Nghiên cứu tính đa hình thái đơn của gen ALDH2 trên bệnh nhân ung thư gan nguyên phát

I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN TRN C TRANH NGHIấN CU TNH A HèNH THI N CA GEN ALDH2 TRấN BNH NHN UNG TH GAN NGUYấN PHT LUN VN THC S KHOA HC H Ni - 2016 I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN TRN C TRANH NGHIấN CU TNH A HèNH THI N CA GEN ALDH2 TRấN BNH NHN UNG TH GAN NGUYấN PHT Chuyờn ngnh: Sinh hc Thc nghim Mó s: 60 42 01 14 LUN VN THC S KHOA HC NGI HNG DN KHOA HC: TS Trn Huy Thnh PGS.TS Hong Th M Nhung H Ni - 2016 LI CAM OAN Tụi xin cam oan kt qu khúa lun ny c thc hin ti Trung tõm nghiờn cu Gen v Protein,Trng i hc Y H Ni cha c ng ti trờn bt c mt hay mt cụng trỡnh khoa hc no khỏc Cỏc bi trớch dn u l nhng ti liu ó c cụng nhn H Ni, ngy 29 thỏng 12nm 2016 Hc viờn Trn c Tranh i LI CM N hon thnh lun ny, tụi xin gi li cm n chõn thnh v sõu sc n PGS TSHong Th M Nhung v TS Trn Huy Thnh - ngi thy ó tn tỡnh ch bo, hng dn v b sung cỏc kin thc cn thit cho tụi sut quỏ trỡnh thc hin lun tt nghip ny Tụi cng xin gi li cm n n GS.TS T Thnh Vn - Giỏm c trung tõm Gen v Protein, Trng b mụn Húa sinh Trng i hc Y H Ni v PGS.TS Trn Võn Khỏnh - Phú giỏm c trung tõm Gen v Protein trng i hc Y H Ni, cựng ton th cỏc thy cụ, cỏc anh, cỏc ch v cỏc bn hc viờn ó to iu kin v hng dn, giỳp tụi quỏ trỡnh hc k thut, hon thin quy trỡnh k thut v phõn tớch kt qu ti Trung tõm Xin cỏm n cỏc thy cụ b mụn Sinh hc T bo, cỏc thy cụ khoa Sinh hc trng i hc Khoa hc T nhiờn, to iu kin v cng c thờm rt nhiu kin thc cho tụi quỏ trỡnh thc hin v hon thnh lun ng thi cng xin gi li cm n sõu sc n gia ỡnh, ngi thõn v bn bố ó ng h v ng viờn tụi sut quóng thi gian va qua H Ni, ngy29 thỏng 12 nm 2016 Hc viờn cao hc khúa Trn c Tranh ii MC LC T VN CHNG TNG QUAN TI LIU 1.1 S lc v ung th gan nguyờn phỏt 1.1.1 T l mc UTGNP trờn th gii v Vit Nam 1.1.2 Cỏc yu t nguy c 1.1.3 Lõm sng v cn lõm sng ca UTGNP 1.1.4 iu tr v tiờn lng UTGNP 1.2 H thng enzym ADH v ALDH v chuyn húa ru ti gan 1.2.1 H thng enzym ADH v ALDH 1.2.2 Chuyn húa ru ti gan 11 1.2.3 Chuyn húa ru v ung th gan 12 1.3 S lc v gen ALDH2 16 1.3.1 V trớ v liờn quan 16 1.3.2 Chc nng ca gen ALDH2 16 1.3.3 Tớnh a hỡnh ca gen ALDH 17 1.3.4 Gen ALDH2 v bnh ung th 19 1.4 K thut xỏc nh a hỡnh n gen ALDH2 20 1.4.1 Hin tng a hỡnh n nucleotide (SNP) 20 1.4.2 Cỏc k thut sinh hc phõn t xỏc nh a hỡnh n 22 2.1 i tng nghiờn