Y HQC VigT NAM THANG 8 - SO 2/2011
PHAN TICH CAC PROTEIN BIEU HIEN KHAC BIET cClA MO GAN 6 BENH NHAN UNG THU GAN
Biii Phuong Thao, Le Lan Phuong, Trinh Hong Thai (*)
TOM T^T
Ung thu- te bao gan la mgt trong nam benh ung thu* pho bien nhat d Viet Nam. Tuy nhien, cho den nay, chi cd mgt sd it nghien ciiu proteomic du'dc thi/c hien tren benh nhan ngu'di Viet Nam. Sii dung ky thuat dien di 2 chieu de phan tach protein, 39 spot bieu hien khac biet giiJa mo gan ung thu" va mo gan lan can u cua cung benh nhan da du'dc xac djnh. Trong so cac spot tren, 32 protein da du'dc nhan dang, trong dd 26 protein da du'dc djnh danh va 6 protein gia thiet.
Nhu'ng protein da djnh danh du'dc chia thanh nam nhdm chiic nang: Nhdm cac protein lien quan den chu trinh te bao, tang sinh te bao va chet theo chu'dng trinh (4 protein, 15,4%); nhdm protein lien quan den qua trinh miln djch va bao ve cd the (5 protein, 19,2%); nhdm protein tham gia vao cac qua trinh trao doi chat (9 protein, 34,6%); nhdm protein tham gia vao qua trinh phien ma, djch ma va cai bien sau phien ma, djch ma (6 protein, 23,1%); nhdm tham gia cau true cua te bao (2 protein, 7,7%). Dac biet, mgt so protein cd tiem nang trd thanh cac chi thj ung thu' nhu': Annexin V (bieu hien tang) va p53 (bieu hien giam) d md gan ung thu' cua benh nhan ung thu'gan.
Tijrkhda:D\en di hai chieu (2-DE), ung thiT gan (HCC), proteomics,
SUMMARY
ANALYSIS OF DIFFERENTIALLY EXPRESSED PROTEINS IN LIVER TISSUE OF HEPATOCELLULAR CARCINOMA PATIENTS
Hepatocellular carcinoma (HCC) is one of the five most common cancers in Vietnam. However, up to now, only a few proteomics studies have been done in Vietnamese patients. Using 2- dimentional electrophoresis to separate proteins, 39 spots of proteins that differentially expressed between the tumor tissue and matched adjacent tissues were detected. Among the above spots, 32 proteins were identified, corresponding to 26 named proteins and 6 hypothetical proteins. These proteins can be divided into five functional groups, including: Proteins involving in cell cycle, cell proliferation and apoptosis (4 proteins, 15,4%);
proteins involving in immune response and body protection (5 proteins, 19,2%); proteins involving in metabolism process (9 proteins, 34,6%);
proteins involving in transcription, translation and modification of post-transcription, post-translation (6 proteins, 23,1%); proteins involving in cell structure (2 proteins, 7,7%). Especially, some potential proteins which can be a candidate of biomarker are annexin V (up-regulated) and p53 (down-regulated) in HCC liver tumor tissue.
Keywords. 2-dimentional electrophoresis (2- DE), hepatocellular carcinoma (HCC), proteomics
I. OAT V X N €>E
Ung thu gan te bao gan (Hepatocellular Carcinoma-HCC) la dang ung thu xukt phat tir cac te bao bieu mo gan, chiem hon 90%
cac trudng hgp ung thu gan nguyen phat.
Viet Nam la nude co ty le mac ung thu gan nguyen phat cao, do nude ta thuoc khu vuc cd ty le nhiem virus viem gan B (HBV) cao.
(*) Phdng Proteomics va Sinh hoc can tnic, thuoc Phdng thi nghiem Trong diem Cong nghe Enzyme va Protein,TriCdng Dai hoc Khoa hoc Ttf nhien, DHQG Hd N6i
HQI NGH! KHOA HQC COA HQI HOA SINH Y DUQC HA NQI VA CAC TINH PHIA BAC LAN THifXVII
Nhiem HBV. viem gan C (HCV) man tinh va xo gan dugc cho la cac nguyen nhan chinh dan den ung thu gan [7]. Do do, viec phat hien sdm ung thu gan rat quan trgng doi vdi viec dieu tri va giam ty le tur vong d loai img thu nay.
