich nghi de tang sinh tot D Ong th6i, vii phucrng phap chuyen djch not to bao sau 24 gity nued cAy la phis hqp yen thin diem bam dinh dm bio tien thin nei m8 nen so luqng to bao barn dinh ban ditu co the duqc xern la cac to bao nei m6 img vien Ngoai ra, ket qua ve hinh anh cling Ong he cho quan diem nay, cac bio a nghiem thirc co clang hinh vien s6i nh6, kha dong nhit va co bieu hien ctia su tang sinh cua cac to bao quan sat dual kinh hien vi Ket qua a hinh la met mirth hoa ding co chic chin ring to bio nuei cAy a nghiem thirc F la tot nhat, thich nghi mei tnrang, barn dinh tot, tang s6 luqng theo cap se nhin va deng thai sau thin gian khao sat, bac tiep toc tang sinh va ce the tien hinh city chuyen sau 40 ke tir bit dint nuoi city so cap Cac nghiem thirc lai cho they stir giam se luqng to bac, barn dinh qua cac thai diem khao sat, khong phit hop de tien hinh thu nhn dong bao Nan cAy tang sinh to brio tien thin nOl mfi Ling vien Quin the to bio nen than On m8 img vien nghiem thirc F duqc sir dong de nuoi city ging sinh, phoc vu cho cac thi nghiem tie, theo Th6ng qua lin city chuyen d au tien, luqng to bao du qc pha bang lin, ti met binh nuoi duqc chia birth nu8i mai Toc de tang sinh cua to bao img vien nuoi city thir clip lit twang d 6i gieng gitra cac lin city chuyen va nhanh hon so yeti qua trinh nuoi cAy sit cap So yin num city so cap, thin gian to bao tang sinh nhanh hon nuoi city thi cif) (sau — 12 gia city chuyen, to bio di barn lui), dong thai, t6c tang truimg cua to bao cling nhanh bon (CO the city chuyen lin tiep theo sau — ngay) Te bio nen thin On m8 img vien tang sinh on djnh qua cac lin cAy chuyen va khong c6 stir bien del ye hinh thai TO bao truerc city chuyen tuin va tuin cho thAy kha nang tang sinh manh, dien tich barn dinh.cita bio lan reng nhanh Sau 24 gia city chuyen, to bio di barn dinh tit loi vi cho they co su phin chia nhim tang sinh ve met s6 'thong to bac Sau win tien hinh city chuyen lin din tien, ngoii viec tang sinh cua tong ca the to bao, to cac cum to bio, chimg cho they so hqp dong, gin ket 806 cac cum to bao lai vii va den tuAn tiep theo di dat m(it de city chuyen lin Sr tang sinh duqc tri cita to bac tien thin nei m6 ting vien qua cac lin cAy chuyen cang cho they to bao thu nh#n nghiem thirc F the hien kha ning phan chia tact cac the he tiep theo va diy la met d c diem the hien tinh g6c cua to bao qua trinh nu8i city in vitro (Deschaseaux et aL, 2007; Mikirova et al., 2009) Nhu v#y, quirt the to bao tien thin nei m6 img vien tren di bieu hien met s6 ilk diem dm to bao tien thin On m6 Quin the to bao tiep toc duqc kiem tra cac thi nghiem tiep theo de kiting dinh Ching chinh la to bao tien thin On me Xic djnh d$c tinh to bio tien thin n(ti nth Theo ket qua nghien ciru bang 2, to bao tien thin nei m6 img vien sau lin cAy chuyen bieu hien am tinh yen marker CD45 (3,09%) va CD73 (6,47%), am drill hoan tom vii marker CD14 (1,06%) va CD90 (0,58%), d Ong thai bieu hien ducmg tinh yin marker CD34 (83,13%), CD44 (87,03%) va CD133 (90,01%) Ket qua nghien ciru cho ditty ring, to bao tien thin nei m6 thu nhOn tir mau cuong ten ngutri nghien dm chua tinh such hoan Wan, quail, the to bao tap nhiem met luqng nho cac clang to bao khac Thuc viy, cac marker CD14 vi CD45 thtrong bieu