Top chi THONG TIN Y DUOC S6 12 nam 2008 CONG NGHE SINH KHOI TE BAO THUC VAT - HUGG MGI TRONG SAN XUAT NGUYEN LIEU LAM THUOC Vai Binh Duong’, Nguyén Vin Long” Hoang Van Luong’, *, Lé Bach Quang” ing nghé sinh kh6i t& bao thyc vat véi nhieu wt diém vupt tr6i so v6l gieo tring céy goa ty nhién d& va dang tao ra cic san phéin c6 gid tri cao, dng: doc phéim, thuc pham, mj phém, néng nghié ing dung trong mhieu linh vut ciia det Vi vy, sinh khol té bio thyc vat dang 1 fusing méi dy trién vong, dic biét trong sén xuat dutoc pham. 1. DAT VAN BE Cho dn nay, 26 khodng >30.000 hoat chat dude téch chiét tiv ngun gc thyc vat, trong 6 6 rat nhiéu chat c6 gid tri cao. Nhu cau vé cdc hoat chat ngudn g6c thye vat ng’y cing tng, trong: khi ngudn thyc vat cung cp 6 hhan va phy thuge vao nhigu vn d8: diéu kién thé nhu@ng, thién tai, dich bénh, thdi vy,... Sinh khdi t& bao thyc vat tao ra nhing sin pham 06 gid tr cao, (ng dyng trong nhiéu finh vut clla déi sng: dugc phém, thyc phim, my pham, néng nghiép,... Sinh khdi t8 blo thyc vat gop phan to ra nguon nguyén liu di 30, 6n dinh tiv nhiing duc ligu quy higm. II. CONG NGHE SINH KHOI TE BAO THY VAT 1. Khai niém Sinh khdi t8 bao thyt vat [a ky thuat nudi cy, duy tri, tao kh6i fugng Idn t& bao thyc vat trong méi truting dinh du@ng phi hgp va diéu kién nui cy v6 Khuén ma khéng phal tham canh gieo tréng, Tir kh6i t& b3o 46, tach chiét cc hoat chat (sin phm thif cp) ly nguyén ligu cho sn xudt thudc, thyc phim, my phdm,... Nghién cfu dau tién vé céng nghé sinh khéi t& bao thyc vat dude thy hién va0 nnhiing nm 20 cia thé ky XX, khi cdc nha khoa hoc 48 thinh cing trong viée nhan giéng v8 tinh cay Lan (orchid). Tuy nhién, chi toi nm 1962, hai nha khoa hoc Murashige va Skoog GS.TS., Hoc vign Quin ¥ tim ra méi truéng thich hdp cho nudi cay thi cing nghé sinh kh6i mdi that sy mang lai ¥ ghia kinh té (8). Tir nim 1990 dén nay, nhidu san phém nghién atu thanh cng di dugc 4p dung réng rai trong sin xuat cdc san phém phuc vu _nganh cBng nghiép due phém va ‘thuc pham nhu' san xuat paclitaxel-taxol tir té bao cSy thy ting (Taxus sp) ding lam thudc iu tri ung thu, san xuat chat khng khuan Shikonin tif ré cy Lithospermum erythrorhizon, sn xuat chit fam thom Vani tif nudi cay té bao cay Vanilla planifolla va rét nhiéu cic ong trinh nghién cit kde dang duigc nghién city san xust quy mé cing nghiép (8). Ly thuyét aia cng nghé sinh khéi dua trén 9 sd tinh toan ning (totipotent) va tinh biét héa cla mét 6 t& bao thyc vat. Céc té bao nay khi dude dua vao méi trudng dinh du@ng vva diéu kién thich hgp, ching cd thé phat trién thanh khdi lugng Ién ding t& bao 4. Qué trinh nay bat dau tir vigc tao ra céc mé seo (callus) tir nhiing t bao, mé khéc nhau cla thyc vat, sau d6 ching dugc lam giam hoc mat tinh biét héa [3) vi dugc thuan héa trén méi truding dinh duGing nhat dinh, cudi cling la tng khdi lung trén hé théng cic binh mudi By I6n (bioreactor), Trong qué trinh 46, dc tinh cia t8 bio giff nguyén nhy ban dau, céc qué trinh sinh hoc cla té bao van xay ra nhu Bi vdi t& bo khi con tn tal trong cay tyTap ché THONG TIN ¥ DUQC Sé 12 ném 2008 nhign, trong dé cé vie tng hdp va tich tly hoat chat [4]. 