THÔNG TIN TÀI LIỆU
Tgp chi Cong nghe Sinh hoc 10(3): 431 -440, 2012 NGHIEN at GAY HOAI TU XISONG BANG GLUCOCORTICOID VA THir NGHItM GHEP TE BAO DON NHAN TUY XVONG TREN MO HINH BtistH LY THI/C NGHIVM Ding Thi Tung Loan', Nguyen Thi Dieu Hang', Pham Van Phicl, Nguyen Thi Bay2, Phan Kim Noel Truemg Dgi hoc Khoa hoc to nhien, Doi hoc Quac gia pha Ha Chi Minh Dgi hoc Y- &roc pha Ho Chi Minh TOM TAT Glucocorticoid tong hqp la thu6c steroid duce sir dung ring rai dm song ngutri hormone c6 tic dung dieu tri nhieu chimg, h6i chimg, benh nhu giam dau, khang viem,,hen suyen, chap Icherp, gay Tuy nhien, glucocorticoid ding gay nhieu tic dung phi, d6, suy giam mien dich (trong ghep tang), hoai tir xtrang la bien chimg thtrang gap va nghiem nhat Viec dieu tri hoai tir xtrang gap nhieu khe khan khe phat hien serm benh va phugng phap pheu thuat (thay Icherp) bi gied han Nghien dm thir nghiem dieu tri hoai tir xuang bang ghep to bio don nhan ray xuang dung loai tren mo hinh chuat benh 1y nham dot den hieu qua ngan ngira benh tot nhat Chuat twang duqc dem dexamethasone (Dex) hang tuan vii cac lieu 41 6, mg/kg tuan Asparaginase (Asp) duqc sir dung nham tang hieu qui gay hoai tir xuang a cac 16 Lieu dexamethasone co hieu qua gay hoai tir xuong cao Oat duqc xac dinh qua phuong phip nhu6m h6a mo xuang, phan tich su hoai tir cita to bio my xuomg bang flow cytometry (FCM) va phan tich su bleu hien dia gen osteocalein, Runx2, BMP2 a mire phien ma (bang RT-PCR) Hieu qua dieu tri hoai tiricucmg cila phuong phap ghep to bao sau tuan ding duqc dinh gia qua nhu6m H&E, FCM va RT-PCR Ket qua: qua phuong phap nhuam H&E, tjr mo xuong chuat hoai tir duqc nhan thay co su gia tang theo lieu Dex tang; do, lieu Dex mg/kg duqc danh gia co hieu qui gay hoai tir xtrang tot nhat, vai tjr lg chuat hoai tir la 50%, tS, 14 chu6t chet 13,3% Sr chez theo chuong trinh (apoptosis) va chet hoai tir (necrosis) ala bao ray xuong ding bi anh huong beri lieu dung Dex tang (4,6; 5,3 va 9,7% cac 16 tuang (mg doi chimg, Dex4, Dex6) Cac gen too xtrcmg ding Om bieu hien mire phien ma a cac 16 co sir dung Dex Hieu qua caa viec ghip bio tiny vao chu6t hoai tir xuang sau hai tuan duqc xac dinh co kha ming ngan ngira su tien triOn ctia bOh Tir khea: chugs benhlje, ghip to bao, glucocorticoid, hogi to xuong, ti bac)* xtrang Men THIEU Glucocorticoid nham cac hormone steroid vimgvo tuyen &rot san xuit tir cac to bio lop b6 thuong then va duorc sir dung rang fel de dieu tri nhieu benh nhu viem thap khop, hen suyen, u bach huyet, ghep tity, ghep mo/ca quan, cac benh tu mien hay than thuang cat song (Yang et al., 2008; Matsuya, et al., 2008) Tuy nhien, viec sir dung cac loai thuoc co ban chat glucocorticoid lieu cao hay thoi gian dai den den nhieu tfic dung phi va bien chimg nhu hOi chimg kieu Cushing, tang huyet ip, loot hanh to tang, loang xuang, hoai tir xuang v Trong do, hoai tir wan IA met nhimg bien chimg thuing gap va nghiem tong nhat Dien tien va trieu chirng benh hoai tir xuang khong ro rang giai Clop dau nen viec phat hien sem benh rat 166 Bente khong gay tit vong nhung lai lam cho cac sinh hoot hang ngiy cita nguii benh gap nhieu kho khan, kha nang lao Ong giim sin, them chi la mit hin kha nang lao ding Benh thuing tae ding den, nhimg ngtrii tit de tuoi 20 — 40 Met xuong glucocorticoid van khac, co che „hoai chua duor hieu rO nguiri truing thanh, kha nang tlr phut hoi cue cac benh ve xuang khOp not chung va hoai tir xuang not rieng deu khong cao Do 4y, viec nghien cuu ve ,benh va cac bien phfip phong, dieu tri va ho tro dieu tri benh la can thiet Nghien cuu ve ca che benh cimg nhu phuini phfip dieu tri phis hop de &roc tien hanh tren nhieu doi ttrong nhu nguii va &Ong vet thi nghiem (tho, chuOt, chim, ciru, de, ch6, ) Tuy nhien, cac