Thiết lập quy trình xác định hoạt tính sinh học của hg CSF tái tổ hợp trên dòng tế bào m NFS 60

Tgp chi Cling nghe Sinh hoc 11(1): 19-26, 2013

THIET LAP QUY TRINH XAC DINH HOAT TINH SINN HQC CUA hG-CSF TAI TO
HOP TREN BONG TE BAO M-NFS-60
Vmmg Cat Khanh, Tran Thanh 1-16a, HuYnh Lam Chau Duyen, Nguyen Thi Phtrcrng
Phyang Thao, Trim Linh Thu.&

Thi

Triding Dgi hoc Khoa hoc ty. nhien, Doi hoc Odic gia Thanh pha Fla Chi Minh
TOM TAT
Nhan to kich thich tao clang bach cau hg cua ngtr6i (hG-CSF) la mOt thanh vien cua Ito cac glycoprotein
dieu hoa su song, tang sinh, biet hoa va chirc nang cua cac to bio mau, Ong vai tr6 quan trong trong su kich
thich tang sinh va biet hoa cua cac to bao tien than dm bach cau trung tinh, dOng th6i lam tang mr6ng mOt so
chirc nang cua bach cau trung tinh tnrong thanh., hG-CSF tir lau d'a ducm sir dung rOng trong dieu tri benh
giam bach au hat, (lac biet la clang giam bach cau hat gay ra bai h6a tri lieu. Trong so cac clang to bao dung
cho thir nghiem G-CSF, Ong M-NFS-60 di duck sir dung de ,xac dinh hog tinh sinh hoc san pham thuOc
thuong mai chira G-CSF (Neupogen - Amgen) do FDA,phe chuan. Day la co so de imp dung dOng t6 bao nay
dinh hog tinh G-CSF tai to hop nham phat trien thanh thu6c clang generic. Den nay, chua 05 cong b8
trong
chinh thirc nao doi v6i chart h6a quy trinh thir nghiem tang sinh G-CSF tren (long M-NFS-60. Dic biet, tai
Viet Nam, chua c6 mOt nghien ciru xay dung, chub hoa quy trinh thir nghiem hog tinh sinh hoc nao duqc
cong be) tren cytokine. Trong nghien dru nay, chimgtoi xay dung quy trinh thir nghiem hoat tinh sinh hoc hGCSF dua tren mirc dO dap ung tang sinh cua Ong to bao M-NFS-60 doi yen hG-CSF tai to hop. Cac khao sat
bao gOm dai Ming dp G-CSF, mat dO to bao ban dau va thori gian thir nghiem. Ket qua thtrc nghiem cho thay
cac tham so tei uu cho quy trinh thir nghiem bao g6m diy neng d8 hG-CSF thir nghiem la 10'2 - 106pg/ml, mat
clO 4 bao ban dau la 105 tobao/ml, thOi gian thir nghiem la 48 gib. Chung t6i di buerc dau xac nhan hieu luc va
ap dung thanh cong qui trinh de xac dinh hog tinh cua protein hG-CSF tai to hop trong qua trinh san mit gem
cac mau truck tinh che, sau tinh cite va bao quan. Ket qua cho thay cac mau thir nghiem deu c6 cling hieu qui
tac Ong tang sinh va hog tinh gAn tucmg ducmg v6i mitt hG-CSF chitin (Neupogen) bleu thi qua cac gia tri
don vi hog firth IU xac dinh tir quy trinh.

Tir khoa: M-NFS-60, G-CSF, gicim bach cau hat, hoot tinh sinh hgc, tint nghiem tang sinh
M6 DAU
Nhan to kich thich tao clang bach cam hat cua
ngtriti (hG-CSF) la met thanh vien cua ha cac
glycoprotein tiet dieu hoi su song, tang sinh, biet
hoa va chirc nang cua cac to bao mau. hG-CSF co
ban chit glycoprotein gOrn 174 acid amin vai khOi
Itrong phan tit la 18,8kDa, chira 2 cau not disulfide
not phan tir (Cys36-Cys42 va Cys64-Cys74) va met
cystein to do (Cys17) (Hill et al., 1993). 0 (lieu kien
binh thutmg, !long de hG-CSF trong huyet thanh
nguOi b mirc khong phat hien duqc hoac thap han
100pg/r11. Khi có su xam nhiem cua tac nhan gay
benh, 'long de hG-CSF tang len va co the dat den
2000pg/ml. hG-CSF duqc sari xuat tai hau het cac
mo va ca quan khi duqc kich thich. G-CSF duqc san
xuat chu yeu bai bach cau mono va dai thuc bao;
ngoai ra hG-CSF con ductc san xuat bai to bao Wei
mo, nguyen bao sqi, cac to bio trung mo va cac to
bac, chat nen My xuang (Avalos et al., 1996; Garland
et al., 1997). Trong ca the hG-CSF dOng vai tre quan

