Báo cáo " Tạo tấm tế bào sừng nhiều lớp trên màng collagen từ màng ối người " pot

Tgp chi Cong nghe Sinh hoc 7(2): 155-160, 2009 TAO JAM TE BAO SUlVG NHIEU LOHP TREN MANG COLLAGEN TlT MANG 6I NGlToa Tran Le Bao Ha', Tran Gong Toai^ Hoang Nghia Sffn^ 'Tru&ng Dgi hoc Khoa hoc Tu nhien, thdnh pho Ho Chi Minh ^Truong Dgi hoc Ykhoa Phgm Ngoc Thach, thdnh pho Ho Chi Minh ^'Vien Sinh hoc nhiet d&i . ,;; ,i , ^ TOMTAT Bieu bi la lop ngoai cung cua da, cau thanh tit bieu mo vay phan ting biet hoa cuoi ciing. Bieu bi dong vai tro bao ve dau tien cua ca the chong lai nhiing tac dong bat lgi cua moi tradng nhu cac tac nhan sinh hoc, hoa hoc, vat ly. Mo bieu bi gom nhieu lop te bao svmg co the dugc tao ra in vitro de umg dimg trong chiia tri cac ton thuang da cap va man tinh. Trong nghien ciiu nay, tam te bao simg nhieu lop dugc tao ra tit te bao sirng nguai truong thanh va mang collagen tir mang oi. Te bao simg dugc phan lap, nuoi, nhan trong moi truang khong huyet thanh SFM (Serum Free Medium). Mang collagen dugc thu nhan ttr mang oi da dugc loai bo lop te bao bieu mo va danh gia bang phuang phap nhupm HE. Sau khi dugc cay chuyen Ien mang collagen, te bao simg dugc kich thich tang sinh va biet hoa thaiih nhieu lop bang each de tiep xuc khong khi. San pham tam te bao simg nhieu lap dugc danh gia bang RT-PCR, hoa mo mien dich. Ket qua cho thay, mang oi da dugc loai bo hoan toan lap bieu mo sau khi xit ly enzyme va ca hpc. Cac te bao simg da tang traang, tao 4 - 5 lap teen mang collagen va bieu hien marker te bao simg da va chua biet hoa sau 7 ngay tiep xiic khong khi. Cac te bao simg duang tinh vai marker tang sinh (p63) nam sat tren mang collagen, cac te bao ben fren la nhiing te bao sirng da biet hoa. » « is I ! • I C • • •• Tit khoa: Bieu bi, collagen, mdng oi, moi truang khong huyet thanh, te bdo simg I MdDAU Sy khep vet thuang can mgt vat lieu de hoi phuc chiic nang hang rao cua bieu bi va sap nhap vao vet thuang dang lanh. Hien nay, tu ghep vat da van la "tieu chuan vang" de tai tao be mat cac vet thuang lan. Tuy nhien, vimg da eho de ty ghep bi han ehe frong cac vet thuang rgng. Dieu nay thuc day phat trien cac lieu phap khac nhu la ky thuat ghep tam te bao bieu bi nuoi cay (Cultured epithelial autograft - CEA)(Raymundefa/., 2005) Cac tam te bao bieu bi nuoi cay thudng gom 3 - 5 ldp te bao va rat khd cam giii. Do do, can cd mgt cong cu de thao tae tam te bao nay. De khac phuc, mgt so vat lieu da dugc su dung de mang tam te bao simg nhu mang collagen loai 1 ciia bo, pHEMA (poly(2-hydroxyethyl methacrylate)), PIAAm (N- Isopropylacryamide), PPS (plasma polymerised fiinctional surface) (Barbora et al, 2003; Jones et al, 2003; Masayuki et al, 2001; Zhu et al, 2005). Mang li bao gom mgt lop dan te bao bilu md, mgt mang ca ban, mgt ldp dac vo bao, lop nguyen bao sgi va lap xdp ben duoi Mang ca ban ciia mang 6i gdm laminin 5, collagen loai IV, VII, XVII, tuang ty vdi mang co ban eua da ve hinh thai va sieu cau tnic (Nakamura et al, 2004) Mang di cd tinh khang viem, khang xa hda, khang seo, kich thich bieu md hda va tinh sinh mien dich thap (Hassan et al, 2008; Oyama et al, 2003). Mang oi loai bieu mo da dugc sit dung lam gia the frong cdng nghe