nghien tiro ve ca the benh ciing nhu nghien ciru ve lieu phip dieu tri thir nghiem mang tinh xam lan lai khe thuc hien tren nguai so vii viec thirc hien ten m8 hinh den vet benh 1St (Irisa et al., 2001; Assouline-Dayan, 2002; Boss et aL, 2003; Cui et aL, 2006; Yang et aL, 2008; Kerachian et aL, 2009; Fan et al., 2010) Viet Nam, phan lin cac nghien tiro tren doi ttrong chit yeu la tai tien cac phuang phap dieu tri hien hanh Cho den nay, viec dieu cac benh ve xuang 431 DAng Thi Tung Loan et aL khOp gAp nhieu Iche khan vi (1) chua co phic d6 dieu trj chinh xic, (2) benh ve xuong khap kho phat hien hoAc , dtrqc phit hien mu6n, d6 benh da tri nang von hoai tir chum xuong dui, phuong phap dieu tri hien chi nham muc dich lam giam dau va lam chim qui trinh hoai dr, hay thay the town phAn khap hang hoai tir hoan toan Khi de, nguai benh, phai chili dung tan tit suot dai hoc chi duat thay the m6t phin chirc nang binh thuang (Conzemius et aL, 2002; Biswas et aL, 2004; Cui et al., 2006; Malizos et al., '2007; Fan et al., 2010) Vi viy, dang vat thi nghiem yin la lua chon tot hon cho viec nghien ciru cic phuong phip dieu trj hay h6 trq dieu tri benh Trong do, chu6t la doi tactile duqc sir dung bien, thao tic thuin lqi va co tinh tuang d6ng tinh tang vii ngutri cao Nghien ciru dieu tri hoai tir xuong hien co xu huang tiep can vii lieu phip to bao Ben vi gia thuyet benh co can nguyen tir to bao &rot nhieu nha khoa hoc d6ng thuin MOt so nghien ciru gin day chime minh ben earth su hoai tir su hu hai cua mach miu, su chet theo chuang trinh hay chet hoai tir cua cic to bao xuong su suy giam so luone, su tang sinh cua cic to bao g8c trung mo va c,ic to bao tien than xuong Vi the, lieu phip to bao duqc dinh gii nhu la mat huang dieu tri trien vong can benh (Cui et al., 2006; Asada et aL, 2008; Jones et al., 2008; Matsuya et al., 2008; Yan et al., 2008; Lee et aL, 2008; Gangji et aL, 2009; Sun et al., 2009; Wang et aL, 2010; Gao et aL, 2010; Ogawa et al., 2010; Li et al., 2011) tic Ong cua Dex, ip ltrc xuong tang cao cling von su hu hai cua cic mach miu nuoi xuong va su mat can bingv'e so lucmg va chirc nang buy cot bao va tao cot bao, cot bao; tir do, tinh tang mat xuong xay va xucmg chet din Mirc da hoai tir xuong Dex gay tSr le thu'in vii lieu va thai gian dune Yang va done tic gii (2009) ba sung Dex von n8ng d6 va mg/L vao nuac wing cua chuat 12 tuan cho ket qua giy hoai tir xuong khoang 45% N8ng da ming mg/L tuang duang vii lieu tiem Dex mg/kg (tinh theo tong luqng Dex tich Asparaginase (Asp) — m6t enzyme co the clay manh tic dung cua Dex thong qua viec irc the su tong hop protein,dieu chinh duqc Ong cua Dex, lam bien d8i su bien du-One lipid (Yang et al., 2008) Ngoai ra, Asp co the gay benh done miu (Hanada et aL, 1989) Vi vay, viec sir dung Asp ket hop voi Dex co the nang cao tan suit mac benh hoai tir xuong Hoai tir xuong, die biet la hoai tir chum xuong dui thuong xay a nam gioi dO tuoi tir 20 — 50 (rSt le mac benh gilta nam: nit la 8:1) va c6 xu huong gia tang (Biswas et al., 2004) Su !chic biet ve tan suit mac benh hoai tir xtrong giaa hai gion tinh cling xay cic nghien ciru tren d6ng vat (Yang et al 2008) Cac loaf thuoc duqc pha vao nuac cat vo trung de dat dirge nong d6 thich hop The tich.dich thuoc duqc tiem vao chuOt Ichong qui 0,05 ml/ Ian tiem VAT LIEU — PHYONG PHAP Dua ten nhimg ca sa tren, thi nghiem duqc thiet lap nhu sau: Gay hoai tar mon chuOt bing Dexamethasone (Dex) Chuat nhAt due, chime Swiss, 4,tuAn tu6i, khOe manh duqc nuoi djnh tuan Inrac thi nghiem Sau thin gian nay, chuat dat tong lucmg trung binh khoing 20 gram va duqc tiem cic thuoc glucocorticoid de giy hoai tir xuong Ngoai ra, cic 18 thi nghiem duqc tiem bo sung asparaginase de thiic day six hoai tir xtrang