CHUYfeN BJ: HJNH THAI HQC VA CONG NGHg T H 6 N G TIN
TAO MANH GHEP TUTTl BAO GOC TRUNG MO VA SAN H 6 l)NG DUNG o f TAI TAO MO XUUNG
Hu^nh Duy Thao', Tran Le Bdo Hd^ Le Thanh Hung*, Pham Xuan Khiem*, V6 Quoc Vu', Hoang Kc Hu-<rag', Thdi True Quynh', Nguyen Khanh Hoa', Cao Mgng Phi An^ Tran Cong Toai'
T6M TAT
o a t vSfn d e : Ngay nay, viec nghien CLTU va uYig dung te bao goc rat pho bien trong y hpc ddc biet la trong cong nghe tai tao mo xu'dng. Do dd viec tim kiem nguon te bao goc de thu nhan, phan lap va utig dung la mpt cau hoi du'dc ddt ra cho cdc nhom nghien CUXJ. Ngoai ra, de cai thien chat lu'dng cho cac manh ghep su' dyng trong cong nghe mo bang each bo sung them nguon te bao goc tren cac gia the de tang hieu qua cua manh ghep cong nghe.
Muc tieu nghien cCtu: Tao manh ghep tiT te bao goc trung mo tiJ' mo m d va gia the san ho de hu'dng den cac litig dung trong y hpc tai tao va cong nghe mo xu'dng.
Phu'dng phap nghien c i h i : T h i nghiem thi/c nghiem mo ta. Mo md ngu'di du'dc thu nhan va sir dung de phan lap te bao goc trung mo.
Sau do, te bao goc trung mo du'dc sir' dung de tao manh ghep bang each sir dung san ho lam gia the.
Ket qua: Quan the te bao sau lan cay chuyen thil" hai du'dc dem di djnh danh te bao goc trung mo. Do la cac te bao bam dinh vao chai nuoi, co hinh thai giong vdi nguyen bao sdi.
Cac te bao nay biet hoa du'dc thanh nguyen bdo xu'dng va te bao md trong dieu kien in vitro. Cac te bao nay du'dng tinh rat cao vdi cac marker CD44, CD73, CD90 va CD105 va bieu hign rat thap cac marker CD45 va HLA-DR. Tao du'dc
^ Bg mon Wo - Pho/ - Di truyen, DH Y Khoa Pham Ngoc Thach
^ Phong Nghien cdu va lfng dung Te bao goc, DH Khoa hoc TWnhien TP.HCM
^ Khoa RSng - Ham - P4at, DH Y DWOc TP. HCM
^ Giam ddc Benh vien EMCA5, TP.HCM Phan bi$n khoa hgc: PGS.TS. Quan HoSng LSm
manh ghep tiT san ho va t e bao goc trung mo, manh ghep du'dc ddnh gia vdi cac phu'dng phap nhupm mo hpc (H8iE) va alkaline phosphatase de danh gia kha ndng biet hoa cua te bao thanh nguyen bao xu'dng.
Ket l u $ n : Quan the te bao nuoi cay tiJ* mo m d la cac te bao goc trung mo. Cac te bao nay CO the ket hdp vcfi san ho de tao manh ghep ilTng dung trong cong nghe tai tao mo xu'dng dang co nhu cau rat ldn trong xa hpi.
T u ' k h o a : Mo m 3 , te bao goc trung mo, gia the san ho, manh ghep.
S U M M A R Y
Background: Nowadays, the study and application of stem cells in medicine is very popular especially in bone tissue regeneration technology. So finding easy sources of stem ceils collected, isolated and applications is a question for many research groups. I n addition, to Improve the quality of grafts used in tissue engineering by adding a source of stem cells on scaffolds to increase the effectiveness of the technology grafts.
Objectives: Creating grafts from mesenchymal stem cells from adipose tissue and coral scaffold toward applications in regenerative medicine and tissue engineering bone.
M e t h o d s : The experiment was arranged as described experiments. The adipose tissue is
Y HQC V | £ T MAM THANG 10 - SO DAC BI$T/2013
collected and used for the isolation of mesenchymal stem cells. Then, mesenchymal stem cells are used to create grafts using coral as the substrate.
Results: Cell populations In the second subculture are identified mesenchymal stem cells.
