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

Y HOC VIET NAM THANG 8 - SO 2/2010

dudng huyet a chudt" Hpi nghi KHCN toan
quoc, Thai Nguyen, NXB DH Thai Nguyen,
tr.877-878.
3. Nguyen Van Mui (2002), Xdc dinh hoat do
TAI LIEU THAM KHAO
1. Vo Van Chi (1999), Tie diin cay thudc Viet enzym, NXB Khoa hpc va ky thuat, Ha Npi
4. Courtney C.H., Jerrold M.O. (2007), Insulin
Nam, NXB Y hpc, Ha Npi.
Resistance, Mechanisms of Insulin Actions,
2. Trinh Thj Thu, Le Thj Hong Nhung, Nguyen
Landes Bioscience and Springer Science +
Van Mui, Phan VanChi (2009), "Tao tieu
duang typ 2 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 trong 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 thanh 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
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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
trong co the truong thanh, co kha nang biet hoa dupe loai bd sau 7 - 9 ngay. Thay mdi trudng 2
tao nhieu dong te bao khac nhau, thupc trung bi lan / tuan. Cay chuyen khi 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 trong y hoc tai tao cua nhieu loai benh bang mdi trudng nudi cay, li tam lay can te bao
li rieng biet, trong do co benh co tim do 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 trong 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, khi chung di chuyen trong
nguyen be mat duy 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 tuy xuong ngudi
phan chinh: He thdng ddng chay, he thdng quang
hpc va he thdng dien tii'.
Tien hanh: Mdi dng do 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, trong 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 2 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 khi
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 trong 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
2

144

Y HOC VIET NAM THANG 8 - SO 2/2010
paraphin la 5 p.m. Loai bo paraphin. Bdc lp
khang nguyen bang dem pH= 6,0 va nhiet dp ciia
hoi noi ap suat trong 10 phiit. Rira bang PBS.
Khu H 0 npi sinh blng H 0 3%. Rua PBS x 3
lan. U khang the thii nhat (pha loang 1/400) tai
4°C qua dem. Rua PBS 5 phut/Ian x 3 lin. 0
khang the thir hai x 60 phiit, trong buong giir am,
2

2

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
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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
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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,
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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.

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//?«/» 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 trong ciy ghep te bao gdc mau, cac
cells) hay te bao dem tuy (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 thanh 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 trong
nhau 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 khi len tdi 10 te bio [2] la can
nam gan day, MSC la loai te bao goc rat duoc thiet trong 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 trong cic md trudng thanh. Trong tiiy xuong,
Hien nay 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 trong 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 trong ITnh vuc
ciru Idn, trong do 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 nay ngudi ta vin chua xac dupe marker be mat
MSC tip trung vao nhieu loai benh li cd tinh duy nhit cho loai te bao gdc nay. Dieu niy giy
9

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Y HOC VIET NAM THANG 8 - SO 2/2010
kho khan cho djnh danh MSC, nhit la do 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
nhau va dugc nghien cuu d nhieu phong nghien mau (CD34 va CD45) tren be mat te bao nhu da
ciiu khac nhau 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 khi cac nha khoa hgc chua tim ra
trong dieu kien nudi cay tieu chuan;
dugc marker duong tinh dac hieu cho MSC nhu
- MSC bieu hien duong tinh vdi CD 105, hien nay 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 thanh cac te baoqui trinh phan lap va nudi cay MSC tir tiiy xuong
tao xuong, tao sun, tao md trong dieu kien in vitro. ngudi. Viec chgn lira kT thuat xac dinh marker be
6 Viet Nam, mdt sd tac tac gia da tien hanh mat cung can duoc dat ra. Phan ling mien dich
nghien ciiu phan lap MSC tir cac ngudn khac dac hieu, sir dung cac khang the don ddng cd gan
nhau nhu tiiy xuong, mau cudng rdn, thanh 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 do FASC duoc sir dung de danh gia cac marker
dieu kien trang thiet bi. De tai nghien ciiu nay 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 (<5%) cac marker thudc te thuan nhat ve bieu hien mpt so khang nguyen be
bao gdc tao mau (CD34) va cac ddng te bao mau mat cu the nao do.
trudng thanh hon (CD45) tren ca hai kT thuat IHC
IHC (Immunohistochemistry), tuy khong
va FACS ngay tir cac giai doan nudi cay ban phai Ia mpt kT thuat duoc ua chudng khi nghien
dau, da chiing td qui trinh phan lap - nudi cay da cuu marker be mat te bao nudi cay, nhung do cd
loai bd duoc su cd mat cd mat ciia ddng te bao Igi the la khdng ddi hoi cao ve thiet bi, va trong
mau trong tap hgp MSC. Cac marker CD13, tung trudng hop cu the cd the thuc hien d nhieu

