La Duy Anh vd Dtg Tgp chf KHOA HQC & CONG NGHE 177(01): 197-201
C H U A N H O A M O I T R l T O f N G V A K t T H U A T N U O I C A Y T E B A O G O C P H O I C H U p T T A I P H O N G T H I N G H 1 £ M TRlTOfNG D A I H O C Y D l T O C T H A I N G U Y E N
Ld Duy Anh', Nguyen Phuong Sinh Truang Dqii hpc Y Duac - DH Thdi Nguyin TOM T A T
NghiSn cuu ve te bdo gic ftng dyng trong jTnh vyc y hgc diu dugc khdi dau tft nuoi cay thdnh cong t l bao gic phfii Tuy nhien tuj' vdo dilu kiSn thyc tl cda moi phfing chi nghiem md cdc nhd nghien ciiu da xdy dyng cho minh mfli tnrdng vd ky thugt nufii cay t l bdo gfle rieng.
Trong pham vi nghifin cftu ndy chung tfli dd nghien cun, tim hilu nhftng dilu kifin va ky thugt nuoi cay te bdo g i c tft nhftng phong thi nghiem trong vd ngodi nude. Sau do chudn hfia moi trudng, kJ thuat dp dung vdo phfing thi nghifim tgi trudng Dgi hoc Y Dugc Thdi Nguyen.
Chung tfii da tifin hdnh chudn hod thanh cong mfii trudng vd kJ thudt nuoi cdy tl bao gfic phoi chugt. Kit qud nufli cay thdnh cflng dong t l bao gfle phfli chugt cho thay. T I bdo sinh truong tot, hinh thdi dien hinh, du dieu kifin de djnh hudng biet hod thanh cdc te bdo mong mufln.
TIT khoa: Ti bdo gdc, li bdo gdc phdi chuot. nuoi cdy, mdi trudng nudi c^, ky thudt nudi cdy D A T V A N D E
Te bao dgng vat tdch tft mfl cfi the d u g c nuoi cay tren cdc logi mfli truong nufii cay tong hgp ben ngoai c a the, chung sinh trudng bdng each tang sfl lugng va kich thudc t l bao [1].
KJ thudt nuoi cay te bao dgng vat ddc biet Id cac te bao goc phdi da tgo c o hpi de nghien cuu ve sy phan hod cua te bao gdc thanh cac logi t l bao khac nhau tft dd cd nhung iing dyng vao cdc nghien curu trong ITnh vuc y hpc [4].
Te bdo goc phdi Id mpt logi te bdo co khd ndng ty lam mdi va phan hoa toan nang, dudi dieu kien nhat djnh cfi kha nang hinh thanh mgi logi te bao ciia cac to chiic trong c o the dgng vgt [5]. Chi'nh vl vgy te bao goc phoi cd tiem ndng nghien cftu rat ldn vd cd khd nang ling dung vdo ITnh vyc y hpc.
Trong nhung nam gan day te bao gdc da chiing minh d u p c tam quan trpng va tiem nang chua benh cd thfi ftng dyng rat Ion trong y hgc. Chinh vi vgy nghien cuu v l te bao goc dd dupc nhieu nhd khoa hgc vd nhieu phdng thf nghiem tren t h i gidi tien hanh nghien cun.
Tuy thupc vao d i l u ki^n thyc tiln md moi phdng thi nghiem cfi the xay dyng d u g c mfli trudng va ky thuat nudi cay rieng. Dfch d i n
' Tei 0912 866006, Email: [email protected]
ciia viec nay Id co the nudi cay thanh cflng ddng te bao goc phfli fln djnh, de lam co sd tien hanh cac nghien cuu t i l p theo. Chinh vi vay ehftng tdi tien hanh thuc hien nghien cftu:
"Chuan hod mdi trudng vd ky thudt nudi cay te bdo goc phfii chudt tgi phdng thi nghiem truong Dgi hpc Y Dupc Thai nguyen" vdi muc tieu nhu sau:
- Chuan hoa thanh cflng moi trudng nudi cay te bao goc phoi chupt;
- Chudn hoa thanh cdng ky thugt nufli cay t l bao gfic phdi chugt;
- Nuoi cay thdnh cfing te bao goc phoi chupt, DOI TU'ONG V A PHU'ONG P H A P N G H I E N C U U
Boi tirgng nghien ciru Te bao goc phdi chupt Dia diem va t h o i gian nghien c m i Thf nghigm d u g c tien hanh d phdng thf nghiem Sinh hgc phan t u trudng Dai hgc Y Dugc Thai Nguyen,
Thdi gian: Tft thang 1/2017 den thdng 12/2017
P h u o n g p h a p nghifn ciru
Trong pham vi nghien cuii ndy, chiing toi tham khao cac ky thuat nuoi cay te bao gdc 197
La Duy Anh vd Dtg Tgp chi KHOA HQC & CONG NGH$ 177(01): 197-201 phfii chupt d cac phdng thi nghiem tren the
gidi vd Viet Nam. Sau do chudn hda mdi trudng va kJ thugt nudi cay dp dyng vdo nudi cdy tl bao gdc phfli chugt tgi phdng thi nghiem trudng Dai hgc Y Dugc Thai Nguyen.