hien tren cac to bio thuec dimg to bao too mau CD14 bieu hien chi yeu tren cac d thuc bac va Inch Cali trung tinh, d Ong thiri, ne cling dtrqc bieu hien a to bio tua Ngoai ra, hai marker, CD73 va CD90 cling dtron xem la tieu chi ding de dinh gia kieu hinh mien djch curt to bio g6c trung rno ducrc ding de lam dei thing am phin tich to bio tien thin nei me Chinh vi the, dart!) gia so bieu hien kieu hinh mien djch dui to bio tien thin !lei m8 mau cuong ren ca marker deu bieu hien nhung a ti duang tinh rat nho D Ong thai cac marker CD34 CD44 va CD133 la marker dung de dinh gia dong to bio tien thin nei m6 sir de tai den cho ket qua throng tinh nhung chua hoan toan Ket qua ttrcmg din phi hop vi quitn the bio gee mau cang rOn rat da clang, sau lin city chuyen, cac to bao tap nhiem chua ducirc thai loci hoin tom nen ket qua ?hin tich chua kiting dinh duqc diy la dong to bio tien than On mo thuc thu Ket qua phin tich to bio tien than nei m6 img vien sau lin city chuyen (Bang 2) cho day rang, to Top chi Cong nghe Sinh hpc 13(3): 801-810, 2015 bao van am tinh vai marker CD14 (2,20%), CD45 (0,25%), CD73 (2,17%) va CD90 (3,00%) va tieing thili Ling Well hien ducmg tinh manh vai marker CD34 (97,66%), CD44 (97,89%) va CD133 (99,90%) Quin the to bao tien thin nei mo img vien c6 nguon gec tir mall cueing ron nguoi nen sv bieu hien marker CD34 a (long to ban la di eu kien tien quyet That viy, CD34 bleu hien tren cac bao A goc tao mau nguyen thily va giam Neu hien chung biet hea cac to bao chic nang truong thanh, cac bieu hien ctia marker cho thtly vai tro quan tong cua chimg qui trinh too mau ban du Ngoai ra, CD44 duqc bieu hien tren nhieu dung bao d Ong vit c6 vu khfic nhu bao trung mo nguai, to bao thuec 16p nei mo Deng thai, CD133 la marker dien hinh dimg de phin biet ding bao tien thin nei mo vai cac quAn the to bao CD133 bleu hien tren to bao gOc too mau, to bao tien thin nei mo, bao g6c thAn kinh va then kinh dem Ngoai ra, nghien c(ru cua Bonanno va (long tac gia (2004), cho thAy quilt the to bao ducmg tinh CD133 china met nhom nh6 quAn the to bao tien thin HLA-DIVCD117+CD90+ Khfing the khang CD133 duqc sir dung de chon loc cac to bao tien thin nei mo va to bao g6c tao matt chua truang mau cuing ron va mau ngooi vi cho nghien ciru dieu tn cay ghep (Hirsch et al., 2008), b6i cac to bao CD34+CD133+ c6 vai tro quan trong stn phvc hei cac m8 bi ten thtrcmg thong qua qui trinh sinh mach mai (Yang et al., 2004) Sau len lin cay chuyen, sir bieu hien cac marker ducmg tinh clang to bao rat math (Ca marker CD34, CD44 va CD133 deu tren 95%), ding thei, marker am tinh bieu hien yeu (dm% 5%) va dic biet la CD45 (0,25%) Ket qua cluing to tinh (Jong nhit quirt the to bao tien thin mei mo la rat cao Dieu hoan toan phis hop vi sau cac lin cay chuyen, quAn the cac to bio top nham di bi looi be va d Ong thai, quan the to bio tien thin nei m6 tang sinh va bieu hien monh cac marker mong mal KET LUAN Khao sat cling quy trinh thu nhin hieu qua ngtfon to bao tien thin nei mo tir mau cuing ron ngtr6i nghiem thirc duqc tien hanh de tai vai moi tnrong nuoi city: EBM-2 + SingleQuots: 12 tg/ml BBE, 10 ng/ml hEGF, lig/m1 hydrocortison & antibiotics, 5% FBS (Sigma) + 50 ng/ml VEGF, 50 ng/ml hIGF, 50 ng/ml hFGF va phuung phi') chuyen.dich