tong trong viec hinh thinh va tang sinh cua (long to
bao bach cau trung tinh. Dong thori hG-CSF cling
tang cu0ng cac chirc nang cua bach cau trung tinh,
nhanh yen nhieu tac nhan
giirp no to. nen phan
kich thich va nang cao kha nang mien dich cua ca
the (Basu et al., 2002; Garland et al., 1997).
Dtra tren nhimg dac tinh 80, tir lau G-CSF,duqc

sir dung pho bien de dieu tri benh giam bach cau hat
(neutropenia),dac biet la benh giam bach cau hat do
h6a tri lieu dieu tri ung thu gay ra. Viec sir dung GCSF lam giam 50% nguy co nhiem khuan vas ro
tuong to del vori cac benh nhan giam bach cau hat,
disc biet doi vai benh nhan dieu tri ung thu. Viec sir
dung G-CSF ket hop vai cac loai thuoc hOa tri lam
giam 50% nguy co bi benh giam bach cau hat cap de
Ong. Ngoai ra, G-CSF con duqc sir dung cho cac
benh nhan cay ghep tuy xuang, cac benh nhan bi
benh to mien, bi thieu but mien dich va cac benh
nhan AIDS (Frampton et al., 1994; Welte et al.,
1996).
19


Vucmg Cat Khanh et al.
Hien nay, nha vao den be dm ding nghe gen, 10011g/1W streptomycin, 10 µM ii-mercaptoethanol
nhieu quy trinh san xuat hG-CSF tai to hop vai hieu va 62ng/m1 M-CSF, nu& trong khong khi am, vai
suit cao, don gian va chi phi thap da ra
nham dap thanh phan khong khi: CO2 (95:5), nhiet de 37°C
img nhu cau benh nhan giam bach cau hat ngay cang trong to am CO2, duqc cay chuyen va thay mai mei
gia tang. Tai Viet Nam, pheng thi nghiem Cong nghe truing 3 ngay/lan.
Sinh hoc Phan ter,, Truemg Dai hoc Khoa hoc Tu
Te bao M-NFS-60 phu thuec vao M-CSF, IL-3
nhien, Dai hoc Quoc gia TP.HCM ding da tien hanh
va
G-CSF.
Khi mei truing nuoi cay khong be sung
nghien ciru xay One quy trinh san xuat hG-CSF tai
cac

cytiokine
nay thi to bao se chet trong khoang 24to hop vai nhimg ket qua dat duqc ban au rat kha
quan (Hieu et al., 2009; .Hea et al., 2009). Vai mtic 72 gia. Khi bo sung G-CSF vao mei trtthmg .nuoi
thuec vao hog tinh
G-CSF ma to bao
dich phat trien thanh thuoc clang generic, hG-CSF tai cay,
to hop san xuat can phai dap img cac tieu chufin ye M-NFS-60 se tang truang va !lit trien. Hoot tinh Ghoat tinh va tic Ong tuong throng vai clang thucmg CSF cang manh thi t6 bao M-NFS-60 tang truing NIA
mai hien nay la Neupogen (Amgen). Do de, viec phat trien cang manh. Do vay c6 the dva ten str tang
nghien cuu xay dtmg quy. trinh xac dinh hog tinh twang va /lit trien cua to bac) M-NFS-60 de xac
sinh hoc vai cac thong so duqc chuan hoa IA can dinh hoat tinh cua G-CSF.
thiet. Trong bao cao nay,. thong toi trinh bay ket qua Phtrang phi!) xac dinh mit di) to bao
cua viec nghien
thiet lap va ap dung quy trinh
Mit TO to bao duce xac dinh ttrong clei thong
the. nghiem sinh hoc hG-CSF in vitro dtra tren tic
qua
1,45
kit CCK-8 (Dojindo, Rockville, ,MD). Kit
deng kich thich tang sinh deng to bao M-NFS-60.
CCK-8
c6
china thanh phan chinh la muoi WST-8,
Day la deng to bio da.throc sir dung xac (firth hog
WST-8
co
the
bi khir bai cac dehydrogenase trong to
tinh sinh h9c san pham thuoc thuong mai chira GCSF (Neupogen 7 Amgen) do Cvc quan ly thtrc bao song tao ra san pham la formazan, met chat c6
pham va duce pham Hoa KS, (US Food and Drug mau yang. Luang formazan tao ra la tuong eng vai
Administration — FDA) phe chuin, tao co sa de (mg so lucong to bao song, do vay Oa ten sv dcpc mau

