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 Khang nguyen b
Trang 1Y HOC VIET NAM THANG 8 - SO 2/2010 cong nghe protein-enzym, DH Khoa hpc Tu
nhien, DH Quoc gia Ha Npi
TAI LIEU THAM KHAO
1 Vo Van Chi (1999), Tie diin cay thudc Viet
Nam, NXB Y hpc, Ha Npi
2 Trinh Thj Thu, Le Thj Hong Nhung, Nguyen
Van Mui, Phan VanChi (2009), "Tao tieu
duang typ 2 a chudt de nghien cim hoat tinh ha
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 enzym, NXB Khoa hpc va ky thuat, Ha Npi
4 Courtney C.H., Jerrold M.O (2007), Insulin Resistance, Mechanisms of Insulin Actions,
Landes Bioscience and Springer Science + Busuness Media
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
Khang nguyen be mat te bao la mpt trong cac
tieu chuan xac dinh te bao goc trung mo
(Mesenchymal stem cells, MSC), bao gom marker
dUdng tinh va marker am tinh Xac djnh cac marker
cung la nham chu'ng minh sU thanh cong ciia qui trinh
phan lap, nuoi cay MSC tir tiiy xUOng ngUdi Muc
tieu: Xac dinh cac marker CD13, CD73, CD90 va
CD34, CD45 tren be mat MSC nudi cay dupe phan lap
tii tiiy xUdng ngUdi PhUOng phap nghien cu"u: Sir
dung khang the ddn ddng xac dinh cac marker be mat
te bao nudi cay bang phUdng phap FACS va IHC Ket
qua: MSC nudi cay d giai doan cay chuyen PI dUOng
tinh vdi CD 13, CD73, CD90, va hau nhu" am tinh vdi
CD 34 va CD45 Ket luan: MSC nudi cay dupe phan
lap tii tiiy xUdng ngUdi bieu hien day dii va chinh xac
kieu hinh khang nguyen be mat dac trUng cho te bao
goc trung md
Tir khoa: Te bao goc trung md (MSC),
marker be mat MSC, FACS, IHC
SUMMARY
EXPRESSION OF CELL - SURFACE MARKERS ON
* Trudng Dai hoc Y Hd Ndi
CULTURED MESENCHYMAL STEM CELLS FROM
HUMAN BONE MARROW Cell -surface antigens are one of minimal criteria
to define mesenchymal stem cells (MSC), including both positive and negative markers Defining the expression of markers to show the successful of isolation and culture methods of MSC from human bone' marrow is necessary Objective: Defining markers: CD13, CD73, CD90 and CD34, CD45 on cell -cultured surface after isolation and culture of MSC from human bone marrow Method: Cultured MSC at
PI were stained with the monoclonal antibodies thereafter analyzed with FACS and IHC Result: Cultured -MSC from human bone marrow at PI express CD 13, CD73, CD90, and lack expression of CD
34, CD45 Concludes: : Cultured-MSC from human bone marrow at PI express phenotypic characterization of Mesenchymal stem cells to cell-surface antigens
Key word: Mesenchymal stem cells (MSCs),
cell-surface markers of MSCs, FACS, IHC
Te bao goc trung md (Mesenchymal stem
Trang 2cells, MSC) co the dugc phan lap tir nhieu mo
trong co the truong thanh, co kha nang biet hoa
tao nhieu dong te bao khac nhau, thupc trung bi
(xuong, sun, mo, co, co tim), npi bi (gan, tuy),
ngoai bi (da, than kinh) [1], MSC da va dang la
ung cu vien sang gia cho cac lieu phap te bao
ung dung trong y hoc tai tao cua nhieu loai benh
li rieng biet, trong do co benh co tim do thieu
mau Tiiy xuong la mpt nguon phan lap va nuoi
cay MSC co hieu suat cao Cac tieu chuan de xac
