JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY Vol 8 - N 2/2013
Nghien cuu clac di?m cSu true va quan thg te bao cua mang 6i nguoi Vict Nam
Research on Structure and Cellular F'opulations of Vietnamese Amniotic IMenibrane
I'liniii Villi TrSn ^('"'^ ^'C'" Qi'"'0' 103. Hoc vien Qudny
T o m t a t
Muc tieu de tai la nghien cdu cau true vi the va quan the te bao cua mang 6i. Phuang phdp nghiin cdu: Thu nhan mang di tU cac s i n phu mo 6k xet nghiem am tinh vdi HIV, HBV, HCV va giang mai. Van chuyen va bao quan mang 6i trong dung dich dem PBS IX. Nhudm mang di theo phUOng phap Hematoxylin-Eosm (HE) va hda md mien djch vdi Oet-4, SSEA-4, ICAM, CK-5, collagen type 111, IV, hbronectin, laminin. Ket q u i : Mang di khdng cd than kinh, mach m i u hay bach huyet va bao gom 3 mang chinh: mang bieu md don, mang nen day va mang vd mach. Mang di va cac te bao mang 6i dUOng tinh vdi cac dau an CK-5, collagen type III, IV, fibroneetin, laminin. Mdt sd it te bao duong tinh vdi Oct-4, SSEA-4, ICAM Ket lugn: Mang di la mang vd mach chda nhieu cac thanh phan gidng m d lien ket vdi cac te bao bieu md, trung md va cac te bao goc,
TUkhda: Mang di, te bao, Oct-4, SSEA-4, collagen.
S u m m a r y
The aim of this rpsearch is to study the structure and cellular population of amniotic membranes.
Methodology. Collection of amniotic membrane from women with cesarean section and negative test for HIV, HBV, HCV and syphilis. Transport and store membranes in a phosphate buffer solution (PBS) IX Amniotic membranes were stained with Hematoxylin-Eosm, Oct-4, SSEA-4, ICAM, CK-5, collagen type III, IV, fibroneetin, laminin. Results: The amniotic membrane is composed of three major layers: single epithelial layer, basement membrane and avascular mesenchyme. There are not nerves, blood vessels or lymphatic in the membranes. Amniotic membranes and the most of their cells are strongly positive with CK-5, collagen type III, IV, fibroneetin, laminin and some cells positive w i t h Oct-4, SSEA-4, ICAM.
Conclusions: Amniotic membrane is a avascular own that contains many of the composition of basement membrane associated with epithelial cells, mesenchymal and stem cells.
Keywords: Amniotic membrane, cells, Oct-4, SSEA-4, collagene.
riuin bicn kho,i lior POS I S TRINH TUAN DUNG
T/^P CHi Y DU'O'C L A M S A N G 108 Tap 8 - S o 2/2013
I.Datvande
Mang 61 duoc hinh thanh t d la phoi ngoai cd nguon goc tU khoi npi b i o vao ngay thU 8 sau thu tinh. Mang 6i ddng vai trd quan trong t r o n g q u i trinh phat trien thai nhi va chuyen da. Trong khdi 3iem va duy tri co t h i t t d cung, prostaglandin ddng vai tro cdt yeu. Bieu md mang oi khong chi I I mpt trong nhiJng nguon cung cap prostaglandin, dac biet la prostaglandin E2 [1], ma nd cdn tao ra cac enzym sinh t o n g hop prostaglandin n h u phospholipase, prostaglandin synthase, va cyelooxygenase. Cac enzyme nay dUoe dieu hda bdi hCG, cac thu e l m the cua chung cung duac tim thay trong bleu mo mang di. Bieu mo mang di ed mdc dp hoat dpng chuyen hda cao trong q u i trinh thai nghen va no CO chUe nang dieu hda pH cua dich mang 6i, giU nodtrangthli hang dinh I I khoang 7,1.
