Hpl NGHj KHOA HQC COA HQI H6A SINH Y Dl/QC HA NQI VA CAC TINH PHIA BAC LAH THLT XVlll technology in the detection of solid
tumors. Cancer 82:1419-1442. 1998 . Watson MA, Darrow C, Zimonjic DB.
Structure and transcriptional regulation of the human mammaglo-bin gene. Oncogen 1999:16:817-24.
. Zach O, Krufcger WH, Kasparu H, ct al: Mammaglobin might become a more useful marker than cytokeratin 19 for the
detection of circulating breast cancer cells.
Onkologie 20:181, 1997(suppl l)(abstr) 1. /{ppclius A, Kufer P, Honold MW, et
ah Limitations of reverse-transcriptase polymerase chain reaction analyses for detec-tionof micrometastatic epithelial cancer cells In bone marrow. J Clin Oncol 15:2701-2708.1997.
XAC DjNH H | PROTEIN NOfrC TI^U B | N H N H A N GHEP T H A N
c b BIEN CHlifNG THAI GHEP CAP TiNH
Phgm DiJrc Dan*, Li Thj Bich Thdo*, Nguyin Thj Minh Phuffng*, Nguyin Tien Ddng*, D6 Hiru Chi*, Phgm Dinh Minh*, Hd Phan Hdi An**, Nguyin Bich Nhi*, Phan Van Chi*
T 6 M TAT:
Nudc tiSu Id mpt trong nhOng djch sinh hpc hdp ddn trong nghidn cuu proteomics idm sdng vl nd cd {hh Idy nhi^u mdu, mdu dn djnh vd ddc bi$t Id khfing xdm Idn vdo ca th^. H$ proteome nudc ti^u dd dupc nghidn ciru bdi cdc k9 thudt khde nhau, tuy nhidn sir dyng Wj thudt khdi phd dd nii ISn nhu mpt phuong phdp phu hpp Trong nghidn eiru ndy SDS-PAGE dupc su dgng d^
phdn tdch protein sau khi sir dyng Kit ProteoSpin™ Unne Protein Concentration Micro de tmh sgch Hdn hpp peptide chi^t ti> cdc mdnh gel 6uqic phdn tich bdng sdc ky Idng nano 2 chi^u k^t n6i hd thing khdi phd lidn tidp (NanoESI-Q-TOF-MS/MS). Trong tdng sd 132 protein nhdn didn dupc, cd 11 protein n6ng dO cao, 72 protein ning d i thdp vd mit vdi ding phdn cua cdc chuii immunoglobulin
rt> kh6a Khdi ph6. nudc ti4u. proteomics.
thii ghip thin c&p tinh.
ABSTRACT:
IDENTIFICATION OF URINE PROTEOME FROM PATIENTS WITH ACUTE RENAL
ALLOGRAFT REJECTION Urine has become one of the most attractive biofluids in clinical proteomics as it can be obtained noninvasively, In large quantities and is stable compared to other biofluids. The urinary proteome has been studied by almost any proteomic technology, but mass spectrometry based unnary protein and peptide profiling has emerged as most suitable for clinical application.
In this study, SDS-PAGE was used to separate urine proteome after purification by ProteoSpin™
Urine Protein Concentration Micro Kit The ' Viin Cdng nghi sinh hge. Viin Khoa hQC vi Cdng nghi Viit Nam
" Khoa Thin & Lgc miu, Binh viin Huu nghi Viit DOc 32
Y HQC VigT NAM THANG 8 • 2/2012 peptide mixtures extracted from each gel slice
were analyzed by two-dimensional nano liquid chromatography (20 nanoLC) coupled online with tandem mass spectrometry analysis (NanoESI-Q-TOF-MS/MS). In total, 132 proteins were identified, 11 hight abundant concentration proteins, 72 low albudant concentration proteins and several isoforms of immunoglobulin
Keywords. Acute renal allograft rejection, mass spectrometry (MS), proteomics. urine
I. O A T V X N o f :
Hi?n nay sinh thiet dupc coi Id tieu chuan vdng de chan dodn thdi ghep cap tinh d bpnh nhan ghep thgn. Tuy nhien phuang phdp ndy xam lan vdo ca the benh nhan cd the dnh hudng den chat lupng chan dodn vd dieu tri. Do dd can phdt trien mpt phuang phdp cd dp nhay cao, ddc hieu vd dae biet la khdng xam Idn vdo ca the benh nhdn.