Mo gan cua benh nhan ung thu gan cd kha nang tong hgp nhieu protein lien quan den khoi u. Hien nay, alpha-fetoprotein (AFP) da dugc sii dvmg rgng rai nhu mgt chi thi img thu gan. Tuy nhien, do dac hieu cua cac xet nghiem chan doan dua vao AFP con thap. Vi vay, gan day, cac nghien ciiu proteomics dang hudng den phan tich he protein md gan nham tim ra nhiing chi thi mdi dac hieu hon [2,4,6]. Trong nghien ciiu nay, chiing toi su dung ky thuat dien di hai chieu 2-DE (2-Dimensional Electrophoresis) de phan tich bieu hien khac biet ciia protein md gan d benh nhan ung thu gan.
II. Odi TU0NG VA PHUONG PHAP NGHIEN COU
2.1. Doi tirong nghien cihi
Mau nghien ciiu la mo gan tai vi tri khdi u cua benh nhan ung thu tk bao gan va md gan lanh each 5-10cm tren la gan dd tai vi tri khong cd khdi u. Mo gan do Khoa Tk bao- Giai phau benh, Benh vien K Tam Hiep cung cdp.
2.2. Phu-ong phap nghien cihi
Chuan bi mau cho buac dien di hai chiiu. Mau mo gan (O.lg) dugc nghien trong dem PBS 0,05M (pH 7,4), sau do bo sung dem lysis chiia 7M urea, 2M thiourea, 30mM Tris-base, 4% w/v CHAPS, 65mM DTT.
Ham lugmg protein trong mlu dugc dinh lugng bang phuong phap Bradford sur diing albumin huySt thanh bo lam chuin.
Dien di hai chiiu (2-DE). TiSn hanh dien di chieu mgt tren thanh gel IPG Strip (11cm, pH 3-10 va 17cm, pH 4-7) dk phan tach
protein theo diem dSng dien sii dimg he thdng Protean lEF cell (BioRad). Sau dd, thanh gel dugc can bSng ikn lugt trong dem can bang I va II rdi dugc dien di chieu hai dl phan tach cac protein theo khoi lugng phan tii tren ban gel polyacriamide 10%. Ban gel dugc nhugm vdi Coomassie Brilliant Blue G- 250 qua dem.
Phan tich hinh anh. Ban gel da nhugm dugc quet va phan tich bSng phan m§m phan tich hinh anh dien di 2 chieu Phoretix (Shimadzu, Nh$t). Cac protein dugc dinh danh thong qua co sd dur lieu ve ban gel dien di hai chieu tren trang web ExPASY [8] va ddi chieu vdi cac ket qua nghien ciiu trudc dd.
III. KET QuA
Su dung thanh gel IPG strip I Icm pH 3- 10 de phan tach cac protein, chiing tdi thSy cac spot protein tap trung d phkn giiia ban gel trong khoang pH 4-7, hon nfta, tren ban gel van cd cac vung spot tap trung vdi mat do cao va chua phan tach dugc. Do dd, chiing tdi tiep tuc tien hanh dien di 2-DE su dung thanh gel IPG strip 17cm pH 4-7, khi do, cac protein trong mau mo gan dugc phan tach tot hon.
Khi so sanh sir bilu hien cua cac spot tren cac cap ban gel da xac dinh dugc 27 spot cd bilu hien khac biet d ban gel 1 Icm pH 3- 10 (Hinh 1) va 36 spot protein khac biet ro rang d ban gel 17cm pH 4-7 (Hinh 2). Trong s6 do cd 24 spot khac biet cd d ca hai cap ban gel dien di tren, gdm: 8 spot chi cd d ban gel mo ung thu, 5 spot chi cd d ban gel md doi chiing, 8 spot cd bilu hign giam va 3 spot cd bilu hien tang d ban gel mo ung thu so vdi md binh thucmg; 3 spot khac bigt chi cd d ban gel 11cm, pH 3-10; 12 spot khac biet chi cd d ban gel 17cm, pH 4-7.
Y H Q C V i g T NAM T H A N G 8 - S O 2/2011
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A: Mo gan binh thuong B: Mo gan ung thu
Hinh 1. Cac spot protein bilu hien khac biet giiia hai ban gel 2-DE mlu protein mo gan ung thu so vdi mo gan binh thuong. IPG strip 11 cm, pH 3-10.
