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Khoa hoc Y - Duo° Danh gia hiOu qua dieu tri xd gan cua vi0c ghep to bao goc trung ma xu'Ong tren mo hinh chuEit Twang Hai Nhungu', Le Van Trinh', Nguyen Hai Nam', NO Quac Data, Le Minh Huy' Huynh Nghia', Nguyen Van Thanh4, Pliant Van Phficil, Phan Kim NgW Thong thi nghi Nghien cent va ang dung to bcio goc, Trwang Dgi hoc Khoa hpc Ty nhien, Doi hpc Qudc gia TP lid Chi Minh :Khoa Sinh hpc, Trzterng Dgi hoc Khoa hoc Tv nhien, Doi hoc Quac gia TP Ho Chi Minh 3Tnrong Doi hoc Y &tor TP He, Chi Minh 'Tryon Dgi hoc Nguyin Thcinh, TP Ho Chi Minh NO), nhan bai 3.11.2015, ngsly chuyen phin bien 10.11.2015, ugly nhan phin bien 11.12.2015, chip nhan dang 21.12.2015 Tren the giel, lieu phi!) to bac, gOc cho duly tiem nang lan cac nghien cuu dieu tri benh xa gan Tay nhien, hien yin chin thong nhit phic dieu tri ve ngutin bac), phucrng phi') trim, ding thei ir nu& ta china ce cac ding bo ve linh vyc Nham myc dich nghien cuu phut trien lieu phap mai dieu tri xcr gan a lurk ta, ale tfic gia tien hinh gay to mo hinh chuOt xor gan bang CCI4 va thu nghiem dem 106 bko goc trung mo tay string de melt tri Ket qua cho thiy, sau 21 ngily tiem 106 to bko vao tinh mach duoi ciia chuOt bi benh xa gan, nh6m chuOt dieu tri phyc hOi chi se Albumin tir 1,7531-0,042 len 2,1161-0,120, grim din bieu hien cac gen xa h6a nhv TGF, giam 13 din Procollagen, giam 3000 lAn Integrin, giam sir bieu hien ciia collagen-1, SMAa ve giam dien tich cac dui xoo teen hinh thai mo hyc so voi nh6m doi chirng Tit khoa: benh xo gan, lige phap to boo gbc, mo hinh chubt gan, d bao goc trung me, to boo giic xtrang Chi so plain logi 3.5 CUM thileu Xa gan tinh trang ton thuang gan mon tinh, dAn tori hinh nhUng m6 xa xam chiem kh8ng gian cua to bao gan Theo se lieu thong ke cua Mokdad va Ong six [I], chi num 2010, xa gan IA nguyen nhan gay tir vong cho,hcm mOt trieu ngtrai Deng thei, xa gan IA yeu to nguy ca cao cua ung thu gan [2] Hien nay, y hoc van chua ce lieu pita!) dieu tri khei benh xa gan vi: (1) xa h6a gan IA qua trinh Ice° dui va rat kh6 phoc hoi m8 gan xufit hien dui xa [3]; (2) lieu phap hea tri phai tri sir dong lau dii NIA kitting mang lai hieu quA cao dung thuoc giup irc the qua trinh xa hea (anti-TGF, anti-PDGF) thi hog (king chirc 'tang gan khb hei phoc, dung thutic co tic dung irc the mien dich bao v'e gan thi kh6 ngan chin so tien trien cua xa hea [4], va dung ca hai loai thu6c ten van chua cho thay hieu qua [5]; (3) ghep gan van chua giai quyet duTic tinh trang thieu but nguZin me, nguy ca thai loai sau ghep cao, chi phi dieu tri cao va gay xam Ifin [6] ce, the thay the cac phtrcmg phap dieu tri xa gan truyen thong nhtr ten vi: (1) to bao etc co the thu nhin tir nhieu nguon nhu m6 ma, my xuang, mau day rem [7]; (2) ghep to bao gOc tix than giam nguy ca thai loai mien dich; (3) to bao g6c ghep giup irc the qufi trinh xa hoa nhe tier cac cytokine, cal thien hog deng chirc fling gan vi to bao