Trong nhihig nam gan day, xu hudng nghien ciiu cua the gidi tap trung vdo tim kiem ehi thj sinh hpc bang cac ky thugt proteomics thdng qua cdc dieh ca the, trong dd nudc tieu cd uu the ddc biet ddi vdi thai ghep than cap tinh. Nghien euu tdng the h?
protein nude tieu la mpt cdng cu manh gdp phdn thuc ddy qud trinh tim kiem lya chpn chi thi sinh hpc mdt each dl dang. Mot trong nhiJng no lye ddu tien de xac dinh he protein nudc tilu da duac cdng bd ndm 2001 (Sparh et al, 2001). Sij dyng LC-MS, cde phdn manh peptide cua cdc mau nudc tieu da dupc thuy phan bdng trypsin duoc gdp lai de phan tich va 124 protein da dup^c xac dinh. Sd lupng protein duoc nhan dien tang len dang ke ndm 2006 la 1500 peptide bdng kk hop dien di 1 chi6u vd sdc ky ldng nguac pha kit hcrp vdi khdi phd (Adachi J et al. 2006), da tilp tuc nhdn manh su da dang va phirc tap trong he protein nudc tilu con ngudi. Trong mot nghien cuu gdn day ndm (2008) da xac dinh
dupc 100 000 peptide khde nhau vd cd ft nhat 5000 ldp igi vdi tan suat cao (Coon JJ et al.
2008). Dieu ndy cho thay sy phong phii cua he protein trong nudc tieu, do dd phdn tich hp protein nudc tieu sfi cung cap cho cdc nhd khoa hpc mpt cdng cy mgnh cho vi?e tim kiem, tuyen chpn cdc chi thj sinh hpe cua thdi gh^p cap tinh.
II. 0 6 l TUpNG VA PHUONG P H A P NGHIEN CCU:
1. Vat lieu:
Mau nudc tieu b?nh nhan ghep than cd bien chirng thai ghep cap tinh tuoi tir 20 - 60, duoc cung cap bdi Khoa than & lpc mdu, Bpnh vicn Hiru nghi Viet Dire, Ha Npi.
Cdc hda chat sir dung deu co dp tinh sach can thiet cho sinh hpe phan tir, bp kit phan tdch protein nudc tieu ProteoSpin^'^ Urine Protein Concentration Micro Kit (Norgen Biotek, Canada). He thdng may sir dung ed dp tin cay cao ciing vdi cac trang thiet bi khde ciia Phdng Thi nghiem Trpng diem Cong nghe Gen, Vien Cdng nghe Sinh hpc, Vien Khoa hpc vd Cdng nghp Viet Nam.
2. PhuoTig phap:
Thu nhan hp protein nudc tieu bdng ProteoSpin^'^ Urine Protein Concentration Micro Kit.
Lay 1 ml mau nude tieu, dieu ehinh ve pH 3,5 bdng 40 pi Dem dieu chinh pH. Hoat hda cdt 2 Ian vdi 500 pi Dem rira vd hogt hda cot. Dua 600 pi mau nudc tieu dupc dieu chinh pH vao cdt da boat hda vd ly tam trong thdi gian 2 phut vdi toe dp 7500 vong/phut, thyc hien cho den khi het 1 ml mau. Rira cpt bang 500 |j.l Dem rira \a hogt hoa cot va ly tam 2 phut d toe dp 7500 vdng/phiit, qua trinh rira cdt thuc hien 2 lan. Chuyin cot da dupe rira sang dng'eppendorf ed ehua 9,3 pi 33
HOI NGHI KHOA HQC cCiA H Q I H 6 A SINH Y DUqC H A NQI V A C A C T I N H P H I A B A C L A N THCT XVlll
Neutralizer vd thdi vdi 100 pi Dung djch thdi. Dung djch dupc thdi ra cd chira protein ting s6 da dupc tinh sgch.
Phuang phdp di§n di SDS-PAGE SDS-PAGE dupc thyc hi§n theo Laemmli.
1970 vdi cdc thilt bj ciia hdng Rio-Rad.
Thuy phdn protein trong gel
Cde dilm protein dupc cdt ra vd dua vdo ong eppendof 1,5ml. Protein dupc thiiy phdn bdng trypsin theo quy trinh dd dupc md td bdi VQ Minh Thiet (2006).
Phdn tfch va ghi pho 2D nanoLC- i:Sl- MS/MS.
Hon hpp peptide sau khi thiiy phdn dupc phan tdch tren hp thong sdc ky ldng 2 chieu nanoLC. Qud trinh thyc hipn khoi pho lien tilp dupc tien hdnh tren hp mdy QSTAR'^^
vdi nguon ESI (2D nanoLC - ESI- MS/MS) theo md td bdi Vu Minh Thiet vd cs, 2006.