md giac mac, sun, than kinh (Jin et al, 2007; Tseng, 2001; Ueno era/., 2006). Trong nghien ciiu nay, chiing tdi da sit dung mang collagen tii mang di loai bieu md de lam gia the nuoi te bao simg nham tao tam te bao simg nhieu lap in vitro. VAT LIEU VA PHUONG PHAP Doi tu-ffng nghien cihi Da lanh ciia benh nhan cd ehi dinh ghep da va mang oi ciia san phu sinh mo. Phuong phap thu nhan mau Thu nhan mau da, mang di ciia san phu vita sinh da dugc xet nghiem am tinh HIV, viem gan sieu vi B, viem gan sieu vi C, giang mai, giii frong PBS 155 Trin Le Bao Ha et al (Gibeo) latih cd khang sinh va chuyen vl phong thi nghiem, thao tie frong vong 4 h. Phirong phap xfr ly mang oi (Hassan et al, 2008) Mang oi duge rira frong dung dich PBS cho den khi sach mau, sach eac md song khong can thiet. Sau do, lac mang oi frong dung dich trj'psin 0,25%) - EDTA 0,02% (Sigma) frong 30 phiit a 37°C va dimg mgt dung cu mem cao bo lap te bao bieu mo HAM, franh lam va mang day. Hieu qua boc tach lap te bao bieu mo mang oi dugc danh gia bang phuang phap nhuom hematoxylin va eosin (nhuom HE). Phuong phap phan lap te bao simg (Susan, 2003) Mau da dugc rira 3 lan frong PBS va loai bo phan thira (long, ma). Sau do, eat mau da thatih manh nho va u vai Trypsin-EDTA (Gibeo) frong 18 h, 4°C. Lop bieu bi va lap trung bi duge tach rai bang ea hgc, thu nhan te bao simg rai tit lap bilu bi bing each dung pipet de huyIn phu. Mat do tl bao, t>; le %> te bao song dugc xac dinh bing each dim te bao sau khi nhuom trypan blue. Phuong phap nuoi, nhan tl bao sirng (Susan, 2003) Te bao tach rai dugc nuoi frong moi truang sa cap: DMEM bo sung FBS (Fetal Bovine Seram), EGF (Epidermal Grov^fth Factor), hydrocortisone, cholera toxin (Sigma) vai mat do 3 x 10^ te bao/cm^ be mat dia nuoi. Sau hai ngay, thay moi traang sa cap bang moi traang khong huyet thanh SFM (Seram Free Medium - Gibeo) danh cho te bao simg. Tiep tuc nuoi a 37°C, 5% CO2 eho den khi tl bao tang tradng dat 70 - 80% be mat dia nuoi va ciy chuyen. Te bao sirng Mang collagen Loc cua dia long Moi tru'd'ng nuoi Hinh 1. Minh hoa phuang phap nuoi cay te bao trong dTa I6ng. Cac tg bao sCrng dugc phai ngoai khong khi, dinh du&ng du'gc cung cap tCr moi tru'ang ben duai qua cac 16 lpc cua dTa I5ng. Phuofng phap nuoi te bao simg tren mang collagen Mang collagen dugc frai fren day ciia dia ling (dia insert) va cay chuyin tl bao simg len mang collagen vai mat do 3 x lO'' tl bao/cm^bl mat mang, u a 37°C, 5% CO2. Moi traang dugc thay ben ngoai va ben frong dia long mli 2 ngay/lin. Phuong phap tao tam nhilu Idfp tl bao simg Khi te bao simg tang sinh kin bl mat collagen, hiit het moi tradng frong dia ling ra. Tilp tuc thay moi tracmg ben ngoai dia ling mli ngay, keo dai 7 ngay. Phuong phap xac dinh te bao simg bang RT-PCR RNA ting dugc ly trich bing Trizol (Sigma). Sy bilu hien gen duge danh gia bing One step RT-PCR (Promega). Su bieu hien gen P-aetin nguai duge su dung lam doi ehiing. Trinh ty eua cac primer: p63 (5'- CAG ACT CAA TTT AGT GAG -3' (sense), 5'- AGC TCA TGG TTG GGG CAC -3' (antisense)), 440 bp; involuerin (5'-TCC TCC AGT CAA TAC CCA TC- 3' (sense), 5'- CTT CAT TCC CAG TTG CTCA. TC-3' (antisense)), 373 bp; K14 (5'- ATG ATT GGC