glucocorticoid Bao gam cic 18 thi nghiem sau: Dex la dang thuoc glucocorticoid duqc sir dung rang rai cu6c song nh6 vao tic dung phone phu cua chime, ttr giam dau, khang viem den irc.che phan ling mien djch Lieu Dex thtrang dime de trj benh la — mg/kg, mat so truing him dAc biet, dung co the cao han Vai cic lieu dung nay, ngoai tic dung dieu tri benh, Dex giy nhieu bien chime tic dung phu, dien hinh nhu hoai tir xuong Nhieu nghien ciru da chime minh , Dex lam giam lucmg va chirc nang cua tao cot bao, lam tang su biet boa va keo dpi vong dal dm cac hity cot bao, lam bien doi va giy su chet cua cic cot bao Ngoai ra, Dex lam gia tang su tao ma ttly, truc tiep lam ton thuong cic to bao nai m8 mach va lam tang tinh done miu Nhu viy, dun 432 + 45 chuat chia deu 18 duqc tiem Dex a lieu tiem 4, 6, mg/kg (qua duang ca chi dun) + 45 chu6t chia deu 10 duqc tiem Dex lieu tiem 4, 6, mg/kg ket hop vii Asp (7500 IU/kg) (qua duang ca chi dun) + 10 chuOt d6i chimg duqc tiem NaCI 0,9% Sau tuAn, lieu Dex sir dung duqc d6i chieu vii Tap chi Cling ngh'e Sinh hoc 10(3): 431-440, 2012 kit qua kiem tra mo xucrng, phan tich sv chit dm to bio tiny xucmg va sv bieu hien dm cac gen tao xucmg mire phien ma Khao sat MO qua ghep to bao don [than day • won teen chu(lt hoai to won Te bao don nhan dugc thu nhan tir tily,xucmg tu tail bang cua nhOng chuet khee6mphb phuang phi), li tam clang ti tong Ficoll, sau dugc rira bang dem phosphate sinh IS, (PBS) Cu6i ding, te bao duqc giu PBS NIA dugc sir dung de ghep Viec ghep te bao don nhan tay xucrng vao chuet bi hoai tir xucmg str b6 sung nguiin te bao ngoai sink vao ca thE nham thay the hay 1)6 sung cho ngtion te bao n'ei sinh da,bi hu hai KEt qui co the din den cac tic dung: so lugng te bao ngoai sinh thay the cac te bao nei sinh thuc hien chirc nang von co ciia no, hay cac san pliant tiEt cua cac to ban ngoai sinh co tic dung huy Ong te bao nei sinh Cic chit tiet co the cac cytokine hay cac protein c6 vai tro sty tao mach va xtrang 15 dwelt benh ly (duqc gay hoai tir xuong bang lieu tiem Dex hieu qui nhat) dugc ghep te bao tiny xucmg veri mat de 5x105 t4 bao/0,01 ml/con vao tuan thir dm lieu trinh, xir vai Dex (8 tuin) Chuet duqc tiem ntrac muoi sinh 15, (PBS) va chuet hoai tir kitting dugc gild!) te bao dugc sir dung lain dOi chimg Hieu qua cua viec ghep to bao test xtrcmg dugc danh gia sau hai tuin Phtron phap danh gia hoai tie 'twang Danh gib cau true mo hpc qua mein xuying nhuOnt hematoxylin va eosin Sau thai gian khio sat, tit ca xuang dui chuet duqc kiem tra mo hoc bang nhuem H&E Dac diem nhan Het xtrcmg bi hoai dr la c6 nhieu xtrang rang vi nhieu te bao xtrang chit viing xuong xop viing Xtrcmg dui chuet kern theo str hoai tir sau loai 1)6 het ca, dugc co dinh formalin Sau do, xtrcmg chuet duqc khir canxi, nhang vao paraffin va cat lit pm Cac lit xuong dtrac nhuem veri Hematoxylin va Eosin va kiem tra dueri kinh hien vi quang hoc Hwang pluip phan tich to boo try xuong Ming Flow cytometry Plurcrng pap cho ghep phan tich,nliCmg thay dOi ye hinh thai va tinh chat dm te bio bang each sv bien d6i tan xa anh sang te bio di qua tia laser dm may Trott? nghien ciru nay, flow cytometry dugc sir dung de phan tich chu ki va str apotosis cua te bao, tir xic dinh str thay doi hinh thai va tinh chat dm te bao glucocorticoid Thong thuang, truac dua vao may, cac te bio duqc nhuern vai cac Ma chat dac (thuoc nhuem, khang the, ) Trong phan tich chu ki va str chit cua to bio, cac te bao dugc xir ly veri thuoc nhuem huYnh quang nhu propidium iodide (PI) Chat gan xen vao DNA; nhimg te bio nio co nhiem sac the gan vai thuoc nhuOin PI, di qua may flow cytometry, phat huSinh quang btrac song 488 nm Tir des, may flow cytometer c6 thE xic dinh cac pha cua chu ki te bao thong