dinh chinh xac chat luong MSC sau phan lap va
nuoi cay bao gom dac diem hinh thai va phat
trien cua MSC trong moi truong nuoi cay, kha
nang da biet hoa in vitro va cac khang nguyen be
mat duong tinh va am tinh Hien chua co khang
nguyen be mat duy nhat duoc sir dung de xac
dinh MSC
Muc tieu cua nghien ciru:
- Xdc djnh su- bieu hien cua cdc marker CD
13, CD73, CD90 vd CD34, CD4S tren be mat MSC
nudi cay duac phan lap tie tuy xuong ngudi
II 06l TUONG VA PHUONG PHAP NGHIEN CUXJ
2.1 Doi tirong nghien ciru:
- MSC dupe phan lap, nuoi cay theo qui
trinh phan lap - nuoi cay MSC tiiy xuong ngudi,
tai giai doan cay chuyen thir nhat (Pl).( n=3)
2.2 Hoa chat, thiet bi:
Khang the khang: CD34 PE, CD45
ECD, CD 13 PE (Beckman Coulter), CD73
-PE, CD90 - FITC (B&D), CD 45 (Dako)
- Mouse IgGl PE Mouse IgGl FITC (B&D)
- Trypsin EDTA 0,25% (Invitrogen)
- PBS (Phosphat buffer salin) (Invitrogen)
- Thiet bj dem dong chay te bao (Flow
cytometer) Beckman Coulter F500
- Kinh hien vi quang hpc
2.3 Phuong phap nghien ciru:
- Chuan bi mdu phdn tich:
MSC dupe phan lap tu tiiy xuong theo qui
trinh ciia Shim W.S.N va cdng sir [5] Cac te bao
don nhan tiiy xuong dupe nudi cay trong mdi
trudng DMEM - LG, 10% FBS, 1% khang sinh
Cac te bao khdng cd kha nang bam nhua nuoi cay dupe loai bd sau 7-9 ngay Thay mdi trudng 2 lan / tuan Cay chuyen khi mat dp te bao dat 80% chai nudi dien tich 25 cm2 Tai lan cay chuyen thir nhat (PI), tach te bao bam dinh chai nuoi bang Trypsin - EDTA 0,25%, bat hoat enzym bang mdi trudng nudi cay, li tam lay can te bao
2000 g x 15 phut d nhiet dp phdng, rua lai bang PBS Cho them 500 u.1 PBS tao djch huyen phii te bao
- Ki thuat FACS (Fluorescene activated cell sorting):
Nguyen li: Dem te bao ddng chay (Flow
cytometry) la kT thuat dem va phan tich dong thai cac dac tinh vat li ciia nhiing phan tir rieng le, thuong la cac te bao, khi chung di chuyen trong mpt ddng dich qua mpt chiim tia lase, bao gom kich thudc ciia phan tir, xac dinh muc dp cac phirc hop va cac hat ben trong, cac dac tinh phat huynh quang
Mdt flow cytometer dupe cau tao bdi ba phan chinh: He thdng ddng chay, he thdng quang hpc va he thdng dien tii'
Tien hanh: Mdi dng do cd khoang 500.000
te bao, dupe u vdi 100 p.1 khang the don ddng chupt khang ngudi, ndng dp 50u.g/ml Co the ii rieng biet tirng loai khang the hoac ii vdi ddng thdi hai khang the neu chiing dupe gan vdi cac chat phat huynh quang khac nhau, vi du khang the khang CD34 PE vdi khang the khang CD 90 FITC, khang the khang CD 45 ECD vdi khang the khang CD73 PE, trong 30 phut, d nhiet dp phdng Mau chirng am la mau te bao ii vdi khang the khang chupt cd gan huynh quang tuong ling
Phan tich tren Beckman Coulter F500, sir dung phan mem chuyen dung su li ket qua, dua tren sir so sanh vdi ket qua te bao chiing am (Moi mau dupe xac djnh 2 lan)
Ki thuat hda md mien dich (Immunohistochemistry - IHC):
Tien hanh: Li tam lay can te bao sau khi
tach khdi chai nudi cay bang trypsin-EDTA 0,25% Co dinh bang paraformaldehyd 4%/PBS Due paraphin can te bao DP day lat cat mlu due