AGC GTG GAG -3' (sense) 5'- GTC CAG 156 Tgp chi Cong nghe Sinh hoc 7(2): 155-160, 2009 CTG TGA AGT GCT T -3' (antisense)), 390 bp; K5 (5'- CCC AGT ATG AGG AGA TTG CCA ACC -3' (sense), 5'- TAT CCA GAG GAA ACA CTG CTT GTG -3' (antisense)), 475 bp; P-actin (5'- CCA AGG CCA ACC GCG AGA AGA TGA C -3' (sense), 5'- AGG GTA CAT GGT GGT GCC GCC AGA C -3' (antisense)), 587 bp (Adriana et al, 1998; Adriana et al, 2001; Mazlyzam et al, 2007; Virve et al, 2007). Phan ling RT-PCR dugc thuc hien nhu sau: 45 phiit a 45°C, 3 phut d 94°C, PCR 35 chu ki (45 giay a 94°C, 30 giay d 55°C, 90 giay d 72°C), ii 10 phut d 72°C bang Thermocyeler cua Eppendorf. San pham PCR dugc dien di fren gel agarose 1,5%. Phuong phap nhan dien te bao simg con kha nang tang sinh Cac te bao simg cdn kha nang tang sinh frong tam te bao dugc nhuom hda mo mien dich vdi marker te bao goc bieu bi la p63. Sau khi tao ldp, tam te bao simg dugc cat lat va nhudm vdi khang the p63 ddng 4A4. Ket qua dugc quan sat va chup hinh dudi kinh hien vi quang hgc Olympus. KET QUA VA BAN LUAN Ket qua nuoi, nhan te bao simg Sau khi dugc phan lap va nuoi trong mdi tradng khdng huyet thanh, cac te bao simg bat dau tao thanh cum vao ngay 3-4. Sau dd, cac cum nay ldn dan va hgp vao nhau tao thanh ldp dan te bao tren be mat dia nuoi sau 7 ngay. Ket qua xir ly loai bieu mo mang oi Sau khi xu ly vdi enzyme (trypsin-EDTA) 30 phiit va ca hgc (cao) toan bg ldp bieu md mang di da dugc loai bd. Ket qua nay phii hgp vdi mgt sd edng frinh da duge cong bd (Hassan et al, 2008; Ishino et a/, 2004). Ket qua nuoi te bao simg tren mang collagen Sau khi xit ly, mang collagen dugc frai len dia long vdi mat bieu md dua len fren. Dieu nay giiip eho tl bao simg dugc cay len se tiep xuc tryc tilp vdi bl mat mang ca ban eiia mang li. Kit qua eho thay mang collagen kich thich tl bao bam dinh nhanh ban. Mang ca ban cua mang li gdm laminin 5, collagen loai IV, VII, XVII, tuang ta vdi mang ca ban cua da ve hinh thai va sieu cau tnic (Nakamura et al, 2004). Do dd, cac protein ngoai bao nay se giup te bao bam dinh nhanh va di cu. Sau 7 ngay nudi tren mang collagen, eac te bao simg da tao thanh ldp dan. Ket qua xac dinh te bao simg bang RT-PCR Sau khi tao nhieu ldp fren mang collagen, cac tam te bao dugc xac dinh bang phuang phap RT- PCR vdi cac marker te bao simg. Ket qua cho thay, cae te bao bieu hien ea 4 marker te bao simg, frong do cd 3 marker te bao chua biet hda la p63, k5, kl4 va marker te bao da biet hda la involuerin. Cac te bao simg frong tam te bao in vitro bieu hien duang tinh eac marker nhu cac te bao simg trong md da ddi ehiing. Dieu nay cho thay cae te bao dugc biet hda tao ldp van cdn giii tinh gde. tao nhieu lop te bao simg tren mang Ket qua collagen Sau 7 ngay tiep xuc khdng khi, mau md duge nhudm hda mien dich vdi khang the p63. Ddi ehiing la mau da nguyen. Ca hai mau deu ed te bao duang tinh vdi marker tang sinh, chua biet hda p63. Mau tam te bao in vitro ed 4 - 5 ldp te bao simg vdi cac te bao duong tinh p63 nam sat fren mang collagen, eae te bao ben fren la nhiing te bao simg da biet hda. Ket qua nay phu hgp vdi dac diem md hgc eiia da vdi cac te bao gde bieu bi duong tinh p63 nam d ldp day, ngay fren mang