• Nghien cii'u - Ky thuat
Tac dung tang cwifng chirc nang miin dich chuyen nhiem chong ung thu* ctia Crilin T
Nguyin Thi Ngoc Tram', Phan Thi Phi Phi\
Phan Thi Thu Anh^ Nguyin Van D6^*
' Cong ty TNHH Thien Duac
^ TnrdmgDai hgc Y Ha Noi 'E-mail: [email protected]
Summary
Criiin T was prepared by combination of the total alcaloids and total flavonoids from the leaves and flowers of Crinum latlfolium L var. crilae Tram & Khanh. var. n. for use in treatment of prostatic hypertrophy and uterine fibroid. Crilin T was tested for antitumor properties through the immunity- enhancing effects using an in vitro model. The expression of CD4, CDS and CD3 of the 48h cultured lymphocytes isolated from both healthy donors and the patients with nasopharyngeal carcinoma was done by flow cytometry analysis. The IL-2 and TNFa expression in the supernatant of cultured cells at the hour 48 were determined by ELISA. At both low and high concentration, i.e. 0.25mg/ml and 0.5mg/ml. respectively. Criiin T exhibited immune-enhancing effect comparable to that oflevamisol. by increased CD4, CDS expression. However, effect was somewhat higher for the high concentration than the low one. The effect level of Crilin T on lymphocytes in the patients was higher than in the healthy subjects. Both cytokines, IL-2 and TNFa. were highly expressed; but TNFa level was muct) higher than IL-2. Overall, by this in vitro model. Crilin T exhibited an immunity-enhancing influence.
asserting desirable antitumor properties.
Keywords: Crinum latifolium. alcaloids, flavonoids, Crilin T, test the antitumor properties.
Dat van de
Cay trinh nO Crila Crinum latifolium L. var.
erilae Tram & Khanh, var. n da d u g c Nguyin Thj Ngoc Tram phat hien va nudi trong tO nam 1990 cho den nay '^'. Cym cdng trinh nghien cOu ve cay trinh n u Crila da tao ra san pham thuoc Cnia va mpt so thyc p h i m chOc nang khac. Sdn pham
Crila dup'c bdo c h l tO eac alcalold cd hogt tinh sinh hgc d u g c e h i l t xudt tO Id cay Tnnh nO Crila Crinum latifolium L. var crilae Tram & Khanh, var. n d u g c Bo Y te cho phep luu hdnh toan quoc t o nam 2005 vdi chl dinh d i l u trj bdnh phi dai lanh tinh t u y l n tien liet vd nam 2007 dugc bo sung chi dinh dieu tri u x a t u cung.
Hinh 1 : Cay trinh nw Crila - Crinum latifolium L. var. crilae Tram & Khanh, var. n.
T ^ CHI DirOC HQC - 05/2013 (SO 445 NAM 53)
• Nghien CIFU - Ky thuat
D l cd thuoc h i trg vd d i l u trj ung thu, N g u y i n Thj Nggc Trdm da t h y c hipn nhidm vu hgp tdc quoc t l v l Khoa hpc va cdng nghp theo Nghi djnh t h u giua Vidt Nam va Bungari '^'. K i t qud cOa Nghj djnh t h u da chOng minh mdt so phan dogn alcalold d u g c e h i l t xuat t u Id va mgt phdn dogn flavonoid d u g c c h l l t x u i t tO hoa la ed tdc dyng chong oxy hda va tang c u d n g m i l n djch c h l n g khoi u gidn t i l p hay cd tac dyng gay ddc t l bao ung t h u t r y c tiep tren cac ddng ung thu nghien cOu in vitro va in vivo. TO k i t qua nghidn c u u nay Nguyen Th] Ngoc Trdm da bdo e h l ra mOt san p h i m mdi d u g c dgt ten la Crilin T, thdnh phdn cd chOa cae alealoid vd eac flavonoid eua Trinh n u Crila. Cdng trinh nay tap tnjng nghidn euu vao tac dung tang c u d n g m i l n dich cCia Crilin T. Nghidn cOu d u g c t i l n hdnh tryc t i l p trdn cac t l bao lympho tdch t u ngudi binh thudng ve lam sdng va t u cac bpnh nhan ung t h u vdm mui hpng (UTVMH) va nudi cay in vitro 48h. Ddnh gid s y tdng sinh ede t l bao TCD3, TCD4, TCDa va mOc dp c h l t i l t 2 cytokin chlng ung thu chQ y l u Id IL2 vd TNFa .
D o i t u ' a n g v a p h u ^ c n g p h a p n g h i e n CLPU
T h u l c
- Vidn nang Crilin T cd hdm lugng 250 mg do cdng ty TNHH Thidn Dugc cung d p , d u g c dCing vdi nong dg cao (Crilin T NDC) la 0,08 mg/ml mdi trudng nudi cay (tuang ung vdi l i l u 80 mg/kg cdn ndng) vd vdi nong dg t h l p (Crilin T NDT) Id 0,04 mg/ml mdi trudng nudi cay (tuang Ong vdi l i l u t h u d n g dung trong ^ l u trj cho ngudi la 40 mg/kg cdn ngng). M l Id Crilin T dupc sdn xuat d Id mdl.
