4. Noriko Tanifuji - Terai, et al. (2006), S.Takahiro Nakamura, et al. (2003), "The
"Expression of Keratin 12 and Maturation of Successful Culture and Autologous Transplantation Corneal Epithelium during Development and of Rabbit Oral Mucosal Epithelial Cells on Postnatal Growth", Investigative Ophthalmology Amniotic Membrane", Investigative Ophthalmol- and Visual Science, 47, p.545 - 5 5 1 . ogy and Visual Science, 44, p.l 05 - 116.
Summary
DETERMINATION THE CELL TYPE OF CULTIVATED CORNEAL EPITHELIAL SHEET
After successful culture of the epithelial sheet from stem cells of limbal corneal area, determination the cell type is very important. Objective: to determine the cell type of cultivated epithelial sheet from stem cells of limbal corneal region of rabit. Methods: using light, electron microscopy, histochemistry, immunohistochemistry to examine microstructure, ultrastructure and chemical structure of cultivated epithelial sheet. Result: the structure of cultivated epithelium sheet was a nonkeratinizied stratified squamous epithelium, with four to five layers of stratified. Transmission electron microscopy revealed that the basal epithelial cells adhered well to the amniotic membrane. The epithelial cells in all cell layers were closely attached to the neighboring cells by numerous desmosomes and sprouts of cell membrane. Immunohistochemistry confirmed the presence of the K3 and K12 in the cultivated epithelial cells. Conclusion: the cultivated epithelial sheet from stem cells of limbal corneal area of rabit was a corneal epithelial sheet.
Keywords: cultured epithelial sheet, desmosome, K3, Kl 2
CHAN DOAN T R U ^ C SINH MOT HG\J6\ ME MANG GEN DYSTROPHIN CO NGUY CO SINH CON Bl BENH
Nguyin Thj Bang SUdng^ Tran Van Khanh\ Nguydn Thj H a \ Ta Thanh Van^'^
^Trung tim nghien cdu Cen - Protein - Tnfdng Dai hgc Y Ha Ngi.
-Bd mdn Hoi sinh - Tntdng Dai hgc Y Hi Ndi
Ngudi me mang gen dystrophin dot biin cd kha nang truyen benh cho 50%o con trai va truyen gen benh cho 50% con gii ciia hg. Muc tiSu: sd dung ky thuit sinh hgc phan td di chan doin trUde sinh cho mdt ngifdi me dj hgp td (mang gen) cd nguy ca cao sinh con bj benh. Bo'i tUgng va pbuang phap nghien cdu: DNA dtfgc tich chiet td te bio di ciia ngudl me mang gen dystrophin bj dot biin mat doan. Sd dung phtfang phap PCR de xic djnh gidi tinh vi xic djnh dot bien xda doan gen dystrophin ciia thai nhi. Kit qua: thai nhi dugc cha'n doin la gidi tinh nam, tuy nhien ket qui xac djnh dot bien cho thi'y thai nhl khdng cd dot bie'n xda doan. Kit luan:
Chan doin trUdc sinh bing ky thuat sinh hge phin td qua phan tich DNA ciia te bao di li phtfang phip ngan chan benh sdm vi hieu qui ddi vdi cic ngUdi me mang gen benh dystrophin d Viet Nam.
Td khoa: loan dudng CO Duchenne, chan doan trudc sinh, ky thuat PCR
10
LDATVANDE
Loan dtfdng cd Dtichenne (Duchenne Muscular Dystrophy, DMD) la mpt trong nhflng benh ly than kinh cP do di truyen thtfdng gap n h i t d Viet Nam cung nhfl d c i c ntfdc k h l c tren the gidi, tan suit mac benh: 1/3500 tre trai. Benh gay nen do dot bien gen dystrophin (nam tren nhiem sac the X) dtfa den sfl b i t thtfdng trong q u i trinh tdng hop protein dystrophin [2]. Gen dystrophin la mpt trong nhflng gen ldn n h i t cua co the vdi c i u true gdm 79 exon, dang dot bien thifdng gap n h i t la dot bien m i t doan, chiem 50 - 5 5 % [1]. Theo nhieu nghien cflu, 2/3 benh nhan D M D la do n h | n gen dot bien di truyen td ngifdi me di hdp td, chl 1/3 benh n h i n la do dot bien mdi p h l t sinh |3]. Do vay, viec chan d o l n ngtfdi me di hpp tfl, chan d o l n trifdc sinh phat hien cac thai nhi b i t thtfdng va ngan chain benh bang phifong phap p h i thai chu dpng la mpt g i i i phap hieu q u i n h i t de l l m g i l m ty le m l c benh. Muc tieu:
Chan doan trUdc sinh nban mgt ngUdi me di harp td co nguy ca cao sinh con bi benh loan dudng ca Duchenne.