qua so luqng nhiem sac the va nhan biet str chet cua te bao thong qua sv phan minh DNA Danh giu sF bieu hien ciia cac gen Igo xuong (BMP2, Runx2, osteocalcin) mac phien ma Str bieu hien cua mRNA BMP2, Runx2, osteocalcin dugc dant' gia bang RT-PCR RNA tong so dugc tich tir xuong chuet bang Ma chat tach RNA Trizol (Sigma) theo quy trinh cua nhksan xuat Urging RNA dugc dat vao cac phin img bang RNA dugc khuyech dai voi nhang mei chuyen biet cho BMP2, Runx2, osteocalcin, GAPDH Trinh ttr moi xuoi BMP2 5'-GTG TTG CTG CTT CCC CAG GTC-3' va trinh ttr rn6i nguqc 5'- TCC CTG TGT GGT, CCA CCG CAT-3', kich thuerc san phim khuyech dai la 627 bp Trinh ttr mei xuoi Runx2 5'AGC AAC AGC AAC AAC AGC AG-3' va trinh ttr mei nguqc 5'-GTA ATC TGA CTC TGT CCT TG3', kich thuac san pham khuyech dai la 685 bp Trinh ttr mei xuoi osteocalcin 5'-CAG ACA AGT CCC ACA CAG CA-3' vi trinh ttr mei ngtrqc 5'CTT TAT TTT GGA GCT GCT GT-3', kich thuoc san phim khuyech 4i 456 bp GAPDH duqc sir cliing lam doi chimg Wei vai trinh ttr mei xuoi 5'AAG TTG TCA TGG ATG ACC-3' va trinh ttr mei ngtrqc 5'- ATC ACC ATC TTC CAG GAG C -3', kich think san pham khuyech dai la 139 bp RTPCR bulk dtrqc den hanh vai dieu kien khuyech dai nhu sau: pan irng duqc giu o nhiet de 45°C 40 phiit de mei bat cap vao sci khuon RNA va enzym reverse transcriptase tong hop dugc cDNA, ding th6i RNase cat be RNA dich khoi cDNA; sau 46 Wen tinh enzym reverse transcriptase, RNase 94°C phut; Nen tinh 94°C 45 giay, bat cap 55°C 45 giay, keo dai a 72°C I phut, giai doan dove Win hanh veri 35 chu kS,; giai doan cuei ding keo dai them chu ky 72°C 10 ph6t 433 DOng Thi Ting Loan e KET QUA - BAN LOAN Eat qua too mo hinh chuOt bi hoai ti' zircon Ty lg chufh chit , So limns chuOt chez khong gift cac 18, cu the, tjf le chit tang dan theo nong 40 Dex sir dung va co su khac bigt ro ret siira lo có va kitting sir dips kit hqp vai Asp L8 doi chimg khong co chuot thet (Hum I) Ket qua theo dui the tiro: ehuit't Dex va nhimg Um& glucocorticoid CO tac dOng phirc top den sinh IS+ cua chuOt Su hien thien Irons, DC Dex-I Dex4+ luqng cua chuOt glucocorticoid cling khong theo mot chieu hu6ng ro rang Trong mot nira thiri gian dau, phan Ian chuOt deu có ltrgng tang monh; khong CO su khac bier giira 16 d8i chimg va thi Tir tuan thir nain cita thiri gian khao sat, Itrong chuOt tang tucmg d8i deu 610 doi chimg Trong do, cac 10 tient (Dex4, 6, 8), cac so it thay 401 hay giatn,nhe I, titan dau, sau d6 tiep tic tang dn th diem cuoi Su hien thien lucmg cita chuOt a cac lo Dex4, Dex6 khalif; khac bit so vai 16 dOi chimg Vie'c sir dung Asp ding vai Dex co xti hinnig lam giant can Ong cult chuOt O clic 10 tient Dex va Asp, su tang city chu0t chi bang klmang 60 70% so vai 16 chi tiem Dex (Ilinh 2) Dexo Devi+ 1-.),_Ns Dex8+ Hinh T9 10 chuOt ch4t & cac 18 (%) 22.4 21.5 21.1 15.5 15.1 16.6 10.8 D Dex4 Dex4+ Dex6 Dex6.i Hinh Sur gia tang luvng trung binh chuOt trtrac va sau thi nghiOrn (g) 434 DexS Dex8.4 Tap chi C O ' ng nghe Sinh hoc 10(3): 431-440, 2012 Kit qua kilm tra ma kw 10 Dex6 cao han hart (9,7%) (Hinh 5) Trong kiem tra vi the, vimg dAu va than xtrang did chuOt dirge cat ming um va nhuOm voi H&E Quan sat mat, nhuOm H&E duai kinh hien vi cho thay, mo xtrang ciia lo thi nghiem da c6 sy bien dOi so vai mo xuang,cna chuOt binh thuang Cie hoc xtrang rat it den khong te bao xtrang Cac to bao xtrang bi chet va hoc xtrang tra nen rang Dong thai, vimg nhan te bin) xtrang khong ro rang nhan te bao bi thoai h6a (5 IC) thi nghiem, cau true sun bi pha ver Wing dia sun dau xtrang (hay dia tang twang) hi Inr hOng nhieu han phan dau sun MOt so vimg xtrang xop thtiOc dau xtrang khong co sy hien dies cua te bao xtrang hoc xtrong va bat mau thuiic nhuOm ding nhat Cac be xtrang xop dan xen Wong