- Levamlsol dup'c cung cap bdi BO mdn Dugc I'if - T r u d n g Dgi hpc Y Hd Npi, nhgn t u Vipn K i l m djnh Trung u a n g .
M l u n u d i c i y
M l u nudi cay Id cdc lympho bdo d u g c tach t o mdu ngogi vi cDa ngudi binh thudng vd bdnh nhdn UTVMH:
Ngwdi binh thwong vi lim sang: Id can bid, sinh vidn Trudng Dgi hpc Y Hd Ndi, t u l i tO 25-60.
B$nh nhan UTVMH: t u giai dogn III d i n IVA, IVB, Ti_4, No-3, Mo da dup'c xac c h i n bang md bdnh hge tgi Bpnh vign K Trung u a n g . Bdnh nhdn dup'c phdn logi TNM vd giai dogn Idm
T^P CHi DUOC HQC - 05/2013 (S6 445 NAM 53)
sang theo AJCC (American Joint Committee on Cancer) nam 1997.
- Giai dogn III g I m Tv2 N2 hogc T3 N0-2 M2.
- Giai dogn IVA gom T4 N0.2 MQ.
- Giai dogn IVB: T b i t thudng N3M0.
Vgt lieu tach iympho vd n u o i e l y Mdl tm-dng nudi e l y RPMI 1640 cd 10% FBS (foetal bovin serum- cua Hang GIBCO, Mexico).
Dung djch tach lympho la Ficol 1070 (GE healthcare, Uppsala, Sweden)
P h u v n g phap n u o i cay te bao i y m p h o 10 ml mdu c h l n g ddng EDTA eiia ngudi binh thudng va benh nhdn UTVMH ehua d u g c d i l u trj d u g c pha loang gap ddi vdi RPMI (ed 10% FBS) vd tach t l bao lympho bang Ficol 1070. ROa vd d i m so lugng t l bdo sdng thu duge, s u dung dung djch xanh trypan de phdn bidt t l bdo chet.
Cho 1 ml mdi trudng RPMI 1640 cd 10^ t l bao vdo moi g i l n g cDa philn nhya logi 12 g i l n g . Do s l lup'ng t l bdo thu duge khdng nhilu nen nghien cuu d u a c t h i l t k l nhu sau:
+ G i l n g chOng dm: chl eo t l bao vd mdi trudng nudi e l y .
+ G i l n g chOng d u a n g . t l bao vd levamisol ( c h i t chuan kich thfch midn djch, n i n g dd 25 mg/ml).
+ 2 g i l n g thO nghidm eho moi logi:
- T l bao + Crilin T NDT (2 gilng) - T l bao + Crilin T NDC (2 gilng) - Te bao + Crilin T Mi NDC (2 g i l n g ) Tat ca cac g i l n g d i u cho thdm PHA nong dp thap (3 pg/ml moi trudng) d l m l i cho dap Ong.
Nudi cdy d 3 / 0 / 5% CO2, thdi gian 48 h.
- Thu hogch t l bdo vdo dng nghigm 1,5 ml vd ly tdm thu dich n i l (cho djnh lugng ILzVd TNFa) vd egn t l bao d l xdc djnh cdc d i u an CD.
- D i m s l lugng t l bao TCD3, TCD4, TCDe (cd t h i t y tfnh them ra ty Id TCD4/TCD8 ) d u g c t i l n hanh d Trung tam Phdng chong HIV, \ ^ e n Vd sinh phdng dich Bp Quoc phdng, vdi mdy FACS count SV version 1,5 (Hoa Ky).
- Djnh lugng IL2 va TNFa trong djch noi cac mau t l bao lympho nudi cay vdi kit ELISA (Invitrogen, California, Hoa Ky). N i n g dO cytokin trong mau do d u g c xac djnh d y a vao dudng e h u l n bang phan mem cua mdy dpc ELISA (Biorad, USA).
- X u ly k i t qud bdng p h i n m I m GraphPad Prism 6.0.
23
0 Nghien cifu - Ky thuat
K e t q u a n g h i e n CLPU
So lup'ng cac d u d i nhom lympho T/pl mdi truwng t r u d c nuoi cay K i t qua d u g c trinh bay trong bang 1.
Bang 1 : So Iwang cic dwcri nhom lympho T/pl moi trwong sau khi tach qua Ficoll, trwac nuoi ciy Te tiao
TCDa TCD4 TCD,
Nguai binh thuang (n=5) XtSEH/t (TB/pl) 823,3 ± 65,98 359,4 ± 39,40 458,9 ± 36,66
Benh nhan UTVMH (n=9) X±S£M (TB/pi) 779,7 ±109,9 317,5 ±52,85 449,3 ± 69,90 Nhan xet: Cdc trj so nay cung xdp xi cdc trj
so ma t h u d n g ngay chung tdi gap trong nghien cOu d benh nhdn UTVMH glai dogn mudn va d ngudi binh thudng^''l
So lu'p'ng cac d u d i nhdm t l bao T/pl moi t r u d n g sau nuoi cay (te bao tir ngu'di binh thudng)
K i t qua d u y c the hipn trong bdng 2.
