HOI NGH! KHOA HOC CCiA HOI HOA SINH Y DUOC HA NOI VA CAC TJNH PHIA BAC LAN THifXVII
nang sdng sdt va phat tridn tucmg tu nhu cac td bao khdng bi chuydn nhiem. Nhu vay qui trinh chuydn nhidm siRNA ciia chiing tdi la hoan toan phu hgp vdi te bao ung thu vii MCF7 va cd thd dugc nghidn curu dd ap dung trdn cac ddi tugng td bao khac nfta.
L&i cdm an: Chiing tdi xin chan thanh cam on GS. J M. Pezzuto, trudng Dai hgc Hawaii, Hoa Ky da cung cap cac ddng td bao phuc vu nghidn cihi. Chiing tdi ciing xin cam on Vien Cdng nghe sinh hgc va dd tai CS11_02 da cap kinh phi de hoan thanh cdng trinh nghidn cuu nay.
TAI LIEU THAM KHAO
1. Crocker PR (2002) Siglecs: sialic-acid- binding immunoglobulinlike lectins in cell- cell interactions and signalling. Curr Opin Struct Biol \2:609-6\ 5.
2. Haltiwanger RS, Lowe JB (2004) Role of
6.
7.
glycosylation in development. Annu Rev Biochem 13:49\-S31.
Marcos N T. et al (2004) Role of the Human ST6GalNAc-l and ST6GalNAc-Il in the synthesis of the cancer-associated Sialyl-Tn Antigen. Cancer Res. 64: 7050-7057
Moody A M. et al (2003) Sialic Acid Capping of CD8_ Core 1-O-Glycans
Controls Thymocyte-Major Histocompatibility Complex Class I
Interaction. The Journal of Biological Chemistry. 278(9): 7240-72456
Schauer R. (2000). Achievements and challenges of sialic acid research. Glycoconj.
J. 17 (7-9): 485-499.
Spiro RG (2002) Protein glycosylation:
nature, distribution, enzymatic formation, and disease implications of glycopeptides bonds. Glycobiology 12: 43R-56R.
http://www.ncbi.nlm.nih.gov/sites/entrez7db
=gene&cmd=Retrieve&dopt=Graphics&list uids=6482
PHAT HIEN CAC KIEU GEN (GENOTYPES) VA KIEU GEN PHU (SUBTYPES) CUA VIRUS VIEM GAN C LAY NHliM QT KHU VOD HA NOI
B A N GKY THUAT
GIAI TRINH TlTGENTRirC TIEP
Nguyin Nghiem Luat*, Doan Van Thuyet*, Nguyen Hihi Quyen* va Vo Ngoc Lan*
T 6 M TAT
MUC oiCH: muc dich ciia nghien ciiXi nay la phat hien cac kieu gen (genotypes) va cac kieu gen phu (subtypes) ciia virus viem gan C lay nhilm d khu vi/c Ha Ndi bang ky thuat giai trinh t y gen tru'c tiep. OOI TlTOfNG VA PHl/ONG PHAP:
huyet tu'dng ciia 36 benh nhan bj nhiem HCV cd HCVAb du'dng tinh va sd lu'dng virus HCV RNA
>10^ copies/mL d khu vi/c Ha Ndi nam 2010 du'dc sii dung de phat hien kieu gen HCV bang cac ky thuat tach chiet va tinh sach HCV RNA tdng sd, RT-PCR bien HCV RNA thanh cDNA tu'dng li'ng, cDNA du'dc khuech dai bang ky thuat PCR, sau dd du'dc giai trinh t y tryc tiep bang ky thuat giai trinh t y gen (sequencing) ciia viing dau tan 5' khdng ma hoa (5'-non coding region:
Y HQC VlfeT NAM THANG 8 - SO 2/2011 5'-NC) cua virus. KET QUA: ty le cac kieu gen va
kieu gen phu ciia HCV du'dc xac dmh nhu" sau:
kieu gen 1 la 25 (69,44%), kieu gen 2 la 2 (5,56%) va kieu gen 6 la 9 (25%); kieu gen phu la la 9 (25%), kieu gen phu l b la 16 (44,44%), kieu gen phu 2a la 2 (5,56%), kieu gen phu 6a la 8 (22,22%) va 6c la 1 (2,78%). KET LUAN: (1) kieu gen HCV thu'dng gap nhat d khu vyc Ha Ngi la kieu gen 1, tiep theo la cac kieu gen 6 va 2;
(2) kieu gen phu HCV thu'dng gap nhat la kieu gen phu l b , tiep theo la cac kieu gen phu la, 6a, 2a va 6c.