Laminin la thanh phan chinh cua mang nen te bao goc m i n g d i , t h a m gia vao q u i trinh biet hda te bao, duy tri hinh dang v l hoat d d n g te bao, duy tri kieu cau true md dong thdi thuc day sU tdn tai cua cac mo thong qua c l e thu C,:MI the cua te bao nhU integrin v l dystroglyean [2]. Vi v i y , xac dinh dac diem eae ehuoi dudi don vi cua laminin d mang di ngiidi la rat quan trong. Laminin ed nhieu ddng dang khac nhau bao gdm cac ehuoi a-, p-, va y- ma moichuoi nay cd nhieu dang ehuoi dudi don vi : a 1 - 5,pi - 3 , Y l - 3 [1]. Laminin dupc tao ra va bai t i e t t U ngay bieu md nam tren mang nen vi vay bieu hien gencija cac ehuoi dUdi d o n vi laminin trong c l e te bao bieu md mang 6i {human amniotic epithelial cells - HAE) duoc p h i t hien b i n g phan Ung reverse transeription-polymerase chain reaction (RT-PCR).
Phan tl'ch ve mat md hpc, mang di gdm hai Idp bieu fiodon v l trung md, Ldp bieu m d dan la mot Idp te bao bieu md don hinh tru, m p t phan tU do va mot phan tiep xuc vdi mang day. N i m dudi mang day la Idp trung md mang di g o m c l e mo lien ket 6uac tao bdi cac te b i o gidng nguyen b i o soi.
Mang di la r i c t h i i sinh hoc t h u d n g bd di trong qua trinh smh nd. Nhieu tac g i i 6a sd d u n g mang 6i fong dieu tri bong, loet giac mac... Gan d i y nhieu nha khoa hoc da phat hien thay mang 6i cdn la mdt
nguon cung cap te bao goc ly tUdng. Sddung te b i o goc mang di khong gap phai nhu'ng van de ve dao dUc, xa hdi. Cle te b i o goc phan lap tU mang di cd tinh sinh mien dich thap, khdng cd k h i nang ung t h u hda v l cd k h i nang biet hda thanh nhieu loai te bao khac nhau [3]. Vi vay chung toi tien hanh de tai nay n h I m muc tieu x l e dinh cl'u true vi the va eae quan the te bao cua m i n g di de lam tien de cho dng dung mang di va te b i o goc mang di trong cdng nghe t i i tao md.
2. £)6'i tdang va phUang phap nghien cdu + Ddi tdang nghien cdu:
M i n g di cua c l e san phu mo de b i o dam tieu chuan xet nghiem sang Ipc I m tinh vdi HIV, HBV, HCV, HTLV, giang mai.
+ Phddng phap nghien cdu:
- Bdc tach mang di khdi nhau thai. Cat Ipc, rUa sach nhieu lan bang dung djch PBS IX eho tdi khi mang di trd nen trong suot.
- De nghien cdu cau true eCia mang di, c i t nhd mang di thanh nhdng manh nhd cd kich thUde khoang Icm^. Mdt phan mang di duoc dan m i n h mang 6i len lam kinh, de khd trong nhiet dp phdng sau 66 CO dinh mang di bang eon tuyet ddi trong vdng 10 phut. Mdt phan 6uac cd dinh trong paraformaldehid (PFA) 4 % sau 66 chuyen due paraffin va e l t l i t tieu b i n . Nhudm theo phuong phap HE h o l e hda md mien dich vdi cac dau an Oct- 4, SSEA-4, ICAM, CK-5, collagen type .11, IV, Fibroneetin, laminin, Quan sit, chup I n h dudi kinh hien vi quang hpc h o l e kinh hien vl huynh quang.
Mo t l cl'u true dai the va vi the cua mang di.
+ Ky thuat nhugm hoa md mien dich DAB (3, 3 '-diaminabenzidin e)
Mau te bao goc mang di duoc nudi cay tren tam mang 6i, sau khi dUoe due paraffin dUOc eat mdng, dan len lam kinh, lam khd d45''C. KhU parafhn trong xylen (Sigma) va con theo quy trinh khuyen cao cua nha cung cap. Lfe che peroxydase npi bao bang hydrogen peroxide 0,03% 20 phut. U vdi k h i n g the khang vdi khang nguyen nghien cUu trong vong 1 gid va rUa tieu ban b i n g PBS. U vdi khang the t h d
JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY Vol8-N"2/2013
hai cd g i n Biotin 1 g i d . U vdi phUc h o p Streptavidlne peroxydase 20 phut. Hien thi mau vdi ca chat DAB.
