Hpl NGHI KHOA HQC VE T^' BAO G6'C TOAN QU6C LAN THa II
DjNH HlriTNG ONG DUNG CUA TE BAD GOC TUY RANG TRONG NHA KHOA TAI TAO
Tr^n Le Bao Hd*, Dodn Nguyen Vu*, Le Thi Ng9C Huwng*, To Minh Qu^n*, Nguyen Thi Ngoc My*,
Ngd Thj Quynh Lan**, Le Dii-c Ldnh"
TOM TAT
Te bao goc tuy rSng la mpt nguon te bao cd tiem ndng tdng sinh va biet hoa cao, cd the du'dc IUXJ triJ" trong nhleu nSm, tu'dng tac tdt vdi nhieu loai vat lieu sinh hpc, de thu nhan vt it xam lan.
Chung toi da tien hanh thu nhan te bao goc tiiy rang bang phu'dng phap cay manh md va cay te bao ddn. Quan the te bao goc tuy rang du'dc xac djnh thong qua kha nang tang sinh, kha nang biet hoa va bieu hien dau an be mat. Sau do, cac te bao goc tuy rang du'dc cay va nuoi tren hai loai khung nang dd la nga rang va gel fibrin. Ket qua cho thay, chiing tdi da thu nhan va nuoi cay thanh cong te bao goc tuy rang ngu'di. Te bao goc tuy rang co kha nang tang sinh tren khung nang d3 nga va gel fibrin. Vcfi nhUng ket qua da dat du'dc, te bao goc tiiy rang hiia hen se la nguyen lieu quan trpng cho nhu'ng nghien aiu trong ky nghe md cung nhu" trong ITnh viic nha khoa tai tao.
Td'khda: be mdt nga, nudi cay sd cap, gel fibrin, te bao goc, tuy rang ngu'di
SUMMARY
DIRECTIONAL APPLICATION OF HUMAN DENTAL PULP STEM CELLS IN
DENTAL REGENERATION Dental pulp stem cells as a source of cells are easy to obtain for non-invasive, has the highly potential to grow and differentiate, can be stored for many years, interact well with many
* Trudng Dgi hgc Khoa hqc Tu nhien. Dgi hqc Quoc gia-lhdnh phd Ho Chi Minh Dgi hgc Y Dugc, thdnh phd H6 Chi Minh
Phan bifn khoa hgc: GS.TS. Nguyin Anh Tri
types of biomaterials. In this research, we performed the experiments to collect and disinfect human dental pulp tissue. Then, dental pulp cells are isolated and cultured. The presence of stem cell populations in cultured cells is determined by proliferation assay, differentiation and Flow cytometry. Simultaneously, dentin was used as a scaffold for the dental pulp stem cells.
Results shown that we are successful in collection and culture of human dental pulp st^n cells. Dental pulp stem cells can proliferate on chemically treated dentin surface and fibrin gel.
With the results achieved, dental pulp stem cells promises- to be an important material for research in tissue engineering as well as in dental regeneration.
Keywords: dentin surface, primary culture, gel fibrin, stem cells, human dental pulp
I. OAI CUONG
Hien nay, Viet Nam dugc coi la nuac co ty le ngucri mac benh rang mieng cao nhat the gicri. Noi nha la phuang phap pho bien de dieu tri cac tmong hop sau rang den tuy. Tuy nhien, phucmg phap nay chi loai bo tiiy rSng hu chu khong phyc h6i dugc rang sau. Sau mpt thai gian, chat lugng rang sau khi tram sg giam, rang tra nen gidn, de gSy hon.
Trong nhihig nam gan day, vai nhiing ky thuat hien dai, cac nha khoa hgc da co nhiJng buoc tien dang k6 khi hp tien hanh nghien cuu phan l^p va nuoi cay thanh cong tt bao
Y Hpc VIET NAM THANG 4 - S6 PAC BlgT/2013
goc tuy rang [1], [2], [3], [4]. Mpt hud'ng iing dung cac te bao goc tiiy rang trong lam sang la kit hgp chiing vai khung nang dd thich hgp de phuc hoi tuy rSng cung nhu nga rSng trong chiia tri. M6t so nghiSn cuu cho thiy ngi rang la loai v$t li?u c6 cac dac tinh vat ly, hoa hgc thich hgp de tai t^o nga. KJiung nSng dd nay giai phdng cac yeu to tang tnrong kich thich su hinh thanh nga siia ch&a [2]. Fibrin co nhiing tinh nang vugt trgi nhu khd nang kich thich te bao tSng sinh, t6ng hgp chat nen ngogi bao, kha nang tu tieu, chen dugc vao cac khuyet hong nho va tren hk fibrin c6 Xhk dugc sin xuit tir huy6t thanh tu than nen day la mgt vat H?u sinh hgc day hiia hen trong tai tao cac mo rang [5].