It is the rail adhesion to the surface of culture bottles, with morphology similar to fibroblasts.
These cells differentiate Into osteoblasts and adipocytes in vitro conditions. These cells highly positive for the markers CD44, CD73, CD90 and CD105 and low expression of markers CD45 and HLA-DR. Creating grafts of coral scaffold and mesenchymal stem cells, grafts were assessed with histological staining methods (H&E) and ailoline phosphatase staining to evaluate the potential of cell differentiation into osteoblasts.
Conclusion: Populations of cultured cells from adipose tissue are mesenchymal stem cells.
These cells are associated with coral to create grafts for use in bone tissue regeneration technology is currently in great demand in society.
Keywords: Adipose tissue, mesenchymal stem cells, coral scaffold, grafts.
I. OAT VXN D I
Nghien curu ung dung te bao g6c hien nay dang rat phat tri^n va di vao nhi^u ITnh vuc nghien cuu, tir cac nghien cuu khoa hpc CO ban cho den cac umg dung lam sang trong y hoc. Nhieu loai te bao g6c trong ca thS da dupe nghien cihi va trien khai ihig dung. Tuy nhien, te bao goc trung mo (Mesenchymal Stem Cells - MSC) thu nhan tir mo truang thMih nhu tuy xuang, m6 lien k^t, tuySn tieu Hoa... hoSc cac phdn phu cua thai nhu mau cuQng r6n, cuong rdn [24] la cac ik bao g6c oa ti§m nSng, co kha nSng tu ddi mai va biet hoa thanh cac te bao thupc dong trung mo nhu nguyen bao xuang, xk bao m o va t^ bao
s\in trong diiu kien in vitro va phat triln thanh mo xuong, md va sun trong in vivo [15,24]. Do CO tiSm nSng bi$t hda kk hpp vai viec de dang phan l^p va nudi cky lam cho cac MSC trdf th^nh ngudn \k bdo gde dupe iJmg dyng rk quan trpng trong cdng nghe mo vk y hpc tai tao. Mac dii cdc MSC c6 rat nhieu tiem nSng irng dyng nhung chung la quan the te bao rat hi6m, chiem khoang 0.001%-0.01% cua todn bd ta bdo don nhan tuy xucmg [15,24]. Do do, MSC cdn phai cd mdt quy trinh thu nhan phu hpp dd phat triSn in vitro truoc khi cd nhihig ung dung trong y hpc [13].
Trong so cdc ngudn md diing dd thu nhan MSC, ndi bat nhdt la md ma vi day Id loai md cd cdc dac diem thudn Ipi khi tien hanh thu nhan, nghien cuu va ung diing: thu nhat, viec thu nhan dan gidn, it xam lan nen it gay dau don cho benh nhan so vdi viec thu nhan tuy xuang. Thu hai, co chiia cdc td bdo CO tron thanh mach, te bao ndi md, nguyen bao spi ... va dac biet la cac MSC [12]. Cac MSC nay de dang phan lap, nudi cdy va tang sinh in vitro va cd the cdm ung biet hda thdnh cdc te bdo ciia cdc dong te bao khac [4,23]. Thii ba, md mo chua cac MSC voi ti 1? kha cao, moi gam md mo chua 10 MSC [17]. Thii tu, tidm ndng biet hoa ciia cdc MSC thu tir md mo it chiu anh hudng tudi cua ngudi hien mo [1,11].
Md ghep ndi rieng va vat lieu cay ghep ndi chung ngay nay dupe sir dung kha phd bien trong y hpc. Ddi vdi nhihig benh ly khuyet xuong viec dieu tri cd the su dung md ghep t\r than, md ghep ddng loai, mo ghep di loai, cac vat Heu cd ngudn goc polymer, gdm, thiiy tinh sinh hpc...Yeu cdu chung cua cac loai vat lieu tren la cdn co dp bSn co hoc cao vd ban chd ddn miic thap nhdt viec kich iing phdn ling miln dich cua co th6 (3,6,22). Ben
CHUygN Pg: HJNH THAI HQC vA C 6 H G N G H £ T H 6 N G TIN
canh dd, de cdi thi?n chdt luprng mdnh gh6p cdc nhd nghidn curu tgo ra cdc manh gh^p cd mang cac te bao xuong hay te bdo tien than tgo xucmg de tao ra mpt mo ghdp xuong vira cd cdc d$c tinh co hpc ciia md xuong vira mang cac te bdo de giup qud trinh tdj tgo vd sira ch&a md xuong dien ra nhanh chdng vd hi?uqudhon [2,7,16].