ABSTRACT
DETECTION OF GENOTYPES AND SUBTYPES OF HEPATITIS C VIRUS SPREAD
IN HANOI AREA BY DIRECT SEQUENCING TECHNIQUE
OBJECTIVE: the purpose of the present study is to detect the genotypes and subtypes of hepatitis C virus spread in Hanoi area by direct sequencing technique. SUBJECTS AND METHODS: HCV genotypes and subtypes from plasmas of 36 patients infected with HCV with positive HCVAb and HCV RNA >10^ copies/mL were determined by total HCV RNA isolation and purification, RT-PCR, Real-time PCR, PCR and direct sequencing techniques of the 5'-non coding region (5'-NC) of the virus. RESULTS: the rates of HCV genotypes and subtypes were determined as follows: genotype 1 is 25 (69.44%), genotype 2 is 2 (5.56%), and genotype 6 is 9 (25%); subtype la is 8 (22.22%), genotype l b is 16 (45.72%), genotype 2a is 2 (5.71%), genotype 6a is 8 (22.85%) and 6c is 1 (2.78%). CONCLUSION: (1) the most common HCV genotype in the Hanoi area is 1, followed by genotypes 6 and 2. (2) the most common HCV subtype is l b , followed by subtypes la, 6a, 2a and 6c.
I. OJ^T V X N OE
Virus vidm gan C (HCV) la mdt virus nhd loai RNA thudc gia dinh virus
flaviviridae [1]. Bd gen ciia HCV gdm mdt chudi dom RNA cd kich thudc khoang 9.400 nucleotid, ma hda cho 4 protein cdu tnic (cac glycoprotein loi va vo El va E2) va it nhSt 6 protein phi cdu tnic (NS2, NS3a / b, NS4a / b va NS5a / b) [3]. Hien trdn the gidi cd khoang 270 - 300 trieu ngudi bi nhiem viem gan C. Khoang 90% sd bdnh nhan bi nhiem HCV cap tinh thudng khdng bieu hien trieu chiing va khoang 70% cac trudng hgp tidn tridn thanh vidm gan man tinh. Didu nay cd thd dan ddn bdnh gan tidn tridn, vidm gan man, xa gan, suy gan va ung thu bidu md te bao gan (hepatocellular carcinoma: HCC) trong vdng 20 ddn 30 nam tiep theo. Cac ydu td lidn quan ddn tien tridn bdnh sau khi nhiem HCV bao gdm kieu gen cua virus, tieu thu rugu ciia bdnh nhan va sd lugng virus. Vide xac dinh kidu gen HCV giiip phan loai kidu gen HCV, xac djnh thdi gian didu tri va du doan higu qua didu tri [3, 4]. Cac bgnh nhan bi nhiem HCV genotype 1 can dugc didu tri trong thdi gian dai ban (48 tuan) trong khi bdnh nhan bj nhiem kidu gen 2 va 3 cdn thdi gian ngan hon (24 tudn) va cd kha nSng dap ling tdt hon ddi vdi su didu tri kdt hgp cua peg- mterferon va ribavirin [4]. d Viet Nam, mdt sd cdng trinh nghidn ciiu vd kidu gen ciia HCV d mdt sd vimg, midn da dugc cdng bd [5, 6, 7].
Tuy nhidn, kidu gen ciia virus HCV gay nhiem d khu vuc Ha Ndi trong may nam gan day cdn chua dugc nghidn ciiu mdt each ddy du. Vi vay, dd phuc vu cho vide chan doan, didu tri va tidn lugng nhiem HCV, muc dich cua nghidn ciiu nay la phat hien cac kidu gen va kidu gen phu ciia HCV lay nhiem d khu vuc Ha Ndi bdng ky thuat giai hinh tu gen hare tidp.