CD73, CD90 la nhung khang nguyen be mat ciia phdng thi nghiem. Cac mau te bao diroc cd dinh,
nhieu loai te bao mau d cac mire dd biet hda khac bao quan trong paraphin, va do vay duoc luu mau
147


CHUYEN DE : HOI NGHI KHOA HOC CUA H0I HOA SINH Y DUOC HA N0I VA CAC TiNH PHlA BAC I_AN TH

trong cac truong hop can thiet, san sang cho kT
Cells: The Past, the Present, the Future",
Cartilage, 1(1), pp. 6-9.
thuat nhuom mien dich d bat cu thai diem nao.
Day cung la su khac biet co tinh uu viet cua IHC, 2. Chen S.L., Fang W.W., Ye F. et al (2004),
"Effect on left ventricular function of
nhung ciing phai luu y la kT thuat nay ton thai
intracoronary transplantation of autologous
gian va doi hoi kT thuat vien phai co tay nghe
bone marrow mesenchymal stem cell in
thuan thuc khi tien hanh co dinh va vui mau trong
patients with acute myocardial infarction". Am
paraphin. Ket qua phan tich bieu hien CD45 ciia
J
Cardiol, 94, pp. 92-95.
te bao nuoi cay bang hai kT thuat FASC va IHC
cho thay a PI, chi co < 5% te bao nuoi cay co 3. Dominici M., Le Blanc K., Mueller L, SlaperCortenbach L, Marini F., Krause D., Deans R,
bieu hien duong tinh. Ket qua nay the hien sir
Keating A., Prockop D., Horwitz E. (2006),
tuang dong giua hai kT thuat va kha nang sir dung
"Minimal criteria for defining multipotent
IHC trong xac dinh khang nguyen be mat cua te
mesenchymal stromal cells", The International

bao nuoi cay.
Society for Cellular Therapy positionstatement,
Cytotherapy; 8, pp. 315-317.
V. KET LUAN
- MSC nuoi cay duoc phan lap tir tiiy xuong 4. Pittenger M.F., Martin B.J, (2004),
"Mesenchymal stem cells and their potential as
ngudi cd bieu hien kieu hinh khang nguyen be
cardiac
therapeutics", Cirs. Res, 95; 9 - 20.
mat ciia te bao gdc trung md: duong tinh vdi
5.
Shim
W.S.,
Jiang S., Wong P., Tan J., Chua
CD 13, CD90 va CD73, ddng thdi am tinh vdi
Y.L., Tan Y.S., Sin Y.K., Lim C.H., Chua T., The
CD34 va CD45.
M, Liu T.C., Sim E. (2004), "Ex vivo
differentiation of human adult bone marrow stem
TAI LIEU THAM KHAO
cells into cardiomyocyte-like cells", Bioch Biophys
1. Caplan A.I. (2010), "Mesenchymal Stem
Res Commun, 324, pp. 48M88.

NGHIEN CUU TAO KHANG THE DON CHUOI SCFV BIEU L0 TREN THU/C KHUAN
THE m i 3 DAC HIEU KHANG NGUYEN UNG THU*TIEN LIET TUYEN
Le Quang Huan*, La Thj Huyen*

TOM TAT
ung thu tien Net tuyen. Ket qua nghien dhi: Khang

Muc tieu: Tao khang the ddn chuoi scFv bieu 16 the ddn chuoi scFv bieu Id tren thirc khua'n the gom
tren third khua'n the cd kha nang nhan biet va gan ket 170 axit amin dUdc bieu Id tren thUc khuan the va
dac hieu vdi khang nguyen bieu hien sdm trong ung budc dau xac dinh kha nang nhan biet va gan ket vdi
thU tien Met tuyen lam nguyen lieu cho cac nghien ciiu cac khang nguyen bieu hien sdm trong huyet thanh
tao KIT chan doan va thuoc dieu tri hudng dich. benh nhan ung thu tien liet tuyen.
Tit khoar. EPCA, Khang the don chuoi, Phage
PhUdng phap nghien curu: Sir dung cac ky thuat
gen va ky thuat phage display de tao khang the ddn display, scFv, Ung thUtien liet tuyen.
chuoi scFv dac hieu khang nguyen bieu hien sdm trong
* I 'i$n Cong nghi sinh hoc
148