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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 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 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 dieu tri benh giam bach au hat, (lac biet la clang giam bach cau hat gay 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) FDA,phe chuan Day la co so de imp dung dOng t6 bao dinh hog tinh G-CSF tai to hop nham phat trien thu6c clang generic Den nay, chua 05 cong b8 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 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 cong qui trinh de xac dinh hog tinh cua protein hG-CSF tai to hop 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 vien cua 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 cau not disulfide not phan tir (Cys36-Cys42 va Cys64-Cys74) va met cystein to (Cys17) (Hill et al., 1993) (lieu kien binh thutmg, !long de hG-CSF huyet 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 duqc kich thich G-CSF duqc san xuat chu yeu bai bach cau mono va dai thuc bao; ngoai hG-CSF 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 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 bien de dieu tri benh giam bach cau hat (neutropenia),dac biet la benh giam bach cau hat h6a tri lieu dieu tri ung thu gay 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 duqc sir dung cho cac benh nhan cay ghep 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& khong am, vai suit cao, don gian va chi phi thap da nham dap phan khong khi: CO2 (95:5), nhiet de 37°C img nhu cau benh nhan giam bach cau hat cang to am CO2, duqc cay chuyen va thay mai mei gia tang Tai Viet Nam, pheng thi nghiem Cong nghe truing 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 thi to bao se chet 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 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 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 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 to tinh sinh h9c san pham thuoc thuong mai chira GCSF (Neupogen Amgen) Cvc quan ly thtrc bao song tao san pham la formazan, met chat c6 pham va duce pham Hoa KS, (US Food and Drug mau yang Luang formazan tao la tuong eng vai Administration — FDA) phe chuin, tao co sa de (mg so lucong to bao song, vay Oa ten sv dcpc mau ding 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 protein, mat de to bao ban dau, thei buac song 450nm va 595nm tuong img vai buck gian the nghiem Sau buac dinh gia hieu sang kiem tra NIA buac song loai nhieu cea kit CCKlvc, quy trinh dtroc ap dung dedexac dinh hoat tinh Gia tri ODTB tuong img vai mat de to bao duqc tinh toan theo ding elk: ODTB = (013450 — 0D595) cua cac mau hG-CSF 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 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 qua trinh xay dtmg quy trinh tong, te am 37°C, 5% CO2, va am 85% 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 caa mat dQ to bho den qua (No CRL-1838TM) Day la deng nguyen toy bao trinh thin nghit'm thu tir chuet titan (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 x 104 to Te bao &roc tri mai truing RPMI bao/ml, 105 bito/ml, va x 105 bao/ml Thu nhan 1640 c6 be sung 10% fetal bovine serum (FBS), 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 to bao va tiling de hG-CSF, tir 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 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 mau: mau hG:CSF thucmg mai la Neupogen, Leukocim va 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 tinh duqc gia tri LU (gia tri don vi hoat tinh rieng theo phong thi nghiem) theo cong thirc: x 109 Units/mg LU = ED50 LU 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& can sir dung met mau hG-CSF chuan (6, day mau hGCSF duqc sir dung Neupogen) de tir 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 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 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 tien hanh thuang duqc thirc hien v6i met clai nong d6 dm chat can lciem tra, 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 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 thi, chimg toi nhfin thay to bao bat On tang truerng tir ming de 10°pg/m1 va di vao pha On dinh noting 104pg/ml Qua trinh thin nghiem sinh hoc tot va On dinh 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 t.0 0.5 I -4 -2 Log1G-CSF] Hinh 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 to bao sir dung quy trinh vai gia tri la x 104, 105, va x 105 to bao/ml (nam 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 nhay cang cao thi can phai c6 bien 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 nhay cao co gii tri Hill slope gan va co bien de dao Ong cao Bang Anh htremg cua met de to bao den cac gia tri Hill slope va Nen de dao dung cua duteng cong prang quan Mat to bao (te bao/m1) Gia trf Hill slope Bien dQ dao Ong x 104 0,64 0,57 105 1,10 0,89 x 105 7,00 0,29 -0- 5.105 tb/ml -0- 105 tb/ml -X- 5.104 tb/ml -2 Log[G-CSF] Hinh 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 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) o mit M x 104 va x 105 te bao/ml, gii tri Hill slope kith xa (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, 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 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 dao Ong, duong cong Wang quan gift sir tang tnrang te bao va nong hG-CSF Ket qua cho thAy o thOi gian 48 h, gia tri Hill slope gan viii gia tri nhat va, bien de dao deng cua duOng cong ding la cao nhat Trong Mil:mg hop lai, gia tr.! Hill slope cach gia tri khoing 0,3-0,5 va bien de dao deng cua dtrOng cong kha 169 Dieu cho thay khoang thoi gian 48 h nuoi cay 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 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 2 Log 1G-CSFI Hinh 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 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 to am 37°C 5° u CO2 48 gia B6 sung CCK-8 n 10 10 ( v v gib Do OD bmic song 50inn 595nni I Phan tick k0t qua x-a xac dinli hoat tinh cita inAu Hinh 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 buck nhtr hinh 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) 108 IU/mg, (2) insulin: la mau chirng am thi nghim 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 Ian, xac djnh gia tri LU, sau 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, d6 mau Leukocim cho &rang cong twang quan pan nhu hoan toan 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 qua trinh sin xuat Bang 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 A IO D4 50 - OD595) M5u -X4- (units/mg) Neupogen Leukocim Insul i n Log [pro teins Hinh 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 tinh che; (2) mau Al, A2: mau hGCSF sau tinh ch'e; (3) mau Cl, C2: man hG-CSF sau 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 qua trinh tang truong to boo acing thai cac matt thir nghiem deu co doan tuyen tinh gan nhu song song hoac khop vai doan tuyen tinh ctia matt hG-CSF chufin (Neupogen), 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 cho thay cac tap chat ton mau cac giai doan‘ sin xuat khac 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 de xac dinh hoat tinh cua hG-CSF a cac buck 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 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 -2 1.0 Log 1G-CSFI Log 1C-CS1.1 Neupogen 00455 - O D 595) 0.6 -0- A2 0.5 0.4 0.3 0.2 4 Log 1G-C.S171 -0- Neupogen 1.5 (0D450 -OD sgs) -0- CI 1.0 0.5 Log 16-CSF1 Hinh 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 don gian, de" tien hanh, cho ke't qua nhanh va có the thoc hien quy mo Ian, thin nghiem tang sinh la phtrcmg phip 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 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 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 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 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: thaodp@hcmus.edu.vn 26 ... 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... giffa mat 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 mau: mau hG: CSF thucmg mai la Neupogen, Leukocim va mau... 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& can sir dung met mau hG- CSF chuan (6, day mau
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