2. Uu va nhuge diém aia céng nghé sinh kh6j té bao thy vat 21. Ue diém So vdi nudi trng ty nhién, céng nghé sinh kn6i té bao thyc vat ¢6 nhiing wu diém sau: ~ Khdng chiu tac déng cla cdc yéu to tw nhién nhur dia ly, khi hau, thé nhuGng, benh dich, thién tal... do toan b6 qui trinh sinh khdi duoc tién hanh trong phdng thi nghiém, nha may. Biéu nay gidp kh&c phyc duge anh huGng bat Ii cia diéu kién ty nhién tdi nang sudt va cht Iugng san phim. Ngodi ra, con loal bd duge yéu t6 théi vu nhu khi gieo tréng nén c thé chu déng duc ngudn nguyén ligu phuc vy sén xuat, = Thdi gian san xudt nguyén tigu theo céng nghé sinh khéi té bao rut ng&n hon nhigu so voi gieo trang tu’ nhién. Giai doan nghién city tif khi tién han tao callus (glal doan dau tién cla quy trinh) dén viéc nuéi Sy trén mdi trudng fing thuding mat rat nhigu thdi gian, Tuy nhién, sau khi di lua chon duige diéu kién, méi trudng nudi By thi thdi gian cho san xuat 1 mé sinh khdi thudng rt nhanh, tif 15-50 ngay. ~ Chat ludng san phdm san xuat theo céng nghé sinh khéi t& b3o thuc vat rét én dinh do cdc yeu t6 anh hudng dén téc 46 phat trién qua him lugng hoat chat nhu nhiét 46, pH; néng d6 oxy hda tan, thanh phn méi trutng, digu kién chiéu sdng... du duc kiém soat chat ché, vi vay sin pham thu duge ed chat ludng én dinh, dap dng dude yéu cau san xuat theo tiéu chudin GMP hign nay. - Mét trong nhiing uu diém ndi bat cla céng nghé sinh khdi té bao thyc vat a cd thé diéu khién qué trinh sinh téng hop tao cdc hoat chat cao han so véi nudi tréng ty nhién, vi vay san phdm thu duge c6 ham lung hoat chat cao hdn so véi nudi trong ty nhién, nén rt phil hgp vdi cdc duigc liéu chifa hoat chat ‘66 ham long thép hodc cae chat rét knd téng hgp héa hoc duoc hu’ taxol, vinblastin, vincristin, shikonin, reserpin... Cac ki thuat pho bién dé ting tich ly hoat chat la: dng cdc chat kich thich t&ng oat cht (elicitor) hose cc tin cht cho qua trinh sinh téng hop hoat chat (precursor); ding Ki thuat nudi cy 2 pha (two phase culture); sif dung nghiém phép gen kich hoat... [8]. 2.2. Nhute diém - Ham lugng cdc hoat chat trong sinh khdi 18 bao cdn thip, dc biét Ia véi cdc t& bdo cb cing nhiéu nhém hoat chat tn tai thi vie kich thich t&ng ham lugng dong théi cdc chat vin chya khac phyc duc. ~ Chi phi dau tu day truyén sin xuat tén kém: do dc thi: clia nubi cy té bao thyc vat khée vol nudi cy ndm va vi khudn nén he théng binh nubi cy phai duoc thiét ké riéng cho ting loai té b30. Hién nay, cic nha khoa hoc Nhat Ban, MY, Han Quéc da thiét KE duoc céc hé théng binh nudi cy th? 10.000-20.000 Nit ding cho san xuat cdc san phdm béng cng ingh@ sinh khéi t8 bao thye vat. 3. Quy trinh tgo sinh khéi té bao thyre vit ‘Thuong due tién hanh theo 5 giai doan ké tiép nhau: ~ Giai doan 1: tao callus. Callus la dong t& bao dau, chua biét héa duge tao ra trong pphong thi nghiém, tang ty nhu nhiing t& bao géc dugc tao ra dé han gan vj tri tén thuong cua céy. Callus dude hinh thanh ti céc md, cc 18 bao dugc tich ra ti co thé thuc vat nhd tac dGng ca chat diéu tiét sinh truting. Trong giai doan nay, ngoai thanh phan méi truéng va diéu kién nudi cay thi Ki thuat v6 khugn duge xem [a yéu t8 quan trong nhét. Lua chon chat sat