Hudng tdi chudn hoa dugc mdi truong nuoi cdy tl bao goc phdi chupt on dinh. Cac te bao nufli cdy cd chiic nang gidng vdi te bdo trong CO the dfing vdt.
Sau khi nudi cdy te bao goc phfli thai chugt, chiing tdi su dung kinh hien vi huynh quang (Nikon) de quan sdt tl bao nudi cay. Sau do su dyng phdn mem chyp dnh bang kinh hien vi (NIS-Elements Nikon) dl tien hanh chyp, phan tfch, xft ly hinh dnh te bao.
KET QUA VA B A N LUAN
Chuan hoa mfii trirdng nufii cay te bao goc phfii chugt
Nhftng ylu to chinh anh hudng din qua trinh nudi cay tl bdo goc phfli chugt:
-Gia the nudi cay;
- Thanh phan hoa li va sinh li cua mdi truong nudi cay,
- Thanh phan pha khf;
-Nhigt dp toi uu khi nufii cay.
Do tl bao ddng vgt khfing cd vach nhu te bdo thyc vgt vd vi sinh vat nen chung can mgt gia thi dl cd thi trai rpng ra trong suflt qua trinh tang sinh. Vdi cac te bao binh thudng, khi nuoi cay chung thudng chi mgc thanh lop don va phu kin be mat gia the. Cdc gid the thong thudng bao gom: DTa 96 gieng, 24 gieng va 6 gilng; binh nudi bang nhua dupc su dyng phd bien, chiing cd hai tru diem: (1) Gid the cd be mat phii hpp cho te bao gdn bam vd tham dupc CO2 va O2; (2) be mat bang nhya mdng, thich hpp cho viec quan sat te bao duoi kinh hien vi quang hpc hay dign tu [2].
Thanh phan quan trgng cua pha khi la CO2 va O2. Trong nudi cay, ap lyc oxy d dieu kien khi quyen phu hpp cho hau het cac te bao nufli cay. Dp sau (be day) ciia moi trucmg nufli cay cung anh hufing dfin ti lg oxy khuyech tan vao te bao, thdng thudng dp sau
tfii tru cua mdi trudng vdo khodng 2 - 5 mm (0,2 - 0,5 ml/cm^) trong nudi cay tTnh. Vai tr6 cua khi carbonic trong nudi cay tl bao dgng vat ndi chung kha Id phftc tgp, do nhilu hoat dgng tuong hfi gifta CO2 hod tan, pH va nong do HCO3. He dgm bicarbonat giup duy fti pH mong muon. Sdn pham nudi cdy chiia nhieu acid do CO2 ndi sinh cao, ndp cua binh nudi cdy cho phep CO2 thfta dugc thai ra ngoai ddng thdi CO2 tu tu nudi se di vao dgm cho mfli trudng. Mfii h | dem cfi nong dg CO2 thich hop phy thugc vao timg logi mfii tnidng dung dl nuoi cdy tl bao [2], [4].
Nhigt dp thich hop cho nudi ciy tl bao nguai va dpng vgt cfi vii la 37''C, 5% CO2.