not bao sau 24 gio nu6i city Hinh thai clang to bao thu nhin duqc de tai la hinh clang dec tnrng cua deng to bao tien thin Wei mo mau cuing ron ngtrai Deng to bao ducmg tinh veld CD34, CD44, CD133 va am tinh yeti CD14, CD45, CD73 va CD90 Hinh Su tang sinh c.6a t41 bao tien than nai m6 ting vien (A), (8)-14 bao truac cay chuyan tan va wan; (C)-Ta bao sau cay chuyen 24 gite; (D), (E)-TO bao sau cay chuyen wan va dot mat da cay chuyen hap theo 807 Nguyen Thanh Tam et al Bang Sty bleu hien marker be mat cOa to bao tien than n6i m6 u•ng vien Te bao sau In cAy chuyen Marker be mat Ty IQ % during tinh CD14 CD34 CD44 CD45 T6 biao sau 5In ay chuyen (n • 1) (n = 1) T,Ig % am tinh 11% i % &rang UM 7-)% le % am tinh 1,06 98,94 2,20 97,80 83,13 16.87 97,66 2,34 87,03 12,97 97,89 2,11 3,09 96,91 0,25 99,75 CD73 6,47 93,53 2,17 97,83 CD90 0.58 99.42 3,00 97,00 CD133 90.01 9,99 99,90 0,10 Lai cam an: Nghien criu duvc tai trp bai Dgi hpc Quac gia thank phi Ho Chi Minh, Viet Nam theo de tai cap trpng diem (B2011-18-07TD) TAI Lieu THAM KHAO Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, Kearne M, Magner M, Isner JM (1999) Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization Circ Res 85: 221-228 Asahara T, Murohara T, Sullivan A, Silver M, Van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM (1997) Isolation of putative progenitor endothelial cells for angiogenesis Science 275: 964-967 Chen J, Chen S, Chen Y, Zhang C, Wang J, Zhang W, Liu G, Zhao B, Chen Y (2011) Circulating endothelial progenitor cells and cellular membrane microparticles in db/db diabetic mouse: possible implications in cerebral ischemic damage Ant J Physiol Endocrinol Metab 301: E62-71 Deschaseaux F, Selmani Z, Falcoz PE, Mersin N, Meneveau N, Penfomis A, Kleinclauss C, Chocron S, Etievent JP, Tiberghien P, Kantelip JP, Davani S (2007) Two types of circulating endothelial progenitor cells in patients receiving long term therapy by HMG-CoA reductase inhibitors Eur J Pharmacol 562(1-2): 111-118 Di Santo S, Yang Z, Wyler von Ballmoos M, Voelzmann J, Diehm N, Baumgartner I, Kalka C (2009) Novel cellfree strategy for therapeutic angiogenesis: in vitro generated conditioned medium can replace progenitor cell transplantation PLoS One 4: e5643 Fadini GP, Losordo D, Dimmeler S (2012) Critical reevaluation of endothelial progenitor cell phenotypes for therapeutic and diagnostic use Circ Res 110: 624-637 808 Fan Y, Shen F, Frenzel T, Zhu W, Ye J, Liu J, Chen Y, Su H, Young WL, Yang GY (2010) Endothelial progenitor cell transplantation improves long-term stroke outcome in mice Ann Neurol 67: 488-497 He X, Gonzalez V, Tsang A, Thompson J, Tsang TC, Harris DT (2005) Differential gene expression profiling of CD34+ CDI33+ umbilical cord blood hematopoietic stem progenitor cells Stem Cells Dev 14(2): 188-198 Hirschi KK, Ingram DA, Yoder MC (2008) Assessing identity, phenotype, and fate of endothelial progenitor cells Arterioscler Thromb Vasc Biol 28(9): 1584-1595 Hristov M, Erl W, Weber PC (2003) Endothelial progenitor cells: mobilization, differentiation, and homing Arterioscler Thromb Vasc Biol 23: 1185-1189 Hur J, Yoon CH, Kim HS, Choi JH, Kang HJ, Hwang KK, Oh BH, Lee MM, Park YB (2004) Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis