GVHD: PGS.TS.

Khut Hu Thanh SVTH : Nguyn Th Hin Nguyn Th Lng

K thut gen trong chun on mt s bnh

Ung th vm hng do virut Epstein-Barr

Bnh lao do vi khun Mycobacterium tuberculosis St xut huyt

chn on trc sinh bnh

Teo c ty(SMA) Lon dng c Duchenne(DMD) Bnh alpha- Thalassemia Tng thng thn bm sinh(CAH)
.

Chun on bnh
Trc tip Quan st bnh phm qua knh hin vi Nui cy, phn lp vi sinh vt gy bnh Huyt thanh bnh nhn Khng nguyn c hiu ca vsv trong mu bnh phm ng dng k thut gen, pht hin DNA, RNA ca vsv trong mu bnh phm

K thut gen
Gen l n v chc nng c s ca b my di truyn

chim mt locus nht nh trn nhim sc th v xc nh mt tnh trng nht nh. Cc gen l nhng on vt cht di truyn m ha mt tnh trng nht nh. Cc gen l nhng on vt cht di truyn m ha cho nhng sn phm ring l nh cc mRNA c s dng trc tip cho tng hp cc enzym, cc protein cu trc hay cc chui polypeptide gn li to ra protein c hot tnh sinh hc. Ngoi ra, gen cn m ha cho cc tRNA, rRNA, mRNA

K thut gen - Phng php PCR

PCR (polymerase chain reaction): phn ng chui trng hp hay phn ng khuch i gen Nguyn l phng php PCR Dng nhit cao tho xon thay cho enzim helicase. Kt hp vi enzim DNA polymerase chu nhit tng hp DNA mi trong mi trng thch hp. H thng iu nhit thch hp cng vi cc on mi c thit k chuyn bit, ch ng.

K thut PCR

K thut Multiplex PCR

L phng php PCR s dng ng thi cng 1 lc

nhiu cp mi c hiu khc nhau

Ung th vm hng
Nguyn nhn: c rt nhiu

gi thit, nhng ch yu c chia thnh ba nhm: do virut Epstein-Barr, do di truyn v do cc yu t mi trng, tp qun n ung, ho cht

Virut Epstein-Barr
H gen ca EBV l chui

ADN xon kp, mch h, kch thc khong 172 kb. H gen ca EBV gm 6 t hp gen m ha cho cc loi protein khng nguyn, k hiu l EBNA (EBNA-1, EBNA-2, EBNA-3A, EBNA3B, EBNA-3C v EBNALP) v 3 t hp gen m ha cho protein mng l LMP-1, LMP-2A v LMP-2B

Trnh t mARN gen LMP-1 ca EBV (1.116 bp)

Phn ng PCR
LMP1F (mi xui):

5-ATGGAACGCGACCTTGAGAG-3 LMP1R (mi ngc): 5-TTAGTCATAGTAGCTTAGCTGAAC-3 Sn phm ca ton b gen LMP-1 c kch thc 1,3 - 1,35 kb

Chun on vi khun lao khng thuc

Theo thng bo ca T

chc Y t Th gii, bnh lao ang l vn khn cp ton cu, mi nm trn th gii c thm gn 9 triu ngi mc bnh lao (trong c 1 triu l tr em) v gn 2 triu ngi cht do lao.

Vi khun lao Mycobacterium

tuberculosis
Tch chit DNA trong

mu m bnh nhn

http://giangduongykhoa.wordpress.com/category/lao-ph%E1%BB%95i/

Kt qu kho st kh nng khuch i ca h mi v mu d nhm pht hin t bin

katG/315/AGC ->ACC

rpoB/516/GAC -> GTC

Kt qu kho st kh nng khuch i ca h mi v mu d nhm pht hin t bin

emb/306/ATG ->GTG (biu mu xanh) emb/306/ATG ->ATA (biu mu )

Kt qu kho st nhy ca h mi v mu d nhm pht hin t bin

katG/315/AGC ->ACC

rpoB/516/GAC -> GTC

Kt qu kho st kh nng khuch i ca h mi v mu d nhm pht hin t bin

emb/306/ATG ->GTG (biu mu xanh) emb/306/ATG ->ATA (biu mu )

C ba biu : tn hiu hunh quang vt trn tn hiu nn t mu c nng amplicon 107 copy/ml

vi chu k ngng Ct gim dn n mu c nng 102 copy/ml. Mu c nng 10 copy/ml th c tn hiu hunh quang thp hn tin hiu nn.

Chun on st xut huyt

Stxut thuyt l do virus Dengue (c 4 type: 1, 2, 3, 4).

L bnh l nguy him e do tnh mng cn c chn on pht hin sm. Phng php ELISA pht hin IgM khng th gip chn on sm nhim Dengue (phi sau ngy th 4 ca bnh) RT-PCR Dengue c th gip chn on sm SXH ngay trong nhng ngy u ca bnh, v cn nh c type Dengue Chn on pht hin sm SXH rt c gi tr iutr v theo di bnh

Chn on trc sinh bnh thiu mu huyt tn Alpha - Thalassemia

Alpha -Thalassemia: bnh di truyn ln NST thng do

t bin gen HbA, nm trn cnh ngn NST 16(16p13.3) gy gim hoc mt tng hp chui alpha globin. Alpha -Thalassemia l mt trong nhng bnh huyt sc t ph bin nht, l nguyn nhn hng u gy hin tng ph thai.

