Biểu hiện marker bề mặt tế bào gốc trung mô nuôi cấy nguồn gốc tủy xươn

Y HOC VIET NAM THANG - SO 2/2010 dudng huyet a chudt" Hpi nghi KHCN toan quoc, Thai Nguyen, NXB DH Thai Nguyen, tr.877-878 Nguyen Van Mui (2002), Xdc dinh hoat TAI LIEU THAM KHAO Vo Van Chi (1999), Tie diin cay thudc Viet enzym, NXB Khoa hpc va ky thuat, Ha Npi Courtney C.H., Jerrold M.O (2007), Insulin Nam, NXB Y hpc, Ha Npi Resistance, Mechanisms of Insulin Actions, Trinh Thj Thu, Le Thj Hong Nhung, Nguyen Landes Bioscience and Springer Science + Van Mui, Phan VanChi (2009), "Tao tieu duang typ a chudt de nghien cim hoat tinh haBusuness Media cong nghe protein-enzym, DH Khoa hpc Tu nhien, DH Quoc gia Ha Npi BIEU HIEN MARKER BE MAT TE BAO GOC TRUNG M0 NUOI CAY NGUON GOC TUY XUUNG NGlTOl Tran Thj Thanh Huong*, Tran Van Khanh*, Nguyen Thj Ha*, Ta Thanh Van*, Do Doan Loi* T6M TAT CULTURED MESENCHYMAL STEM CELLS FROM Khang nguyen be mat te bao la mpt cac HUMAN BONE MARROW tieu chuan xac dinh te bao goc trung mo Cell -surface antigens are one of minimal criteria (Mesenchymal stem cells, MSC), bao gom marker to define mesenchymal stem cells (MSC), including dUdng tinh va marker am tinh Xac djnh cac marker both positive and negative markers Defining the cung la nham chu'ng minh sU cong ciia qui trinh expression of markers to show the successful of phan lap, nuoi cay MSC tir tiiy xUOng ngUdi Muc isolation and culture methods of MSC from human tieu: Xac dinh cac marker CD13, CD73, CD90 va bone' marrow is necessary Objective: Defining CD34, CD45 tren be mat MSC nudi cay dupe phan lapmarkers: CD13, CD73, CD90 and CD34, CD45 on cell tii tiiy xUdng ngUdi PhUOng phap nghien cu"u: Sir cultured surface after isolation and culture of MSC dung khang the ddn ddng xac dinh cac marker be mat from human bone marrow Method: Cultured MSC at te bao nudi cay bang phUdng phap FACS va IHC Ket PI were stained with the monoclonal antibodies qua: MSC nudi cay d giai doan cay chuyen PI dUOng thereafter analyzed with FACS and IHC Result: tinh vdi CD 13, CD73, CD90, va hau nhu" am tinh vdiCultured -MSC from human bone marrow at PI CD 34 va CD45 Ket luan: MSC nudi cay dupe phan express CD 13, CD73, CD90, and lack expression of CD lap tii tiiy xUdng ngUdi bieu hien day dii va chinh xac 34, CD45 Concludes: : Cultured-MSC from human kieu hinh khang nguyen be mat dac trUng cho te bao bone marrow at PI express phenotypic characterization of Mesenchymal stem cells to cellgoc trung md Tir khoa: Te bao goc trung md (MSC), surface antigens marker be mat MSC, FACS, IHC Key word: Mesenchymal stem cells (MSCs), cellsurface markers of MSCs, FACS, IHC SUMMARY EXPRESSION OF CELL - SURFACE MARKERS ON I OAT VAN OE Te bao goc trung md (Mesenchymal stem * Trudng Dai hoc Y Hd Ndi 143 CHUYEN BE : HOI NGH| KHOA HOC CliA HOl HOA SINH Y DUOC HA N0I VA CAC TINH PHIA BAC LAN T cells, MSC) co the dugc phan lap tir nhieu mo Cac te bao khdng cd kha nang bam nhua nuoi cay co the truong thanh, co kha nang biet hoa dupe loai bd sau - Thay mdi trudng tao nhieu dong te bao khac nhau, thupc trung bi lan / tuan Cay chuyen mat dp te bao dat 80% (xuong, sun, mo, co, co tim), npi bi (gan, tuy), chai nudi dien tich 25 cm Tai lan cay chuyen ngoai bi (da, than kinh) [1], MSC da va dang la thir nhat (PI), tach te bao bam dinh chai nuoi ung cu vien sang gia cho cac lieu phap te bao bang Trypsin - EDTA 0,25%, bat hoat enzym ung dung y hoc tai tao cua nhieu loai benh bang mdi trudng nudi cay, li tam lay