cu 27 2.2 Thi gian v a im nghiờn cu 28 2.3 Phng phỏp nghiờn cu 28 2.3.1 Thit k nghiờn cu 28 2.3.2 C mu 28 2.3.3 Cỏch thc tin hnh 29 iii 2.3.4 Trang thit b v húa cht 31 2.4 Phng phỏp x lý s liu 34 2.5 o c nghiờn cu ca ti 34 CHNG KT QU NGHIấN CU V THO LUN 35 3.1 c im ca nhúm nghiờn cu 35 3.1.1 c im v tui 35 3.1.2 c im v gii 36 3.2 T l kiu gen v alen ALDH2 nhúm nghiờn cu 38 3.2.1 Kt qu tỏch chit DNA 38 3.2.2 Kt qu khuch i vựng gen ALDH2 cha SNP ti codon 487 40 3.2.3 Kt qu kiu gen v alen ALDH2 41 3.3 Mi liờn quan gen ALDH2 vi mt s yu t nguy c khỏc 47 3.3.1 Gen ALDH2 v tui phỏt hin bnh UTGNP ca cỏc kiu gen .47 3.3.2 Mi liờn quan ca gen ALDH2 vi gii tớnh nhúm ung th gan nguyờn phỏt 48 3.3.3 Mi liờn quan ca gen ALDH2 vi tỡnh trng s dng ru 49 3.3.4 Mi liờn quan ca gen ALDH2 vớ nhim virut HBV 50 KT LUN 52 KIN NGH 53 TI LIU THAM KHO PH LC iv DANH MC HèNH Hỡnh 1.1 Mụ hỡnh ca enzym ADH Hỡnh 1.2 Chuyn húa ru gan v vai trũ hỡnh thnh ung th gan 15 Hỡnh 1.3 V trớ ca gen ALDH2 16 Hỡnh 1.4 S quỏ trỡnh chuyn húa ethanol ti gan 18 Hỡnh 1.5 Minh hin tng a hỡnh n gen (SNP) 22 Hỡnh 1.6 Nguyờn lý k thut PCR 23 Hỡnh 3.1 in di DNA tng s trờn gel agarose 0,8% 39 Hỡnh 3.2 Hỡnh nh minh ho in di sn phm PCR khuch i vựng SNP gen ALDH2 ca nhúm bnh trờn agarose 1,5% .40 Hỡnh 3.3 Hỡnh nh minh ho in di sn phm PCR khuch i vựng SNP gen ALDH2 ca nhúm chng trờn agarose 1,5% 41 Hỡnh 3.4 Hỡnh nh ct on gen SNP codon 487 gen ALDH2 bng enzym MboII trờn mu i chng 42 Hỡnh 3.5 Hỡnh nh ct on gen SNP codon 487 gen ALDH2 bng enzym MboII trờn mu ca bnh nhõn UTGNP 43 v DANH MC BNG Bng 1.1 Cỏc isoenzym ADH ngi Bng 1.2 V trớ ct ca mt s enzym gii hn 24 Bng 3.1 c im v tui ca nhúm nghiờn cu 35 Bng 3.2 c im gii ca nhúm nghiờn cu 36 Bng 3.3 So sỏnh gii ca bnh nhõn UTGNP vi cỏc nghiờn cu trờn th gii v Vit Nam 37 Bng 3.4 c im mc viờm gan B v ung ru ca nhúm UTGNP 37 Bng 3.5 Mt s kt qu o nng v tinh sch ca DNA 39 Bng 3.6 T l kiu gen ALDH2 v alen ca nhúm 44 Bng 3.7 T l kiu gen, alen ALDH2 ca nhúm v nguy c mc UTGNP 45 Bng 3.8 T l kiu gen ALDH2 kt hp ca nhúm v nguy c mc UTGNP 46 Bng 3.9 Trung vtui phỏt hin bnh ca cỏc kiu gen ALDH2 47 Bng 3.10 Gen ALDH2 v gii nhúm bnh 48 Bng 3.11 Gen ALDH2 vi tỡnh trng s dng ru 49 Bng 3.12 Gen ALDH2 vi tỡnh trng nhim virus viờm gan B 50 vi DANH MC CC CH VIT TT ADH : Alcohol Dehydrogenase ADH1A, ADH1B, ADH1C: Alcohol Dehydrogenase 1A,1B, 1C ALDH : Aldehyde