ding trong kiem tra hoat tinh G-CSF tai to hop. Quy san pham formazan c6 the xac dinh ttrcmg doi lam
trinh thin nghiem duccc toi tru vai cac tham . se nhu to bao song. Qua trinh d9c mau dtroc thtrc hien In hai
day nong do protein, mat de to bao ban dau, thei buac song 450nm va 595nm tuong img vai buck
gian the. nghiem. Sau khi buac
dinh gia hieu sang kiem tra NIA buac song loai nhieu cea kit CCKlvc, quy trinh dtroc ap dung dedexac dinh hoat tinh 8. Gia tri ODTB tuong img vai mat de to bao duqc
tinh toan theo ding elk: ODTB = (013450 — 0D595).
cua cac mau hG-CSF trong cac buac san xuat.
Gia tri ODTB.cuoi cling dtroc thu nhan la gia tri trung
binh
ba Ian doc.
NGUYEN LIEU VA PFITIONG PHAP
Khao sit mill twang quan gifra kha nang tang
truing ctia to bit) va ning di) hG-CSF
Hia chit
Deng to bao M-NFS-60 a giai loan tang sinh
M-CSF, Insulin duqc cung cap bai hang Sigma &roc rea yeti mei truing RPMI 1640 de 104i M-CSF.
(MD. hG-CSF diming mai, Neupogen (Roche, Thuy Dem va pha loang to bao de mat de dtra vao gieng
Sy) va Leukocim (CIM, Cuba), ca hoat do 1 x 108 dat 105 te bao/ml. BO sung hG-CSF yeti day king de
Ili/mg. San phim Neupogen &roc sir dung nhu hG- dtrqc pha bang bac 10, tir 104 tai,108pg/ml. U dla
CSF chuan trong qua trinh xay dtmg quy trinh.
tong, te am 37°C, 5% CO2, va do am 85% trong 48
h. Tien hanh xac dinh mat de to bao va phan tich ket
Mktg to bao M-NFS-60
qua bang phan mem GraphPad Prism 5.0.
Deng to bao M-NFS-60 duqc cung cap tir ATCC Khao sit anh hung caa mat dQ to bho den qua
(No. CRL-1838TM). Day la deng nguyen toy bao trinh thin nghit'm
thu tir chuet titan thanh (NFS X DBA/2) da nhfern
Tien hanh quy trinh vai day neng de hG-CSF
virus bach cau clang toy Cas Br-MuLV va c6 sat
phi' hop, co dinh cac tham so the nghiem va khao sat

nhap gen tai vi tri c-myb proto-oncogene.
tham so mat de to bao a cac gia tri la 5 x 104 to
Te bao &roc duy tri trong mai truing RPMI bao/ml, 105 4 bito/ml, va 5 x 105 4 bao/ml. Thu nhan
1640 c6 be sung 10% fetal bovine serum (FBS), 4 IC& qua va phan tich dua tren hai thong se: gia tri
mM L-glutamine, 1001U/m1 benzylpenicil I in, Hill slope va bien de dao Ong dm throng cong
20