Arterioscler Thromb Vasc Biol 24: 288-293 Janic B1, Guo AM, Iskander AS, Varma NR, Scicli AG, Arbab AS (2010) Human cord blood-derived AC133+ progenitor cells preserve endothelial progenitor characteristics after long term in vitro expansion PLoS One 5(2): e9173 Lin RZ, Dreyzin A, Aamodt K, Dudley AC, MeleroMartin JM (2011) Functional endothelial progenitor cells from cryopreserved umbilical cord blood Cell Transplant 20(4): 515-522 Mikirova NA1, Jackson JA, Hunninghake R, Kenyon J, Chan KW, Swindlehurst CA, Minev B, Patel AN, Murphy MP, Smith L, Alexandrescu DT, Ichim TE, Riordan NH (2009) Circulating endothelial progenitor cells: a new approach to anti-aging medicine? 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Med 7: 106 Moubarik C, Guillet B, Youssef B, Codaccioni JL, Tup chi Cong nghe Sinh hQc 13(3): 801-810, 2015 Piercecchi MD, Sabatier F, Lionel P, Dou L, FoucaultBertaud A, Velly L, Dignat-George F, Pisano P (2011) Transplanted late outgrowth endothelial progenitor cells as cell therapy product for stroke Stem Cell Rev 7: 208-220 Murayama T, Tepper OM, Silver M (2002) Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization in vivo Exp Hematol 30: 967-972 Rehman J, Li J, Orschell CM, March KL (2003) Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors Circulation 107: 1164-1169 Rouhl RP, van ORJ, Damoiseaux J, Tervaert JW, Lodder J (2008) Endothelial progenitor cell research in stroke: a potential shift in pathophysiological and therapeutical concepts Stroke 39: 2158-2165 Urbich C, Aicher A, Heeschen C, Dembach E, Hofmann WK, Zeiher AM, Dimmeler S (2005) Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells J Mo! Cell Cardiol 39: 733-742 Wu X, Lensch MW, Wylie-Sears J, Daley GQ, Bischoff J (2007) Hemogenic endothelial progenitor cells isolated from human umbilical cord blood Stem Cells 25(11): 2770-2776 Yang C, Zhang ZH, Li ZJ, Yang RC, Qian GQ, Han ZC (2004) Enhancement of neovascularization with cord blood CD133+ cell-derived endothelial progenitor cell transplantation Thromb Haemost 91(6): 1202-1212 Yoder MCI, Mead LE, Prater D, Krier TR, Mroueh KN, Li F, Krasich R, Temm CJ, Prchal JT, Ingram DA (2007) Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals Blood 109: 1801-1809 Yoder MC (2012) Human endothelial progenitor cells Cold Spring Harb Perspect Med 2(7): a006692 Taniguchi E, Kin M, Torimura T, Nakamura T, Kumemura H, Hanada S, Hisamoto T, Yoshida T, Kawaguchi T, Baba S, Maeyama M, Koga H, Harada M, Kumashiro R, Ueno T, Mizuno S, Ikeda H, Imaizumi T, Murohara T, Sata M (2006) Endothelial progenitor cell transplantation improves the survival following liver injury in mice Gastroenterology 130: 521-531 Loomans CJ, de Koning EJ, Staal FJ, Rookmaaker MB, Verseyden C, de Boer HC, Verhaar MC, Braam B, Rabelink Ti, van Zonneveld AJ (2004) Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type diabetes Diabetes 53: 195-199 Yang N, Li D, Peng J, Chen B, Yao S, Sang H, Yang M, Han J, Zhang Y, and Qin S (2011) The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions Cytotechnology 63: 217-226 Ye C, Bai L, Yan ZQ, Wang YH, Jiang ZL (2008) Shear stress and vascular smooth muscle cells promote endothelial differentiation of endothelial progenitor cells via