II. DOI TUONG vA PHU'ONG
P H A PNGHIEN CLfu
1 . Do'i tuong nghien cdu
- Nhdm ddi chflng: 2 ngfldi nam va 2 ngfldi nfl h o l n t o l n binh thfldng.
- Nhdm nghien ci'fu; Mdt ngifdi me mang thai ed con trai bi m l c benh D M D . Con trai cua b l da dtfdc chan d o l n la cd dot bien m i t doan gen td exon 3 - 8. Ngfldi me n l y dflpc x l c dinh I I ngifdi lanh mang gen benh bang ky thuat multiplex PCR dinh lflpng.
2. PhUdng phap nghien cdu
2.1. Pbuang pbap RT - PCR xac dinb dot biin mat doan gen dystrophin
T i c h chiet RNA tdng sd cua benh n h i n tfl bach e l u m l u ngoai vi va tdng hdp c D N A . Sd dpng 15 cap mdi thifc hien p h l n dng nested PCR de khuech dai t o l n bp chieu d i i gen dystrophin.
San pham PCR lan 2 difdc dien di tren agarose, so s i n h m i u benh nhan va ddi chdng, khi vach dien di cua benh n h i n ed kfeh thfldc nhd hdn m l u dd'i chdng la benh n h i n bj dot bien mi't doan gen.
Tien hanh giai trinh tfl doan gen etia benh n h i n de x l c dinh vi trf exon bi dot bien.
2.2. Ky thuat multiplex PCR dinh luang xac dinb ngUdi lanh mang gen benh
Difa tren ket q u i xac dinh dot bien cua ngifdi eon trai, ehung tdi se lifa chpn 4 exon, trong dd 2 exon khdng bi dot bie'n lam dd'i chdng npi va 2 exon bi dot bien de x l c dinh k h i nang mang gen benh. Bd'n exon se dtfdc khuech dai vdi cung ndng dp va chu trinh p h l n dng PCR d mau D N A cua benh nhan, me va chi g i i .
2.3. Ky thuat chgc hut dich di
Thtfc hien vao tuan thfl 14 cua thai ky difdi sfl htfdng dan eua sieu am, ky t h u i t dfldc tien h i n h tai Benh vien Phu s i n Trung Uong.
2.4. Ky thuat nuoi ca'y ti bao di ' ' Nudi c i y te b l o dieh di ngoai muc dfch lam tang so lfldng mau, nd cdn giup loai bd c l e te bao mau me l i n vao m i u benh pham. Sd dung mdi trtfdng Amniomax de tao c l e te bao sdi b i m dfnh v i o mat binh nudi c i y . Nudi c i y theo phflong p h i p hd tu i m 37°C vdi 5% CO2 va 9 5 % khdng khf cung ngudn am.
Thdi gian trung binh nudi c i y la td 7 - 1 5 ngay.
C I c te b l o di thai nhi thu dfldc vdi so lifpng tfldng ddi sau khi nudi cl'y difoc t i c h D N A theo phfldng p h i p phenol - chloroform va sd dung ky t h u i t PCR de p h i n tfch gen.
2.5. Ky tbuat PCR xac dinh gidi tinb thai nhi Sd dung cap mdi d i e hieu khuech dai vung gen SRY (cd kfch thifdc 254 bp) dac hieu tren NST Y:
Mdi xudi (SRY - F):
5' - CATGAACGCATTCATCGTGTGGTC - 3' Mdi ngflpc (SRY - R):
5' - CTGCCGGAAGCAAACTGCAATTCT - 3' 2.6. Phan dng PCR phan tich gen dystrophin cua tbai nhi
Dfla vao ke't qua x l c dinh dot bie'n cua ngifdi anh trai, chung tdi tien h i n h phan tich gen cua thai nhi bang p h l n flng PCR. P h l n flng PCR ed
the tfch 20|.il gdm c i c thanh phan; 1 00 ng DNA, 10 pmol mdi primer, 200 nmol/L dNTPs, 2 don vj enzym Taq polymerase v l 2|iL GeneAmp 10 x Buffer. Chu trinh nhiet cua p h l n flng nhfl sau; 94°C - 5 phut; [94''C - 30 giay, 58''C - 20 g i l y , 72''C - 20 giay] x 35 chu ky; 72°C - 5 phut. S i n pham PCR se difpc dien di tren gel agarose, ket q u i dflpc so s i n h vdi ke't q u i cua ngifdi anh trai bi benh cung vdi mau dd'i chflng.