Trang 3Y 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 H202 npi sinh blng H202 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,
nhiet dp phong Rira lai bang dem U Streptavidin- HRP x 60 phiit Rua PBS Nhupm 3,3'-Diaminobenzidine (DAB) Nhupm 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)
Ti le dUOng tinh (%)
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 Al»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,
Trang 4Nhan 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%
IV BAN LUAN
Te bao goc trung mo (Mesenchymal stem
cells) hay te bao dem tuy (Marrow stromal cells)
(viet tat MSC) la te bao goc da nang, dupe phat
hien dau tien d tiiy xuong, nhung cung co the
dupe phan lap tu cac mo truong thanh khac
Chiing co kha nang biet hoa tao loai te bao khac
nhau nhu xuong, day chang, co' tim, co xuong,
than kinh, gan, mo, to chiic lien ket [1], Trong 10
nam gan day, MSC la loai te bao goc rat duoc
quan tam nghien ciiu, bao gom nghien ciiu co
ban in vitro, in vivo va cac thii nghiem lam sang
Hien nay nhirng hieu biet kha day dii ve MSC co
dupe tir cac nghien ciiu ve dac diem kieu hinh,
dac diem mien dich, kha nang biet hoa ngoai co
the cung nhu co che tac dung trong lieu phap ciy
ghep te bao v.v Tuy nhien, nghien ciiu co ban
va ling dung MSC d Viet Nam dang d giai doan bat
dau va mdi chi tip trung d mdt sd trung tam nghien
ciru Idn, trong do cd trudng Dai hpc Y Ha Npi
Cac thir nghiem lam sang lien quan den
MSC tip trung vao nhieu loai benh li cd tinh
rieng biet, nhu MSC tham gia vao qua trinh hd trp tao mau trong ciy ghep te bao gdc mau, cac benh li ve chuyen hda tao xuong, khuyet xuong, teo co' xo ciing (amyotrophic lateral sclerosis) va dac biet la nhoi mau co tim [2] Tinh an toan, kha thi va hieu qui ciia viec ciy ghep MSC ddi vdi nhieu trudng hpp benh li dupe thirc hien trong cac khio nghiem nghiem ngat Mpt lupng kha Idn MSC, ddi khi len tdi 109 te bio [2] la can thiet trong cac thir nghiem lam sing Tuy nhien, MSC la loai te bio gdc mi sir hien dien la rat han che trong cic md trudng thanh Trong tiiy xuong, MSC chi chiem 0,001% den 0,01% tong sd te bio cd nhin, bang 1/10 te bao gdc tao mau [4]
Do vay, viec phan lip - nhin nudi MSC la kT thuat ddng vai trd co bin, quan trpng khdng chi cho cic nghien ciru co bin ma cdn ddi vdi cac thii nghiem lim sang MSC
Tuy da cd nhirng nd lyre trong ITnh vuc nghien ciiu dac tinh mien dich ciia MSC, cho den nay ngudi ta vin chua xac dupe marker be mat duy nhit cho loai te bao gdc nay Dieu niy giy
Trang 5Y HOC VIET NAM THANG 8 - SO 2/2010 kho khan cho djnh danh MSC, nhit la do MSC
dugc cho la co the phan lap tu nhieu nguon khac
nhau va dugc nghien cuu d nhieu phong nghien
ciiu khac nhau tren the gidi Hiep hdi qudc te ve
lieu phap te bao (International Society for
Cellular Therapy, ISCT) da qui dinh nhung tieu
chuan tdi thieu nham xac djnh MSC [3], theo do:
- MSC phai bam dinh vao nhua nudi cay
trong dieu kien nudi cay tieu chuan;
- MSC bieu hien duong tinh vdi CD 105,
CD73 va CD90; Bieu hien am tinh (tuy thudc vao