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A: Mo gan binh thucmg B: Mo gan ung thu
Hinh 2. Cac spot protein bilu hien khac biet giiia hai ban gel 2-DE mau protein md gan ung thu so vdi mo gan binh thuomg. IPG strip 17cm, pH 4-7.
Nhu vay, chiing toi da xac dinh dugc tdng so 39 spot bieu hien khac biet giura ban gel md gan ung thu so vdi ban gel md gan binh thudmg, budc dau nhan dang dugc 32 spot protein tuomg ling vdi 26 protein da dugc dinh danh va 6 protein gia thilt, ben canh dd vln con 7 spot chua xac dinh dugc. 26 protein dugc djnh danh dugc chia thanh nam nhdm chiic nSng. Nhdm cac protein lien quan din chu trinh te bao, tSng sinh tl bao va apoptosis (4 protein, 15,4%), gom: Proliferating cell nuclear antigen ( t ^ g sinh tl bao, dilu khien ADN v^ sua chiia ADN); Cellular tumor antigen p53 (protein lic chi khdi u, dilu boa sir tang sinh te b^o, kiem soat chu trinh te
bao); Annexin V (khang lai apoptosis, chdng ddng mau); KIAA0979 (lic chi tl bao tang sinh va lam ben lien ket giiia cac nhiim sic tur chi em). Nhdm protein lien quan den qua trinh mien dich va bao ve co the (5 protein, 19,2%), gom: Hapoglobin (a, P) (Lien ket vdi Hemoglobin); HLA classic I (dap umg mien dich, tham gia tuong tac giura virus va vat chii); HLA-A2 antigen (chi biln va trinh dien khang nguyen); Superoxide dismutase [Cu-Zn], Superoxide dismutase [Mn] (pha hiiy cac gdc tu do, bao ve co thi). Nhdm protein tham gia vao cac qua trinh trao ddi chat (9 protein, 34,6%), gdm: Poly (ADP- ribose) glycohydrolase (tham gia qua trinh
HOI NGHI KHOA HOC C O A HQI HOA SINH Y Dl/QC H A NQI vA CAC T I N H PHIA BAC LAN THIJf XVII
trao doi carbohydrate); Glyceraldehyde-3- phosphate dehydrogenase (tham gia vao qua trinh dudng phan, van chuyen cac chat qua mang te bao); H^ transporting 2-sector ATPase beta chain precursor (van chuyen ion H"^^; Glutamate dehydrogenase (truyen xung than kinh); CATD_ Cathepsin D (proteinase ngi bao); Proteasome subunit beta type-3 (phan cat lien ket peptide);
KYC_UMP_CMP kinase (chuyin gdc phosphate tir ATP sang UMP va CMP, tong hgp Pyrimidin); Phosphoinositide-3-kinase regulatory subunit 3 (enzym chuyen goc phosphate); ECHMEnoyl-CoA hydratase, mitochondrial (trao doi lipid). Nhdm protein tham gia vao qua trinh phien ma, dich ma, cai bien sau phien ma, dich ma (6 protein, 23,1%), gdm: FAM64A protein (tham gia vao qua trinh bien doi sau phien ma); Finger protein HPF4 (lic chi phien ma); MAX protein, isoform b (dilu hoa phien ma);
SUMO-3 protein (pha vo va tai tao lien kit S-S); PDIAl_protein disulfide isomerase 1 (pha vo va tai tao lien kit S-S); Protein disulfide isomerase A3 (tai sip xIp lien kit S-S). Nhom protein tham gia cau tnic cua tl bao (2 protein, 7,7%), gom: ACTB_ Beta actin (cau tnic nen tl bao); MAP IA (protein lien ket va cau tnic).
IV. BAN LUAN
Dua tren ket qua nhan dang protein nhu tren, chiing toi da xac dinh dugc 4 protein tham gia chu trinh tl bao va apoptosis, chiing cd vai tro bet siic quan trgng trong viec tdn tai cua te bao ung thu. Trong do dang chii y la Annexin V. C5 giai doan dku ciia qua trinh apoptosis, tinh bit ddi xiing ciia lop phospholipid mang tl bao bi pha vo lam bgc 16 cac phan tu Phosphatidylserine (PS) tren mang te bao chat, Annexin V cd ai luc cao
vdi PS (khi cd mat ion canxi) lam bat boat chiic nang truyin tin hieu apoptosis cua PS, nhu vay, te bao cd the thoat khdi qua trinh apoptosis [5]. Protein nay bieu hien tang trong mo ung thu [2].