II. OOI Ta^NG VA PHUONG PHAP NGHIEN COU
2.1. Benh nhan va thiit ki nghien ciru:
Day la mdt nghidn cuu md ta cdt ngang. Cac
HQI NGH! KHOA HOC CClA HQI HOA SINH Y DUOC HA NOI VA CAC T I N H PHIA BAC L A N THifXVII
bdnh nhan bi nghi ngd vidm gan virus C d khu vuc Ha Ndi, trong khoang thdi gian tir 3/
2010 ddn 6/ 2011 dugc sang Igc bang each do khang thd chdng vidm gan C (HCVAb) bang ky thuat midn dich hoa phat quang trdn may Cobas 6000. Nhimg ngudi ducrng tinh vdi khang thd khang HCV da dugc lua chgn dd phat hien sd lugng HCV RNA bang each sir dung ky thuat Real-time-PCR. Trong sd 54 bdnh nhan cd HCVAb ducmg tinh, 36 mau huydt tuomg (66,67%) cd sd lugng HCV RNA > 10 copies/mL da dugc lira chgn de dua vao nghidn cuu. Dd bao dam giai trinh tu thanh cdng, 18 bdnh nhan cdn lai cd HCVAb duomg tinh nhung sd lugng HCV RNA < 10^
copies/mL da bi loai khdi nghidn cuu.
2.2. Phuang phdp
Cac mau 2 mL mau tinh mach dugc dugc thu lugm trong cac dng chiia sSn EDTA. Cac mau huydt tuomg dugc tach sau khi ly tam va ngay lap tiic dugc bao quan d - 20 ° C. Huydt tuomg cua 36 bdnh nhan bi nhiem HCV cd HCVAb duomg tinh va sd lugng virus HCV RNA >10VmL dugc su dung dd phat hign kidu gen HCV bang ky thuat giai trinh tu gen true tidp. Trudc tidn, can tach chidt va tinh sach HCV RNA tdng sd tu huydt tucmg. HCV RNA dugc phat hign bdng ky thuat reverse transcriptase PCR (RT-PCR) vdi cac cap mdi dac hieu vdi viing 5' khdng ma hoa 5'-NC (5'- non coding region), bidn HCV RNA thanh cDNA tuomg
ling. Sd lugng HCV RNA dugc xac dinh bdng each khudch dai cDNA, sir dung ky thuat Real-time-PCR, thuc hign trdn may IQ5 ciia BioRad. Dd giai trinh tu gen true tidp, cDNA cung dugc khuech dai bdng ky thuat PCR. Sau dd, cDNA tir san pham PCR da tinh sach dugc giai trinh tu gen (sequencing) true tidp viing ddu tan 5'khdng ma hoa (5'- NC) cua virus bdng kit va thidt bi CEQ 8000 cua Beckman Coulter. Dd xac dinh cac kidu gen va kieu gen phu ciia HCV, cac chudi nucleotid cua kdt qua giai trinh tu dugc so sanh vdi trat tu gen cua HCV tuong ling trong Ngan hang gen Qudc td (GenBank).
III. KET QUA
Nam muoi tu bdnh nhan nghi ngd bi vidm gan C da dugc sang loc de tim khang thd khang HCV (HCVAb). Cac mau tir 54 bdnh nhan duomg tinh vdi khang thd chdng viem gan C da dugc chay RT-PCR dk phat hign HCV RNA. Ba muoi sau bgnh nhan duomg tinh vdi HCVAb va cd sd lugmg HCV
> 10^ copies/mL da dugc su dung dd xac dinh kieu gen va kidu gen phu dua trdn ky thuat giai trinh tu gen true tidp. Kdt qua thu dugc nhu sau:
Vd kidu gen: kidu gen 1 la 25 (69,44%), kidu gen 2 la 2 (5,56%) va kidu gen 6 la 9 (25%) (Bang I v a Hinh 1).