lily ding bi htr hai can talc khong dm nguyen yen Cac o xtrang kho dtrac nhan dung han chat nen xtrong 6day it bi khoang hem han so vai yi% xtrang dec Theo s6 lieu thu dirge, ty le chuOt bi hoai tir mo xtrang tang theo eine,' tang nong dO Dex sir dung Ket qua chi dtrac tinh (reit nhimg dun), song Kit qua clash gia st, biiu bre,' cult Mc gen tao xwang (BMP2, Runx2, osteocalcin) mar phien mn Bang phuong phap nhuOm H&E, tieu chuan ciia Arlet dugc sir dung nhieu nhat; theo do, xtrong dutyc xac dinh bi 114 tur kin co it nhat mOt tinh trang sat, xay ra: cac hOc xtrang khuyet te bao cac te bao xtrong chet; sy thoai boa nhan te bao xtrang va sy hoai tir cua cac vimg gun ke nhtr mown, mo toy, mo co (Hinh 3) Sr gia tang the hien re) a 10 Dex8, Dex4 va Dex6 co ty le tang khong ding ke NhAn dinh ding bi giai han bai so lugng chuOt cho mai 10 khong dii Ion cho mot ket qui ro rang Dong thai, Med ghi nhan dung chit 5, la cac IC) co sir dung Asp cung vai Dex, chuOt bi hoai tir nhieu han; chuOt chi dugc tiem Asp hay ntrac muoi 0,9% khong co dau hien SO Itrang chuOt bi hoai tir nhieu nhat 115 Dex6+ va Dex8+ (100%) Deng thai, ty le chuOt chet hai 10 cling cao Mat (>60%) (1 linh 4) Kit qua phan tklt set• chit theo chtrang trinh vd chit hopi tri call re hao tap xtrang Tir cac hinh anh,ket qua pilau tich FCM, sy khac ve mat dO te bao a vimg P4 biet co the nhan gitra lo doi chimg va hai lo thi nghiem ding nhu giira hai 10 thi nghiem sir dung lieu Dex khac Sy chet hoai tir cila te bao co xu htrang tang lieu dimg Dex cao horn Dieu kha phi, hap vai cac so lieu dugc cung cap bed phan mem CELLQuest coo may FASC Calibur Theo do, so lugng te bao thuOc vimg P4 dm to Dex4 co sy tang nhe so vai 10 diii chimg (DC: 4,6%; Dex4: 5,3%); do, ty le , Sy bieu hien dm GAPDH tat ca cac tau d 'L rat ro rang Dieu chirng to ket qua tach mRNA va phan Ong RT-PCR ved chu trinh thief lap dtrac dive hien Ket qua cho thay rang sir Neu hien dm cac gen BMP2, Runx2, osteocalcin mac mRNA giam theo ming di) Dex tang Ket qua phi, hyp vai gia thief dat ding nhtr cac nghien cat, twat: BMP2 bleu hien my,11, 'that doi chin% va giam 10 Dex4 va Dex6 Xet ve mow dO, sy bieu hien dm BMP2 a doi chimg manh han gap Ian so vai to Dex4 va gap 16 Ian so vai 10 Dcx6 Sr hien hien dm Runx2 giam manh cac 10 ill nghiem c6 sir dung Dex so vai doi chimg, tir mire giam hang chuc Ian (14 Ian Dex4) den hang tram Ian (xap xi 478 Ian 10 Dex6) Sr bleu hien mirc phien ma dm osteocalcin giam re) ret a lo thi nghien, Dex4 so vai 10 doi chimg (Hinh 6) ang di) va,hau nhtr kitting bieu hien a 10 Dex6 Xet truing bieu hien dm GAPDH va osteocalcin gitra cac 10, ty le bieu hien osteocalcin ciia mRNA doi chimg manh ham xap xi 10 Ian so vizi a lo Dex4 (Bang 1) Minh gia hieu qua an phtrong phap ghep tc bito tiiy xtrong tren mo hinh Kith IS, thus nghiem Danh gia ket qua nhuOm haa me) xtrang ding dtrac dcra tren tieu chuan cilia And ChuOt binh thuimg dugc tiem PBS hay dirge ghee te bao don xtrong deu khong co dau hien hoai tir (Hinh nhan 7), la cac doi chang cho phan tich Theo phan tich nay, chuOt binh thuang co hoac khong dugc ghep te bao don nhan tidy xtrang den khong co dat, hien xtrang bi hoai tir Ming filed, ty le chuOt hoai tir a 10 ghep t6 bao (DB) khong giam so vai IC) Dcx6 Tuy nhien, sy hoai tir a cac viingd5 co sy khac biet so vai me) xtrang 10 Dex6 ,Cau Inc mo kha ro rang, dugc xen Ian bai cac can Inc bi v6 ,Trong cot bao hien din vai nhfin bat mat, xuong dac, tim dam va hai ep vao hoc xtrang; do, cac be xucmg xop toy van htr hai MO tily la nai the hiOn sy hai phuc kha re) rang, do, ty le to bao mir va te bao toy gall nhtr can bang Ket qua dinh gin tinh to bao thy xtrang bang FCM: (,Hinh 8) the hien sy khac biet kha nho ve mat dO te bao chet (vimg P4) gitra