Dehydrogenase AA : acetaldehyde bp : base pair (cp ba-z) CI : Confidence Interval ( tin cy) Nu : Nucleotide (hoc Deoxyribonucleotide) A : Deoxyadenosine triphosphate T : Thymidine triphosphate G : Deoxyguanosine triphosphate C : Deoxycytidine triphosphate OD : Optical Density ( hp th quang) OR : Odds ratio (T sut chờnh = t s gia nguy c mc bnh v khụng mc bnh) PCR : Polymerase Chain 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and Derived Gene Frequency" ScienceAsia28: 99-103 PH LC PH LC QUY TRèNH TCH CHIT DNA T MU TON PHN Bc : Loi b hng cu v phỏ v mng t bo - Cho vo ng eppendorf 1,5 ml: 0.5ml dung dch phỏ v hng cu (Lysis buffer) + 0.5ml mỏu ton phn chng ụng EDTA - Trn u, ỏ (10 phỳt) - Ly tõm 8000v/phỳtx 10 phỳt/4C - Loi dch ni, thu cn A Lp li quy trỡnh ny 2-3 ln cho ti ta (nhõn v xỏc t bo) t nõu thnh trng ỏy ng Bc 2: Phỏ v mng nhõn - ng eppendorf1,5ml cha cn A + 0,5ml dung dch K (dung dch h tr phỏ mng t bo v c nh nhõn) - Trn u, 10 phỳt nhit phũng - Ly tõm 8000v/phỳtx 10 phỳt/4C - Loi dch ni, thu cn B - Cn B + 0,5ml dung dch Lysis buffer + 0,5àl SDS 10% + 10àl proteinase K Sau ú em 56C/2h-3h kốm theo lc nh phõn hy protein, hũa tan ta thu dung dch C Bc 3: Ta DNA - Thờm dung dch Phenol: Chloroform: Isoamyl Dung dch C + 0,5ml dung dch Phenol: Chloroform: Isoamyl (25: 24: 1) l dung dch ta protein Trn u v ly tõm 10000v/phỳt x 10 phỳt/4C Hn hp phõn thnh lp: lp trờn cựng cú cha DNA, lp gia l cn t bo, lp di cựng l dch chit Thu lp dch trờn cựng: dung dch D Chỳ ý: Lp li bc ny mt ln na m bo khụng cũn ln cht mu thu dung dch D - Thờm dung dch Chloroform: Isoamyl Dung dch D + 0,5ml Chloroform: Isoamyl (24:1) Trn u v ly tõm 10000v/phỳt x 10 phỳt/4C Hn hp phõn thnh lp: thu lp dch trờn cựng (Dung dch E) Lp li bc ny mt ln na thu dung dch E (ó loi b hon ton protein) - Ta DNA: Trn 1V dch E + 2.5V Ethanol 100% + 0,1V Sodium Acetate 3M Lc u, qua ờm -20C kt ta DNA Ly tõm 13000v/phỳt x 20 phỳt/4Cthu ta F Ta F bng 1ml Ethanol 70%, trn u v ly tõm 13000v/phỳt x 10 phỳt/4Cthu c ta DNA (lp li ln) Ta DNA khụ t nhiờn (56 C), sau ú hũa tan bng m TE hoc nc ct ln - Sn phm DNA tỏch chit c s c kim tra nng v tinh sch bng o mt quang trờn mỏy Nano Drop bc súng 260nm v 280nm - Sn phm t yờu cu t lA260/A280 = 1,8-2,0 Chỳ ý: Sn phm DNA tỏch chit c bo qun 4C thi gian ngn hoc lu gi -20C thi gian di PH LC QUY TRèNH TIN HNH PCR Thnh phn ca mt phn ng (th tớch