Thanh phan mfli trudng nufli cay te bdo dgng vgt phftc tgp ban rat nhieu so vdi nufli cay vi sinh hay tl bdo thyc vat, chung la hdn hop cac ylu tfl dinh dudng va cdc chat khac. Tuy theo timg logi tl bao khde nhau ma dung mfii truong thich hgp, Thanh phin mfii trudng nufii cdy tl bao dupc thiet lap gan gifing nhu thanh phan dich Idng trong ca the sfing bao gom muoi vfi co, carbonhydrate, vitamin, amino acid, nhan to tdng trudng, hormone, cac ylu to vi lugng... va dgc bi?t quan trgng la huylt thanh bd chfta. Cdc chat dinh dudng thiet ylu giiip tl bao phan chia nhu amino acid, acid beo, dudng, cac ion, cac vitamin, cofactor va cac phan tft cdn thiit de duy tri mdi trudng hda hgc cho cdc te bdo.
Tu nhftng nghien cftu kl tren chiing tfii tiln hanh chudn hda mdi trudng nuoi cay te bao gdc phdi chugt. Thanh phdn djch nudi cay te bao goc phdi chupt dugc chuan hoa dn djnh cd thanh phan nhu sau: Mfli trudng ca ban D'MEM (Dulbecco's Modified Eagle Medium-Hang Gibco)+ 15% huylt thanh thai bd chua FBS (Fetal bovine serum - Hang Gibco) -t- 1% Non-Essential Amino Acid/NEAA Solution (Hang Hyclone) + 1%
GlutaMAX™ (Hdng Hyclone) + 1% Sodium
Pyruvate (Hang Hyclone)) + Leukemia
inhibitoiy factor (1:10000 - Hdng Millipore)
-I- 2-mercherapto ethanol (O.I mM - Hang
Gibco) [4], [5].
Ld Duy Anh va Dtg Tgp chi KHOA HQC & CONG NGHE 177(01): 197-201 Dieu kign nudi cdy: Nuoi cay trong tii 37°C,
C02ndngdp 5%i.
Vdi thanh phan ti lg cdc hpp chit, dilu kien nuoi cay nhu tren, chiing toi da tiln hanh nudi cay thanh cong ddng tfi bdo goc phfii chudt trong dieu kign phdng thi nghiem, Thf nghigm da dugc thyc hien nhilu lan va ket qua dugc the hign ro net qua hinh dnh te bao nudi cay dugc chyp.
Trong qua trinh lam thi nghiem ve sau nay chiing tdi se tim ra them nhftng ti lg mfii trudng khac nhau de co the nudi cay dupc cac te bao goc phdi chudt on dinh hon nira.
Chuan hoa ky thu^t nuoi cay te bao gic phoi chugt
Nuoi cay te bao gfic phdi chupt ddi hoi thdi gian va tuan thu nghiem nggt k^ thuat nuoi cay tu tien hdnh lay mdu den nudi cay te bdo sinh truong, tuy vao myc dich nghien ciiu ma chiing ta cd the djnh hudng te bao duy tri tinh todn nang, hodc Id lam te bao phan hfia [3]. Te bao gfic phfii chugt nufli cay duoc khodng 15 ngay thi da phat trien den giai doan can phai chia tach te bao, tuc la can phdi logi bd bdt mgt sd tl bao, giir lai mpt so te bdo de bat dau mgt chu ki nudi cay mdi. Trong mgt so truong hpp can phai bao quan te bdo ngay lgp tuc, khflng lam thi nghiem tiep ma can phai luu giir te bao, hay la cho te bdo ngu ddng thi chiing ta cd the thyc hien qua ky thugt ddng lanh te bao.
Tft dd, chiing tfli da tiln hdnh chuan hda kJ*
thuat nudi cay te bao goc phdi chudt theo ba budc nhu sau;
(I) Phuc hfii tl bao (Cell recovery) Chudn bj flng Iy tam 15 ml, them vao ong I ml dich nufli cay te bao. Hiit te bao dang bao qudn lanh -80°C, cho vao be dn nhigt 37''C ra dfing nhanh cac tl bao. Chuyen t l bao da ra dong vdo flng Iy tam 15 ml da chuan bj trudc do. Dung pipet mix nhe nhang va thgt deu ong nghiem. Ly tam 1000 rpm trong 5 phdt, luc nay trong ong Iy tam se chia hai pha, pha I la phdn djch noi phia tren va pha 2 la phan can
te bdo Idng phia ben dudi. Hut bd phan dich ndi phia ben tren. Dung 2 ml djch nudi cay tl bdo ddnh tan phan te bao lang d ddy ong ly tam, dung pipet mix deu nhg nhang roi chuyen tl bao vao cac dTa nufii cay duong kinh 6 cm/gieng. Ddt cac dTa vao tii nufli cay tl bao (37°C, 5% CO2). Sau 12 h nuoi cdy, tien hanh thay dich nuoi cay te bao, tiep tuc nufii cay cho tdi khi te bao sinh trudng dupc khoang 80-90% gilng nudi cay la c6 thfi chia tach te bao.
