Sau nhleu nghien cuu, d~n nay da khang dinh SI,I'co mat cua TB phoi thai trong rnau ngo~i vi cua me.. TCYtu~n thf 8 cua thai ky, da phat hien dU'Q'Ccac TB cua thai gbm: trophoblast, bach
Trang 1TAP CHi Y - D110C HOC QUANSl1 s66-2014
- ~D"'"":
-f:
Ith Orqanization fi].:TAcH::t::;~l~ . vA PHAN LAp TE BAo PHOI THAI TRONG MAu NGOAI VI
It
f?:'::>"c'
determinants ir ;:.,10"
'aediatr Perinat;;;:'?:'H)M
;-545
'Nam 1893, Schmorl phat hien t9 bao (TB) ph6i thai & ph6i cua phu n(Y mang thai Toi nay, , nhi~u nghi~n cou, oa kh~ng ojnh Sl,l' c6 mat cua TB ph6i thai trong rnau ngo<;1ivi cua me llJ~n th(l'8cua thai ky, oa phat hien oU'Q'ccac TB cua thai gOm: trophoblast, bach cau, hOng nhan, TB gOc DOi tU'Q'ngva phU'O'ng phap nqhien cou: Itla chon 30 phu nO mang thai tli th(l' 8 oEmtuan th(l' 22 cua thai kyo SLYdl)ng phuc hop hat tli va khang the don dong CD71
If Research or hOng C8U c6 nhan ra khoi TB rnau ngo<;1ivi cua m~ Sau khi tach oU'Q'CTB hOng cau co
eua con ra khoi TB mau ngo<;1ivi cua me, dung bQ kit Vysis probes Multicolor MultiVysion
Ifor the period
~.;;; ., nhuom huynh quang nhiem s~c the (NST) 13, 18,21, X va Y Ket qua: 15 te bao hOng
co nMn b~t mau huynh quang vai NST X, cae NST 13, 18, 21 13 TB hOngc8Ue6 nhan b~t huynh quang vai NST XY, NST 13, 18,21 blnh thU'ang o~u co 2 tin hi$u hu}mh quang C6 m~u TB hOng cau c6 nMn ella con kMng b~t oU'Q'ctin hi$u
~Tli kh6a: Te bao phOi thai; Mau ngo<;1ivi; Phan I~p
j completeness
ation in a rura'
lic Health Suppl
introduction: the discovery of the existence of fetal cells in maternal peripheral blood has , ed up a new direction for the early diagnosis of congenital anomalies by means of
non-E:,i!rtsrference Objects, chemicals and research methods: we selected 30 pregnant women in
t~.-::=gneticbeads were attached to streptavidin Complex particles from above C071 binds to red
j: ::::::-Jd cells of the embryo have the principle immune After extraction of isolated embryonic cells
j- maternal peripheral blood cells and staining kits using Vysis probes Multicolor MultiVysion ,=ST probe which binds specifically on the Y chromosome.Results 15 nucleated red blood cells of 'i::talcells carrying XX chromosomesand 13 of nucleated red blood cells carryingXY chromosomes.
c.-:30 nucleated red blood cells, there are2nucleated red blood cells not signal appeared.
* Key words: Embryo cells; Peripheral bfood: fsolation,
EXTRACTION AND ISOLATION OF EMBRYONIC CELL IN MATERNAL PERIPHERAL BLOOD CELL APPLYING IN
NON-INTERFERENCE DIAGNOSIS
~ l-l9C vi~mQulm y
D?i h9CYOIlOC- f)?i h9C Quac Gia Hi! N¢i Hgl.Y&i phim hoi (Corresponding): Tri~u Tie;' Sang (trieusangk83@yahoo.com.vn) Ngay nh~n btli: 28104i2014; Ngay phim bi~n danh gia bai bao: 2810712014
Ngay bai bao dl.Fg'Cdang: 08i0812014
49
-=:::: _~
~ -= ~-;;;,
Trang 2TAP CHi Y - D110C HOC QUAN Sl1 SO 6-2014 .