IILKETQUA \
1. Xlc djnh dot bie'n mat doan gen dystrophin cua benh nhan d mde do mRNA
Exon 2 , Exon 9
1230 bp 600 bp
>
Sequencing
Hinh 1. Kit qua phan tich dot biin mit doan gen dystrophin ciia benh nhan DMD
San pham khuech dai et3NA cua mau ddi chflng (C) va benh nhan (BN).
Ky thu|t nested PCR difpc thtfc hien vdi p h l n flng PCR lan 1 dflpc khue'ch dai bdi cap mdi I A - 1 B, p h l n t'fng PCR lan 2 thtfc hien vdi cap mdi 1C - 1 D. Cap moi I C - 1 D cho phep khue'ch dai doan gen dystrophin tfl exon 1 den exon 1 1 . Hinh anh dien di cho t h i y s i n pham PCR cua mau ddi cht'fng cd kfch thtfdc khoing 1230 bp. Trong khi dd vach dien di cua mau benh nhan chl cd kfch thifdc khoang 420 bp. Nhfl .vay, benh nhan da bi dot bie'n mat doan gen dystrophin nam trong vung exon 1 - 11. Ket q u i g i i i trinh tfl doan gen cua benh nhan cho t h i y dau 3' cua exon 2 (TCTAAG) g i n trflc tiep vdi dau 5' cua exon 9 (ATCACG) va exon tfl 3 - 8 cua benh nhan da bi mat doan hoan toan.
2. Ke't qua xac djnh me benh nhan mang gen dystrophin bj dot bien
Difa tren ke't qua xac dinh dot bien cua ngtfdi con trai, chung tdi lifa chpn 4 exon, trong dd 2 exon khdng bi dot bien lam dd'i chflng npi la exon 41 va exon 48 va 2 exon bi dot bie'n la exon 3 va exon 5. Bd'n exon se dflpc khuech dai vdi cung nong dp va chu trinh phan flng PCR. M l u DNA cua benh n h i n , me va chi g i i se dflpc lfla chpn de tie'n hanh phan flng.
Ke't q u i PCR dinh Iflpng cua me va chi g i i benh nhan eho t h i y , vdi 2 exon benh nhan khdng bj dot bie'n (exon 4 1 , exon 48) thi ndng dp cua chung (tifdng flng vdi chieu cao cua mdi dinh) bang vdi dd'i chflng nfl. Nhtfng vdi 2 exon benh nhan bi dot bien m i t doan (exon 3 va exon 5) thi chieu cao cua mdi dinh chi b l n g 1/2 so vdi cht'fng ntf, chflng td ngifdi me va chj cua benh nhan d dang di hdp td.
Nhfl vay kha nang ngtfdi me truyen gen benh cho thai nhi dang mang thai la 50%. Tien hanh chpc hut djch d'i de chan d o l n trfldc sinh x l c djnh trinh trang benh ly cua thai nhi. „ . . , . ,,;,
12
Do'i chdng nam
3 41
dU
"•^"-"'•""y " - - ^
[FU].
80 60 - 40 - 20 • 0 - \'.\
Dd'i chdng nd
46 3 "*!
Esi
IL 1 1
40 50 60 70 80 90 100110120 40 50 60 70 80 90 100110120
Benh nhan Me benh nhan Chi benh nhan
15 [FU] , ,
80 • I 60 • Il
40 ' '
2 0 , * h 46 0 r~y •••;•
1500 15 [FU]-
80 - 6 0 - 40 • 20 ' 0 •-
1500
Esl
3
41
[FU]
80 60 40 20 0
15
• i
{
.i
15(
46
^r T 41 E5 3 1 1
1 t i
40 5060 70 8090100110120 40 60 80 100 120 40 60 80 100 120
(Mui ten chl vj tri exon bj dot bie'n)
Hinb 2. Pban dng xac dinb kiiu gen di hgp td cua me va chi gai benh nhan 3. Ke't qua xac djnh gidi tinh thai nhi
2 5 4 bp
Hinb 3. Kit qua xac djnh gidi tinh cua thai nhi
Sfl dung cap mdi de khue'ch dai doan gen SRY (cd kich thfldc 254 bp) tren NST Y. Ke't q u i cho t h i y , s i n pham PCR cua thai nhi cd kfch thfldc 254 bp tifPng flng vdi kfch thfldc cua m l u dd'i chflng va cua benh nhan D M D (anh trai cua thai nhi). Trong khi dd d m l u ddi chflng am va m l u ddi chflng nfl khdng x u i t hien vach. Ket q u i tren chflng td thai nhi la nam gidi.