Tap chi Cong nghe Sinh hoc 11(1): 19-26, 2013

tucmg quan giDa mg do to bao va tiling de hG-CSF,
tir do xac dinh mat de to bac, toi uu cho quy trinh.
Khao sat anh hu*ng cua thiri gian nuoi ctty den
qua trinh flax. nghi'C'm
Tren co sof quy trinh vai cac tham s6 da chon,
tien hanh khao sat thoi gian thir nghiem sinh hoc vai
cac gia tri 24, 48, va 72 h. Ka qua cling duqc phan
tich dga ten gia tri Hill slope va bien de dao Ong
cua dtrOng cong Luang quan giffa mat do to bao va
ruing de hG-CSF.
Dinh gia higu luc quy trinh
Quy trinh vai cac tham se da tei tru duce btrac
dau tien hanh danh gifi veil 3 mau: 2 mau hG:CSF
thucmg mai la Neupogen, Leukocim va 1 mau IA
insulin. Trong do, Neupogen duqc xem la mau dOi
chimkdtrong (protein hG-CSF chuan), insulin la
mau doi chirng am va Leukocim la protein hG-CSF
can danh gia hoat tinh. Dim tren ket qua thu nhfin
duoc, tien hanh xac dinh gia tri ED50 cua cac mau
thin, tir do tinh duqc gia tri LU (gia tri don vi hoat

tinh rieng theo phong thi nghiem) theo cong thirc:
1 x 109
Units/mg
LU =
ED50
LU là gia tri don vi hoat tinh rieng theo phong
thi nghiem, no se thay doi tit)/ theo dieu kien cua
timg phong thi nghiem, sire song cua to bao vao tiled
diem thi nghiem, vi vfiy de chuan hem va de c6 the so
sinh hoat tinh cua cac mau hG-CSF kit& nhau can
sir dung met mau hG-CSF chuan (6, day mau hGCSF duqc sir dung là Neupogen) de tir do xac dinh
dtrqc gia tri don vi hoat tinh rieng quoc to IU theo
ding thirc:
L Uma,
IL/01th .
unttsimg
=
LUchuin
Ap dqng quy trinh de dinh luvng hoat tinh hGCSF tai to hqp
Quy trinh sau khi danh gifi hieu tut duqc ap
dung de tien hanh xac dinh hog tinh cac mau hGCSF tai to hop thu nhfin tir quy trinh sin xuat bao
gem 3 log: Mau protein truck tinh che, mau protein
sau tinh che Ira mitt protein (long kh6.
KET QUA VA THAO LUAN
Anh hirfing cua hG-CSF den sq tang sinh cua to
him M-NFS-60
Met thin nghiem sinh hoc ve tang sinh to bao khi

tien hanh thuang duqc thirc hien v6i met clai nong
d6 dm chat can lciem tra, trong de doi hoi phai co

nhimg Ming de ma to bao dot mire de tang truang toi
da va tOi thieu. Do vfiy de tien hanh xay dung quy
trinh thin hoat tinh hG-CSF can xac dinh duqc mei
tucmg quan gitra ming d$ hG-CSF va sg tang truong
cua to bao M-NFS-60. Ket qua khao sat cho thay do
thi the hien mei Wong quan girra kha nang tang
twang to bao va nong de mau hG-CSF chuan
(Neupogen) co hinh sin dang dee won img vai hinh
dang duong cong cua met thin nghiem tang sinh. Duo
vao do thi, chimg toi nhfin thay to bao bat On tang
truerng tir ming de 10°pg/m1 va di vao pha On dinh
noting do 104pg/ml. Qua trinh thin nghiem sinh hoc tot
va On dinh khi bien de thin nghiem bao g6m 2-3 gia
tri nong de irng vai mire tang truang to bio thap nhat
va 2-3 gia tri Wong de img vai mire tang twang to
bao cao nhat. Do vfiy dim teen ket qua khao sat nay,
chimg toi chen day Ming de hG-CSF thich hqp tir 10'
2_ • •-••
lu peal] de tien hanh khao sat cac tham so cho
quy trinh thin nghiem sinh hoc.
1.5

0

t.0

0 0.5

I

-4

-2

4

2
0
Log1G-CSF]