Narn 1893, Schmorl phat hi$n SI,I' ton
tal cua TB phot thai (y ph6i cua phu nCl'
mang thai Sau nhleu nghien cuu, d~n
nay da khang dinh SI,I'co mat cua TB phoi
thai trong rnau ngo~i vi cua me TCYtu~n
th(f 8 cua thai ky, da phat hien dU'Q'Ccac
TB cua thai gbm: trophoblast, bach cau,
hbng cau nhan, TB goc So 1U'Q'ngesc TB
nay r~t thcfip,nhU'ngmang v$t chat di truy~n
clla phoi thai, do v$y rcfitco y nghia trong
ch~n doan di t$t sam cho thai Tuy nhien,
xuat hi~n sam nhat, nhanh b] hllY, kh
co khang nguyen b~ mat d~c hieu; ba cau phci thai tbn tai ca sau thai ky, k
co khimg nguyen b~ m~t d~c hieu: cau nhan xuat hi$n sam va tbn tal suet thai ky, co khang nguyen b~ mat hieu, Do v$y, cac nqhlen ciru tren th~ 9 tap trung vao phan tach h6ng cau nha Tach dll'Q'Crieng bi$t TB phoi thai mau ngo~i vi clla m~ da ma ra mot h m&i trong ch~n aoan trll'&c sinh bang bi phap khong can thi$p
Hinh 1:Hlnh anh cac TB t6n t~i trong mau ngo~i vi clla thai phl,l
(©2009 Artenmis Health Inc.)
eOITVQNG, HOA CHAT, THIET B!vA h$ tlnh dl,lCI~n nao, cLmgdll'Q'c Icfiy5 PHVONG PHAp NGHIEN c(fu Cac mau nghien ClrU all'Q'c lay t~i Tn;
1 eOi tlpgng nghi€m c(l'u
30 phl,l nO'mang thai atuiln thlr 8 d€!n
tu~n th(f 22 cLlathai kyoTat ca Oll'Q'Clay 5
ml mau toan ph~n 2 mau ooi ch(fng am
la 2 nCl'gi&i chll'a mang thai va chll'a quan
50
tam Nghien c(fu Y DlI'Q'c hQc Quan HQcvi$n Quan y
2 H6a ch~t nghien c(l'u
H~t tCYtinh da gan streptavidin,khang CD71 da biotin hoa, PBS 1X, ethanol 70
~~
Trang 3TAP CHi Y • Dl10C HOC QUANSl1 s66·2014 trophoblas·,=-;ICI'1'\!~nrll100%, proteinase K,nuoccllt, b(>kit
huy: kh6 s MultiVysion PGT Multicolor probes,
hieu: bacl kinh, lamen, NP40, SSC20X, antifade II,
Iiky, kh6 ide, nU'&c tinh khi~t, HCI, methanol,
hi$u; ho
)n tal tro
b~ mat d~ 3 Thitit b] may moe
rem th~ g; May I~c 6n nhi$t (Eppendorf, f)(rc), may
diu nhar tam cao t6c (Hettich, f)(rc), pipet man
i thai trorl~lf;i:tGlnson, My), buong thao tac PCR (My),
rO'n,;:i.t:~,e;attYtinh (Promega, My), b~ nuoc 6nnhiet
, h$ th6ng kinh hi~n vi huynh quang
io Zeiss, f)(rc)
4 PhlPO'ngphap nghien ciJ'u
Quy trlnh thl,l'c hi$n
T90 phCtc h<;1ph9t W -hong cau nhan
Tach phCtc h<;1ph9t W - hong cau nhan
Thu TB hong cau nhan
¥>
r ~G~n probe huynh quang va ki~m tra TB
t 'trem h$ th6ng kinh hi~n vi huynh quang
:: 11Iy5m: 1;_' -'
, t9i Trun~'
Quan Sl,!