(2) Chia tach tl bao (Cell Passage) Hiit het phan dich nufli cay te bao trong cac dTa duong kfnh 6 cm/gieng. Them mot lupng thich hpp Trypsin-EDTA rfta mdt lan. Hut het phan Trypsin-EDTA, mdi gieng them khoang 400 ^il 0,25% Trypsin-EDTA, dl Trypsin- EDTA phdt huy hoat tfnh trong 1 phiit. Nhe nhang dung pipet danh bat cac te bao cdn bam vao gilng nufii cay. Them vao 1 ml dich nuoi cdy tl bao, dung pipet mix deu den khi cac dam te bao phan tan thanh cdc te bao don dgc.
Hut phan dich te bao sang cac dng ly tdm 15 ml. Ly tam 1000 rpm trong 3 phut, luc nay trong fing ly tam se chia hai pha, pha I la phan dich ndi phia tren vd pha 2 Id phan can te bao lang phia ben dudi. Logi bo phan djch sau khi Iy tam, thu phan cgn te bao. Them 1 ml djch nufii cay te bdo ddnh tan phan can.
Chuan bj cac dia nudi cdy te bdo, loai duong kinh 6 cm/gieng (Cac dTa ndy da dupc trang 0,I%> Gelatin qua dem), moi gieng them 2 ml dung djch nuoi cay te bao. Hiit te bdo tft dng ly tdm vdo cac gieng nudi cay ddm bao nfing d6l-l,5xI0'tlbao/gilng,
(3) Ddng lanh te bao (Freezing Ceils) Sau khi chia tdch te bao, diing may dim te bao sau dd hut te bdo vao cac dng bao quan dam bao nfing dp 3-5x10^ tl bao/fing. Trong mfli ong bdo quan da chfta sdn chat bao quan vdi thanh phan: 40% D'MEM •+ 50% FBS + 10% DMSO.
Nuoi cay thanh cong te bao goc phoi chugt
trong dieu kien phong thi nghiem
Thanh phdn, ti Ie cac chat cd trong mdi
truong nudi cay, ket hgp vdi ky thuat nufli
La Duy Anh vd Dig Tgp chi KHOA HOC & CONG NGHE 177(01): 197-201 cay thi'ch hpp Id yeu tfl quyet dinh tdi vi^c
nuoi cay thanh cflng te bao goc phfli chupt.
Trong qua trinh Idm thi nghiem chung tdi da gap phdi rdt nhieu truong hpp cdc te bao chi cd t h i sfing d u g c den ngdy thir 7, thu 8. Cd trudng hpp thi te bao Igi bj nhiem khuan...
Nguyen nhan cua hien tupng tren din tft hai nhom chinh; ( I ) Ti Ie cac chat cd trong mdi trudng nudi cdy chua hpp ly; (2) Trong qua trinh Iam thi nghigm khfing tudn thft nghiem cac qui trinh lam thi nghiem da de ra ddn tdi t l bao bj nhiem khuan.
De khdng djnh nufii cay thanh cong ddng te bao gfle phfli chudt chiing tdi da tien hanh nufli cay cac te bao goc phfii chugt 8 lan.
Thyc hien chu ky phuc hfii te bdo, chia tdch te bao, dfing Ignh te bdo tren mgt dfing te bdo khoang 36 ldn. Sau khi tiln hanh thi nghigm n h u tren cdc te bao van gift dupc hinh thai tieu chuin, sinh trudng fin dinh da khang dinh nufii cay thanh cflng te bao gfic phdi chupt.
Vdi mdi trudng, dieu kien nudi cay, ky thugt nufii cdy da dugc chuan hfia d trfin, chung tfli da tiln hanh nudi cay thanh cdng te bao goc phfli chudt, Dudi kinh hien vi huynh quang (Nikon) hinh dnh te bao dung rieng re, hinh thdi rd rang, te bdo sinh trudng dn dinh [6].