6

8

Hinh 1. mei Wang quan giCra kha nang tang trullmg to bao
va n6ng d6 mau Neupogen

Anh hir&ng cua mat di) to him den qua trinh thir
nghRm.
Mat ,dc) to bao IA met nhan to quan tr9ng anh
huOng denqua trinh thin nghiem, mat de to bao qua
thap co the Anh huong den de nhay cua quy trinh,
nhung mat de to bao qua cao lai cling c6 the anh
cua quy trinh. Do vfiy
chinh
huang den
chimg toi tien
tien hanh kith) sat mat do to bao sir dung
trong quy trinh vai 3 gia tri la 5 x 104, 105, va 5 x
105 to bao/ml (nam trong khoang mg de tang

throng tot nhat cua dung to bao M-NFS-60). Ket
qua duqc dinh gia thong qua gia tri Hill slope va
bien de dao Ong dm dm:mg cong. D6i yeti cac thin
nghiem tang sinh nhu thin nghiem sinh hoc G-CSF,
21


Wang Cat IChinh et aL
gii tri Hill slope chuAn la I.
&Ong cong tircmg
quan gift so tang truang te
to bao vi riling de chat
thir nghiem co gii tri Hill slope cang gan 1, thi
Bang tic Ong sinh hoc dm mau kiem tra len te bao
cing gi8ng \Teri dip Ong chuan, quy trinh kiem tra
cho de chinh xic cang cao. Ben canh d6, met thir

nghiem mutm co do nhay cang cao thi can phai c6
bien do dao Ong dm dui/rig cong twang quan gitia
so tang truong te bao vi 'tong M chat thir nghiem
cang km. Do vay met thir nghiem sinh hoc se co de
chinh xic vi do nhay cao khi co gii tri Hill slope
gan 1 va co bien de dao Ong cao.

Bang 1. Anh htremg cua met de to bao den cac gia tri Hill slope va Nen de dao dung cua duteng cong prang quan
Mat do to bao (te bao/m1)

Gia trf Hill slope

Bien dQ dao Ong

5 x 104

0,64

0,57

105

1,10

0,89

5 x 105

7,00

0,29

-0- 5.105 tb/ml
-0- 105 tb/ml
-X- 5.104 tb/ml

-2

0

2

4

Log[G-CSF]
Hinh 2. Anh hue:mg ctia mat dO t6 bao den dub.ng cong tu'ong quan gal'a sty tang tnyang cua t6 bao M-NFS-60 va n6ng dO
hG-CSF

Ket qui cho thAy 0 mat de te bac* ban dAu la 105
te bao/ml, gia tri Hill slope gan vai I nhat (1,10) vi
c6 bien de dao Ong cua duong cong Ion (0.89) trong
khi o mit M 5 x 104 va 5 x 105 te bao/ml, gii tri Hill
slope kith xa 1 (0,64 va 7,00) va bien de dao Ong
&Ring cong nho (0.57 va 0.29). Do 4y, mat de te
bac, ban dau la 105 t6 bio/m1 la phi' hop cho thir
nghiem kiem tra hoat tinh hG-CSF bing dong te bio
M-NFS-60.
Anh hieing cua thoi gian nuoi
thir nghi'Om.

cay

den qua trinh

MZU loaf to bao có the co thOi gian the he thay
doi khac nhau, do 4y tit), thuec vao thOi gian nuoi
cay ma de chinh xic cua quy trinh cling se thay Mi.
Dtra vao khoang thoi gian nuoi cay thich hop cho so

22

tang truemg cua dung t6 bao M-NFS-60, chimg t6i
lora chon 3 gia tri 24, 48 va 72 h de tien hanh, khao

sit sir anh hoeing dm thoi gian nuoi cay den ket qua
cua quy trinh thir nghiem. Ket qui ding dtro.c phan
tich thong qua hai thong so: gia tri Hill slope va bien
do dao Ong,
duong cong Wang quan gift sir
tang tnrang te bao va nong do hG-CSF.
Ket qua cho thAy o thOi gian 48 h, gia tri Hill
slope gan viii gia tri 1 nhat va, bien de dao deng cua
duOng cong ding la cao nhat. Trong khi do 0 2
Mil:mg hop con lai, gia tr.! Hill slope cach gia tri 1
khoing 0,3-0,5 va bien de dao deng cua dtrOng cong
kha 169. Dieu nay cho thay khoang thoi gian 48 h
nuoi cay là th?ri gian phi' hop de xic dinh so inh
huong cua hG-CSF den so tang irtreng cua te bio MNFS-60.