toy xtrang chuOt binh thtrang va chuOt ON dtrac ghep te bao don nhan toy xtrang So Iieu phan tich FCM cho thay co sy suy giam so Itryng te bao chet so vai 18 Dex6 (DB: 6,1%; Dex6: 9,7%; DC: 4,6%) 435 Deng Thi Timg Loan et al •te'rou - 4, „• • L Hinh Sr t khac bitt cau trim xaang 0' 10 dal chirng va thi nghiOm (nhuOm H&E; A, B, E, F: 100X; C, D: 200X) Cu a trim sun cua dau xtrang chuOt 16 d6i chCfng (hinh A, C, E) 6/ 16 liern thu6c (Hinh B, D, F), ceu truc sun bi ver, khang nhan r6 cac to bat) sun va CO cac be xtfang ch6t nem xen len; cec hoc xtrang ch6t khOng CO to bao (c6t bac)) & be xu,ang (Hinh D); phan ma tang m6 cua chuOt hoai tit (Hinh F) 110 /MI Sly T 60 aU 20 : [)C Dex-I Dex4+ Dex6 Dex6 I Dex8 Dex8+ Hinh Ty I chuOt hoai ter & cac 10 (%) ■■•••••••■•■■• P4 P1 Dex4 Hinh Ket qua phan tich flow cytometry to bao xt.rang 436 ■■■111■••• Dex6 Tgp chi aing ngh'e Sinh h9c 10(3): 431-440, 2012 DC Dex4 Dex6 DC DB DB Dex4 Dex6 WM alb GAPDH OOP BMP2 • 111•1111111111•11• morawro osteocalcin 111111111111•11110 wrisapro 61.11.0 411611•111111 Hinh Icet qua diOn di san ph6m RT-PCR Bang Cuteng dO bi$u hiOn cue cad gen mirc phian ma 6, cac 16 thi nghiem DC GAPDH BMP2 Runx2 Osteocalcin 22888 33853 30196 38931 3307 Dex4 18735 13959 1735 Dex6 31575 2923 87 qud plain tich sinh hpc plain Cac vach bleu hien cua BMP2 hal lo doi chUng va DB sang gan nhu deu nhau, chimg to mire bieu hien ttrang &rang (Hinh 6) SO lieu ban Binh Itrong (Bang I) phit hap vai (Hinh 6) Dip tre.n cuing dO.bieu hien GAPD11 va BMP2 mire phial ma, su bleu hien cua BMP2 610 d6i chimg va 10 DB dtrgc nhan thay gan nhu khong co su khac bier (ty le bieu hien DC:DB la 1,1:1) Trong (10, ty le bieu hien gen BMP2 a mire phien ma giira 10 doi chimg va Dex6 'cap xi 16:1 Dip tren su bieu hien oh Runx2 it mire phien ma (Hinh va Bang 1), co the nhanthay rang sty tao xtrong hay sira chira xtrang clang dien mph me a to chuOt dtrgc ghee to bao (DB) Mire bleu hien Runx2 10 DB da tang rat nhieu so vai to Dex6 nhtmg van chua dat mire 10 doi chimg (t$' le bieu hien DC:DB la 1,6:1) Ngoai ra, ket qua bleu hien Runx2 va BMP2 deu tang 610 DB so vai 10 Dex6 va van thAp ham so vai doi chimg Su bieu hien cua osteocalcin a mire phien ma a 10 dieu tri bang to bao don nhan try xuang tucrng throng vai 10 001 chimg (ts, le xap xi 1:1)., Trong do, chuOt hoai tir khong dugc to bao don nhan try xtrang thi bieu [lien rat yeu gen osteocalcin Ty le cbuot hoai tir xtrang co Wong tang theo 'long Dex tang; dieu phi, hgp vai nhieu nghien ciru da bao cao (Wang el al., 2000; Zhang et al., 2008; Yang et al 2008; Powell et al 2010) Ket qua thi nghiem ,cho thay rang hieu qua gay hoai tir ?arcing nhieu nhat sir dung Dex lieu cao va ce ket hap vai ,Asp (Dex6+, Dex8+) Tuy nhien, viec, sir dung phoi hap Asp co tac dOng lam giam sire song va gay chet a chuOt,cao (46,7 — 66%) Ming thin, xet ye !lieu qua kinh, te, viec sir dung Asp khOng thiet thuc Trong ba lieu 4, 6, mg/kg Dex, hieu qua gay hoai lir xtrcmg cao,nhat to Dex8; nhien, 10 nay, ty ie cbuot Chet cao hon han so vai hai 10 Dex4 va Dex6 Do do, sir dung lieu Dex va mg/kg cho hieu qua tot hon 437 Dang Thi Tung Loan et aL bieu hien cua BMP2, Runx2 va osteocalcin giam a cac 18 c6 xu ly vai Dex la dau hieu cho thay mo xtrong cling nhu qua trinh tai to va tri xucmg da bi anh huong Tic deng gay hai xuang co xu Itrqn* Dex tang Dong thoi, twang phat trien theo ket qua phan tich FCM cho thay c6 si,r suy giam sire song dm cac to bao tity xtrong va to &to goc trung mo theo sir tang king de gluccicorticoid sir dung Xet nghiem mo bang phtrong phap nhuem H&E cho thay tji,le hoai tir that su tang sir dung lieu Dex tucrng cao (Dex8) nhtmg Or,lieu nay, tj, chuet chet cao Do do, sir dung Dex lieu thap holt c6 hieu qua phir hqp Tir cac lc& qua tren, Dex6 duqc nhan *ay la lieu gay hoai tir hieu cua nhat Lieu Dex-nay &Km sir dung cho thi nghiem ve danh hieu qua lam phuc Wei ON bang phircrng phap ghep to &to tiry xtrong Hinh IC& qua nhu6m hoa m6 xuang khao sat dieu tri bang ghep to bar) tOy xtrang.