can te bao li rieng biet, co benh co tim thieu 2000 g x 15 phut d nhiet dp phdng, rua lai bang PBS mau Tiiy xuong la mpt nguon phan lap va nuoi Cho them 500 u.1 PBS tao djch huyen phii te bao cay MSC co hieu suat cao Cac tieu chuan de xac - Ki thuat FACS (Fluorescene activated dinh chinh xac chat luong MSC sau phan lap va cell sorting): nuoi cay bao gom dac diem hinh thai va phat Nguyen li: Dem te bao ddng chay (Flow trien cua MSC moi truong nuoi cay, kha cytometry) la kT thuat dem va phan tich dong thai nang da biet hoa in vitro va cac khang nguyen be cac dac tinh vat li ciia nhiing phan tir rieng le, mat duong tinh va am tinh Hien chua co khang thuong la cac te bao, chung di chuyen nguyen be mat nhat duoc sir dung de xac mpt ddng dich qua mpt chiim tia lase, bao gom dinh MSC kich thudc ciia phan tir, xac dinh muc dp cac Muc tieu cua nghien ciru: phirc hop va cac hat ben trong, cac dac tinh phat - Xdc djnh su- bieu hien cua cdc marker CD huynh quang 13, CD73, CD90 vd CD34, CD4S tren be mat MSCMdt flow cytometer dupe cau tao bdi ba nudi cay duac phan lap tie xuong ngudi phan chinh: He thdng ddng chay, he thdng quang hpc va he thdng dien tii' Tien hanh: Mdi dng cd khoang 500.000 II 06l TUONG VA PHUONG PHAP NGHIEN CUXJ te bao, dupe u vdi 100 p.1 khang the don ddng 2.1 Doi tirong nghien ciru: - MSC dupe phan lap, nuoi cay theo qui chupt khang ngudi, ndng dp 50u.g/ml Co the ii trinh phan lap - nuoi cay MSC tiiy xuong ngudi, rieng biet tirng loai khang the hoac ii vdi ddng thdi hai khang the neu chiing dupe gan vdi cac tai giai doan cay chuyen thir nhat (Pl).( n=3) 2.2 Hoa chat, thiet bi: chat phat huynh quang khac nhau, vi du khang - Khang the khang: CD34 - PE, CD45 - the khang CD34 PE vdi khang the khang CD 90 ECD, CD 13 - PE (Beckman Coulter), CD73 -FITC, khang the khang CD 45 ECD vdi khang PE, CD90 - FITC (B&D), CD 45 (Dako) the khang CD73 PE, 30 phut, d nhiet dp - Mouse IgGl PE Mouse IgGl FITC (B&D) phdng Mau chirng am la mau te bao ii vdi khang the khang chupt cd gan huynh quang tuong ling - Trypsin EDTA 0,25% (Invitrogen) - PBS (Phosphat buffer salin) (Invitrogen) Phan tich tren Beckman Coulter F500, sir - Thiet bj dem dong chay te bao (Flow dung phan mem chuyen dung su li ket qua, dua cytometer) Beckman Coulter F500 tren sir so sanh vdi ket qua te bao chiing am (Moi - Kinh hien vi quang hpc mau dupe xac djnh lan) Ki thuat hda md mien dich 2.3 Phuong phap nghien ciru: (Immunohistochemistry - IHC): - Chuan bi mdu phdn tich: Tien hanh: Li tam lay can te bao sau MSC dupe phan lap tu tiiy xuong theo qui trinh ciia Shim W.S.N va cdng sir [5] Cac te bao tach khdi chai nudi cay bang trypsin-EDTA don nhan tiiy xuong dupe nudi cay mdi 0,25% Co dinh bang paraformaldehyd 4%/PBS trudng DMEM - LG, 10% FBS, 1% khang sinh Due paraphin can te bao DP day lat cat mlu due 144 Y HOC VIET NAM THANG - SO 2/2010 paraphin la p.m Loai bo paraphin Bdc lp khang nguyen bang dem pH= 6,0 va nhiet dp ciia hoi noi ap suat 10 phiit Rira bang PBS Khu H npi sinh blng H 3% Rua PBS x lan U khang the thii nhat (pha loang 1/400) tai 4°C qua dem Rua PBS phut/Ian x lin khang the thir hai x 60 phiit, buong giir am, 2 2 nhiet dp phong Rira lai bang dem U Streptavidin- HRP x 60 phiit Rua PBS Nhupm (DAB) Nhupm 3,3'-Diaminobenzidine Hematoxylin nhan va bao tuong Doc ket qua bang kinh hien vi quang hoc Ket qua duong tinh: Te bao bat mau vang nau d viing chu vi KET QUA 3.1 Bieu hien cac marker dirong tinh cua MSC tren be mat te bao nuoi cay