cui 15L) DNA : 1,5 l (50-100 ng/ml) Mi xuụi : pmol Mi ngc : pmol HotStart Taq Polymerase : l Nc ct va :4,5l Chu k nhit ca phn ng : 94oC : phỳt 94oC : 30 giõy 54oC : 30 giõy37chu k 72oC : 30 giõy 72oC : phỳt PH LC IN DI SN PHM PCR CCH CHUN B GEL AGAROSE 3% - Cõn 3g agarose hũa tan 10ml boric acid EDTA (TBE) (s dng lũ vi súng) Sau agarose tan ht, ngui 55- 60C, vo khuụn gel, tựy thuc vo s lng ging cn cho in di m ci lc lm ging t 4- 6- 8- 12 rng - Pha dung dch TBE 10X : Tris 0,89M; acid boric 0,89M; EDTA 0,02M TIN HNH K THUT IN DI Thnh phn ng chun ng bnh nhõn Dung dch TLPT chun (Hae III) 10l Sn phm PCR - 9l Dung dch m 10X - 1l Tng s 10l 10l - a gel agarose vo mỏy in di, cho TBE n ngp gel - Dựng pipet v u cụn nh hỳt ln lt dung dch mi ng a vo ging (5l/ging) - Mỏy in di 85V (Mupid-Nht Bn), in di khong 100 phỳt - Sau in di, gel c ngõm vo Ethidium bromide phỳt, qua nc ct v a vo soi di ốn UV, chp nh PH LC QUY TRèNH TIN HNH PCR -RFLP - Thc hin phn ng khuych i gen vi cp mi c hiu (xem quy trỡnh tin hnh PCR) - in di kim tra sn phm khuch i gen (xem quy trỡnh in di sn phm PCR) - Thc hin ct sn phm PCR vi enzym c hiu tng ng Thnh phn phn ng ct enzym: STT Thnh phn Th tớch Sn phm PCR 10 l m 10x ca enzym 1.5 l Nc Enzym (10 u/ l) 0.5 l Tng th tớch 15 l l - Hn hp c 37 oC 12-18 gi (qua ờm) - Sn phm ct c in di trờn gel agarose 2-3% ging quy trỡnh in di sn phm PCR Phn ng ct enzym t yờu cu cỏc bng in di rừ nột, khụng cú bng ph PH LC QUY TRèNH TINH SCH SN PHM PCR TRấN GEL AGAROSE S dng Promega Wizard SV gel clean-up system (Promega, USA) Chun b dung dch mng (Membrane Wash Solution) (ch thc hin bt u s dng hp sn phm mi) : Thờm ethanol 95% vo l dung dch mng Lng ethanol cho thờm vo ph thuc vo th tớch ca l dung dch mng (c quy nh sn kit) Ct phn gel agarose cú cha sn phm PCR mong mun (hin th di ốn chiu UV) c lng trng lng ming gel Cho ming gel vo ng cú dung tớch 1,5mL, thờm vo 10 àL dung dch bỏm mng (Membrane Binding Solution) cho mi 10 mg trng lng ming gel Nh nhng trn u hn hp ng v ng 50 - 65oC 10 phỳt hoc cho n quan sỏt thy ming gel tan hon ton Ly tõm ng ton b DNA trung xung ỏy ng t ct lc (SV Minicolumn) vo mt ng thu thp (Collection Tube) Chuyn ton b hn hp gel ó hũa tan vo ct lc v phỳt nhit phũng Ly tõm phc hp ct lc-ng thu thp tc 14 000 vũng/ phỳt phỳt G ct lc ra, b phn dung dch ng thu thp Sau ú t ct lc li ng thu thp Thờm vo ct lc 700 àL dung dch mng v