Mpt trong nhfl-ng loai vat li?u sinh hpc dupe ling dyng rat phd bien trong gh^p dieu tri benh ly tdn thuong xuong hi?n nay Id san hd ty nhien. San hd dupe sir dyng trong nghien ciiu ndy la Porites lutea, dupe sii dung nhieu trong y hpc (14,20,21). Ngodi ra, san hd cd cau tnic lo xdp phii hpp cho su bdm dinh vd phdt trien ciia te bdo, duac xem nhu la gia the ly tudng cho nudi cay te bdo (3,9,10,19).
Do dd de cd the thu nhan va iing dung ede MSC tir md ma mpt each hieu qua, chiing tdi tien hanh phan lap, nudi cdy vd dinh danh cac MSC thu tit md md ngudi. Tir do, tien hanh nghien eiru nudi cay cac MSC tir md md va biet hda thdnh nguyen bdo xuong tren gia the san hd nham tao ra mdt md ghep xuong vira mang cac tinh chdt co hpc va tinh chat sinh hpc de cd thd ghep didu tri trong nhung benh ly tdn thuang xuong khi khdng cd du ngudn md xuong cung cdp trong tuong lai.
II. odl TU<?NG VA PHUONG PHAP NGHIEN COU 2.1. Boi tufrng nghien ciiu
Md md ngudi duoc thu nhan tir viec ehpc hut md trong cdc quy trinh phdu thuat thdm my. Mau md dupe thu nhdn tir cdc ngudi hien khde manh, ddng y hiSn md dk tien hanh nghien ciiu va ed cae xet nghiem dm tinh vdi HIV, HBV, HCV va VDRL.
San hd ty nhien {Poriles lutea) dupe thu nhan tir vien Hai Duang Hpe Nha Trang
2.2. Phu-ffng phap nghi€n cihi Thi nghi?m dupe thuc hi?n la phucmg phdp thyc nghi?m md td.
2.2.1. Thu nhgn mo m&
Mau md dupe thu nhan trong dieu ki^n vd triing ciia phdng mo. Sau do, mau dugc van chuydn nhanh chdng ve phdng thi nghi?m.
2.2.2. PhSn l$p va nuoi c^ tSbao gSe tir moms'
Md md dupe eho vao cdc tube 50ml khodng 20-30ml djch md md eho moi tube.
Sau dd bd sung hon hpp enzyme Dispase- Collagenase vao cdc tube vdi ti I? (1 mau: 2 enzyme), mau md dupe u trong enzyme d nhiet dp 3/C, trong thdi gian 90 phiit. Sau d6, ly tam thu nhan phan cdn ldng d ddy chai. Bo sung mdi trudng nudi co ban vdo tube vd huy^
phil phan can lang d day chai. Lpc phdn dich te bao qua pheu lpc te bao (cell strainer, BD Falcon™) vdi dudng kinh 70 |aM.
Sau dd thu phan cdn ldng d day chai va bd sung dujig dich Iy gidi hdng cau de loai bo hdng cdu cdn lan tap trong dich te bdo. Sau dd, quay li tam thu phdn cdn ldng, bo sung mdi trudng nudi va dem mat dp te bao voi dung dich Trypan blue bang budng dfim Neubauer. Te bao dupe nudi trong chai nuoi T-25 cm^ (Coming) va ii trong tu nudi 6 nhietdp37Vvd5%C02.
2.2.3. Dinh danh te bdo goc trung md Theo td chiic The International Socie^
for Cellular Therapy dua ra cdc nguyen tic chung de xdc dinh mpt loai td bdo gde la MSC cdn phdi thda 3 dieu kien sau: thii nhit, cdc MSC phdi Id cac td bao cd khd nang bam dinh khi tidn hdnh nudi edy tren bd mat chai nudi. Thii hai, phai ed khd nang biet hoa thanh nguyen bdo xuang, td bao md hogc xk bdo sun trong didu kien in vitro. Thii ba, cac te bao nay phai bidu hien duoc ede marker bS
Y HOC VigT NAM T H A N G 10 - S 6 D A C BlgT/2013
m^t nhu CD 105, CD73 va CD90, khdng b\ku hiSn cdc marker nhu CD45, CD34, CDM vd HLA-DR. Dya theo cdc tieu chudn trSn, chdng tdi danh gid cdc te bdo bdo nudi cAy tir mo md la cac MSC.