Du d i l u kign de tien hanh djnh hufing biet hod cdc te bao mong mudn.
Hlnh 1. Te bdo gdc phoi chudt ngdy Ihu 14 nuoi cdy KET LUAN
Qua chuan hoa mfli trudng va kJ thuat nudi cay te bao gdc phoi chugt. Chiing tfli nit ra ket lugn nhu sau:
200
- Chuan hod thanh cong mdi t r u a n g nuoi cay te bdo gdc phdi chupt.
- Chudn hod thanh cdng k J thuat nudi cdy tl bdo gfle phfii chugt.
- Nudi cay thdnh cdng te bao gdc phfli chugt trong dieu kien phdng thi nghiem.
KlfeNNGHI
- Chuan hod thanh cflng mdi truong nufii cay t l bao gdc phoi chugt la b u d c khdi dau trong cdc nghien cuu cd lien quan den te bdo goc ling dung trong y hgc. Ben canh te bao goc phdi chugt cdn cd mdt sd te bdo khde phyc vu trong nghien cun can nuoi cay. Vi vay chung tdi se tiep tyc nghien cuu, chuan hod mfii truong nudi cay nhieu loai te bao khac.
- Sau khi chudn hda thanh cfing mdi trudng, nuoi cay fin dinh ddng te bao gdc phoi chugt tilp tuc tiln hdnh cdc huong nghien cuu khac nhau nhdm khai thac triet de tiem ndng nghien cuu ve te bao goc phdi chugt de ung dung vao y hpc.
TAI LIEU T H A M K H A O 1. Duong Thj Bgch Tuylt vd cgng sii (2005).
Budc ddu nghiin ciru qui Irinh tdch vd nudi cdy le bdo goc phdi chudt, Truong Dai hgc Su pham Thdnh phfi Hfi Chi Minh, Hfl Chi Minh.
2. Phan Kim Ngpc, Pham Van Phiic, Truang Djnh (2009), Cong nghi ti bdo goc, Nhd xudt ban Qi&o due. Ha Ngi.
3. Lindvall O., Barker R. A., Brustle 0 . Svendsen, C, N (2012), "Clinical translation of stem cells in neurodegenerative disorders", Cell Stem.
Cell,\0{2),p. 151-155.
4. Lu R., Yang A. and Jin Y (2011), "Dual functions of T-box 3 (Tbx3) in the control of self- renewal and extraembryonic endoderm differentiation in mouse embryonic stem cells", J Biol. Chem., 286(10), pp, 8425-8436.
5. Weiying Lv (2014), The functions and molecular mechanisms of histone deacelylases in the lineages specification of mouse embryonic stem cell, Tong Ji University, Shanghai.
6. Weiying Lv, Xudong Guo, Guiying Wang et al, (2014), "Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells", Plos. One, 10, pp, 1371- 1374.
La Duy Anh vd Dtg Tap cht KHOA HQC & CONG NGH$ 177(01): 197 - 201
A B S T R A C T
S T A N D A R D I Z I N G T H E C U L T U R E E N V I R O N M E N T AND T E C H N I Q U E F O R M O U S E E M B R Y O N I C S T E M C E L L S A P P L I E D IN T H E L A B O R A T O R Y A T T H A I N G U Y E N U N I V E R S I T Y O F M E D I C I N E AND P H A R M A C Y
La Duy Anh', Nguyen Phuong Sinh TNU - University of Medicine and Pharmacy Stem cell research in the field of medicine has been started trom successfiil embryonic stem cell culture. However, depending on the actual conditions of each laboratory, researchers have built their own stem cell culture environment and culture technique.
In this research, we have studied the conditions and techniques of stem cell culture from domestic and foreign laboratories. Then we standardize the environment and techniques of mouse embryonic stem cell culture Is applied in the laboratory in TNU - University of Medicine and Pharmacy.
We have successtully standardized the environment and techniques of mouse embryonic stem cell culture. Successfiil transplantation of mouse embryonic stem cell line. Well-formed, morphologically-normal cells, which are capable of targeting differentiated cells into desired cells.
Keywords: Stem cells, mouse embryonic stem cells, culture, culture environment, culture technique.
Ngdy nh^n bdi: 22/12/2017; Ngdy phdn biin: 23/12/2017; Ngdy duyft ddng: 30/01/2018
' Tel: 0912 866006, Email: [email protected]