Top chi Cong nghe Sinh hoc 11(1): 19-26, 2013
Bang 2. Anh hirdrng cia thai gian nuOi cay den cac gia tri Hill slope ya hien c10 dao dOng cua &rang cong tuvng quan.

Thel gian nutil cay

Gil tri Hill slope

Bien dO dao gong

24 gib,

0,75

0,19

48 gib

0,97

0,62

72 gib

1,57

0,33

0

2

2

4

8

Log 1G-CSFI
Hinh 3. Anh timing cua thei gian nuoi cay clan duteng cong twang quan giDa w tang trip:mg cua to bao M-NFS-60 va n6ng
41$ hG-CSF.

Pha loAng man G-CSF can dish hoot Will tlianh &Ay
'tong do tir 10.2 den 106 pg'ml
B6 sung 50411 G-CSF va 50p1t0 bao vao dia 96 gthig

(mat dote bao ban dam 105 ti bao n il)
1) din trong to am 37°C. 5° u CO2 trong 48 gia

B6 sung CCK-8 n 10 1 10 ( v v trong 4 gib

Do OD bmic song 4 50inn 595nni

I

Phan tick k0t qua x-a xac dinli hoat tinh cita inAu

Hinh 4. Quy trinh thin hoat tinh hG-CSF bAng

t6 bac M-NFS-60.

Dinh gii hi?u luc cua quy trinh.
Qua qua trinh khao sat, va t6i tru,,quy trinh tiler
nghi'e'm hoot tinh hG-CSF bang d6ng to bao M-NFS60 duoc de xuat bao Om 6 buck nhtr hinh 4.

De' buck dAu dinh gia tinh chinh xac cua quy
trinh, quy trinh dtrcc tien hanh danh gia hie;u lye sa b8
vai cac mau: (I) Leukocim: la mau hG-CSF thtrcmg
mai cua cong ty CIM (Cuba) co hoot di) là 108 IU/mg,
(2) insulin: la mau chirng am trong thi nghim nay vi
23


Vtrang Cat Khanh et at.
insulin da dtrgc biet den IA khong co kha nang kich
thich tang truong dung to boo M-NFS-60. Cac mau

ducrc den hanh 3 Ian, xac djnh gia tri LU, sau do dua
ten gia tri LU va gia tri IU da xac dinh (108 IU/mg)
cua mau hG-CSF chufin (Neupogen) de tInh ducrc gia
tri IU.
Ket qua cho thiy mau insulin hoan toan khong
c6 kha nang kich thIch tang truong (long to boo M-

NFS-60, trong khi d6 mau Leukocim cho &rang
cong twang quan pan nhu hoan toan trong khop yeti
mau hG-CSF chuan (Neupogen) va gia tri IU tinh
dtrac cua mau Leukocim la 0,97 ± 0,04 x 108 IU/mg,
chi sai Ichic rat thap so yen gia tri IU da cong be dm
mau Leukocim IA 108 IU/mg. Do vay c6 the sir dung
quy trinh thir nghiem ma chi:mg toi da xay dung de
tien hanh xac dinh hoot tinh cua hG-CSF trong qua
trinh sin xuat.

Bang 3. K6t qua xac nhOn hiOu Itec

IU Ijr thuyet

IU tinh toan
(units/mg)

0{) Rich chuin

Leukocim

0,97 x 108

0,04

108

Insulin

0

0

0

A IO D4 50 - OD595)

M5u

-X4-

(units/mg)

Neupogen
Leukocim
Insul i n

0
4
2
Log [pro teins
Hinh 5. Dung cong twang quan giCya sty tang truemg to boo va ming 1:10 cac mau protein tai to hap.

Ap dung quy trinh d4 djnh luvng hoot tinh hGCSF tai to hup.
Cac mau thin nghim tir cac giai doan cua quy
trinh sin xuat protein hG-CSF tai to hqp duqc chia
thinh cac nhom nhu sau: (1) mau BI, B2: mau hGCSF truck khi tinh che; (2) mau Al, A2: mau hGCSF sau khi tinh ch'e; (3) mau Cl, C2: man hG-CSF
sau khi thing khO.
Ket qua cho thiy cac dm:mg cong Wang quan
giCia mirc dO tang sinh to boo va nOng dO protein c6
Bang hinh sin dlic len, khong c6 bieu hien bat thuong
trong qua trinh tang truong to boo. acing thai cac
matt thir nghiem deu co doan tuyen tinh gan nhu