Quan sdt te bdo dudi kinh hien vi ddo ngupc va nhupm te bdo bang thudc nhupm Giemsa dd quan sdt hinh thdi vd kha ndng bam dinh ciia te bdo.
Bk khdo sat ti§m ndng bi$t hda eiia te bao, tien hdnh thu n h ^ td bdo kk tir sau I ^ ciy chuyen thii hai. Cdc te bdo ndy sg dupe thu: nghifm de cdm iing bipt hda thdnh nguyen bao xuong vd te bdo md.
Quy trinh biet hda thanh nguyen bao xuong tii cdc te bdo gde md m a nhu sau: te bao sau Ian cay chuyen thii hai dupe chuyen len mpt chai nudi mdi va sau mdt ngay nudi cay dupe thay bdng mdi trudng cdm img tao nguyfin bdo xuong gdm DMEM/F12, 10%
FBS, 10'''M Dexamethasone, lOmM/ml p- plycerol phosphate, SOng/ml Ascorbic acid, ]10ng/ml FGF9, l O ' V vitamin D2 vd khdng pinh [5].
] Ket qua sau 14 ngay nudi cay se dupe jquan sat dudi kfnh hien vi ddo nguac de danh pi su thay ddi ve mat hinh thai. Tien hanh fihuom Alizarin Red (AR), Von Kossa (VK) ]de danh gid khd nang tao chat nen xuong va
|Alkaline phosphatase (AP) dd ddnh gid hogt Mi enzyme dde trung cho td bdo xuong, Woai ra, thuc hien phdn iing RT-PCR de -khao sat sy bieu hien gien Osteocalcin (la ttipt trong nhthig gien hien tai dupe sir dung
^ xac djnh cho td bdo xuong).
Quy trinh bi$t hda thdnh td bdo md ttr cac t6 bao goc md m a nhu sau: te bdo sau ldn cSy chuyen thii hai dupe chuyin len mpt chai nuoi mdi va sau mdt ngay nudi cdy dupe thay oang mfli trudng cdm ung tao te bdo md gdm 'DMEM/F12, FBS (10%), Gentamycine
(50ng/ml), HEPES {15 mm), NaHC03 (14nM), Biotin (33nm), D-Panto (17 ^m).
Human insulin (66 nM), Triiodo-L-thyronine (InM), Human transferrin (lO^g/ml) [18].
Ket qua sau 14 ngay nudi cdy se dugc quan sdt dudi kinh hien vi ddo ngupc de danh gid sy thay ddi vk mdt hinh thdi. Tidn hdnh nliupm Oil Red O dd ddnh gid sy tich tuu cdc gipt lipid ben trong te bdo vd nhupm hda md mien dich huynh quang Nile Red (Id mdt trong cac phuang phdp nhupm d^c hi$u hien tai dupe sii dung de xac djnh eho td bao md).
2.2.4. Tgo mo ghip diing di tdi tgo mo xwcrng tit san ho vd cdc MSC
Tach te bao bang dung djch Trypsin- EDTA (Gibco). Dich te bao sau khi tach cho vdo tube sach, vd triing. Bd sung 5ml mdi trudng nudi vd eho mau san hd vdo tube sao cho mat dp te bao khoang 10^ te bdo/ml.
Huyen phii vd quay ly tam d tdc dp 1000 vdng/ 1 phjit. Sau do, Igp lgi huyen phii dich td bdo vd quay Ii tam 1000 vdng/1 phiit eho den lan thii 5.
Lay mdu san hd ra va cho vao chai nudi, bd sung mdi trudng nudi (DMEM/F12, 10%
FBS, khdng sinh) sao cho ngap mdnh san hd.
U trong tu nudi d 37"C, 5% CO2.
Sau mpt ngay nudi edy, mdi trudng dupe thay bdng mdi trudng edm iing tao nguyen bdo xuong nhu tren. Thay mdi trudng mdi mdi 3 ngay/1 ldn.