II. PHUONG PHAP NGHIEN CCOJ Phuang phdp phdn lap va nuoi cdy te bdo tuy rdng
Sau khi tach khoi rang, mau tiiy dugc cat thanh nhiing manh nho (2x2x1 mm); sau do, nuoi trong dia 35nim (nuoi cay manh mo) hay u trong hon hgp coUagenase/dispase (nuoi cay te bao don), bo sung moi truang DMEM/F12 (Sigma) b6 sung 10% FBS (huyet thanh thai bo - Sigma).
Xdc dinh tinh goc cua te bdo tity rdng ngucri
Ddnh gid su tdng sinh cua ti bdo tren be mgt chai nuoi cdy: Cac te bao dugc cay chuyen sang dia 96 gieng vai mat dp 10^ te bao/cm . Meit do te bao dugc xac dinh moi ngay bang buong dem te bao.
Ddnh gid sir biiu hiin ddu dn be mat te bdo: bang phuang phap Flow cytometry, sur dung may FACS Calibur sii dung phSn mim Cell Quest Pro.
Ddnh gid tiem ndng biit hod: Te bao tiiy rang dugc cam iing bi6t hoa thanh xk bao ma bai moi truang DMEM/F12 co bd sung 10%
FBS, 1 nM dexamethasone, 200 pM indomethacin, 1,7 pM insuline, 500 pM isobutyl-methylxanthine (IBMX) va dugc cam ling biet hoa thanh nguyen bao xuang boi m6i truong DMEM/F12 co bo sung 10%
FBS, 100 nM dexamethasone, 50 pg/ml L- ascorbic acid 2-phosphat (AsAP) va 100 mM p-glycerolphosphate. Sau do, te bao dugc danh gia bang each nhupm vai thuoc nhupm Alizarine Red va Oil Red.
Tgo vd cdy te bdo goc tuy rdng len khung ndng da ngd rdng
Nga rang dugc thu nhan va be mat cua nga se dugc xii ly lan lugt vai citric axit 19%
trong 1 phiit, EDTA 17% trong 10 phiit dh loai bo lap mun nga. Te bao goc tuy rang a lan cay chuyen thii tu dugc cay len be mat nga vai mat d6 lO'* te bao/cm^. Sau do, mau se dugc quan sat duoi kinh hien vi dien tii quet (SEM).
Tgo vd cdy te bdo goc tuy rdng len gel fibrin
Cac xk bao g6c tuy rang a lan cay chuyen thli tu dugc cay len cimg liic vai qua trinh tao gel fibrin a mat do 10"* te bao/cm . Sau do, mSu se dugc quan sat kinh hien vi dien tii quet (SEM).
111. KET QUA NGHIEN cCfU
\. Ket qua nudi cay te bao tuy rang
Theo phuong phap nuoi cay manh mo, cac te bao bat dau hgp dong, trai thanh mpt lop don phu kin hh mat dia nuoi vao ngay thii 21.
Theo phuang phap nuoi cdy tk bao dan, cac te bao bat dau hgp dong, trai thanh mgt lap don phu kin hh mat dia nuoi vao ngay thii 14.
Hpl NGH! KHOA HQC Vg T£' BAO G 6 C TOAN QUOC LAN THU II
Hlilh 1. Tc b:io moc lan ra lir nianh ino ; ngay nuoi cay (lOOX): li. 1 c hao iu\ ran.12 smi
cac ngay nuoi cay. A. Te bao tliy .5 ngay nuoi cay (lOOX).
Hlnh 2. Te bao dan sau cac ngay nuoi cay. A. Te bao tiiy rang sau 7 ngay nuoi cay (lOOX); B. Te bao tuy rang sau 14 ngay nuoi cay (lOOX).
K^t qud ddnh gid sir tdng sinh cda te bdo tren be mat chai nuoi cdy
Do thi 1 cho thay sau khi dugc cay chuyen, te bao tang sinh mgnh tir ngay 2 den ngiy 6, dat cao nhat vao ngay 6, sau do giam nhanh den ngay 9.
5 6 7 s 9 Thd'i gian
Do thj 1. Duong cong tSng truang ciia te bao tuy rang nguai nuoi c4y.
Ket qud xdc dfnh ddu dn bi m^l te bdo
KSt qua cho thdy, cac te bao umg viSn duang tinh m ^ vai cdc ddu &i CD73 (99,77%), CD90 (97,34%), CD105 (91,95%) va am tinh voi cac ddu dn CD34 (0,42%), CD45 (0,08%), HLA-DR (0,20%).
Y HOC VlgT NAM THANG 4 - Sd" BAC BIET/2013
S.i
Q k ^ .
Hinh 3. Ket qua phan tich Flow cytometry ciia tk bao tuy rang iing vien.