(A, C: m6 mang cua chuOt hoai tir duvc ghep to bao dan nhan tay xtrang; B, D: m6 xu'ang cua chu6t b Id Dex6; A, C: 100X; B, D: 200X) Hinh Ke't qua phan tich t6 bao xu'ang bang flow cytometry So sanh cac ket qua nhuem H&E, phan tich BMP2, Runx2, osteocalcin a 18 d6i chang Nikki dieu FCM va dinh gia su bleu hien dm gen ma haa cho tri ghep to bao dan nhan lily xucmg cho thay rang 438 Top chi Cling nghe Sinh hoc 10(3): 431-440, 2012 viec ghep to bao xuong c6 hieu qua tich cur len su tang mow cac dau hieu cue su phuc hoi xuang hoai lir Trong cac m6 xuong cila 16 DB, cac cot bao song hien dien vai mat dO nhieu hon, cau tritc sun ding nguyen yen han so yeti 16 Dex6 Ket qui co su tuong (long voi cac nghien ciru ctia tic gia nuac ngoai (Asada et al 2008; Matsuya et al 2008; Sun et al 2009) Bong that su hoai tit tity giam re ret, duqc chimg minh bai ket qua, phan tich FCM Theo do, t9 lete bao tity xuang chet chucmgtrinh hay hoai lir da giam den 37% so vai 16 Dex6 O 16 chuOt hoai tir duqc ghep to bao don nhan my xtrcme, su bieu hien dm gen ma h6a cho BMP2 Runx2 va osteocalcin tra ye mirc gin voi muc binh thtrong cho thay su tao xuong da c6 dau hieu h6i phuc va kha !tang khoing hoa chat nen xuong (la dugc cai thien ding ke Tuy nhien, sir hoi phuc khong the hien CO rang a t9 le ca the Ty 1e chuqt hoai tir a 16 DB khong giam so vii 16 Dex6 Do do, viec ghep to bao dan nhan xtrang chi co the dugc ket luan ve kha nang ngan ngira va lam giam su hoai at xuang KET LUAN 1.M6 hinh chuOt hoai ni xuang Ke't qua thi nghiem cho thiy de tai da thuc hien dugc mo hinh benh 19 hoai tir xuong tren chuOt nhat tang bang glucocorticoid Lieu tiem Dex mg/kg dugc danh gia co hieu qui gay hoai to xuong va hieu qua kinh to tot nhat yen so lugng chuOt chet chi hal (2/15) va ty le chuOt hoai tic la 5/10 Cac phan tich d'e‘u cho thay tic long gay ton hai xuong cue Dex tang lieu dimg thuoc tang Tic dung dm Asp the hien re viec thilc day str hoai ttr xuang nhung cling gay tic dung phi nang ne (chuOt chet 47 — 67%) Hieu qua ghep to bao don nhan tity xuang Viec ghep to bao don nhan tity xuong dugc danh gia la co kha nang ngan ngira str phat trien ale benh va c6 xu huang lam giam su hoai to xuang Theo tieu chuan phan tich cua Arlet, t9 le chugt hoai 16 ghep to bao dam nhan tit), xuong khOng giam so yeti 16 Dex6; nhung tnirc dO hoai lir 016 da giam ding ke so vori 16 Dex6, the hien CO a mo, Ket tich FCM chimg minh viec ghep to bao don qua nhan ttiy xuang lam giam mat de, to bio chet thy xuong Bong thai, su bieu hien dm cac gen tao xuong (BMP2, Runx2 va osteocalcin) a 16 DB tang mph so yeti 16 Dex6 cho thay qua trinh tao meri xuong (kw tang ctrang Cong trinh dugc den hinh to Ltd cam nguein kinh phi ctia Chirceng trinh Virem Coln Sang too Khoo hoc Cling nghe Ire, Thanh dam Tp.HCM TAI LIEU THAM KHAO Boss JH, Misselevich (2003) Osteonecrosis of the femoral head of laboratory animals: The lessons learned from a comparative study of osteonecrosis in man and experimental animals Vet Pathol 40: 345-354 Conzemius M, Brown T (2001) Animal models of osteonecrosis Tech Orthop 16(1): 90-97 Hanada T Horigome Y, Inudoh M (1989) Osteonecrosis of vertebrae in a child with acute lymphocytic leukaemia during L-asparaginase therapy Eur J Pediatr 149: 162163 Kerachian MA (2009) Molecular pathogenesis of osteonecrosis of the femoral head McGill University Powell C, Chang C Gershwin ME (2010) Current concepts on the pathogenesis and natural history of steroid-induced osteonecrosis Clin