2.2.5. Ddnh gid kha ndng bdm dinh, tdng trir&ng vd biet hda cua MSC tren gid the san ho
Td bdo sau khoang thdi gian nudi cay tir 1 4 - 2 1 ngdy, duoc ddnh gid khd nang bdm dinh, tang tmdng va biet hda thdnh nguyen bdo xuong tren gid the san hd.
Tidn hdnh nhupm Giemsa vd chup kinh hiin vi dien tir quet (SEM) d l danh gia kha nang bam dinh cua td bdo tren gid thd san hd.
CHUVeN DJ: HJNH THAI HpC VA C O N G NGHg T H 6 N G TIN
Nhupm md hpe H&H dl ddnh gia khd ndng tdng trudng vd phdt frien phdn bo trong khoi san hd.
Nhupm alkaline phosphatase dl ddnh gid khd ndng bi?t hda ciia MSC trdn gid thd san hd.
2.2.6. Phan l$p vd nudi ciy tSbdo gSc tir mdmd'
Cdc te bdo sau khi phan l$p tir md md bdt diiu xu^t hi?n vd bdm dinh sau hai ngdy nuoi cay trong mdi trudng nudi ca bdn. Cdc
tl bao bdm dinh rieng le, c6 hinh dgng then, dai giong vdi hinh thdi cua nguydn bdo spi.
Nhftng tl bdo ndy bam dinh tren ddy chai nudi. Sau mpt tudn nuoi cay, cdc te bao nay tdng trudng vd phdt trien mgnh, tgo thdnh cac cym (CPU) tren chai nudi. Sau 14 ngdy nuoi cay, cdc td bdo ndy dgt den mgt dp phu hpp de cay chuyen (chiim khodng 75-80% diSn tich be m^t chai nudi).
Hinh 1: Phan lap va nuoi cay te bdo goc tir mo m&. (A) Td bao bdt ddu tdng trudng sau 2 ngay nudi edy. (B) Te bao tdng trudng sau 7 ngdy nudi cdy. (C) Td bdo dupe nhudm vdi thude nhupm giemsa sau 10 ngay nudi cdy.
2.3. Dinh danh te bdo goc md md
2.3.]. Dira vdo khd ndng bi^t hda cua ti bdo gde tit md m&
Biet hda te bdo goc md m& thdnh nguyen bdo xtrcfng
Sau 14 ngay cdm iing biet hda td bdo bdng mdi trudng tgo xuong, tl bao bdt ddu thay hinh thai tir hinh dang thon ddi gidng voi cdc nguyen bao spi, cdc tl bdo bdt ddu xudt hien dang hinh thai da di^n vd chi tilt chdt nin xuong. Trong chai nudi xudt hien eac not xuong la dang ket tuu chdt nen xuong dgc trung trong in vitro. Sau 14 ngdy nudi cay, eac tl bdo nay bieu hien dupe protein cua chdt nen xuong la Osteocalcin cung nhu bilu hidn duac enzyme dac trung la AP.
(B) nhuom AP. (D). Nhuom AR. Tat ca nhupm d k duong tirf, voi thu6c. nhupm (vjt kinh
Y HQC VI6T NAM THANB 1 0 - 8 6 DjiC 8167)2013
XIO). (E) Th\tc hipn phan tag RT-PCR vdi gien Osteocalcin {Ichung miu trSng) b i k hi$n trong ca hai mau nghiSn cilu ( O S T l ; 0 S T 2 ) Idii so s4nh voi mau chimg (MSC).
Biet hoa te b4o gAc mo mS thinh tk bJo mO
Sau 14 ngay cam ilng bi?t h6a tS bao bang m6i trucmg tao ti bao m5, ti bao bat d4u thay hinh thai tir hinh dpig thon dii gi6ng v6i c4c nguyen b i o soi, cdc tS b i o bat diiu xuit hi^n dang hinh tron, ben trong b i o tirong bit diu xuat hi?n cic hat lipid nho. Sau d6, bSn trong bao tuong ti bao cac hat lipid tiep tyc k^t t\ru vio nhau de tao thinh cic hat lipid 16n hon v i chijm gjn h8t bao tuong te bao, nhan te bao bj diy ra phia ngoai vi. Tiin hinh nhu6m Oil Red 0, cac ti b i o duang tinh xuat hi$n v6i ben trong chiia rit nhigu hjt lipid miu d6 v i nho6m Nile Red cho thiy cic h?it lipid hinh thinh ben trong b i o tucmg cua te bio rit nhiiu khi so sanh voi te b i o doi chting khong cim img bi?t hoa tao t^ bao md (hinh 3).