Ket qud ddnh gid tiem ndng bi^t hod
Ket qua nhugm Alizarine Red va Oil Red cac tk bao tiiy rang sau bi?t h6a cho thdy cac tk bao g6c tuy rang iing vidn thu nhan theo quy trinh tren co kha nang biet boa thanh nguyen bao xuong va te bao tao ma.
Elinh 4. T^ bao tuy rang a thS h? thii tu da biet hoa, A. T6 bao tuy rang biet hoa thanh xk bao tao md dugc nhuom vcri Oil Red (thay ro cac gipt ma); B. Te bao tiiy rang biet hoa thanh nguyen bao
xuang dugc nhupm vo'i Alizarine Red (thay ro cac not calci).
Ket qud ve khd ndng bdm dinh cua te bdo goc tuy rdng tren khung nang dd
Hinh 5. Hinh chup SEM t^ bao g6c tuy rang tren khung nang da. A. Te bao goc tuy rang tr&i khung nang d 5 nga (4500X); B. Te bao goc tuy rang trong gel fibrin (4000X).
Hinh 5 cho thdy co su hien diSn va bam dinh cua te bao goc tiiy rang tren khung nang dd nga xii Iy b^ mat va gel fibrin.
HQI NGHI KHOA HQC VJ lfe' BAG GO'C TOM* WUUU LAW I nu ii
IV. BAN LU$N
T6 bdo goc tliy rang c6 bieu hi^n cdc ddu an te bao goc trung mo [2], [3], [4]. K i t qua nghien ciiu ciia chiing toi dd chiing minh rdng tk bdo tiiy ring c6 khd ndng bdm dinh, ldng sinh, bi?t hda trong dieu ki?n nuoi cdy in vitro vd bilu hi?n cdc dau dn CD73, CD90, CD105, day Id cdc ddu an ciia te bdo goc trung mo. Cdc kit qud thu dugc cho thay qudn thk tk bdo goc trung mo tir tuy rdng ngu6i da c6 s\r dong nhdt 6 the h? nu6i cdy thli tu vd cdc te bdo ndy vdn duy tri dugc ddc tinh goc trong qud trinh nuoi cay in vitro.
Huang va d6ng tdc gid (2006) da chiing minh khung ndng d& ngd rdng co khd ndng cam ling t l bdo g6c tiiy rdng biet hoa thdnh nguyen bdo ngd [2]. Tir nhihig ket qud nghien ciiu da dugc cong bo tren the gioi, chua CO cong trinh ndo sir dung nguon fibrin tu than Idm gid d o sinh hgc trong ky ngh? mo rang. Trong nghien ciiu ndy, chiing toi da chiing minh khung ndng d a nga rang vd gel fibrin co kha nang kich thich te bdo goc tiiy rang bam dinh va tdng sinh tot. Day la tien de cho viec phat trien phuang phdp dieu tri mai nham ling dung nguon te bdo goc ndy de tdi tao mo giong tuy rdng.
V. KET LUAN
Chiing toi da nuoi cay thanh cong t l bao goc trung mo tir tiiy rdng nguai. Them nGa, cdc te bdo nay co khd nang bam dinh va tdng sinh tdt tren b l m^t ngd rdng vd gel fibrin.
Ket qud nay hiia h?n khd ndng tdi tao mo Cling va m6 mem ciia rdng.
LM cim am: Nghien ciiu ndy dugc tai trg bdi B ^ Khoa hgc va Cong nghS trong khuon kho d l tdi md so DTDL.20I2-G34 va Dai hgc Quoc gia Thdnh ph6 Ho Chi Minh (VNU-HCM) trong khuon kh6 d l tai ma s6 B2012-18-11TD.
T A I UEU THAM K H A O
1. Batouli S, Miura M, Brahim J, Tsutsui TW, Fisher LW, Gronthos S, Robey PG, Shi S, Comparison of stem-cell-mediated osteogenesis and dentinogenesis, J Dent Res, 2003,82,976-981.
2. George T J H , Wataru S, James C, Sang HP, In vitro characterization of human dental pulp cells: various isolation methods ang culturing enviroment. Cell Tissue Res, 2006, 10, 1-23.
3. Gronthos S, Brahim J, li W, Fisher LW, Cherman N, Boyed A, DenBesten B, Robey PG, Shi S, Stem cell properties of human dental pulp stem cells, J Dent Res, 2002,81,531-535.
4. Jinhua Y, Huixia H, Chunbo T, Guangdong Z, Yuanfei L, Ruooing W, Junnan S, Yan J, Differentiation potential of STRO-I+ dental pulp stem cells changes during cell passaging, BA4^C Cell Biol, 2010, 11,32-39.
5. Takayuki O, Toshimitsu 1, Kazutada U, Yusuke A, Hiroya S, Masaki J H, Minoru U, Hideaki K, Evaluation of scaffold materials for tooth tissue engineering, J Biomed Mater Res Part A, 2010, 94A, 800- 805.