84 KHOA HQC CONG NGHe BUtiC DAU XAY DUNG QUY TRiNH PHAT HOT EBNA-1 O CAC MAU MO THU NHAN 're 13tNH NHAN UNG THU' VOM HONG NO), nhan bai: 16/06/2015 Ngay nh4n loi: 11/07/2015 Ngay duy4t clang: 26/10/2015 Lao Dar Thutini Nguyen Hoeing Anh Tuan2 Nguyen Thi Yein Tuyit3 Nguyen Him Diing4 Le Huy'dn Ai Thay5 TOM TAT TO Viet Nam, ung tint vont hong la clang ung tint pha bd'n nit& cac benh ung tint thuac vitng deiu va c6 Nhieu nghien thu da charzg minh rang su xam nhiem caa Epstein — Barr virus (EBV) co mai quan he mat thiet va la mat nhimg nguyen nhein gay benh ung tint yam hong Nham intang tai viec xely dung quy trinh than clown sant benh ung tint vOm hong, chting toi lua chon gen EBNA-1 (Epstein — Barr nuclear antigen 1), mat gen can thief cho su xam nhilm diet EBV, lam gen muc tieu cho viec xeiy dung quy trinh phat hien su hien dien cita EBV Trong nghien cau buoy dau, chting toi tien hanh thong ke tan so trung binh so ve su hien dien cart EBV nhieu loaf mau benh pheim khac Dong thai, thong toi da xely dung thank tong quy trinh PCR phat , hien gen EBNA-1 tren cac man mo benh ung tint vorn hong thu nhein tit Benh Vin Cho Ray Ket qua nghien thu &tor dew fa tien de cho viec pheit trien cac pingang phap than clown khong xam lan nham than clown sem: benh ung tint yam hong duct tren Icy thuat sinh hoc phein to tren claf tztong cac gen lien quan den su xam nhiem adz EBV Tir khoa: EBV, EBNA-1, ung tint vOm hong ABSTRACT In Viet Nam, Nasopharyngeal carcinoma (NPC), originating in the nasopharynx, is the most common cancer of the head and neck cancers The association of NPC with Epstein — Barr virus (EBV) firmly demonstrated that EBV played an important role in nasopharynx carcinogenesis For the aim to establish the method for early diagnosis and prognosis of NPC, EBNA-1 (Epstein — Barr nuclear antigen 1) which is necessary for infection of EBV, was chosen as target gene to establish the method for detection of EBV In this study, we systematically analyzed the mean of percentage weighted of the presence of EBNA-1 in various sources of sample Moreover, we initially succeed to establish the PCR method for detection of EBNA-1 in biopsy tissues collected from nasopharyngeal cancer patients in Cho Ray hospital These results indicated that, in the future, it will be useful in developing a non-invasive method for early diagnosis and prognosis of NPC based on molecular techniques in detection of concerning genes which were EBV infection required Keywords: EBNA-1, EBV, Nasopharyngeal carcinoma ' ThS, Trutmg Dai hoc Ma TP.HCM Tnrtmg Dai hoc Ma TP.HCM Trutmg Dai hoc Ma TP.HCM TS BS, B'e'nh vi4n Chq Ray TP.HCM PGS.TS, Truting Dai hoc Ma TP.HCM Email: thuy.lha@ou.edu.vn TAP CHI KHOA HOC TRUONG DAI HOC MO TP.HCM - 56 (45) 2015 Giiri thieu Ung thu yam hong (Nasopharyngeal carinoma) mot khoi u dc tinh xuat phat tir lap bleu mo cua yam hong DOng thai, day la loai ung thu pH) bien nhat cac benh 15, vimg dAu (Phiic va Ong su, 2008; Yang va cong su, 2014) TS, le mac benh ung thu yam hong c6 su khdc