:^;-,.^
'» \ . f ' *t It
».,.^A. . , ,B'^^ • ' . ^ „ ^ , , - - C
Hinh 3: Biet hda te bdo gde md md thdnh t l bdo md. (A). Te bdo ddi chiing dupe nhupm Giemsa. (B). Td bao nhupm Oil Red sau 7 ngdy nudi trong mdi trudng biet hda, td bdo thay d6i hinh dgng va bao tuong bdt dau tich tru lipid nhupm mdu dd vdi thudc nhupm. (C).
Nhupm Nile Red cho t l bdo md sau khi bi^t hda, kdt qua cho thay bdo tuong eua te bao chiia rat nhieu hgt lipid bdt mau do cam, cdc hgt lipid ndy chiim hdu het dien tich te bdo, nhan t l bao hi ddy lech ve mdt phia cira td bdo.
2.3.2. Dira vdo khd ndng bieu hi^n cdc marker be mat
Te bdo sau Idn cdy chuydn thii hai, sg dupe thu nhgn bdng enzyme Trypsin-EDTA (0.25%
- 0.02%). Sau dd, t l bdo sd dupe thu nhdn bdng each quay ly tdm d tde dp 3000 vdng/phiit trong 5 phiit. Td bdo se dupe nia 2 ldn vdi dung djch PBS d l tinh sach vd d l tdeh t l bao thdnh cac te bao don nhdm phuc vy eho ddnh gid t l bao ky thugt Flow Cytometry (dem te bao ddng chay). Ket qud cho thdy, qudn thi t l bdo khao sat bidu hi?n mot so marker gdm CD105, CD73, CD90, CD45, CD34, C D M vd HLA-DR. Sy bilu hi?n cua cdc marker dupe thue hi?n ttong hinh 4.
CHUYEN BJ: HiNH THAI HgC VA C 6 N 0 NGHC T H 6 N 0 TIN
.M$^ lA .
ii'
a.
U ,».
R.
= 0 ' M l
10'
1
..^^Hi
C073PE 97.7(m
.03 10^
Hinh 4: Ket qua phdn tich marker cho quan the te bao goc tir md mo'. Ket qua phan tich cho thay, quan the te bao am tinh hoan toan vdi cdc marker CD45 va HLA-DR (bilu hien ducmg tinh khdng qud 2%). Te bao ducmg tinh hoan toan vdi cac marker CD44, CD73, CD90 va CD105 (bieu hi?n duong tinh tren 95%).
Tgo md ghep tie MSC vd gid the san ho
Mdu san hd cd mang ede MSC sau khi biet hda thanh nguyen bdo xuong dupe xir ly trong dung dich cd dinh (Neutral Buffer Formalin 10%) vd nhupm Giemsa va H&E.
Chung tdi tien hanh nhupm khdi san hd vdi thudc nhupm Fast Red violet LB salt (sigma), neu cdc MSC biet hda dupe thdnh eac nguydn bdo xuong thi cac t l bdo nay se phdn img Iai vdi thude nhupm vd trong bdo tuong xuat hi?n mau hdng, do s u hoat ddng eiia enzyme alkaline phosphatase. Day Id mpt trong nhung enzyme dupe su dyng phd b i i n de dinh danh cho cdc nguydn bdo xuong.
Mau san hd dupe ed dinh va lam khd (khu nuoc) theo timg giai doan bdng ethanol. Sau do, se dupe chup dnh dudi kinh hiin vi di?n tir quet.
Y Hpc VI$T NAM T H A N G 10 - SO PAC BI$T/2013
Hinh 6: Danh gia khd ndng bam dinh, phat triln va biet hoa cua cac MSC tren gia thi san ho, (A).