Nhan dipn protein bdng phan mem Mascot vl.8
Phd khoi MS/MS dupc phdn tich bdng phan mem Mascot vl.8 (Matrix Science Ltd., London, Anh) vd Ngan hdng D\i lieu Protein NCBI (My) tren he thdng mdy chii Workstation XW6200 BASE UNIT vdi hon 7.3 Uipu trinh ty protein cua cdc loai khac nhau.
III. KET QuA NGHIEN CLfU VA BAN LUAN:
1. Tinh sach \a phan doan h^ protein nude tieu bfnh nhan ghdp than cd bien chung thai ghep cap tinh.
Mudi vd cdc tap chat khac trong nudc tieu sg gay dnh hudng rat Idn den qud trinh thi nghiem. Do dd, de loai bdt nhihig chat ndy ben canh cdc phuang phap tinh sach thdng thudng, mau s5 dupc chgy qua ProteoSpin^"^ Urine Protein Concentration Micro Kit. Sau dd se dupc kiem tra tren gel poly acrylamide 12.6 %.
11$ protein nudc tieu b?nh nhan gh^p th$n cd biln chung thdi ghip dp tinh sau khi tinh sgch duprc phdn tdch tren gel polyacrylamide n6ng di 12.6%. Tiep dd, cdc dudng chgy ciia bdn dipn di s5 duprc phdn dogn nhu trfin hinh 1.
kDa M 1 2
'''^•^ BUH^H
62.6-i^ll^ki^
45.0-i w.
35.0-1 — 2 5 5 - ! » -
1 6 4 - — 1
II III
IV
Hinh 1: Dipn di do SDS-PAGE va cac phan dogn cdt gel
M: Thang marker protein (Fermentas) Gieng 1,2: Dudng cha\ protein nudc tieu Phdndogncdtgcl:!, 11,111, IV
2. Nhan diyn MI phiin tich h^ protein nudc tieu b^nh nhan ghep than cd bien chung thai ghdp cap tinh.
Pho khdi thu nhan duprc sau khi phan tich 2D nanoLC-ESI -MS MS sg dupc tim kilm tdng h9p tren eo sd diJ lipu ciia NCBInr va SwissProt bdng phan mem Mascot Vl.8.
Tdng so 5786 manh peptide dugc xdc dinh.
Sau khi sang Ipc nhihig protein vdi dp tin cay eao da thu nhgn dupe tdng sd 132 protein.
Trong tdng sd 132 protein nhgn dipn dupc, cd 12 protein cd ndng dp cao (Bang 1), 72 protein cd ndng dp thdp, vd mdt vdi dong phan ciia cdc chuoi Immunoglobulin.
34
tan Bing/l
STT 1 2 3 4 5 6 7 8 9 10 11 12 72 pro 5cuong
>!<i n Y HQC VlfT NAM T H A N G 8 - 2/2012
: Danh sachpti&tein'nong-d^xao tronR nudc tieu benh nhan thai gh^p than cSp tfnh.
"Tin protein Alpha-1-acid glycoprotein 1 Alpha-1-acid glycoprotein 2 Alpha-1 -antitrypsin Alpha-2-HS-glycoprotein Protein AMBP Serum albumin Zinc-alpha-2-glycoprotein Prostaglandin-H2 D-isomerase Kininopcn-1
Nidogen-1
Alpha-1 -antichymotrypsin Haptoglobin-related protein
So UniProt Q8TC16 Q61B74 Q9UCM3
Q9P152 Q9UDI8 Q9UJZ0 Q8N4N0 Q9UCD9 Q7M4PI Q86XD7 Q9UNLJ9 Q9ULB0
Kh6i lirgne 23725 23873 46878 40098 39886 71317 34465 21243 479253 139142 47792 39518
pl 4.93 5.03 5.37 5.43 5.95 5.92 5.71 7.66 6.34 5.12 5.33 6.63 ein nong dp cao dugc phan loai theo vj tri ton tgi trong te bao. Su phan loai kha nSng hieu biet ve s\r tham gia ciia cac protein vao qua trinh b^nh ly.
nay
Hinh 2. Bieu dd phan loai he protein nudc ghep cap tfnh cd ndng dp thap
Trong nghien cuu proteomics, nhimg protein hdm luong thdp thudng dupe quan tdm nghien cuu do nhihig biln ddi trong qua trinh hinh thdnh benh ly. Trong nghien cuu nay, trong tdng sd 72 protein ham luong thap thi protein mang chilm ty le cao nhdt (25%).