song song hoac trong khop vai doan tuyen tinh ctia
matt hG-CSF chufin (Neupogen), do do. CO the not
mau kiem tra va mau chuan co ding kieu tac Ong
tang sinh len dong to boo M-NFS-60. Dieu nay cho
thay cac tap. chat con ton
trong mau cac giai
doan‘ sin xuat khac nhau co anh htrang khong ding
ke den qua trinh thir nghiem hoat tinh bang (long t8
boo M-NFS-60, va co the sir dung quy trinh thir
nghiem nay de xac dinh hoat tinh cua hG-CSF a cac
buck trong qua trinh san xuat. Val cac gia tri don vi
hoat tInh IU xac dinh ducrc ding btrac dau cho thay,
cac mau hG-CSF sau tinh che va d8ng kilo ma chung
tai thu nhandtrac co hoat tinh kha tot, gan twang
duong vai mau hG-CSF chuan (Neupogen).

Bang 4. Gia tri hoot tinh riang cua cac mau hG-CSF tai tó hap.
PAOLI

B1

B2

Al

A2

Cl

C2

IU (units/mg)

0,29 x 108

0,01 x 108

1,49 x 108

0,81 x 108

0,76 x 108

0,57 x 108

24


Top chi Cong nghe Sinh hoc 11(1): 19-26, 2013

-0- Neupogen
-0- 131

1.5

0
0

-2

1.0

4
0
2
Log 1G-CSFI

0

6

2

Log 1C-CS1.1

-- Neupogen

00455 - O D 595)

0.6

-0- A2
0.5
0.4
0.3
0.2

2

4

4
2
0
Log 1G-C.S171

-0- Neupogen

1.5
(0D450 -OD sgs)

8

-0- CI
1.0

0.5

4

2
0
Log 16-CSF1

0

2

4

8

Hinh 6. Dutnig cong Wring quan gift ski, tang triserng to bar) va n6ng d6 protein hG-CSF cua cac mhu can xac dinh
hoat thin.

KET LUO
Vol uu diem là don gian, de" tien hanh, cho ke't
qua nhanh va có the thoc hien 6 quy mo Ian, thin
nghiem tang sinh la phtrcmg phip pho bien de xac
dinh hoat tinh cua G-CSF tai to hop. Nhieu (long to
bao da &roc Oat trien cho 111ir nghiem tang sinh GCSF nhtr NFS-60, Kasumi-1, M-NFS-60... Ling v6i
moi dong to bao, quy trinh thir nghiem hoat tinh can
phai duce chufin h6a v6i cac thong so toi uu nham
mang !al ket qua thin nghiem nhay va chinh xac nhat.
Bang viec sir dung dong to bao M-NFS-60, mOt
(long to bao phu thuOc vao G-CSF de tang sinh va
phat trien, chung toi da thiet lap va toi tru thanh ding

quy trinh xac dinh hoat tinh hG-CSF. Quy trinh da
&roc toi uu ve cac tham so: day n6ng dq th6 nghiem

(10-2 - 106 pg/ml), mat dO to bao ban dau (105 to
Wimp, thoi gian nuoi cay to bio trong thin nghiem
(48 h). Buck dau da xac nhan hieu toe cua quy trinh
va thir Ap dong thinh ding quy trinh de xac dinh hoot
tinh cua hG-CSF trong cac bu6c san xuat. Ket qua
thin nghiOm ding cho thay cac mau hG-CSF sau tinh
the va &mg kho cho hoot tinh kha tot, gin tucmg
throng v6i mau hG-CSF chuan (Neupogen).
101 cam on: Cong trinh clugc thus hien biing kinh
phi de led: "Nghien cciu tong nghe son xucit
Granulocyte colony stimulating factor (G-CSF)
ngudi tai to hop de h6 try dieu tri benh glom bath
25