ea ban. Sau khi tao ldp, tam te bao simg dugc tach ra de dang khdi be mat dia nudi bang each keo lot mang collagen. Nhu vay, mang collagen tit mang di la vat mang ly tudng eho tam te bao simg. KET LUAN Mang di da duge loai bd hoan toan ldp bilu md sau khi xir ly enzyme va ca hge, thu duge mang collagen. Cae te bao simg da tang trudng, tao 4 - 5 ldp tren mang collagen va bieu hien marker tl bao simg da va ehua biet hda sau 7 ngay tiep xue khdng khi. Cac tl bao bieu hien marker te bao gdc bilu bi (p63) nim ngay fren mang collagen. Vdi ket qua thu dugc, hudng nghien cim ed thl tien xa ban frong iing dung lam sang dl dilu tri cac tdn thuang mit da fren benh nhan. 157 Trin Le Bao Ha et al Hinh 2. A. Cac cum (40X) va lap dan te bao sOng trong dTa nuoi; B Cac cum (100X) va lap dan te bao sOng trong dTa nuoi; C. Mang ol nguyen ven (100X); D. Mang 61 da loai hoan toan lap bieu mo (100X). E. Te bao sCrng bam tren dTa nuoi sau 3 h cSy chuygn (100X); F. T§ bao sCrng bam tren mang collagen sau 3 h cay chuyen (100X). Hinh 3. Lap dan te bao sOng tren mang collagen (40X). 158 Tgp chi Cong nghe Sinh hoc 7(2): 155-160, 2009 M 1 2 3 4 5 Hinh 4. Ket qua RT-PCR cac marker cua te bao sOng. M. Thang marker; 1, 3, 5, 7, 9. San pham khuech dai cac gen cua t4 bao sirng nuoi cay tu'ong iing p63 (440 bp), k14 (390 bp), k5 (475 bp), involuerin (373 bp), p-actin (587 bp); 2, 4, 6, 8, 10. San pham khuech dai gen cua mau da binh thuang doi chung tuang u'ng p63, k14, k5, involuerin, p-actin. Hinh 5. Ket qua nhupm hoa mo mien djch vdi khang the p63 mo bleu bi tren mang collagen (A) va mau da doi chCrng (B) (400X). Cac te bao bat mau nau: duang tinh p63. Ldi cam on: Chiing toi chdn thdnh cdm cm Benh vien Chdn thuong chinh hinh, Binh vien Phu sdn Hiing Vuong da cung cdp mdu da vd mdng oi nguai de tien hdnh nghiin cuu ndy. TAI LIEU THAM KHAO Adriana C, Arzu S, Sandrine W, David FF, Stan PCB (2001) SPRR4, a novel comified envelope precursor; UV- dependent epidermal expression and selective incorporation into fragile envelopes. J Cell Sci 114: 3837- 3843. Annie Y, Mourad K, Yunmei W, Emily G, Mark DF, Volker D, Nancy CA, Daniel C, Frank M (1998) p63, a p53 Homolog at 3q27-29, Encodes Multiple Products viii'tx Transactivating, Death-Inducing, and Dominant-Negative Activities. Mot Cell 2 : 305-316. Barbora D, Zuzana H, JiRi V, Radana, Zuzana K, JiRi M, Martin P (2003) Reconstraction of epidermis by grafting of keratinocytes cultured on polymer support - clinical study. Inter J Dermatol 42: 219-223. Hassan N, Habibollah P, Abolhassan A (2008) Properties of HAM in tissue engineering. Eur Cell Mater 15: 88-89. Ishino Y, Sano Y, Nakamura T, Connon CJ, Rigby H, Fullwood NJ, Kinoshita S (2004) Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol Vis Sci 45: 800-806. Jin CZ, Park SR, Choi BH, Lee KY, Kang CK, Min BH (2007) Human amniotic membrane as a delivery matrix for articular cartilage repair. Tiss Eng 13: 693-702. Jones I, James SE, Rubin P, Martin R (2003) Upward migration of cultured autologous keratinocytes in Integra artificial skin: a preliminary report. Wound Repair Regen 159 Trin Le Bao Ha et al. 11:132-138. Masayuki Y, Mika U, Ai K, Chie K, Akihiko K, Terao O (2001) Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheet without dispase by reducing temperature. Tissue Eng 7: 473-480. 