khuan, phudng phap tiét Khudn dung sé gitip cho té bao phat trién tét ma khdng bi nhiém nim cling nhu' vi khugn [11]. - Giai doan 2: duy tri nuéi ey callus trong mdi truting thach mém. Viée duy ti nubi cay trén_méi trvéng thach mm dam bao cho té bao thich nghi hoan toan véi diéu kién séng mdi (tach toan toan khoi cd thé me), ngoai ra am cho té bao ¢6 hinh thal mém, xp, 66 dui site s6ng va d&c biét lam mat kha nang biét héa thanh cdc cg quan bé phan khac. Bigu nayTap chi THONG TIN Y DUOC eee eee ee ———_—_—_——_———— S6 12 nam 2008 tao thuan Igi cho cy chuy€n t& bao sang méi trutng nudi cy long. ~ Gat doan 3: nuéi céy té bio trong méi truéng long. Bay la bude rat quan trong, quyét dinh t6c 46 phat trién cing nhu’ ham lugng hoat chit ciia sinh khéi. Trong giat doan nay phai kho sét day di cdc yéu t6 anh hung nh digu kién nudi cy, thinh phan méi truting nhm lam cho sinh khéi phat trién nhanh nhat, ham Iugng hoat chat trong sinh kndi cao nhat [3], ~ Giai dogn 4: khuy8ch dei quy mé nudi cy (scale-up). Sau khi da tim duge céc digu kién va méi truéng nuéi cy thich hgp, dé ting khéi lugng sin pham, cdc t& b2o phai duge nudi cy trong hé théng cic binh nudi ey thé tich khdc nhau tiy theo quy m6. © giai doan nay, céc yéu 16 vé digu kién sn xugt nhur loat binh nui cy, kigu cénh khudy, phan dp oxy hoa tan 18 nhiing yéu t8 cn phai khdo sit. Hién nay, san xuat taxol va shikonin nguéi ta da st dung c&c binh nudi cy 10.000 va 20.000 lit. + Gial doan 5: Thu hoach khéi té bio. Thu hoach sinh khéi té bao [a buéc cudi cling trong guy trinh nui cy. Théng thutng, cde hoat chat dugc tich Idy chi yéu trong t& bao, vi vay phuong php loc I8y t& blo [a cach phé bién dé thu san phém. Tuy nhién, cd mét sé truéng hgp hoat chat lai ly gidi ra ngodi méi truéng ‘trong qua trinh phat trign, vi vay viée thu hét cdc hoat cht mt nhi8u céng site va chi phi. Céc oat cht sau khi tach chiét sé duoc nghién ctu téc dung duge Ii va b&o ché cdc ssan pham, 4, Thanh tyu cia céng nghé sinh khéi 18 bao thyc vat trong sin xuat duge Cho dén nay, rat nhiéu hoat chat nguon Gc thuc vat c6 gid tri kinh té cao la san phém ca céng nghé sinh khéi t& bao thyc vat 13,4,8,11}. 4.1, Cic alkaloid = Ngu@i ta 06 thé thu dude cde chat nh’ caffein tiv nudi cay té bao cay Coffea arabica, betalain trong callus cli cai duéng, berberin tiy t8 bio cy Coptis japonica (loai cay nay phai tréng ty 4-6 ndm mdi thu duge him long dang ké berberin trong r&, trong khi ham lugng nay cé thé thu dude sau 4 tuan bing phutong phép nud! cy t& bao)... Nhiing chat nay dugc sif dung rng rai trong céng nghiép hung liéu va trong y hoc. - Reserpin la alkaloid chiét xuat tir cy Ba ‘gac (Rauwolfia sp) c6 téc dung diéu tri ting huyét dp va cdc bénh réi loan tun hoan, 43 duc san xudt bang phuong phdp nudi cay té bao cay Rauwoffia serpentina. Ty hé théng binh nuéi cy 10.000 lit c6 thé sin xudt dug 3.500kg reserpin trong 30 ngay, tudng duong vdi lung hing n&m cia ca thé giéi thu dugc tire cay dé, - C4c nha nghién cifu thuéc t6 hop duge pham Gibageigy (Based, Thuy Si) d3 san xuat dugc loai alkaloid 18 scopolamin tir t& bao cay Hyoscyanus aegypticus nudi cy trong hé én men khéng 6 canh khudy. Bang céch chon loc