RCfa tieu b i n vdi nude cat. N h u p m vdi Hematoxylin tao nen t u o n g phan 2 phut. RUa tieu b i n vdi nudc eat. KhU nUdc b i n g eon v l xylen. D i n lamen va quan s i t , chup luu lai hinh I n h tren kinh hien vi. Ket q u i sau khi n h u p m hda m d mien dich, ket q u i I m t i n h chi cd mau xanh t i m cua Hematoxylin n h u p m nhan.
Duong tinh neu cd hien dien cua khang nguyen tren te bao, phUe h p p khang nguyen - k h i n g the - streptavidlne se eho mau vang nau (mau cua DAB),
3. Ket q u a n g h i e n c d u
J.T. Ket qui cau true mang 6i ngddi Viet Nam.
Mang 6i cd nguon goc tU I I phoi ngoai (ngoai phoi bi) v l ed 5 Idp, lan luot I I : 1. Bieu m d ; 2. Mang d i y ; 3. Ldp d i e ; 4. Ldp nguyen bao sOl; 5. Ldp xop.
Oe x i c dinh ranh gidi glUa c l e Idp cua m i n g 6i, chung tdi 6a tien hanh n h u p m HE m i n g di da p h i n tach duoc (Hinh 1).
Man^rvnfl . i « j g ^ ^
. 1 ^ N^ang 6i
C
100X j ^
^ T^Motungmfl
5>-^^^^^
D
200X
Hmh 1: Cdu true mdng 6i trin tieu bdn nhudm HE.
A: Nhau thai. B- Mdng 6i cdt ngang. C, 0: Mang 6i todn phdn ddn tren lam kinh, co dinh vd nhugm chup dnh dUdi kinh hien vi vdi do phong dgi
tan luot Id WO vd 200 lan.
3.2. Ket qua nhugm hoa mo mien djch Ket qua n h u p m hda m o mien dich m a n g di bang c l e marker dac hieu d u o c t h e hien t r o n g hinh Hinh ?.
CK-5 Oct4
TAP CHi Y DU'O'C L A M S A N G 108
Tap 8 - S 6 2/2013
agentype IV
Fibroneetin Laminin
Hinh 2: Nhudm hda md miin dich mdng di.
Mdng dichda nhieu CK-5 vd collagen type III, laminin mot so tebdo bieu md mong 61 dUang ti'nh vdi
Oct-4, SSEA-4, collagen type IV, ICAM, Fibroneetin, laminin (mui tin). Hinh dnh long ghep
vdi do phdng dai Idn han.
4. Ban luan
4. T. Cau true mang 61 ngddi Viet Nam Trong nghien eUu cua chung tdi, mang di cung CO the duoc p h i n chia m p t c i c h dan gian hon nhu sau (Hinh 1 A v a B ) :
Ldp bieu md; gom Idp te bao bieu md va mang
^ay, la Idp cd the cho phan l i p duoc c l e te bao goc bieu md.
Ldp nen dac gom Idp d i e , nguyen bao soi va ldp xdp la Idp cd the cho phan lap duoc cac te bao goctrungmd.
Ldp te bao bleu md la Idp trong cung va tiep xuc vdi djch di. Ldp nay la m d t Idp te bao bieu mo dar\ det, hinh hdp hay t r u , phan tU do hUdng ve dieh oi tao thanh cac vi nhung mao. Cic te bao bleu md
duoc nudi cay sau 3 ngay phan lap cd hinh da dien
hoac hinh thoi. -Day cua eae te bao bieu md don nay tiep xuc vdi m i n g d i y . Phan d i y cua ldp bieu md don nay ed hinh d i n g phUe tap va khdng deu, nhd vay bam c h i t duoc vao Idp mang day.
Mang nen: La mdt ldp mang mdng ehUa cac soi ludi, dan xen vdi nen te bao cua ldp bieu md don.
Ldp dac: La mdt ldp d i e chUa cac mang cac soi ludi dan xen phUe tap, sU thay doi ve mat cau true cle soi ludi cua idp nay so vdi ldp mang nen nham mang lai tinh deo dai cua mang di va chong lai sU tham n h i p cua bach cau khi mang di bi viem.