Vucmg Cat Khanh et al.
cau hgt" thuOc chuang trinh nghien cziu va phcit
trien Cong nghe sinh hQc KC.04.
TAI LIEU THAM KHAO
Avalos BR (1996) Molecular Analysis of the Granulocyte
Colony-Stimulating Factor Receptor. Blood 88: 761-777.
Basu S, Dunn A, Ward A (2002) G-CSF: function and
modes of action. Int .1 Mol Med 10: 3-10.
Chandavarkar N (2009) Novel use of an indicator cell line
for bioassay. US 2009/0197293 Al. Patent application
publication.
Frampton JE, Lee CR, Faulds D (1994) Filgrastim a
review of its pharmacological properties and therapeutic
efficacy in neutropenia. Drugs 48: 731-760.
Garland JM, Quesenberry PJ, Hilton DJ (1997) Colonystimulating factors: molecular and cellular biology. New

York: M. Dekker.
Hammerling U, Kroon R, Sjodin L (1995) In vitro
bioassay with enhanced sensitivity for human granulocyte

colony-stimulating factor. J Pharm Biomed Anal 13: 9-20.
Hill CP, Osslund TD, Eisenberg D (1993) The structure of
granulocyte-colony-stimulating factor and its relationship
to other growth factors. Proc Nail Acad Sci U S A 90:
5167-71.
Nguyen Thi Phucmg Hieu, Vucmg Cat Khanh, Iran Thanh
D4ng Thi Phuong Thao, Tran Linh Thu& (2009)
Thu nhan hG-CSF cb hag tinh tar the yid non-classical a
Escherichia coli. Tuyen tap bao cao khoa hQc hai nghj
Cong nghe Sinh hQc wan qutic, Thai Nguyen. Nha xuat
ban DH Thai Nguyen: 791 - 793.
Tran Thanh Hee, D4ng Thi Phuang Thao, Tran Linh
Thu& (2009) To dOng va bieu hien Granulocyte Colony
Stimulating Factor ngtroi a nim men Pichia pastoris.
Tuyen tap biro ccio khoa hQc heii nghi Ging nghe Sinh hQc
loan qucic, Thai Nguyen. NM xuat ban DH Thai Nguyen:
794 - 797.
Welte K, Gabrilove J, Bronchud MH, et al (1996)
Filgrastim (r-metHuG-CSF): the first 10 years. Blood 88:
1907-1929.

ESTABLISHMENT OF A BIOASSAY FOR RECOMBINANT HUMAN GRANULOCYTE
COLONY STIMULATING FACTOR (hG-CSF) IN M-NFS-60 CELL LINE
Vuong Cat Khanh, Tran Thanh Hoa, Huynh l,am Chau Duyen, Nguyen Thi Phuong Hieu, Dang Thi
Phuong Thao*, Tran Linh Thuoc
University of Science, VNU-HCMC

SUMMARY
Human granulocyte colony stimulating factor (hG-CSF) is one of a family hematopoietic growth which
stimulates the proliferation and differentiation of neutrophil precursor cells and enhances some of the
functional properties of mature neutrophils. hG-CSF has been well known to widely use to treat different forms
of neutropenia, especially neutropenia caused by chemotherapy. Currently, some reports show that an
increasing of neutropenia patient impulses the development of production of G-CSF. Among the cell lines used
for biological assay of G-CSF, MNFS-60 has made it possible to estimate levels of G-CSF with great ease.
This cell line has been used to determine bioactivity of commercial G-CSF (Neupogen-Amgen) which is
approved by US Food and Drug Administration (FDA). Until now, there is no report on validation of
biological assay of G-CSF using M-NFS-60 cell line. Especially, in Viet Nam, there is no study on validation
of biological assay of cytokine as well. In this study, we investigated the bioactivity of G-CSF samples in MNFS-60 cell line by using proliferation assay. We studied some parameters as the concentration of hG-CSF,
cell density, and time of hG-CSF treatment to optimize these conditions. Our results showed that the optimal
concentration of hG-CSF uses for this bioassay is from 1012 to 106pg/ml, the cell density is 105cells/ml and the
time to treat with hG-CSF is 48 hours. This process was efficient and used to determine the recombinant hGCSF's activity, including samples before and after purification as well as lyophilization. The unit activities of
these samples determined by using our process showed that these samples have the same effect on M-NFS-60
cell line comparing to commercial sample (Neupogen).
Keywords: G-CSF, neutropenia, bioassay, M-NFS-60, proliferation assay.

• Author for correspondence: Tel: +84-8-38307079, E-mail:
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