kh~ng th~
lanol70%
Khang th~ C071 dU'Q'c biotin hoa gan vai h9t ttY tinh da gan vai streptavidin
Ph(rc hQ'p h?t ttY trem c6 C071 gan vai
hong diu nhan clla ph6i thai theo nguyen
t~c mien dich,
Cl,Ith~ cac buoc nhu sau:
+ BU'ac 1: 11Iy10 1-11khanq th~ da biotin h6a vao 6ng 1,5 ml
+ Buoc 2: them 20 1-11hat tlr da gan streptavidin, Ll(y4°C trong 30 phut
+BU'ac 3: them 100 fJl PBS 1X, lac nhe, d~t vao gia tlr tinh, hut be dich
+ BU'ac 4: them 100 fJl PBS 1X, lac nhe, hut h~t djch cho vao 6ng chua maungo?i
vi cua meda lay tnroc do Lac quay tron
15phut(y4°C
+ BU'&c 5: hut tlrng 1 ml mau cho vao ong 1,5 ml f)~t len gia tLl' tinh hut be toan b(> djch, giCr I?i ph(J'c hQ'p h?t tLl' - hong c~u nhan da bi g~n I€m tren thanh bng phia gia ttY tlnh
+ BU'&c 6: them 100 fJl PBS, lac d~u, d~t len gia ttY tfnh, hut be ph~n dich long (lam f?i 3I~n)
+BU'ac 7: them 20 1-11PBS 1X, Voltex 5 giay, lU'u mau TB trong tu -20°C,
* Quy trinh gan probe len TB hong du
co nhan:
BLl'ac 1: hut djch TB ph6i da tach dU'Q'C sau khi tach (y quy trlnh tr€m d~t len lam kinh 8anh dllu vi tri d~t TB b~ng cach khoanh vung m~fdU'ai cLla lam kinh
BU'ac 2: d~ kh6 lam kinh trong kh6ng khf, sau d6 chuy~n lam kfnh vao dung dich nhLl'Q'c trLl'O'ng 0,2% Tween20 in 0,01 M HCI, pH 2,0 b(>c'0nhan TB
BU'ac 3: nia lam kfnh b~ng dung dich PBS 1X (pH 7,0: 0,14 M NaCI, 3 mM KCf,
10 mM Na2HP04, 2 mM KH2P04) trong
5 phut (y nhi$t dO phong RLra s?ch trong nLl'ac cllt v6 trung 2 I~n Lo?i nLl'ac b~ng dung dich con 70%, 90%, 100% trong 2 phut
(ynhi$t d(>phong va lam kh6 trong kh6ng khi
51
Trang 4TAP CHi Y - DU'OC HOC QUAN SU' 566-2014
BU'ac 4: them 2 j.J1hen hop probe va
phu len tren b~ng 1 lamen kich thuo'c 9x
9 mm Bao phu xung quanh b~ng lap
nhva g~n cao 5U d~ tranh thoathO'iruroc
BU'ac 5: utrong block nhi$t 75°C trong
5 phut, sau do lai tLi' 16 - 22 gia trong tu
tIm 37°C
BU'ac 6: tach bo lamen va keo gan lam
kinh, rLra lam klnh trong dung djch SSC
4X10,05%Tween20 (pH 7,0)&nhi$t dOphong
BU'ac 7: nra nghieng mat lam kinh bang dung dich O,4X SSC/O,3% NP40 & 73°C trong 5 phut
BU'ac 8: rLra lam klnh trong dung die!", SSC 2X&nhletdQ ph6ng trong 1phut BU'ac9:lay lam kinh ra d~t trong phon~ t6i, dung giay m~m lau kho Them 3 ~ antifade II,sau do d~t lamen IEmtren BU'ae 10: quan sat tin hi$u huynh cua TB phoi tren kinh hit§n vi huynh quang
KET QUA NGHIEN cuu vA BAN LU';N Sau khi tach chiet phan I~p dU'Q'cTB ph6i trong mau nqoai vi me va nhuorn TB b~n~
bQ kit Vysis MultiVysion PGT Multicolor probes, trong do co probe gan d~e hieu trer NSTY
Hinh 2: Hlnh anh nhuorn huynh quang mau TB phoi thai 56 555
(NST 56 13: ph6 mau do, NST 56 18: ph6 Aqua xanh nU'ac bit§n, NST 56 21: ph6 xanh
18cay, NST Y: ph6 mau vang, NST X: ph6 mau xanh reu)
52
Trang 5TAP CHi Y • DU'OC HOC QUAN SU' s66·2014
Hlnh anh tren cho th~y day laph6i mang b¢ NST 46, XY cacNST 13, 18, 21 d~u c6 2 hieu, rnau vang c6 1 tin hieu cua NST Y va mot tin hieu rnau xanh reu cua NST X
d~t trong ,,,.~~""",
:h6 Them 3
mien tren
~u huynh quar
ii huynh quan;
Hinh3:Hinh anh nhu¢m huynh quang NSTcuaTB h6ng diu c6nhanph6i thai56 556 Ket qua trenTB h6ng cauc6 nhan cuaph6i thai 0benh nhannghien cuu 56 556 c6 J-JSTX va cac NST 13, 18, 21 d~u c6 2 tin hieu binh thLl'cYng.Trong tieu ban nay kh6ng
thien tin hieu rnauvang cuaNST Y.