4. Ke't qua phan tich gen dystrophin cua thai nhi
Ngtfdi me va anh trai cua thai nhi mang gen dystrophin bj dot bien mat doan tfl exon 3 - 8. Tie'n hanh phan flng PCR vdi 8 cap mdi khue'ch dai 8 exon tfl exon 2 - 9 t u a gen dystrophin. Kdt qua the hien d hinh 4. , ,: .;,. ... . , • ,, , - ' -.
Exon 2 Exon 3 Exon 8
V >i~
•I
I
i-s-v > *• '^H/n/i 4. /Cef qua phan ticb gen dystrophin ciia ti bao thai nhi
Trong dd: MS 1: Chdng am (nddc); MS 2: Chitng ditang (miu binh thtfdng); MS 3: Miu ti bao thai nhi; MS4: Miu benh nhan DMD (anh trai thai nhi)
Nhin xet: hinh inh dien di cho tha'y: d benh nhin DMD, cic exon 3, exon 8 khdng xui't hien vach khi dien di tren gel agarose, trong khi dd miu DNA ciia thai nhi vin xua't hien vach cd kich thtfdc bang, vdi vach Cila miu ddi chdng, diiu nay chdng td thai nhi khdng bj dot biin gen dystrophin.
I V . B A N L U A N
Benh nhan dfldc phan tfch gen d mt'fc dp mRNA va p h l t hien cd dot bien mi't doan tfl exon 3 den exon 8. Ke't q u i djnh Itfpng tren m i y dien di mao q u i n eho t h i y me v l chj g i i benh n h i n la ngfldi I l n h mang gen benh. Theo c i c t i e g i l , ngfldi me di hdp td cd k h i nang truyen benh cho 50% con trai va truyen gen benh cho 5 0 % con g i i cua hp [5]. Tren thflc te d gia dinh nay ngfldi me da truyen gen benh cho 100% c i c con (gdm c l ngtfdi eon trai va con g i i da sinh). Dieu n l y khang djnh k h i nang mang gen b e n h c u a thai nhi ma ngtfdi me dang mang thai lan thd ba I I ri't cao [2, 5). Tien hanh tfl vi'n cho gia dinh benh n h i n va c h i n d o l n trtfdc sinh dflpc tie'n hanh nham loai bd thai nhi neu mang benh.
X l c djnh gidi tfnh cua thai nhi la bfldc dau tien can thifc hien sau qua trinh t i c h chiet D N A d mau te b l o d'i ciia thai nhi. Sfl dung cap mdi cho phep khuech dai doan gen SRY (cd kfch thifdc 254 bp) nam tren NST Y, trinh tfl nucleotid cua cap mdi dtfoc chpn tifdng tfl nhfl nghien cflu cua Hussey [4]. P h l n dng PCR difpc tien hanh vdi mau DNA cua thai nhi, kem theo c l e chflng nam, chflng nfl va DNA eua benh nhan D M D (la anh
trai eua thai nhi). S i n pham PCR difdc dien di tren gel agarose ndng dp 1,5%. X l e djnh gidi tfnh cua thai nhi dfla vao sfl x u i t hien vach dien di d i e hieu ed kfch thfldc 254 bp, hoac so s i n h vdi m l u dd'i chflng nam. M d i m l u DNA thai nhi, lap lai p h l n flng 3 lan de t r i n h sfl nham l l n . ., :
Ke't q u i dien di cho t h i y d mau te bao thai nhi x u i t hien vach dac hieu cd kfch thtfdc b l n g vdi vach cua m l u dd'i chdng nam va eua ngfldi anh trai thai nhi. Trong khi dd m l u ddi chdng I m va dd'i chdng nfl khdng x u i t hien vach PCR tfldng dng. Dieu n l y chflng t d ' c d sfl hien dien cua NST Y trong te b l o thai nhi va chung tdi khang djnh gidi tfnh cua thai nhi la nam. Theo ly thuye't, ngfldi me dj hdp td ed k h i nang truyen benh eho 5 0 % con trai eua hp, nhfl vay k h i nang mac benh D M D cua thai nhi ri't cao, vdi tan suit I I 5 0 % [2, 6].