md phan lap) vdi CD45, CD34, CD 14, hoac
CDllb, CD79 (ddng tuy), hoac CD19 va
HLA-DR (ddng lympho);
- MSC cd kha nang biet hda tao thanh cac te bao
tao xuong, tao sun, tao md trong dieu kien in vitro
6 Viet Nam, mdt sd tac tac gia da tien hanh
nghien ciiu phan lap MSC tir cac ngudn khac
nhau nhu tiiy xuong, mau cudng rdn, thanh
cudng rdn Tuy nhien, chua cd cdng bd de cap
den viec djnh danh MSC bang cac marker be mat
mdt each day dii Viec lua chgn cac marker am
tinh - duong tinh va ki thuat xac dinh de chirng
minh tap hop MSC sau qua trinh phan lap va
nudi cay, tiiy thudc vao ngudn phan lap cung nhu
dieu kien trang thiet bi De tai nghien ciiu nay sir
dung hai marker am tinh CD34 va CD45 dac
trung cho te bao gdc tao mau va cac te bao mau
trudng thanh, ba marker duong tinh ddi vdi
MSC, bao gdm hai marker duoc dua vao tieu
chuin toi thieu (CD90 va CD73) va CD13 la mdt
marker duoc nhieu nghien ciiu chiing minh la
bieu hien duong tinh tren be mat MSC [4] Ket
qua cac MSC nudi cay khdng bieu hien hoac bieu
hien vdi ti le rat thap (<5%) cac marker thudc te
bao gdc tao mau (CD34) va cac ddng te bao mau
trudng thanh hon (CD45) tren ca hai kT thuat IHC
va FACS ngay tir cac giai doan nudi cay ban
dau, da chiing td qui trinh phan lap - nudi cay da
loai bd duoc su cd mat cd mat ciia ddng te bao
mau trong tap hgp MSC Cac marker CD13,
CD73, CD90 la nhung khang nguyen be mat ciia
nhieu loai te bao mau d cac mire dd biet hda khac
nhau, nhung sir bieu hien ciia cluing ket hop vdi sir vang mat cac khang nguyen thudc ddng te bao mau (CD34 va CD45) tren be mat te bao nhu da trinh bay d tren, la kieu hinh khang nguyen be mat dac trung cho MSC Tuy nhien d lan cay chuyen thii nhat, miic dd bieu hien ciia chiing chua cao mdt each tuyet ddi (>95%)
Trong khi cac nha khoa hgc chua tim ra dugc marker duong tinh dac hieu cho MSC nhu hien nay thi viec chimg minh cac te bao nudi cay
am tinh vdi marker ddng te bao mau va duong tinh vdi cac marker duong tinh dac trung ciia MSC cd y nghTa gdp phan khang dinh tap hop MSC thu duoc sau tirng qui trinh cu the, d day la qui trinh phan lap va nudi cay MSC tir tiiy xuong ngudi Viec chgn lira kT thuat xac dinh marker be mat cung can duoc dat ra Phan ling mien dich dac hieu, sir dung cac khang the don ddng cd gan cac chat phat hien huynh quang hoac mang man,
de phat hien cac dac tinh khang nguyen tren be mat te bao la mpt sir lira chon cd tinh kha thi va hieu qua cao Nghien cuu sir dung hai phuong phap FACS va IHC de danh gia sir bieu hien cac khang nguyen be mat tren te bao nuoi cay Trong
do FASC duoc sir dung de danh gia cac marker duong tinh va am tinh, IHC dugc sir dung xac dinh bieu hien marker am tinh (CD45)
KT thuat ddng chay te bao hay FASC (Fluorescence activated cell sorting) la mpt phuong phap co dp nhay va dp chinh xac cao duoc sii dung phd bien nham xac dinh sir bieu hien cac marker tren be mat va cung cap nhieu thong tin ve te bao Ngoai ra, FACS cd the dugc sir dung de phan lap tap hop MSC cd dac tinh thuan nhat ve bieu hien mpt so khang nguyen be mat cu the nao do