Qua phan tich ket qua cua 30 ph6i thai nqhien cuu, chunq t6i thu dLl'Q'cbang ket qua rotype cua 5 NST 13, 18, 21 va X, Y nhu sau:
2
277
CHAN E)OANADN PHOI TI,1DO TREN GEN SRY
CHANE)oAN FISH
269
270
46,XX 46,XX
+
46, XX
+
J.
46, XX
KhOng xuat hien tin hi~u
21: ph6 xanh&.
8
i~
53
Trang 6TAP CHI Y - DU'OC HOC QUAN SU' s66-2014
46,XX
+
+ + +
+ +
+
+ +
+
46,XY 46,XY 46,XY 46,XX 46,XY
NG
46,XY 46,XY
+ +
+ + KhOngxu~t hientin hi~u
46,XX 46,XX 46,XX 46,XX 46,XX 46,XY 46,XX 46,XY 46,XY 46,XX 46,XX
+ +
+ +
+
15 T8 hOngcau co nhan cua phoi thai mang NST XX va 13 h6ng cau co nhan cu: ph6i mang NST XV, Trong 30 ph6i nay, 2 phoi kh6ng xuM hi$n tin hieu, Co th~
ph6i nay kh6ng pha va mang T8 va mang nhan, do v$y cac probe kh6ng th~ gan
vao NST Trong 15 phoi oU'Q'c ki§m chLrng bang ADN ph6i thai tl,l' do mang NST dU'Q'c pnan I$p tLl' T8 phoi, chung t6i nhuQm thanh c6ng cho 13/15 trU'cmg hop kl" nhuorn FISH cac phol dU'Q'c ki§m chLrng bang ADN ph6i thai tl,l' do mang NST dU'Q'cnhuorn FISH d~u thay bQ NST xuat hlen 2 NST X, di~u nay phu hop v&i k~t cua ADN phoi thai tl,i' do va sleu am sau nay
cac bat thU'ang NST va benn di truyer bang phuonq phap kh6ng can thlep
TAl LIeU THAM KHAo Qua nghi€mCLrUnay, chLrng minh duoc
trong mau ngo<;livi cua m~ tLl' tu~n thLr 8
co xuat hi$n T8 ph6i thai tl,l' do bang ky
thu$t nhuQm huynh quang d~c hi$u NST
Y Nghien CLrUnay gop ph~n rna ra mQt
hLl'ang mai trong ch§n doan trLl'ac sinh
54
1 Bianchi OW, Flint AF, Pizzimenti MF Knoll JH, La.tt SA Isolation of fetal DNA
fror-nucleated erythrocytes in maternal blood Pre: Natl Acad Sci USA 1990, 87, pp.3279-3283
=::::::::."""
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Trang 7TAP CHi Y • DU'OC HOC QUAN SU' s66·2014
Bianchi DW, Williams JM, Sullivan LM,
FW, Klinger KW, Shuber AP PCR
~t2ticm of fetal cells in maternal blood in and aneuploid pregnancies Am J Genet 1997,61, pp.822-829
S, DeMaria MA Male fetal progenitor
ist in maternal blood for as long as postpartum Proc Natl Acad Sci USA
, pp.705-70B
(hongxu~t
hi~-I tin hi~u
K Oligonucleotides with fluorescent
opposite ends provide a quenched system useful for detecting PCR product hucleic acid hybridization PCR Methods
,4, pp.357-362
5 Lo YMD, Fleming KA, Wainscoat JS.
~:!eQlleS for the detection of autosomal fetal sequence from maternal peripheral blood
NY Acad Sci 1994,731, pp.204-213
th~ g~n
due-lang NST X'
rang hoop
k-lang NST
'P voi k~t qL
Inthiep,
-fAo
lal blood Pre:
J.3279-3283
6 Lo YMD, Lo ESF, Watson N, Noakes L Sargent u: Thilaganathan B, Wainscoat JS.
Two-way cell traffic between mother and fetus: biologicand clinicalimplications.Blood 1996,88, pp.4390-4395
7 Simpson JL, EliasS Isolating fetal cells from maternal blood: advances in prenatal diagnosis through molecular technology.JAMA 1993,270, pp.2357-2361
8 Sohda S, Arinami T, Hamada H, Nakauchi H, Hamaguchi H, Kubo T The
proportion of fetal nucleated red blood cells in maternal blood: estimation by FACS analysis Prenat Diagn 1997, 17, pp.743-752
9 WalknowskaJ, Conte FA, GrumbachMM Practical and theoretical implications of fetal/maternal lymphocyte transfer Lancet 1969,1, pp.1119-1122
55