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Susan SY (2003) Current Protocols in Cell Biology. John Wiley Sons, Inc. : 155-160. Tseng SC (2001) Amniotic membrane transplantation for ocular surface reconstraction. Biosci Rep 21: 481-489. Ueno M, Matsumura M, Watanabe K, Nakamura T, Osakada F, Takahashi M, Kawasaki H, Kinoshita S, Sasai Y (2006) Neural conversion of ES cells by an inductive activity on human amniotic membrane matrix. Proc Natl Acad Sci USA 103: 9554-9559. Virve P, Jussi V, Tuula S, Leo T (2007) Effects of TGF-bl on interleukin profile of human dental pulp and odontoblasts. Cytokine 40: 44-51. Zhu N, Warner RM, Simpson C, Glover M, Hemon CA, Kelly J, Eraser S, Brotherston TM, Ralston DR, MacNeil S (2005) Treatment of bums and chronic wounds using a new cell fransfer dressing for delivery of autologous keratinocytes. Eur J Plast Surg 28: 319-330. CREATION OF MULTILAYERED KERATINOCYTE SHEET ON COLLAGEN FROM HUMAN AMNIOTIC MEMBRANE Tran Le Bao Ha'' *, Tran Cong Toai^ Hoang Nghia Son^ 'University of Natural Sciences, Hochiminh City ^Pham Ngoc Thach University of Medicine, Hochiminh City ^Institute of Tropical Biology SUMIVIARY Epidermis composed of terminally differentiated stratified squamous epithelium is the outermost layer of the skin. The epidermis is the major barrier protect the body from inhospitable environment factors such as biological, chemical, physical factors. Epidermis which consists of the multilayered keratinocytes could be created in vitro for the freatment of acute burn injuries and chronic wounds. In this study, the multilayered keratinocyte sheets were created from the adult human keratinocytes and the amniotic collagen membrane. Human keratinocytes were isolated, cultured, amplified in seram free medium. Collagen membrane was derived from human amniotic membrane of which the epithelium was removed and evaluated by HE staining. After subcultured on collagen membrane, keratinocytes were stimulated for proliferation and differentiation to create multilayer by airlifting. The multilayered keratinocyte sheet characteristics were evaluated by using RT- PCR and immunohistochemistry. The results showed that epithelial cells were removed completely from the human amniotic membrane by enzyme and mechanical treating. Keratinocytes proliferated and formed 4-5 layers on collagen. They also expressed some differentiation and proliferation markers at 7 days after airlifting. The keratinocytes which were positive with proliferative marker (p63) lied closely on the collagen membrane and upper ofthe cells differentiated. Keywords: Amniotic membrane, collagen, epidermis, keratinocyte, serum free medium ' Author for correspondence: Tel: 0988575507; Fax: 84-8-38350 096; E-mail: tlbha&.hcmuns.edu.vn 160 . te bao simg tren mang collagen Mang collagen dugc frai fren day ciia dia ling (dia insert) va cay chuyin tl bao simg len mang collagen vai mat do 3 . nam sat tren mang collagen, cac te bao ben fren la nhiing te bao sirng da biet hoa. » « is I ! • I C • • •• Tit khoa: Bieu bi, collagen, mdng