cdc dang t& bao cao sin nhg kj thudt dét bign t& bao tn, bién dj don dong va ky thuat gen, ngudi ta da téng ‘duge sin lugng scopolamin lén gp hang ngan fan. Sikuli va c6ng sy’ (1997) sau khi gay nhigm cdy Datura stramonium —vdi Agrobacterium rhizogenes d& nhan thay ham lugng hyoscyamin & r& dat cyc dai 100mg/| sau 6 tuan nudi cy. Nhiéu nghién aifu cho thay nudi cy callus va té bio alia cy Catharanthus roseus a6 him lugng serpentin ngang voi céy duc ligu binh thyéng. MBt $6 nghién edu d& phan lap duc céc ding t& bio Cantharanthus san xudt serpentin va ajmalacin tif nudi cy in vitro. Bling ogi méi truéng sin xuat dic biét, nguéi ta d3 dua dude san luong alkaloid cla hai dong té bao t6t nhat [én mét mie cao hon nifa, trong dé mét dang tao duc 162mg/I sempentin, cin dong kia tao dugc 72mg/l serpentin cing véi 264mg/! ajmalacin. Méi day, nguéi ta dé hoan thién duge cng nghé nudi cy t& bao cla cdy C. roseus 68 san xuat viblastin va vincristin (Id hai ch&t chéng ung thir *3t manh, hién dang diing chifa ung thu’ mau).Tap chi THONG TIN Y DUOC Sé 12 nam 2008 4.2, MBt 56 chat khdc - Thi dy ign hinh nh&t 1 céng nghé sn xu8t shikonin, mot logi sc t6 6 c6 kha ning diét khudn, cb trong r& cy Lithospermum erythrorhizon. Binh thudng, shikonin tich Idy kkhéng nhiéu trong ré. Tuy nhién, cdc nha khoa hoc Nhat d& tao dugc ding té bao ré cay Lithospermum c6 kha néng tich dy dén 15% shikonin va d& hoan chinh c6ag ngh8 nudi cy t€ bao san xuat shikonin. Céng nghé nay cho phép trong mét chu ky nuéi cy thu hoach tdi kg hoat cht va gitip gkim rt nhiéu gid thanh cla shikonin, Bén nay, toan b6 ngubn nguyén ligu shikonin du duge sin xudt bing cing nghé nay, ~ Paditaxel |& chat tach chidt ti wo va la kim ca cay thiy ting-théng 46 (Taxus sp) dang duc st? dung hiéu qua trong diéu tri nhiéu loai ung thu. Vie cung cp paclitaxel gp Khd khn vi ban than cy thy ting khan hiém va ham lugng hoat chat rat thép. Cong ty Escagenetics (California, Mj) 48 thinh céng trong san xuat Paclitaxel b&ng céng nghé sinh khéi té bao thyc vat. Ham lugng taxol trong sinh khéi t& bao cao hon so véi cy ty nhién, tiv dé bao ché thud diéu tri ung thu’ Taxol va Taxotae. Hi8n Taxotae la sin pham déc quyén sd hit tri tué toan cau ‘aia hang duigc pha Bristol-Myers-Squibb dang ban vdi doanh sé mdi nim hang ty dé la. © viét Nam, céng nghé sinh khéi t& bao thuc vat da va dang duge dp dung thinh céng dé san xuat cdc san phdm tiv nhiing duge liéu quy him nhy sém Ngoc linh (Panax vietnamensis); s’m VG dip (Panax bipinnatifidus), théng dé nam (Taxus wallichiana) [1]. D&c biét, tai Hoc vign Quan y 3 nudi c&y thanh cbng sinh khéi t& b3o sm Ngoc linh trén h8 théng binh nudi cBy 100 lit [2], bute d3u da tao ra cdc sin phim bao ché tir sinh khdi t& bao sm Nggc tinh, NhGing thanh céng nay md ra trién vong sin xuat cc ‘san ph3m 6 gid tri tif ngudn dude liu quy hiém trong nuéc bang cong nghé sinh Khéi té bao thu vat. IIL. KET LUAN Céng sinh khéi té bao thy vat véi nhiéu uu diém vudt trdi so vdi gieo tréng cdy ngoai ty nhién da va dang tao ra céc san phdm cé gid tri phyc vy nhigu finh vu ctia dat sing, dc biét 18 c6ng nghiép dudc phaém. 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