goc da duqc chimg minh kha nang biet hi:5a to bao chirc nang ging to bio gan, kich thich sv phan chia cua to bao gan truong [8-12] Do do, to bio goc dangsduqc sir dung nhieu cac nghien dm lam sang dieu tri benh xa gan va mang lai hieu qua tich cor [4, 13] Te bao etc trung mo tir toy xuang (BM-MSC) dang dyer sir dung nhieu tren lam sang (26/31 nghien cim sir dung to bao g8c) de dieu tri xa gan [14] Ket qua ghi nh'in, ghep BM-MSC giap benh nhan cii thien chirc Wang gan va chi so MELD [14, 15] Deng thei, BM-MSC a thu nhin va c6 the cay ghep tir than Do de, BM-MSC met ngulin to bao tiem nang Ion dieu tri benh xa gan Viet Nam yang c6 ty le benh gan rat cao [16] Nham gap phan nghien ciru tim lieu phip hieu qua Ghep bac* g6c lieu phap moi dang dtrqc nghien cho dieu tri benh xa gan, cung cap ca se cin lam sang ciru dieu tri benh xa gan Lieu pita') to bac) gee cho cac nghien ciru xa han tren lam sang, chting toi *Mc gia lien he: Email: rhnhung@hcmus.edu.vn WHOA HOC\ CONG NGHE V am 4(4) 4.2016 55 Khoa hoc Y - Duoc Effectiveness of the treatment of liver cirrhosis by using bone marrow mesenchymal stem cell in a mouse model Summary In the world, stem cell therapy has shown a potential in liver cirrhosis treatment Up to now, we have not found out the best regimen of cell types, injection methods, while Vietnam has not yet published any result in this field For that reason, this research aims to developing a new therapy for liver cirrhosis treatment in Vietnam The authors conducted experiments using bone marrow mesenchymal stem cell for treatment in the mouse model with liver cirrhosis by CCI4 The result showed that 21 days after injected 106 stem cells into tail vein, the treatment group was improved with the Albumin index from 1.7531-0.042 to 2.1161-0.120; the expression of genes related to fibrosis was decreased such as TGF (by times), Procollagen (by 13 times), Integrin (by 3000 times); and the result also showed the reduction in the presence of collagen-1, SMA-a and the smaller fibrotic areas in histology compared with the control group musculus var, albino)duc, tan tuoi, can nang khoang g, khee manh duqc mua tir Vien Pasteur TP He Chi Minh Tien hinh thu dui chuOt, loci be phan ca dui, thu toy ttlr xuang dui va xuang tang chin chutt, nuoi cay tang sinh va chen lqc BM-MSC mei trueng DMEM/F12 (Gibco) be sung 10% FBS (Sigma) va 1% Antibiotic (Gibco), dieu Icien 37°C, 5% CO2 Chip ti bit o: chi* mo hinh benh xa gan dtrqc chia ngau nhien nhom: nhem doi cluing (n=5) tiem 0,15 ml PBS, nhom thi nghiem (n=5) tiem 106 huyen phit bao dan 0,15 ml PBS-/con qua tinh mach duoi, chuOt binh thueng duqc sir dung lam doi chimg &mg Sau 21 ghep, hieu qua dieu tri benh se duqc dinh gia dua tren cac chi tieu: Men gan: thu 0,2 ml huyet Ming, cac chi tieu nhu AST/ALT bang KIT GOT(ASAT)/GPT(ALAT) IFCC Mod.liquiUV test (GERMANY), Albumin va Billirubin bAng Kit QuantiChrom BCG Albumin/ Billirubin Assay (Bioassay Systems, CA, USA) theo quy