Bang 1. Cac kieu gen va Cac kieu gen
So bgnh nhan Kieu gen phu Sd benh nhan
cidug 1
25 la
9
lb 16
Ic 0
3n phu cua HCV phan bd d khu vuc 2
2 2a
2
2b 0
3 0 3a
0
4 0 4a
0
Ha Ndi 6 9 6a
8
6b 0
6c 1
7
7a 0
8
8a 0 Nhu vay, vd kidu gen, kieu gen HCV thudng gap nhat d khu vuc Ha Ndi la kidu gen 1, tidp theo la cac kidu gen 6 va 2.
Y HQC VigT NAM T H A N G 8 - SO 2/2011
• Tyit%
3 « 6 6 U c kiiu gen HCV
80 70 ; 60 , 50 I 44.44 40 fl 30 >2S 1 20
oTyl«%
10 5.56
0 0 0 0 0 , EJ u EJ
la lb 1c 2a 2b 3a 4a 5a 6a 6b 6c 7a 8a Cic k>4u gen phu cCia HCV
(a) (b) Hinh 1. (a) Ty Id % cac kidu gen va (b) kidu gen phu ciia HCV phan bd d khu vuc Ha Ndi
Ve kidu gen phu: kidu gen phu la la 9 (25%), kidu gen phu lb la 16 (44,44%)), kidu gen phu 2a la 2 (5,56%), kidu gen phu 6a la 8 (22,22%) va 6c la 1 (2,78%) (Bang 1 va Hinh 2).
Nhu vay, vd kieu gen phu, kidu gen phu HCV thudng gap nhat la kidu gen phu lb, tidp theo la cac kidu gen ph\i la, 6a, 2a va 6c.
IV. BAN LUAN
Vigc chan doan nhiem vidm gan vi rus C thudng bdt ddu vdi cac xet nghigm mau huydt thanh dugc sir dung dd phat hien khang thd khang HCV (HCVAb). Thdng thudng, HCVAb cd thd dugc phat hign d 80% so bgnh nhan sau 15 tudn phai nhiem vdi HCV, d > 90%) sau 5 thang phoi nhiem va d >97%
sau 6 thang phoi nhiem. Nhin chung, cac xet nghigm HCVAb cd gia tri chan doan duomg tinh manh ddi vdi su phoi nhiem vdi virus vidm gan C, nhung cd thd bd sdt nhumg bdnh nhan chua phat tridn khang thd (chua chuydn ddi huydt thanh) hoSc nhihig bdnh nhan cd mgt miic do khang thd khdng dii dd phat hign. d cac bgnh nhan suy giam mien dich bi nhiem HCV khdng bao gid cd thd phat tridn khdng thd khang virus va do do, khdng bao gid cd HCVAb duomg tinh khi sang Igc khang thd HCV Vi v^y, xet nghiem dinh lugng HCV RNA phai dugc xem xet khi HCVAb am tinh nhung nghi ngd vd bdnh vidm gan C la cao (yi du, khi cac enzym nguon goc gan nhu ALT trong huydt tuomg
tang cao d ngudi cd ydu td nguy ca vidm gan C). HCVAb duomg tinh cho thdy cd sir phoi nhiem vdi virus, nhvmg khdng thd xac dinh hien tai cd dang bl nhiem HCV hay khdng.
Tdt ca nhumg ngudi cd HCVAb duomg tinh phai dugc xet nghiem bd sung de phat hign su hign didn ciia virus vidm gan C de xac dinh hien tai dang bi nhiem HCV Sir hign didn ciia virus dugc phat hign bang each dinh lugmg so lugmg virus HCV, chdng ban nhu bdng ky thuat Real-time PCR. Sd lugmg virus vidm gan C la mdt ydu td quan trgng trong vigc xac dinh su dap ling vdi didu tri bdng peg-interferon va ribavirin, nhung khdng chi ra miic do nang cua benh cQng nhu kha nang tidn tridn ciia bdnh.