WW« I(JG-P?Cn3,MiDfOTIC(»iKWa]15lf Mn:ri1l ^ ' ^ * '(WaCO.CD/3PE fW ' atqJMDU i Mdlll Q i Cuiaoim ito n vij.' M)l I rt , l ( B J ID (, U Hinh 3.1 Phan tich dong chay te bao cac khang nguyen be mat CD 13, CD 90 va CD 73 MSC nuoi cay duoc nhupm voi cac khang the don dong gin vdi PE va FITC, Bang 3.1 Miic dp bieu hipn marker be mat CD13, CD90, CD73 tren MSC nuoi ciy PI (n=3) Marker be mat Ti le dUOng tinh (%) ( X ± SD) CD13 CD90 CD73 80,2 ±2,1 78,4 ± 1,5 60,3 ± 2,0 Nhan xet: Ket qua d hinh 3.1 va bang 3.1 cho thiy MSC nuoi ciy d giai doan PI duong tinh voi cac marker be mat CD13, CD90, CD73 a muc dp tuong doi cao tir 60 - 80 % 3.2.Bieu hien cac marker am tinh cua MSC tren be mat te bao nuoi cay ff 1»tin^ea] Huaaj i:at'i niJIlOWIt iMIi.i M>|: FI at nn A»l"t Hinh 3.2 Phan tich dong chay te bao cac khang nguyen be mat CD 34 va CD 45 MSC nuoi ciy duoc nhupm vdi cac khang the don dong gan vdi PE va ECD, 145 CHUYEN BE : Hpi NGH| KHOA HOC CUA jjOl HOA SINH Y DUOC HA N0I VA CAC TlNH PHlA BAC LAN T Nhan xet: MSC nuoi cay d giai doan PI co co bieu hien bieu hien am ti'nh > 95% doi voi CD34 va CD45 •3> • \ ** # A -ft I * - » • i •* > »* //?«/» J.i Tieu ban IHC - khang nguyen be mat CD45 MSC nuoi cay duoc nhupm voi khang the don dong, danh dau bang enzym peroxidase ( xlOO) Nhan xet: - Tren tieu ban nhupm IHC doi voi CD 45, MSC nuoi cay tai giai doan cly chuyen PI duong tinh < 5% rieng biet, nhu MSC tham gia vao qua trinh hd IV BAN LUAN Te bao goc trung mo (Mesenchymal stem trp tao mau ciy ghep te bao gdc mau, cac cells) hay te bao dem (Marrow stromal cells) benh li ve chuyen hda tao xuong, khuyet xuong, (viet tat MSC) la te bao goc da nang, dupe phat teo co' xo ciing (amyotrophic lateral sclerosis) va hien dau tien d tiiy xuong, nhung cung co the dac biet la nhoi mau co tim [2] Tinh an toan, kha dupe phan lap tu cac mo truong khac thi va hieu qui ciia viec ciy ghep MSC ddi vdi Chiing co kha nang biet hoa tao loai te bao khac nhieu trudng hpp benh li dupe thirc hien nhu xuong, day chang, co' tim, co xuong, cac khio nghiem nghiem ngat Mpt lupng kha than kinh, gan, mo, to chiic lien ket [1], Trong 10 Idn MSC, ddi len tdi 10 te bio [2] la can nam gan day, MSC la loai te bao goc rat duoc thiet cac thir nghiem lam sing Tuy nhien, quan tam nghien ciiu, bao gom nghien ciiu co MSC la loai te bio gdc mi sir hien dien la rat han ban in vitro, in vivo va cac thii nghiem lam sang.che cic md trudng Trong tiiy xuong, Hien nhirng hieu biet kha day dii ve MSC co MSC chi chiem 0,001% den 0,01% tong sd te dupe tir cac nghien ciiu ve dac diem kieu hinh, bio cd nhin, bang 1/10 te bao gdc tao mau [4] dac diem mien dich, kha nang biet hoa ngoai co Do vay, viec phan lip - nhin nudi MSC la kT the cung nhu co che tac dung lieu phap ciy thuat ddng vai trd co bin, quan trpng khdng chi ghep te bao v.v Tuy nhien, nghien ciiu co ban cho cic nghien ciru co bin ma cdn ddi vdi cac va ling dung MSC d Viet Nam dang d giai doan bat thii nghiem lim sang MSC dau va mdi chi tip trung d mdt sd trung tam nghien Tuy da cd nhirng nd lyre ITnh vuc ciru Idn, cd trudng Dai hpc Y Ha Npi nghien ciiu dac tinh mien dich ciia MSC, cho den Cac thir nghiem lam sang lien quan den ngudi ta vin chua xac dupe marker be mat MSC tip trung vao nhieu loai benh li cd tinh nhit cho loai te bao gdc Dieu niy giy 146 Y HOC VIET NAM THANG - SO 2/2010 kho khan cho djnh danh MSC, nhit la MSC nhau, nhung sir bieu hien ciia cluing ket hop vdi dugc cho la co the phan lap tu nhieu