Mau san ho dupe dupe chyp SEM d kich thuac 200fim, khong nudi te bao. (B) Mau san hd dugc dupe chyp SEM a kich thudc lOOum co mang cac MSC da biet hoa thanh nguyen bao xuong, ket qua cho thSy cic te bao bam dinh va phat trien tren be mat khoi san ho, cac MSC tao idp va phat trien ben trong cung nhu phia ngoai cac hdc san hd. (C). Mau san hd dupe nhupm H&E, cho thdy tl bao phat triln phan bl b^n trong khoi san hd. (D). Mau san ho nhugm AP, cac t l bao duong tinh vai thuoc nhupm chiing to bilu hi?n dupe en zyme AP. Dieu nay cho thdy cac MSC da biet hoa dupe thanh nguyen bao xuong tren gia the san ho.
IV. KET l\)fyN
Voi quin the te bdo gde thu nhan tir md md da phdn lap va nudi cdy, chiing tdi dd I danh gid cdc chi tieu d l xac djnh cho MSC, ket qua cho thdy cdc te bdo nudi cay deu la c^ t6 bdo bam dinh tren chai nudi, cd hinh dang thon dai ddc trung eho t l bdo gde trung mo. Qudn the t l bdo nay ed khd nang biet hda dupe thanh 2 ddng td bdo khac nhau thudc ngu6n goc tmng md dd la nguyen bao xuang va te bao md vdi phuong phdp xde dinh ddng tinc^y. Thii ba, chiing tdi dd khdo sdt su bilu fii^n cua cdc marker dung d l xdc dinh cho quan the MSC tir md md, kit qua cho thdy t l bao duong tinh khd cao (trdn 95%) vdi cdc
marker dupe CD73, CD90 vd CD105. Ddy Id ba marker phd bien duoc chap thuan de xdc dinh eho ede MSC. Cdc t l bdo nay bieu hien rdt thdp dudi 2% ede marker CD45 vd HLA-DR.
Cdc MSC thu tir md md cd the tien hdnh phdn lap vd nudi cay tren gid the san hd.
Chiing cd khd ndng bdm dinh, tang trudng rat nhanh va biet hda thanh nguyen bdo xuong trong dieu kien nudi cdy in vitro. Ket qud ndy dem lgi mdt hudng iing dung mdi sir dung san hd ghep thay xuang cd bd sung te bao nudi cdy, day la mdt trong nhimg hudng iing dung cua cdng nghe md vd vdt lieu y sinh hpc trong tuong lai.
CHUVeN B^! HiNH THAI HQC VA CflNG NGHj THflNG TIN
Nhu v4y, day li ngu^n t^ bio g6c rit quan trpng vi tilm ning cho cic umg dyng lim sing. Tuy nhiSn, de c6 th^ img dyng cic te bio niy mOt cich an toin vi hi^u qui, cin thg-c hi$n th£m cic thi nghidm khic ii khio sit s\r an toin ve m$t di truydn cOng nhu ti^m ning bi?t h6a ciia te bio theo th6i gian nu6i ciy vi s6 lin nhin d6i th^ h?, di tit d6 c6 thi sir d\mg cic ti bio niy trong cic diiu trj g6p phin nang cao chit lu^g diiu trj cho ngu6i b$nh.
TAI LtlU THAM K H A O
1. Brian M Strem et al (2005). Multipotential differentiation of adipose tissue-derived stem cells. The Keio journal of medicine. 54:132- 141.
2. Carl A. Gregory (2008), Mesenchymal stem cells: from culture to clinic, Stem Cell Repair and Regeneration, Imperial College Press, Singapore, 2:21-45.
3. Chun Y, TM Lee, KH Chiu, SY Shaw, CY Yang. (2009). A comparative study of the physical and mechanical properties of three natural corals based on the criteria for bone- tissue engineering scaffold. J. Mater. Sci.
Mater Med, 20:1273-1280.
4. Coleman SR (2006). Structural fat grafting:
more than a permanent filler. Plasl Reconsir Surg. 118:I08S-I20S.
5. Cong Toai Tran, Ciro Gargiulo, Huynh Duy Thao, Huynh Minh Tuan, Luis Filgueira and D. Michael Strong (2011).
Culture and differentiation of osteoblasts on coral scafford from human bone marrow mesenchymal stem cells. Cell and Tissue Banking. 12:247-261.