Ci nhiimg ngudi bi nhiem HCV da dugc xac nhan, vide xac dinh kidu gen va kidu gen phu thudng dugc khuydn cao. Thu nghigm kidu gen dugc su dung dd xac dinh thdi gian didu tri cdn thidt va kha nang dap ung doi vdi didu tri bdng peg-interferon va ribavirin, trong khi kidu gen phu cho ngudi thdy thudc bidt vd kha nang dap ung doi vdi didu tri va
HQI NGH! KHQA HQC C O A HQI HOA SINH Y Dl/OC HA NOI VA CAC T I N H PHIA BAC LAN T H Q XVII
nguy ca ddi vdi ung thu td bao gan (HCC) d cac bdnh nhan xa gan cua cac kieu gen phu trong ciing mdt kidu gen. Chang ban, trong so cac kidu gen HCV khd didu tri, kidu gen phu la dap ling dieu tri vdi peg-interferon va ribavirin kem hem cac kidu gen phu lb, 4a va 4d [4], cdn d cac bdnh nhan xa gan, bdnh nhan nhidm HCV kidu gen phu lb cd nguy ca ung thu td bao gan cao hom nhihig bdnh nhan nhidm cac kidu gen phu khac [1].
Su phan tich trinh tu nucleotid (sequencing) cd thd dugc coi nhu la tidu chuan vang (gold standard) cho vide xac dinh cac kidu gen va kidu gen phu ciia HCV. Su phan bd cac kidu gen va kidu gen phu cua HCV khac nhau theo cac viing dia ly: kidu gen la chii ydu thay d Bac va Nam My, Uc, lb chii yeu dugc thdy d Chau Au va Chau A, 2a phd bien nhdt d Nhat va Trung Qudc, 2b phd bidn nhdt d My va Bdc Au, 2c phd bidn nhat d phia tay va nam Chau Au, 3 a phd bidn
d Australia va Nam A, 4a phd bidn d Ai Cap, 4c phd bidn d Trung Phi, 5a phd bien d nam Chau Phi, 6a phd bidn d Hdng Kdng, Ma Cao va Viet Nam, 7a va 7b phd bidn d Thai Lan, 8a, 8b va 9a dugc thdy d Viet Nam, 10a va
1 la dugc thay d Indonesia [2, 3 ].
Cd mdt sd bao cao da dugc cdng bd vd sir phan bd cua kidu gen va kidu gen phu ciia HCV d cac vung khac nhau Viet Nam [5, 6, 7]. Vd kidu gen cua HCV, d khu vuc Ha Ndi, chiing tdi hign mdi chi phat hign dugc 5 kidu gen, dd la cac kidu gen 1, 2 va 6, vdi ty Id kidu gen I la 24 (68,57%), kidu gen 2 la 2 (5,88%) va kidu gen 6 la 9 (25,71%). So vdi cac kidu gen da dugc cdng bd d khu vuc midn Bdc ciia Vign Quan Y 108 nam 2007 va d khu vuc Mien Nam cua Cdng ty Nam Khoa, kidu gen I ciing chidm uu the, tidp sau la kidu gen 6 va kidu gen 2, ridng kdt qua ciia Vign quan y 108, cd thdm kidu gen 7 (8%) va kidu gen 8 (1,33%) (Bang 2).
Bang 2. Cac kidu gen cua HCV dugc phat hign tai Bdnh vign MEDLATEC, Quan Y vign 108 va Cdng ty Nam Khoa
Cac kieu gen ( % )
MEDLATEC (2011) 69,44 5,56 25
BV108 (2007) 65,33 6,67. 18,67 1,33
NAM KHOA (2009) 71,02 10,31 0,05 0,05 18,62
Vd kidu gen phu ciia HCV. d khu vuc Ha Ndi, chiing tdi hign mdi chi phat hign dugc 5 kidu gen phu, dd la cac kidu gen phu la, lb, 2a, 6a va 6c, vdi cac ty Id kidu gen phu la la 9 (25%), lb la 16 (44,44%), 2a la 2 (5,56%), kidu gen phu 6a la 8 (22,22%) va 6c la 1 (2,78%).