Protein mdng thudng la phirc he protein cd khdi lupng phan tu cao. Do dd vipc dupc loc qua cdu than vd cd mat trong nudc tieu loat sau phdn anh nhiing thay doi bdt thudng ve sinh ly than. Ddc biet trong nhdm nay nhieu protein lien quan din thai ghep cdp tinh da duoc cac nhd khoa hoc tren thl gidi nghien ciru: Beta 2 - microglobulin (Getting WS el
tieu benh nhan ghep than cd bien chimg thai al, 2006) Uromoduiin (Susan JA el al, 2010).
IV. KiT LUAN:
Da thu nhan va tinh sach dupc hp protein nudc tieu bdng sir dung ProteoSpin Urine Protein Concentration Micro Kit. Sii dyng ky thuat dien di SDS-PAGE de phan doan he protein vd ket hprp vdi he thong sac ky long nano da chieu ket ndi vdi khoi pho da nhan di?n dupe 132 protein trong nude tieu benh nhan ghep than ed bien chimg thai ghep cap tfnh. Trong do ed 12 protein ndng dp cao, 72 protein ndng dp thap vd mpt sd dong phan cua cac chudi immunoglobulin.
35
Hpl NGHI KHOA Hpc CIJA Hpl H 6 A SINH Y DtUfC HA NQI VA CAC TINH PHIA BAC LAN THg XVlll
TAI U | U THAM KHAO:
1. Adachi, J., Kumar, C , Zhang, V., Olscn, J.V., and Mann, M. (2006). The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins, Genome Biol. 7, pp. 80.
2. Coon, J. J., Zurliig, P and SchmitI, K.P.
(2008), CE MS analysis of the human urinary proteome for biomarker discovery and disease diagnostics. Proleomic! Clin Appl. 5(9) pp, 34-.18
3. Getting, W.S., Rogers, T.B., Krick, T.P., Mai:is, A..I., and llirahim, H.N.
(2006), Urinary beta2- microglobulin is associated with acute renal allograR rejection, Am J Kidney Dis, 47(5), pp, 898-904,
, Spahr, C.S., Davis, M.T., McGinley IM.D., ct al. (2001), Towards defining the urinary proteome using liquid chromatography-tandcm mass spectrometry. 1. Profiling an unfractionated tryptic digest, Proteomics.
1(1), pp. 93-107.
. Susan ,I.A (2010), Transplantation:
Integrative peptidomics identifies acute renal transplant rejection biomarker^, Nature Reviews Nephrology. 6, pp. 249.
. va Minh J hilt, Tran The Thinh, Nguyen Thj Minh Phiromg, Nguyen Bich Nhi, Phan Viin Chi (2006) Phan tich cdc glycoprotein trong huyel thanh ngu6i. Tgp chi cong nghe sinh hoc, 4(1):
13-22.
NGHIEN Ciiu MOT SO CHi S6 LIPID HUYil TimNG Of BENH NHAN SAU GHEP T H A N TAI B | N H V I E N V I | T Oljnc
NguySn Thj Hoa*. Ha Phan Hal An**, Phgm Thifn Sgoc TOM TAT:
Tang lipid trong HT \i biin chi>ng thui/ng gap sau ghSp than, c4c thu6c ire chk m\hn djch (LTCMD) c6 anh hifong khic nhau din n6ng se>
mjt si5 chi s6 lipid trong huyit tirong (HT) Mi,ic tieu: 1) Khio sit ndng af mfl s6 chi s6 lipid trong HTib$nh nhin sau ghip thin 2) So sinh 31/ thay aSl mit s6 chi s6 lipid trong HT d binh nhin sau ghip thin Oiiu trj bing cyclosporin hoic tacrolimus. B 6 | tu'i/ng phu'vng phip nghiin cipu: NghiSn ciru mO ta, tiin ciru trSn 26 b$nh nhan suy thjn man giai Jogn cu6i da dupc
* Truing Bai hoc Y Duvc Thii Nguyin.
"Trudng Dai itQc Y Hi Nti 36
ghSp than tai Banh vian Viat Oirc nam 2009- 2010 nhSm 1) 9 banh nhan diiu trj bang cyclosporin (Neoraf- Novartis-CsA). nh6m 2) 17 banh nhan diiu tri bang tacrolimus (Prograf', Astellas-Tac), banh nhan dirvc I4y mSu tai thin diim trudc ghap than, sau ghap than 3 thang vi 6 thang Kit q u i : Ndng JJ cholesterol trong HT tai thiri aiim trudc ghap than, sau ghap than 3 thang, 6 thang tuong irng la 3,83±0,93mmol/L, 5,88±1,24mmol/L, 5,32±1,09mmol/L, nhbm banh nhan diiu trj bang CsA c6 xu hudng cao hon so vdl nhdm banh nhan diiu tri bang Tac. Ning dO