activation of Akt Clin Biomech (Bristol, Avon) 23(Suppl 1): S118—S124 Ingram DA, Mead LE, Tanaka H, Meade V, Fenoglio A, Mortell K, Pollok K, Ferkowicz MJ, Gilley D, Yoder MC (2004) Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood Blood 104: 2752-2760 Kibler CM, Wechselberger J, Hilbe W, Gschwendtner A, Colleselli D, Niederegger H, Boneberg E, Spizzo G Wendel A, Gunsitius E, Patsch JR, Hamacher J (2007) Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury, Respiratory Research 8: 50 ESTABLISHMENT OF A PROTOCOL FOR ISOLATION AND IN VITRO PROLIFERATION OF HUMAN UMBILICAL CORD BLOOD - DERIVED ENDOTHELIAL PROGENITOR CELLS Nguyen Thanh Tam', Pham Le Buu Truc143, Vu Bich Ngoci , Nguyen Minh Holing', Duong Thi Bach Tuyet2, Phan Kim Ngoc', Pham Van Phuc' 'University of Science, Vietnam National University, Ho Chi Minh City 2Ho Chi Minh City University of Pedagogy SUMMARY Currently, the progenitor cell is important issue in the development of cell therapies for the ischemic disease treatment In particular, endothelial progenitor cells also have ability to differentiate into endothelial Author for correspondence: E-mail: plbtruc@hcmus.edu.vn 809 Nguyen Thanh Tim et al cells Therefor, they play a key role in the process of vascular repair and regeneration Hence, we studied the process of establishing this cell line obtained from human umbilical cord blood Mononuclear cells (MNCs) were collected by Ficoll-Hypaque density gradient centrifugation MNCs were then resuspended in three types of different media by two different methods aimed at establishing the optimal process for isolating and culturing of endothelial progenitor cell line The experiment cells were assessed by morphology changes, adhesion and proliferation at the time of the survey 7, 14, 21 and 28 days after admission The endothelial progenitor candidate cells were also analyzed by flow cytometry with markers: CD14, CD34, CD44, CD45, CD73, CD90 and CD133 The result showed that culturing the candidate cells in EBM-2 medium add SingleQuots (12 1.1g/m1 BBE, 10 ng/ml hEGF, µg/ml hydrocortison & antibiotics, 5% FBS, 50 ng/ml VEGF, 50 ng/ml hIGF, 50 ng/ml hEGF) by transfering supernatant cells after 24h culturing was the most effective method to obtain endothelial progenitor cell line This cell line simultaneously expressed positive for CD34, CD44, CD133 and negative for CD14, CD45, CD73, CD90 In addition, they had the ability to proliferate invitro and the cell morphology was stable in culture Keywords: cell culture, cell isolation process, endothelial progenitor cells, stem cells, umbilical cord blood ... khfic, viec thu nhen lai phirc tap horn mang tinh xam len cao Do do, viec phan lop loai te bao thuOng it co hieu qua Gan day, mau cluing ron ngoai duqc chirng minh la met ngu6n phong phis de thu nhOn... nang cita quan the te bao tang sinh D6I TUQNG VA PHUONG PHAP NGHIEN OYU Thu MO miu cuong ron MAu matt cuOng ron ngoai duqc thu nhOn tir Benh vien Hung Vuang — Thanh phO Ho Chi Minh 802 Cfic mkt... nguyen yen va duqc thu nhOn tir tre sa sinh kb:5e manh, di thing; (2) — MAu mau sach, duqc xet nghiem am tinh vai HIV, HBV, HCV va met se benh truyen nhiem khac; (3) — Mau ducre thu nhOn sau sin
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