6. Davide Zafre.(2005). Some considerations on biomaterials and bone. Micron, 36:583-592.
•7. F Chen et al (2007), Segmental bone tissue engineering by seeding osteoblast precursor cells into titaniummesh-coral composites
scaffolds. Journal of Oral and Maxillofacial Surgery, 36: 822-827.
8. F Chen, S Chen, K Tao, X Feng, Y Lin, D Lei, T Mao.(2004). Marrow derived osteoblasts seeded into porous natural coral to prefabricate a vascularized hone graft in the shape of human mandibular ramus:
experimental study in rabbits. Joumal of Oral and Maxillofacial Surgery, 42:532-537.
9. F. Chen, X. Feng, W. W u 4 I . Ouyang, Z, Gao, X. Cheng, R. Hou, T. Mao. (2007).
Segmental bone tissue engineering by seeding osteoblast precursor cells into titaniummesh-coral composites cafTolds.
lnt.J.OralMaxillofac.Surg, 36:822-827.
10. Fulin Chen, Tianqiu Mao, Kai Tao, Shujun Chen, Guicong Ding, XiaoDiing.(2002). Bone graft in the shape of Human mandibular condyle reconstruction via seeding marrow-derived osteoblast into porous coral in a nude mice model. J Oral Maxillofac Surg, 60:1155-1159.
11. J o et al (2009). Comparison neural cell differentiation of human adipose mesenchymal stem cells derived from young and old age. Dev Repord. 13:227-237.
12. Kalz AJ, Mesenchymal cell culture: Adipose tissue. In: Atala A, Lanza R (2002) Method of Tissue Engineering. Academic Press San Diego.
13. Michelle Locke, John Windsor, P. Rod Dunbar (2009), Human adipose-derived stem cell: isolation, characterization and application in surgrey, ANZ Surgrey Joumal, 79:235-244.
14. Nguyin Tri Dung, Trinh Dinh Vn Linh.(2004). Khao sit diln hien linh xircmg 6 vit thucmg thirc nghiem nho a xucmg ham dudi CO dimg san ho Porites lutea. Tap Chi Y hoc TP.HCM, tap 8, so 1, 28-32.
Y HQC VigT HAM THANG 10 - SO 0^0 Bl|Tf2013
15.Pittenger MF, Mackay AM, Beck SC et al (1999). Multilineage potential of adult buman mesenchymal stem cell. Science; 284:143- 147.
16. Ralph E. Holmes (1979), Bone regeneration within a coralline hydroxyapatite implant, Plastic and Reconstructive Surgery, 63 :626- 633.
17. Sen A et al (2001). Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogenous. J Cell biochem. Z\:3\2-3\9.
18. Tran Cong Toai, Huynh Duy Thao, Nguyen Phuong Thao, Ciro Gargiulo and Phan Kim Ngoc, et al (2010). In vitro culture and differentiation of osteoblasts from human umbilical cord blood. Cell and
• Tissue Banking. \\:269-2SQ.
19. Trk Cong To^i, Nhan Nggc Hien.(2008).
Xay dung quy trinh nu6i dy nguyen bao s(?i tren gia the san ho. T^ip chi Y hpc TP.HCM, Tap 12, phy ban s6 1,31-42.
20. Tran Giao H6a, Do Thu Hkg.(2002).
Budc dau danh gia ket qua lam s^ng vi§c sit dyng che pham san ho Viet Nam dk dieu tt-i sang thuong trong xuang. Tuy^n t§p cong trinh nghlSn curu khoa hpc Rang Ham Mat, D^i hpc Y Dupe TP.HCM, 131-138.
21. VQ Ng9c Huy, NguySn Danh, Trkn BSc Hai.(1999). Khdo sit qua trinh lien xuong qua manh ghep san h6 Porites lutea d dau dudi xuong diii tho. Hinh thai hpc, tSp 9, s6 2, trang 80-89.
22. Waese EY, R Kandel.(2007). Application of stem cells in bone repair. Skeletal Radiology, 37:7, 601-608.
23. Welter TP, Von Heimburg D, Stoffels I et al (2005). Cryopreservation of mature human adipocytes: In vitro measurement of viability, Ann Plasl Surg. 55:408-413.
24. Znk P.A, Zhu M, Ashjian P. et al (2002), Human adipose tissue is a source of multipotent stem cells. Molecular Biological Cell, 13:4279-4295.