Trong nghien ciiu nay, chiing toi da nhan thay protein lic che khdi u (Cellular tumor antigen p53) bieu hien giam d mo ung thu so vdi mo doi chiing. Protein nay boat dgng nhu mgt chat lie che khoi u. p53 cd thi gay ra apoptosis theo hai co che hoan toan ddc lap.
Con dudng thii nhat phu thuoc vao chiic nang phien ma ciia p53. Con dudng thii hai doc lap vdi chiic nang phien ma cua p53 nhung phu thuoc vao tuong tac protein- protein cua p53: protein p53 cd the lien kit vdi cac protein cua qua trinh sinh tong hgp ADN [3].
Ben canh do, chiing toi ciing danh gia vai trd ciia cac phiic he phd hgp td chiic mo (HLA). Trong nghien ciiu nay, protein HLA lop I giam, dieu do cd thi dugc gia thuyet rang qua trinh tien trien thanh ung thu d te bao gan da lam anh hudmg tdi su dap ling miln dich qua trung gian te bao, te bao ung thu khdng bi tieu diet do thilu hut sir nhan dien khang nguyen ngi sinh.
Hai enzym: superoxide dismutase [Mn]
(SODM) va superoxide dismutase [Cu-Zn]
(SODC)-SOD la cac chSt khang oxi hda quan trgng ciia co thi. Nam 2006, kit qua nghien ciiu cua Takashima va cgng su da chi ra rang SODM cd phan img mien dich vdi do dac hieu cao vdi huylt thanh ciia benh nhan HCC [4]. Cac SOD bilu hien giam d md ung thu cho ta thiy vai tro ciia cac phan ling oxi hoa ddi vdi te bao ung thu.
Nhdm protein cd biln ddi nhilu nhat khi tl bao bi tdn thuong dac biet la khi chuyen sang trang thai ung thu la protein tham gia vao cac qua trinh trao ddi chat nhu Glyceraldehyde 3-phosphate dehydrogenase
Y HOC VIET NAM THANG 8 - SO 2/2011
(GAPDH hay G3PDH). Day la mgt enzym (~37kDa) xiic tac phan ling thii sau cua con dudng duong phan. Tuy nhien, gan day cac nha khoa hgc con kham pha ra vai trd cua nd trong qua trinh apoptosis. Nam 2005, nghien ciiu ciia Hara va cac cgng su chi ra ring 2 GAPDH cd thi gay ra apoptosis. Nghien ciiu da chumg minh ring GAPDH bi S-nitrosyl hda bdi NO khi tl bao 0 trang thai stress, khi do, nd se lien kit vdi protein Siahl-mgt ubiquitin ligase. Phiic nay di chuyin vao trong nhan va Siahl se phan giai cac protein trong nhan lam cac tl bao bi apoptosis [1].
Dilu nay giai thich cho kit qua cua chiing toi ring GAPDH khong bilu hien tren ban gel mo ung thu.
V. KET LUAN
32 spot protein tren tdng so 39 spot bieu hign khac biet gifta ban gel mo gan ung thu so vdi ban gel mo gan binh thuomg da dugc xac dinh, trong do 26 protein da dugc dinh danh va 6 protein gia thiet. 26 protein nay dugc chia thanh nam nhdm chiic nang: Nhdm cac protein lien quan den chu trinh te bao, tang sinh tl bao va apoptosis (15,4%); nhdm protein lien quan din qua trinh mien dich va ^•
bao vg co thi (19,2%); nhdm protein tham gia vao cac qua trinh trao ddi chit (34,6%); nhdm protein tham gia vao qua trinh phien ma, dich ma, cai biln sau phien ma, dich ma (23,1%);
nhdm protein tham gia cau tnic ciia te bao (7,7%). Trong s6 cac protein nay cd mgt so protein bilu hien khac biet ro rang, cd tiem g nang trd thanh cac chi thi ung thu nhu Annexin V cd bilu hien tang hay p53 cd bieu hign giam 0 ban gel mo ung thu so vdi md binh thucmg.
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