Rev Allergy Immunol :1-12 Sun Y, Feng Y, Zhang C (2009) The effect of bone marrow mononuclear cells on vascularization and bone regeneration in steroid-induced osteonecrosis of the femoral head Joint Bone Spine 76: 685-690 Wang BL, Sun W, Shi ZC, Lou JN, Zhang NF, Shi SH, Guo WS (2008) Decreased proliferation of mesenchymal stem cells in corticosteroid-induced osteonecrosis of femoral head Orthopedics 31: 444 Wang GJ, Cui Q, Balian G (2000) The pathogenesis and prevention of steroid-induced osteonecrosis Clin Orthop Relat Res 370: 295-310 Yang L, Boyd K, Kaste SC, Kamdem LK, Rahija RJ, Relling MV (2008) A mouse model for glucocorticoidinduced osteonecrosis: Effect of a steroid holiday J Orthop Res 27: 1-12 Zhang NF, Li ZR, Wei HY, Liu ZH, Hemigou P (2008) Steroid-induced osteonecrosis: The number of lesions is related to the dosage J Bone Joint Sung Br 90(B): 12391243 439 Ding Thi Tung Loan et al STUDY ON OSTEONECROSIS INDUCED BY GLUCOCORTICOID AND PREVENTING POTENTIAL OF BONE MARROW MONONUCLEAR CELL TRANSPLANTATION TESTED IN MICE Dang Thi Tung Loan'', Nguyen Thi Dieu Hang2, Pham Van Phucl'2, Nguyen Thi Bay3 , Phan Kim NgocU Lab of Stem cell Research and Application, University of Science, VNU-HCM of Animal physiology and biotechnology, HCM University of Science 3University of Medicine and Pharmaceuticals — HCMC 2Department SUMMARY Glucocorticoids is a synthetic steroid drug used widely in human life because this hormone has diverse effects in the treatment of disorders, syndromes, diseases such as pain, inflammation, asthma, arthritis, and causing immunosuppression (in the transplant), However, glucocorticoids also causes many side effects Osteonecrosis is one of the most common and serious complications The treatment of osteonecrosis is not quite effective because early diagnosis is difficult and surgical methods (joint replacement) may be limited This study investigated the effective prevention of osteonecrosis by bone marrow mononuclear cells transplantation Adult mice were injected with dexamethasone (Dex) with weekly doses of 4, 6, mg/kg for weeks At once, asparaginase (Asp) is used to enhance bone necrosis Osteonecrosis was identified through staining method of bone tissue, analysis of the necrotic cells in the bone marrow by flow cytometry (FCM) and gene expression of osteocalcin, Runx2, BMP2 by RT-PCR Results: in the H&E staining method, osteonecrosis rate increases together with Dex dose increasing The dose of Dex mg/kg was assessed effectively causing osteonecrosis, with osteonecrosis mice rate was 50%, death rate was 13.3% The apoptosis and necrosis of bone marrow cells are also affected by increasing Dex dose (4.6, 5.3 and 9.7% respectively in the control, Dex4, Dex6) The expression of osteogenic genes also decreased, which induced by Dex The effect of bone marrow mononuclear cell grafting after two weeks was determined as a potential of preventing the progression of the disease Keywords: bone marrow cells, cell transplant, glucocorticoid, mice, osteonecrosis Author for correspondence: E-mail: pvphuc(ihennins.edit.vn 440 ... 20 gram va duqc tiem cic thuoc glucocorticoid de giy hoai tir xuong Ngoai ra, cic 18 thi nghiem duqc tiem bo sung asparaginase de thiic day six hoai tir xtrang glucocorticoid Bao gam cic 18 thi... dui the tiro: ehuit't Dex va nhimg Um& glucocorticoid CO tac dOng phirc top den sinh IS+ cua chuOt Su hien thien Irons, DC Dex-I Dex4+ luqng cua chuOt glucocorticoid cling khong theo mot chieu... tich chu ki va str apotosis cua te bao, tir xic dinh str thay doi hinh thai va tinh chat dm te bao glucocorticoid Thong thuang, truac dua vao may, cac te bio duqc nhuern vai cac Ma chat dac (thuoc
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