Ldp nguyen bao soi: Day la Idp day nhi't cua mang di, cau true cua Idp n i y la mdt mang ludi eic nguyen bao spi long nhung trong khdi chat lUdi
Ldp xdp: cac phan cua mang oi cung mang dem nen eae md cua khoang ngoai phdi dan den hinh thanh Idp xop. Cau tao cua ldp xdp 11 eae bd spi ngam trong chat nhay, cac bd soi nay thudng bi truong len lam tang dp day cua mang oi, Chdc nang cua ldp xdp la lam cho mang di trUdt de dang tren
JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY V o l 8 - N ° 2 / 2 0 1 3
mang dem t r o n g khi mang d e m b I m chat vao mang rung.
4.2. Quan the te bao cua mang oi ngddi Viet Nam
Oe x l e d m h quan the te bao cung nhU thanh phan cua chat nen eo b i n ngoai b i o , chung toi tien hanh n h u p m HE, n h u d m hda m d mien djch. Cle te bao goc mang 61 d u o n g t i n h vdi Oct-4, SSEA-4 1^ c l e marker cua te b i o goc phdi (Hinh 2). Cac te bao g i n ket duoc tren m i n g di la n h d c l e thanh phan cua chi't nen gian bao {extracellular matrix - ECM) [4].
Trong do, chat nen chda nhieu CK-5 va collagen type 111, collagen type IV (Hinh 2). Su hien dien cua cac phan tU collagen, laminin, fibroneetin va vitronectin cd nhdng I n h hUdng t o Idn den k h i nang g i n ket va p h i t trien cua te bao [5, 6]. Vdi nhUng yeu cau tren thi ro rang mang di I I m o t g i i the phu h p p eho edng nghe m d vdi cau true cua khudn gian b i o dien hinh [5]. Cac te b i o bleu mo mang oi tiet ra collagen type 111 va IV v l cac glycoprotein non-collagenous (laminins, nidogen va hbronectin) hinh t h i n h mang day (Hinh 2). Trong Idp xdp cd nhieu cua proteoglycans v l cac glycoprotein v l ed m p t mang lUdi nonfibrillar chu yeu collagen 111 (Hinh 2). Trong mang day, ton tai m o t lupng ldn perleean, la yeu to quan t r o n g cho tang trudng v l t u a n g t i e vdi cac phan tU protein ngoai bao v l ket dinh te bao [7]. V\ vay khi nuoi cay cac te bao bieu md va trung mo v i o g i i the mang di da cho thay su lien ket tot gida chung cung n h u vdi ea'u true cua mang oi [8].
Kef ludn: Mang di ed cau true cua md lien ket t r o n g d d cd nhieu te bao bieu m d , trung m d va te bao goc, Mang 6i la mang vd mach khong cd mach
mau, t h i n kinh va mach bach huyet. Vi vay, m a n g oi hda hen I I m o t s i n p h a m co nhieu t r i e n v p n g cua c d n g nghe m o t r o n g t u o n g lai.
Tai tieu t h a m khao
1. NIknejad, H., et al„ 2008. Properties of the amniotic membrane for potential use in tissue engineering. Eur Cell Mater. 15: p. 88-99.
2. Lee, PH., et al., 2012. A prototype tissue engineered blood vessel using amniotic membrane as scaffold.
Acta Biomater
3. Parolini, 0., et al., 2008. Concise review: isolation and charaaerization of cells from human term placenta:
outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells. 26(2): p. 300-11.
4. Ml, S„ et al., 2012. Tissue engineering a fetal membrane. Tissue Eng Part A. 18(3-4): p. 373-81.
5 Niknejad, H., et al., 2011. The effects of presen/ation procedures on amniotic membrane's ability ta serve as a substrate for cultivation of endothelial cells.
Cryobiology. 63(3): p. 145-51.
6. Redondo, P., et al„ 2011. Amniotic membrane as a scaffold for melanocyte transplantation in patients with stable vitiligo. Dermatol Res Praet. p. 532139.
7. Murdoch, A.D., et al., 1994. Widespread expression of perleean proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization. J Histochem Cytoehem. 42(2): p.
239^9.
8. Portmann-Lanz, C.B., et al., 2007. Manufacture of a cell-free amnion matrix scaffold that supports amnion cell o u t g r o w t h in vitro. Placenta. 28(1) : p. 6-13