nguon khac sir vang mat cac khang nguyen thudc ddng te bao va dugc nghien cuu d nhieu phong nghien mau (CD34 va CD45) tren be mat te bao nhu da ciiu khac tren the gidi Hiep hdi qudc te ve trinh bay d tren, la kieu hinh khang nguyen be lieu phap te bao (International Society for mat dac trung cho MSC Tuy nhien d lan cay Cellular Therapy, ISCT) da qui dinh nhung tieu chuyen thii nhat, miic dd bieu hien ciia chiing chuan tdi thieu nham xac djnh MSC [3], theo do: chua cao mdt each tuyet ddi (>95%) - MSC phai bam dinh vao nhua nudi cay Trong cac nha khoa hgc chua tim dieu kien nudi cay tieu chuan; dugc marker duong tinh dac hieu cho MSC nhu - MSC bieu hien duong tinh vdi CD 105, hien thi viec chimg minh cac te bao nudi cay CD73 va CD90; Bieu hien am tinh (tuy thudc vao am tinh vdi marker ddng te bao mau va duong md phan lap) vdi CD45, CD34, CD 14, hoac tinh vdi cac marker duong tinh dac trung ciia CDllb, CD79 (ddng tuy), hoac CD19 va HLA- MSC cd y nghTa gdp phan khang dinh tap hop DR (ddng lympho); MSC thu duoc sau tirng qui trinh cu the, d day la - MSC cd kha nang biet hda tao cac te baoqui trinh phan lap va nudi cay MSC tir tiiy xuong tao xuong, tao sun, tao md dieu kien in vitro ngudi Viec chgn lira kT thuat xac dinh marker be Viet Nam, mdt sd tac tac gia da tien hanh mat cung can duoc dat Phan ling mien dich nghien ciiu phan lap MSC tir cac ngudn khac dac hieu, sir dung cac khang the don ddng cd gan nhu tiiy xuong, mau cudng rdn, cac chat phat hien huynh quang hoac mang man, cudng rdn Tuy nhien, chua cd cdng bd de cap de phat hien cac dac tinh khang nguyen tren be den viec djnh danh MSC bang cac marker be mat mat te bao la mpt sir lira chon cd tinh kha thi va mdt each day dii Viec lua chgn cac marker am hieu qua cao Nghien cuu sir dung hai phuong tinh - duong tinh va ki thuat xac dinh de chirng phap FACS va IHC de danh gia sir bieu hien cac minh tap hop MSC sau qua trinh phan lap va khang nguyen be mat tren te bao nuoi cay Trong nudi cay, tiiy thudc vao ngudn phan lap cung nhu FASC duoc sir dung de danh gia cac marker dieu kien trang thiet bi De tai nghien ciiu sir duong tinh va am tinh, IHC dugc sir dung xac dung hai marker am tinh CD34 va CD45 dac dinh bieu hien marker am tinh (CD45) trung cho te bao gdc tao mau va cac te bao mau KT thuat ddng chay te bao hay FASC trudng thanh, ba marker duong tinh ddi vdi (Fluorescence activated cell sorting) la mpt MSC, bao gdm hai marker duoc dua vao tieu phuong phap co dp nhay va dp chinh xac cao chuin toi thieu (CD90 va CD73) va CD13 la mdt duoc sii dung phd bien nham xac dinh sir bieu marker duoc nhieu nghien ciiu chiing minh la hien cac marker tren be mat va cung cap nhieu bieu hien duong tinh tren be mat MSC [4] Ket thong tin ve te bao Ngoai ra, FACS cd the dugc qua cac MSC nudi cay khdng bieu hien hoac bieu sir dung de phan lap tap hop MSC cd dac tinh hien vdi ti le rat thap ( ... gia cac marker dieu kien trang thiet bi De tai nghien ciiu sir duong tinh va am tinh, IHC dugc sir dung xac dung hai marker am tinh CD34 va CD45 dac dinh bieu hien marker am tinh (CD45) trung cho... thiy MSC nuoi ciy d giai doan PI duong tinh voi cac marker be mat CD13, CD90, CD73 a muc dp tuong doi cao tir 60 - 80 % 3.2.Bieu hien cac marker am tinh cua MSC tren be mat te bao nuoi cay ff... ling dung MSC d Viet Nam dang d giai doan bat thii nghiem lim sang MSC dau va mdi chi tip trung d mdt sd trung tam nghien Tuy da cd nhirng nd lyre ITnh vuc ciru Idn, cd trudng Dai hpc Y Ha Npi