Tren ed sd bie't difde vj trf dot bie'n m i t doan cua anh trai thai nhi cung nhfl cua ngifdi me (tfl exon 3 de'n exon 8), chung tdi tien hanh p h i n tfch doan gen dystrophin td exon 2 de'n exon 9 cua thai nhi bang p h l n flng PCR. Ke't q u i dien di d hinh 1 cho t h i y ; ddi vdi exon 2, vach dien di x u i t hien d t i t e l e l e m l u phan tfch (trfl mau chdng
14
I m ) la m l u ddi chflng difOng, m l u te bao d'i va mau l5cnh nhan. Nhflng vdi cap mdi khtiech dai exon 3, vach D N A tflpng t'fng ehi xuit hien d m l u chflng dtfong va m l u DNA thai nhi ma khdng xuat hien d benh nhan. Dieu nay I I phu hop vl benh nhan bj dot bien m i t doan exon 3 nen se khdng x u i t hien vach D N A ttfong dng vdi exon 3. Ket qua nay chi ra rang, thai nhi khdng bj dot bien m i t doan exon 3. Tie'n hanh tflong tfl vdi c i c exon tfl 4 - 8 chung tdi difa ra ke't luan, thai nhi nam khdng mang gen dot bien va gia dinh dtfpc khuyen gifl thai.
V. KETLUAN
Day la tnfdng hpp dflpc nghien ct'fu chan d o l n trifdc sinh benh loan difdng co Duchenne dau tien d Vict Nam. Ket qua btfdc dau nay se md ra trien vpng flng dpng trong thflc hanh lam sang benh di truyen ndi chung hay benh D M D ndi rieng. .
T A I LIEU
THAM
K H A O1. Alcantara M.A., Cavazos R.G., Hernandez U.E., Gonzalez - del A.A., Carnevale A., Orozco L. (2001), "Carrier detection and prenatal molecular diagnosis in a IDtichenne muscular .dystrophy family without any affected relative
available", Ann Genet, 44(3), pp. 149 - 53.
2. Behrman J., Kliegman L., Arvin E. (1998),
"Neuromuscular Disorders", Nelson lexbooks of pediatrics. Edition 15th, pp. 1739 • 53.
3. Hung C C , Chen C.P., Lin C.P., Chen S.C.
et al (2006), "Quantitative assay of deletion or duplication genotype by capillary electrophoresis system; application in Prader - W i l l i syndrome and Duchenne muscular dystrophy", Clin Chem, 52, pp. 2203 - 10.
4. Hussey N.D., Donggui H., Froiland D.A., Hussey D.|., Haan E.A., Matthew C D . , Craig J.E.
(1999), "Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells", Moi Hum Reprod, 5(11),1089 ^ 94.
5. Mendell R.J., Griggs C R . (1991), "Muscular dystrophin", Harrison's principles of internal medicine, 1 2th edition, pp. 2 1 1 2 - 8 .
6. Polani P.E. (1989), "Prenatal diagnosis and genetic screening". Royal College of Physicians Report, London.
*Chu thich: Nghien citu dttac thtfc hien tai Trung tam nghien cifu Gen - protein, Trififng Dai hoc Y Ha Ngi, vdi stf hd tra kinh phi eiia de tai
"Nghien cifu phit bicn ngtfdi mang gen benb, chan doin tnfdc sinh, ifng dung trong vice sang Ioc benh loan ditdng ca Ouchenne/Bccker d ccing dong"
trich td ngan sach si/ nghiep khoa hgc cap Bo Y te.
•.'•'.' S u m m a r y
PRENATAL DIAGNOSIS OF DUCHENNE MUSCULAR DYSTROPHY IN VIETNAM
A heterozygous mother can transfer the mutated allele to 5 0 % of her sons and 50°/) of her daughters.
Objectives: to perform the first molecular prenatal diagnosis in a DMID carrier woman in Vietnam.
Materials and Methods: chorionic villus sampling (CVS) was performed in a D M D carrier woman in which partial deletion mutation were present. Result: presence of PCR products corresponding to Y chromosome - linked loci in D N A CVS sample was compatible with a male fetus. Fortunately, the fetus was confirmed not to receive the mutation from the mother. Conclusions: Molecular prenatal diagnosis through CVS could be an early reproductive prevention strategy applicable to Duchennc/Beckcr muscular dystrophy carrier women in Vietnam.
Keywords: Duchenne muscular dystrophy, prenatal diagnosis, PCR technique