trinh cua nha san xuat Danh gia mik de) bi& hien cac gen xa hoas bang qRT-PCR: sinh thief mo gan chu6t, tach mRNA tong tir me gan bang b45 Kit Easy-BLUE Total RNA Extraction (iNtON Biotechnology, Korea) Dinh gia mirc de bieu hien gen bang qRT-PCR (qRT-PCR Master Mix Kit, 1-Step Agilent, CA, USA), Om cac gen: Bang trinh tK cac mo' sri dang phan sing qRT-PCR ciia nghien mitt Keywords: bone marrow derived stem cells, liver cirhosis, mouse model of liver cirrhosis, mesenchymal stem cells, stem cell therapy Ten m6i Fibronectin 3'-TGAAGAGGGGCACATGCTGA-5' 5.-GTGGGAGTTGGGCTGACTCG-3' Procollagen 3'-CCTGGACGCCATCAAGGTCTAC-5' 5'-CAAGTTCCGGTGTGACTCG-3' Integrin 3'-GCCAGGGCTGGTTATACAGA-5' 5'-TCACAATGGCACACAGGTTT-3' Nt5e 3'-TTTGGAAGGTGGATTTCCTG-5' 5'-CCTCTCAAATCCAGGGACAA3-' TGF-6 3'-CTTCAGCTCCACAGAGAAGAACTGC-5 5'-CACAATCATGITGGACAACTGCTCC-3' GAPDH (ch(mg n¢i) 3'-AAGTTGTCATGGATGACC-5' 5'-ATCACCATCTTCCAGGAGC-3' Classification numbers 3.5 tor 4101P-n1111111111111111111111101.1111111111111.1.1•111111=1.0 tien hanh thir nghiem dieu tri benh xa gan bang BMMSC tren mo hinh chuOt bj xa gan bei VOt hOu Ira phuung phap MO hinh chu'Ot xa gan: thuc hien theo quy trinh cua Tnrcmg Hai Nhung va tong su [17], chuet duqc ming CC14 bang cach pha yeti dau olive (3:7 v/v), thong qua duCmg thuc quan, lieu lucmg ml CC14/kg, lin/ tuan 11 tuan Danh gia cac chi tieu thuc hien m6 hinh gem men (AST, ALT, Albumin, Bilirubin), bleu hien gen (Fibronectin, Integrin, TGF, Procollagen, Nt5e), mo hpc nhuOm H&E va Trichrome, bieu hien collagen-1 va SMA-a Chutin bj BM-MSC: chuOt nhat tang (Mus Danh gia mo hoc: ce dinh me gan Parafolmardehyde 4% (Merck Millipore, Germany), nhuOm H&E va Trichrome theo quy trinh cua Benh vien Chq Ray (TP He Chi Minh) Danh gia bi"juhien collagen-1 va SMA-a bang nhuOm hew ma mien dich: mo gan duqc có dinh TAP 011 WA HOC LNG1■KAIEVIet am 4(4) 4.2016 Tani' tkr 56 Khoa hoc Y - Ducc 4% paraformaldehyde (Merck Millipore, Dar) Lat cat (slide) paraffin duqc kilt paraffin va tai hydrate h6a bing sodium citrate (10mM, pH 6) a 95-100°C 20 phitt Cac slide duqc lam lanh xuang khoang 20-30 phut a nhiat d4 phong Sau do, cac slide duqc block 30 phut a nhiat dO phong vai blocking buffer (2% huyet de, 1% BSA, 0,1% Triton X100, 0,05% Tween 20, 0,01M PBS pH 7,2-7,4, 0,05% sodium azide) Slides duqc nhuOm khang the khang collagen I (Santa Cruz Biotechnology, Inc) hoc antisma-Anfa (Santa Cruz Biotechnology, Inc) vai ding dO pha bang 1:100 TBS 1% BSA qua dem 4°C Peroxidase nal sinh duqc block bang peroxidase blocking buffer (3% H202 PBS) 10 phut a nhiat dO ph6ng Horseradish peroxidase-conjugated secondary goat-anti-rabbit antibody TBS 1% BSA duqc nhuOm gia a nhiat dO ph6ng Ket qua nhuOm khang the duqc kiem tra bang cach sir dung ACE kit (Sigma-Aldrich, St Louis, MO) theo hiking dAn Nhan to bao ducx nhuOm bing herrratoxylin-Gill eoo su giam nang dO Albumin huyet tuang so yeti chi* thuong (p
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