So vdi cac kidu gen va kidu gen phu da dugc cdng bd d khu vuc midn Bdc ciia Vign Quan Y 108 nam 2007 va d khu vuc Midn Nam ciia Cdng ty Nam Khoa nam 2009, kidu gen phu lb cung chidm uu thd, tidp sau la kidu gen phu la, 6a, 2a va 6c, ridng kdt qua cua Vign quan y
108 cd thdm kieu gen 7a (8%) va kidu gen phu 8a (1,33%).
Bang 3. Cac kidu gen phu cua HCV dugc phat hign tai Bdnh vign MEDLATEC, Quan Y
vign 108 va Cdng ty N Kieugenphu(%)
MEDLATEC Vien 108 Nam Khoa
la 25,00 20,00 7.80
a m K h o a
lb 44,44 45,33 57,8
Ic 0 0 5,4
2a 5,56 6,67 9,9
2b 0 0 0,4
3a 0 0 0,05
4a 0 0 0,05
6a 22.22 18,67 17,40
6b 0 0 1,2
6c 2,78
0 0
7a 0 8 0
8a 0 1,33
0
Y HQC VIET NAM THANG 8 - SO 2/2011
Dac biet, rieng Bdnh vign MEDLATEC cd phat hien 1 hirdng hgp nhiem HCV kieu gen phu 6c (2,78%) ma trudc do chua dugc phat bidn d Viet Nam (Bang I va Hinh lb).
C4c ty Id kidu gen va kidu gen phu cua HCV cac viing cdng bd cimg cd khac nhau chiit it, didu nay cd thd do vung dia ly, thdi didm nghidn cuu va sd lugng bdnh nhan dugc nghidn ciiu cd khac nhau.
Vide xac dinh cac kidu gen va kieu gen phu cua HCV cd y nghia rdt quan trgng trong vigc giiip ngudi thdy thuoc quyet dinh chgn each kdt hgp thudc khang virus trong didu tri, thdi gian didu tri, danh gia dap ung ddi vdi didu tri va tidn lugng kha nang xa gan ciing nhu ung thu te bao gan.
V. KET LUAN
(1) Kidu gen HCV thudng gap nhat d khu vuc Ha Ndi la kidu gen 1, tidp theo la cac kieu gen 6 va 2;
(2) Kidu gen phvi HCV thudng gap nhdt 1^ kidu gen phu lb, tidp theo la cac kidu gen phy la, 6a, 2a va 6c.
TAI UEU THAM K H A O
1. Bruno S, Silini E, Croslgnani A, et al (1997).
Hepatitis C vims genotypes and risk of hepatocellular carcinoma in cirrhosis; a prospective study. Hepatology, 25 (3): 754-758.
2. Dev, A T., McCaw, R , Sundararajan, V., 7.
Bowden, S. & Sievert, W. (2002). Southeast Asian patients with chronic hepatitis C: the impact of novel genotypes and race on treatment outcome. Hepatology 36,1259-1265.
Doi H, Apichartpiyakul C, Ohba K I, Mizokami M & Hotta H (1996). Hepatitis C virus (HCV) subtype prevalence in Chiang Mai, Thailand, and identification of novel subtypes of HCV major type 6. J Clin Microbiol 34: 569-574.
Legrand-Abravanel F, Colson P, Leguillementte H, et al (2009). Influence of the HCV subtype on the virological response to pegylated interferon and ribaverin therapy.
J Med Virol; 81 (12): 2029-2035.
Nguyen Dang Manh, Bui Dai, Nguyen Trong Chinh (2007). Nhiem virus viem gan C, cac ydu to nguy ca nhiem HCV va genotype HCV d mgt so doi tugng nguy ca cao. Du(rc Ldm sdng 108; 2.
NguySn Thanh Bao va Pham Hiing Van (2009). Ap dyng ky thu|it giai trinh t\f tryrc tiep san pham PCR thu nh?ln dugc tir thir nghigm RT Realtime PCR viing 5'-NC de lam xet nghiem djnh kidu gen HCV. Y hoc Thanh phd Hd Chi Minh; 13 (1): 243-248.
Song P, Due DD, Hien B. et al. Markers of hepatitis C and B virus infections among blood donors in Ho Chi Minh City and Hanoi, Vietnam. Clin Diagn Lab Immunol 1994; 1(4): 413-318.