IHC (Immunohistochemistry), tuy khong phai Ia mpt kT thuat duoc ua chudng khi nghien cuu marker be mat te bao nudi cay, nhung do cd Igi the la khdng ddi hoi cao ve thiet bi, va trong tung trudng hop cu the cd the thuc hien d nhieu phdng thi nghiem Cac mau te bao diroc cd dinh, bao quan trong paraphin, va do vay duoc luu mau
Trang 6trong cac truong hop can thiet, san sang cho kT
thuat nhuom mien dich d bat cu thai diem nao
Day cung la su khac biet co tinh uu viet cua IHC,
nhung ciing phai luu y la kT thuat nay ton thai
gian va doi hoi kT thuat vien phai co tay nghe
thuan thuc khi tien hanh co dinh va vui mau trong
paraphin Ket qua phan tich bieu hien CD45 ciia
te bao nuoi cay bang hai kT thuat FASC va IHC
cho thay a PI, chi co < 5% te bao nuoi cay co
bieu hien duong tinh Ket qua nay the hien sir
tuang dong giua hai kT thuat va kha nang sir dung
IHC trong xac dinh khang nguyen be mat cua te
bao nuoi cay
V KET LUAN
- MSC nuoi cay duoc phan lap tir tiiy xuong
ngudi cd bieu hien kieu hinh khang nguyen be
mat ciia te bao gdc trung md: duong tinh vdi
CD 13, CD90 va CD73, ddng thdi am tinh vdi
CD34 va CD45
TAI LIEU THAM KHAO
1 Caplan A.I (2010), "Mesenchymal Stem
Cells: The Past, the Present, the Future",
Cartilage, 1(1), pp 6-9
2 Chen S.L., Fang W.W., Ye F et al (2004),
"Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in
patients with acute myocardial infarction" Am
J Cardiol, 94, pp 92-95
3 Dominici M., Le Blanc K., Mueller L, Slaper-Cortenbach L, Marini F., Krause D., Deans R, Keating A., Prockop D., Horwitz E (2006),
"Minimal criteria for defining multipotent mesenchymal stromal cells", The International Society for Cellular Therapy positionstatement,
Cytotherapy; 8, pp 315-317
4 Pittenger M.F., Martin B.J, (2004),
"Mesenchymal stem cells and their potential as
cardiac therapeutics", Cirs Res, 95; 9 - 20
5 Shim W.S., Jiang S., Wong P., Tan J., Chua Y.L., Tan Y.S., Sin Y.K., Lim C.H., Chua T., The
M, Liu T.C., Sim E (2004), "Ex vivo differentiation of human adult bone marrow stem
cells into cardiomyocyte-like cells", Bioch Biophys Res Commun, 324, pp 48M88
NGHIEN CUU TAO KHANG THE DON CHUOI SCFV BIEU L0 TREN THU/C KHUAN
THE mi 3 DAC HIEU KHANG NGUYEN UNG THU*TIEN LIET TUYEN
Le Quang Huan*, La Thj Huyen*
TOM TAT
Muc tieu: Tao khang the ddn chuoi scFv bieu 16
tren third khua'n the cd kha nang nhan biet va gan ket
dac hieu vdi khang nguyen bieu hien sdm trong ung
thU tien Met tuyen lam nguyen lieu cho cac nghien ciiu
tao KIT chan doan va thuoc dieu tri hudng dich
PhUdng phap nghien curu: Sir dung cac ky thuat
gen va ky thuat phage display de tao khang the ddn
chuoi scFv dac hieu khang nguyen bieu hien sdm trong
ung thu tien Net tuyen Ket qua nghien dhi: Khang the ddn chuoi scFv bieu Id tren thirc khua'n the gom
170 axit amin dUdc bieu Id tren thUc khuan the va budc dau xac dinh kha nang nhan biet va gan ket vdi cac khang nguyen bieu hien sdm trong huyet thanh benh nhan ung thu tien liet tuyen
Tit khoar EPCA, Khang the don chuoi, Phage
display, scFv, Ung thUtien liet tuyen
* I 'i$n Cong nghi sinh hoc