Chn on trc sinh bnh Alpha-Thalassemia

Vng gen -globin gm 2 gen: HbA1 v HbA2 Cc t bin thng gp gy Alpha-Thalassemia gm: mt on ln dng SEA, thailand, philipin, 4.2, 3.7

v cc t bin im HbQs, HbCs t bin trn vng gen -globin gm 2 nhm: gy mt hon ton s lng chui globin v lm gim s lng chui globin

Chn on trc sinh bnh Alpha-Thalassemia

i tng nghin cu
Sn ph c thai t tun 15-25
Hnh nh siu m thai bt thng Tin s sinh con bt thng Tin s gia nh hoc ngi thn mc cc bnh

nh teo c ty, tng sn thng thn bm sinh..

K thut tin hnh

Nui cy t bo dch i: dch i c nui cy trong dung

dch Amniomax Fluid. Sau 10-14 ngy, thu hoch cc t bo dch i v ra sch bng dung dch PBS 1X Tch chit DNA t mu ngoi vi v t bo dch i sau nui cy K thut Multiplex PCR: nhn bit s mt on ca 5 t bin mt on ln

K thut tin hnh

C-ARMS-PCR( combine Amplification Refratory Mutation System Polymereas Chain Reaction): l k thut Multiplex PCR c s dng sng lc cc t bin. Dng cc mi c hiu c trnh t u 3 b sung vi alen t bin v mt mi chung ngc chiu vi mt c hiu alen. S c mt ca t bin c th hin bng sn phm DNA khuch i vi cc kch thc khc nhau.

K thut thc hin

Gii t vng gen alpha-globin v phn tch kt qu:

gii trnh t c thc hin trn my ABI 3130. Kt qu c phn tch bng phn mm chromas v chromas pro, sau c a ti ngn hng gen so snh vi trnh t gen alpha thalassemia chun.

Kt qu

Kt qu

Nhn xt
Trong 7 t bin sng lc, alen t bin dng

SEA gp vi tn sut cao nht 59%, t bin HBQs 17,6%, HbCs 11,8%, 4.2: 5,9%

Chn on trc sinh bnh tng thng thn bm sinh

Tng thng thn bm sinh (TSTTBS-CAH) l

bnh dii truyn ln trn NST thng, ch yu do thiu ht hot ng ca enzyme 21-OH, gen CYP21A m ha cho enzyme 21-OH. S thiu ht hot ng ca enzyme 21-OH dn n s thiu ht cortisol hoc thiu ht aldosternone cng nh tha adrogen thng thn.

Chn on trc sinh bnh tng thng thn bm sinh

Vng gen m ha cho E. 21-OH gm 2 gen: gen

CYP21 v gen gi CYP21P. Vng gen ny nm trn cnh ngn NST6 Gen CYP21P l gen bt hot, gen CYP21 m ha cho E. 21-OH. Qua qu trnh phn bo gim nhim, s bt cho khng ng u gia cc cp NST lm cho trnh t vng gen gi chuyn sang gen CYP21 gy nn t bin trn gen. 95% t bin trn gen CYP21 l do t bin chuyn on t gen gi CYP21P, 5% cn li l do t gen CYP21 b t bin

i tng nghin cu
2 mu dch i ca 2 gia nh c con u

c chn on lm sng b bnh tng thng thn bm sinh

Phng php nghin cu

Mu bnh phm: 15ml dch i
Phn tch gen CYP21 Nui cy t bo dch i: trong dung dch Amniomax

fluid l mi trng c hiu ch cho t bo dch i. Thu v ra sach bng dung dch PBS sau 10-14 ngy. Tch chit DNA t t bo dch i: bng cc kit tch DNA ca Qiagen c.

Phng php nghin cu

Sng lc cc t bin thng gp bng k thut MLPA: s dng kit MLPA P050B2 sng lc 6 t bin thng gp: P30L(exon1), mt on 8bp(exon3), I172N(exon4), exon 6 cluster, Q318, mt ton b gen CYP21, mt on 30kb.
Gii trnh t gen CYP21 v phn tch kt qu

Kt qu
Gia nh 1:

Kt qu
Gia nh 2:

Nhn xt
Gia nh 1: thai nhi l b gi, mang ng hp

mt on 30kb, t bin thng gp trn tr mc bnh tng thng thn bm sinh th mt mui Kiu gen ny ph hp vi kiu t bin c tm thy trn b m Gia nh 2: thai nhi l b trai, mang t bin d hp t mt on 30kb-l ngi lnh mang gen t bin

Kt lun
K thut PCR v k thut gii trnh t gen l

nhng k thut di truyn hin i nht. ng dng thnh cng k thut ny, gip cho vic chn on mt s bnh do vi khun gy ln v chun on trc sinh bnh tng thng thn bm sinh, l c s cho iu tr trc sinh, lm gim t l nam ha bt thng i vi thai nhi n mc bnh

Ti liu tham kho

PGS.TS. Khut Hu Thanh. K thut gen nguyn l v ng dng. Nh xut

bn khoa hc v k thut. 2006. Nguyn nh Phc v L Thanh Ha. Virut Epstein-Barr gy ung th vm mi hng v mt s phng php hin i ng dng trong chn on. Tng quan. Tp ch Cng ngh Sinh hc. 2008, 6 (2), tr.1-18. H Th Thanh Thy, Nguyn Vn Hng Pht hin nhanh vi khun lao Mycobacterium tuberculosis khng cc thuc isoniazid, rifampin v ethambutol bng realtime PCR. H Mnh Tng, Trng nh Kit, ng Quang Vinh. Chun on di truyn tin lm t http://ditruyen.com/c3/chan-doan-di-truyen-truoc-khi-sinh.html http://www.nihe.org.vn http://www.ykhoanet.com/NCKH/bn04.HTM http://www.medinet.hochiminhcity.gov.vn/ttyh/bshkhkt/sotxuathuyet.htm http://www.xaluan.com/modules.php?name=News&file=article&sid=61494