ly tõm tc 14 000 vũng/ phỳt phỳt Lp li bc Thờm vo ct lc 500 àL dung dch mng v ly tõm tc 14 000 vũng/ phỳt phỳt 10 Chuyn ct lc sang mt ng 1,5 mL mi Thờm vo ct 50 àL Nuclease-Free Water nhit phũng phỳt, sau ú ly tõm tc 14 000 vũng/ phỳt phỳt 11 B ct lc,dung dch ng cha DNA ớch ó c tinh sch Tip tc thc hin cỏc k thut hoc tr ng -20oC PH LC PHIU THễNG TIN BNH NHN Hnh chớnh: H v tờn bnh nhõn: Gii tớnh: Nam:1N: Tui: (Sinh nm): a ch liờn h: S in thoi Chn oỏn lõm sng: Bỏc s iu tr: Cỏc yu t nguy c Viờm gan B1Thi gian mc: Viờm gan C2Thi gian mc: X gan3Thi gian mc: Ru4 Nu ỏnh du thỡ hi tip Anh (ch) cú thng xuyờn ung rucúkhụng Anh (ch) ung ru bao lõu ri 12 thỏng trc anh (ch) cú ung ru khụngcú khụng a Anh (ch) ung bao nhiờu ln thỏng va qua >1 ln ngy ln ngy n ln tun n ln tun ln tun n ln thỏng ln thỏng t hn ln thỏng b Anh ch ung bao nhiờu cc ln. c Loi cc anh (ch) s dng 30ml 50ml 100ml 200ml 330ml d Loi ru anh (ch) s dng Vodka hoc ru chng ct Ru vang Bia Loi ru khỏc Lõm sng Nng AFP: Kớch thc u: Siờu õm Doppler Tỡnh trng u v di cn ( MRI, CT) Hỡnh nh gii phu bnh hc Hỡnh nh t bo hc Kt qu gen ALDH2 Alen ALDH2*1 Alen ALDH2*2 Kiu genALDH2*1/*1 ALDH2*1/*2.ALDH2*2/*2 Cỏch tớnh gram ru tiờu th trung bỡnh/ngy Bng h s chuyn i t cõu hi a Cõu hi a H s chuyn i >1 ln ngy 30 ln ngy 30 n ln tun 20 n ln tun 11 ln tun n ln thỏng ln thỏng t hn ln thỏng 0.5 Cụng thc tớnh s gram ru trung bỡnh / ngy: H s chuyn i* cõu hi b* cõu hi c= s ml ru (1 thỏng trc) S ml ru (1 thỏng trc)/30 ngy = s ml ru trung bỡnh ngy Chuyn i ml ru sang gram ru da vo bng lng ru nguyờn cht Bng hm lng ru nguyờn cht ca mt s loi ru Loi ru Lng ru Ru nguyờn cht Cc tiờu chun (ml) cn Hm lng Vodka 50 40%/1 V 20g Ru vang 100 12.5%/ V 12,5g 1,3 Bia 330 4-5%/ 1V 15g 1,3 15,16,18,22,23,33,39,40,41,42,43 2-14,17,19-21,24-32,34-38,44- ... nay, nghiên cứu mối liên quan đa hình thái gen mã hóa cho enzym ALDH2 với UTGNP chưa nghiên cứu tìm hiểu cách đồng Chính vậy, đề tài Nghiên cứu tính đa hình đơn gen ALDH2 bệnh nhân ung thư gan nguyên. .. 1.3.2 Chức gen ALDH2 16 1.3.3 Tính đa hình gen ALDH 17 1.3.4 Gen ALDH2 bệnh ung thư 19 1.4 Kỹ thuật xác định đa hình đơn gen ALDH2 20 1.4.1 Hiện tượng đa hình đơn nucleotide... nhân ung gan nguyên phát CHƢƠNG TỔNG QUAN TÀI LIỆU 1.1 Sơ lƣợc ung thƣ gan nguyên phát 1.1.1 Tình hình mắc UTGNP giới Việt Nam Ung thư gan nguyên phát loại ung thư phát sinh từ tế bào gan thư ng