biet dia ly gicra cdc khu vuc khdc Trong do, Viet Nam norm khu vuc c6 tS/ le mac benh cao, cu the the theo thOng ke norm 2012 (WHO), thAy tj, le mac benh ung thu vom hong va tir vong Ian lugt len den 3,9% va 3,3% dan so (World Health Organization, 2012) EBV (Epstein - Barr virus) la, virus co tinh nhiem rung cong dOng dugc phat hien a 90% ngtthi tnrang thanh, thuOc nh6m Herpes virus type va dugc nghien cim rat sam tir norm 1980 (Philc va Ong su, 2008; Niedobitek, 2000) Su xam nhiem cua EBV la mot ba nguyen nhan chinh bao gem: ,(1) su xam nhiem EBV; (2) moi truong song thOi quen sinh hoot; (3) bien doi di truyen dAn den hinh thinh ung thu vom hong (Frappier, 2012; Tao va cong su, 2007) Tren the giai, rat nhieu cong trinh nghien ciru dor chimg minh su hien dien cua phan tir DNA, RNA, cac protein cua virus bieu hien a cac to bao ung thu yam hong va cac to bAo bf nhiem EBV có nguy ca tie'n trien ung thu vom hong Chang Ilan, nghien ciru dm Chang va Ong su (1990) thuc hien ten 50 mh ,m13 yam hong khong biet hoa, ket qua cho thay EBVDNA hien dien cac mh mo vai lugng ban rat cao (Thompson va cong su, 2004) EBNA-1 (Epstein - Barr nuclear antigen 1) la mot gen thuOc ho EBNAs ram bO gen cua EBV, co vai tro quan trong quA trinh )(am nhiem, nhan len va tri bO gen EBV mo ung thu vom hong [Xing thai, EBNA-1 clang vai tre ‘quan trong viec tri thdi tiem tan cua EBV (Chang va cong su, 1990; Frappier, 2012; Tao va cong su, 2007) Cu the horn, EBNA-1 ma hoa cho protein EBNA-1, EBNA-.1 tuong tdc vai vi tri OriP, diem tri khai dau chdp, khoi phat su nhan len cua EBV-DNA 85 (Frappier, 2012; Thompson va cong su, 2004) Hon nita, EBNA-1 c6 vai tro viec kich hoot su bieu hien dm cac gen khdc giai doan tiem tan nhu LMP-1,LMP-2, BARTs, (Brooks va cong su, 1992; Frappier, 2012; Thompson va cong su, 2004) Do do,,gensEBNA-I va san phAm cu n6 &Km tim thy hau het cac khoi u vom hong lien quan den EBV (Brooks va cong su, 1992; Phiic va cong su, 2008; Zhang va cong 2012) Chinh dAc diem dor ma mot khuynh huang sir dung EBV-DNA hay cac gen lien quan nham phdt trien mot ky thuAt chan dodn sam khong xam Ian benh ung thu yam hong, tir nang cao hieu qud dien tri va khA nang song sot cua benh nhan Trong nghien ciru nay, chimg toi he thong hoa 14i cac du lieu khoa hoc nghien dm lien quan ten doi tugng ung thu yam hong va gen EBNA-1 cac loaf benh pham khdc Dong thai, bulk dau xay dung quy trinh PCR phat hien su hien dien cua gen EBNA-1 cac mo sinh thiet tir benh nhan yam hong dugc Itru gift va bao quan hai dung moi khac Formalin va PBS V4t lieu va phtrang phi') Mau nghien cuv , mh mo sinh thiet tir benh nhan dugc chan doan mac benh ung thu vom hong thu nhAn tir benh vien Chg RAy Thanh phO HO Chi Minh Cdc mh mo dugc Itru gift bao quan a nhiet dO °C hai loci dung moi khac nhau: formalin va PBS (Phosphate buffer saline) Khai thac dir lieu Cdc bai bdo du.' lieu khoa hoc lien quan dugc khai thac tren ca sor dft lieu NCBI (www.ncbi.nlm.nih.gov) bang cac tir kh6a nhu: nasopharyngeal carcinoma (ung thu vein hong), EBNA-1, EBV (Epstein - Barr virus), non-invasion method (phucmg phdp khong xam ran), v.v Dit lieu khai thdc duce sir dung de thOng ke, xac dinh tan so trung binh co so cua su hien dien cua gen EBNA-1 tren nhieu loci mau benh pham ung thu khdc nhu mau dich phet, mo sinh thiet, mdu, v.v bang cac ky thuAt sinh hoc phan tir khac 86 KHOA HOC CONG NGHe nhu PCR, Real-time PCR, v.v 'rich chiet DNA Cac mh benh pham dtroc tach chiet theo phuong phap Phenol/Chloroform theo quy trinh tach chiet cua Chomczynski va Sacchi (1987) c6 su cai Nen Phenol tir pH=4 pH=8 (Chomczynski, 1987) NOng dO va dO tinh sach DNA tir cac mh benh pham sau tach chiet &roc xac dinh bang phuong phap mat dO quang thong qua ti le A260/A280 nm Sau tach chiet, DNA mh &roc luu TE (Tris-EDTA) va bao quan a nhiet dO -20 °C cho den sir dung sau Phin irng PCR khuech did gen EBNA-1 Trinh to cap moi sir dung phan PCR khuech dai gen EBNA-1 duoc tham khao tir cong trinh nghien ciru cita Adam va cOng su (2011) Trinh to cap moi cluoc the hien O Bang Bing Trinh tir cep moi sir dung nghien cirti Ki hieu Trinh to EBNA-F (M6i xtoi) TGAATACCACCAAGAAGGTG EBNA-R (MOi ngugc) AGTTCCTTCGTCGGTAGTC Chieu dai san pham 223 by Chu trinh phan *rig PCR &roc thiet ke nhu sau: chu ky yen 95°C-5 phut, 40 chu kS, 14p lai yeti 95°C-30 giay, 42,5°C-30 gifiy, 72°C-30 giay va chu kS' vai 72°C-5 phitt Tiing the tich phan ung la 15 bao gOm: 7,5 Mastermix 2X, 1.t1 moi (0,5 IA moi xuoi, 0,5 IA moi ngugc), 5,5 IA ddH2O, 1.11 DNA San pham phan ung PCR dugc kiem tra bang phuong phap dien di teen gel agarose 1,5% va gui giai trinh to tai cong ty Nam Khoa Ket qua giai trinh to dugc tier' hanh hieu chinh vi kiem tra mirc dO tucmg dOng tren co sa dt lieu Genbank (NCBI) Ket qui vi thio luin Khai thic dir lieu Cho den nay, chimg toi khai thac dtroc 13 ding, trinh nghien ciru ten the giai lien quan de tin hanh thong Ice danh gia ve loai mAussir dung, ca mh, phuong phap sir dung nham phat hien su hien dien cita gen EBNA7 I tren berth ung thu vOm hong Ket qua thong ke dtrac the hien ei Bang Bing ThOng la loai mau vi phirorng phip dime sir dung phit hien EBNA-1 MO sinh • thiet , Dich phet Mau Mo (tic parraffin TOng Ong n(%) Multiplex Methylation Specific PCR 1 - - (15,4) Nucleic Acid Sequence — Based Amplification - - - (7,7) - (15,4) Real — Time PCR PCR 1 - (30,7) Nested PCR - - - (7,7) 87 TAP CHI KHOA HQC TRLIONG DAI HOC M6 TP.HCM - S6 (45) 2015 MO sinh , thiet , Dich phet Mau MO Mc pang& T6ng cOng n(%) Nested PCR and Sequencing - - - (7,7) Quantitative PCR 1 - - (15,4) T6ng Ong (46,1) (30,8) (15,4) (7,7) 13 (100,0) Ghi chic - khong co sti clung nghien thu Ket qua th6ng ke cho thy phucrng phap sir dung chit yeu viec phat hien EBNA-1 cac nghien ciru tren the Oen la phucmg phap PCR (chiem 30,7%) Ben canh do, ve loai mh sir dung, chit yeu la mo sinh thiet (46,1%) va dich phet (30,8%) ,Tir cor o.sdir lieu thu thap dugc, chang Vol tien hanh thong ke tan s6 trung binh phat hien gen EBNA-1 nhieu loai mh mo khac dugc the hien a Hinh Tin so phiit hien EBV (%) 100 n=