Bang 2: Si Iwang cac dwal nhom te bao T/pl moi trwimg sau nuoi ciy 4Sh/37°C cua chwng am vi chimg dwang (te bio tir ngwoi binh thwdng)
So l u g n g eac d u d i nhdm lympho T sau nuoi cay ( t l bao tO ngudi benh UTVMH)
Ket qua nghien euu d u g c t h i hidn trong bdng 3.
Bang 3: So Iwang cic dwai nhdm lympho T sau nuoi cay 48 h cua chirng im va chimg dwang a ngwai benh UTVMH
Teljao
TCDs TCD4 TCD,
Chung am (n=5) XtSEM (TB/pi)
115,2±10,18 25,25 ± 7,603 104,2 ±13,77
Chung duang (n=9) X*SEM (TB/pi)
293,7 ± 80,69 82,13 ±23,94 238,7 ± 58,83
P
0,04 0,03 0,04
r e bao
TCDs TCD.
TCDa
Chung am (n=5) XtSEI/l (TB/pi)
172,6± 12,35 37,78 ± 8,594 139,5 ±12,61
Chung duang (n=9) XtSEM (TB/pi)
326,2 ± 75,65 108,2 ±24,58 139,5 ±12,61
P
0,04 0,02 0,04
Nhan xet: Sau nudi cay 48 h thi:
+ 0 cac mau ehung am, so te bao c h i t di n h i l u (hien t u g n g sinh hgc), s6 cdn lai chf cdn t o 1/4 den 1/7 hay 1/14 so l u a n g t l bao d u a vdo nudi cay ban ddu: chet nhieu nhdt la TCD4
+ 0 cdc mau chOng d u a n g , cd chat chuan kich thich m i l n dich Id levamisol, mdt so t l bao da tang sinh lam sd l u g n g t l bao thu d u g c sau 48 h nudi c i y tang cao han n h i l u ede mau Chung am, deu cd p <0,05.
Nhan xet: T d bao tang smh sau nudi ely a chOng d u a n g cao gap tO 2 lan d i n 3 lan so vdi chOng am. ChOng d u a n g cua t l bao lympho tach t o bdnh nhan UTVMH cd dap ung mgnh ban chOng d u a n g cua te bdo lympho tdch ttf ngudi binh t h u d n g (bang 3 so vdi bdng 2), tri>
t l bdo TCDa. Khi nghien cOu so sanh tac dyng tang c u d n g m i l n djch giua cdc che pham Crilin T d cae nong dd cao, ndng do thap vd Crilin T Ml NDC vdi levamisol va dap Ong cua t l bao lympho tach t u ngudi binh t h u d n g (bang 4) vd ngudi benh UTVMH giai dogn mudn (bang 5)ta thay cdc logi Crilin T dCi vdi cac dot san xu^t khdc nhau cung d i u cho k i t qua ddp Ong tang mien djch nhu levamisol hay cao han levamisol.
Dap Ong cua te bdo lympho T ngudi binh t h u d n g vdi cac c h i t nghien cu-u
K i t qua d u g c t h i hien trong bang 4.
Bang 4: Dap
Tebao
TCD3 TCD4 TCDB
ung ctia tS b^o 1 Chung duang
(n=5) X±S£M (TB/pi)
293,7 ± 80,6 82,1 ±23.9 238,7 ± 58,8
'ympho T nguvi binh Criiin T NOT
XtSEM (TB/pl) 208,6 ± 52,9 65,5 ±17,3 137,3 ±33,4
thu^g V&I cac chat nghien Criiin TNOC
(n'5) XtSEM (TB/pi)
290,3 ± 87,5 66.7 ± 33.5 208,9 ± 44,4
cuu Crilin T Ml
(n'S) XtSEM (TB/pl)
161.2±8,6 29.0 ± 14.1 135,4 ±7,6
T A P C H I D l / O C H O C - OS/Ml •» r s n 44« N A M *
• Nghien CLFU - Ky thuat
Kit qud cho thiy ca hai logi ehl phim Crilin T ning dO thlp cung nhu nong dp cao, deu cd tdc dyng tang cudng mien djch nhu levamisol, vdi p diu >0,05.
Dap ung cua t l bdo lympho T tach tu>
ngudi bgnh UTVMH
Dap Ong cua t l bdo lympho T tach tO ngudi bpnh UTVMH gial dogn mupn vdi cac chat nghien cOu duac thi hidn trong bang 5.
Bang 5: Dip wng cua ti bio lympho T tich tir ngwai binh UTVMH giai do$n mugn vdi cic chit
nghiin ciruTebio TCDs TCD, TCD,
Chung dtfcmg (TB/pi) 326,2 ± 75,65 108,2 ±24.58 139,5 ±12.61
Criiin THDT (TB/pi) 273,1 ±47,3 59.3 ±16.50 238.6 ±44,09
Crilin TNOC (TB/pi) 423.4 ± 88,5 120.71± 39.20 284,5 ± 52,93
Criiin T NBC M, (TB/pi) 387,0 ± 74,46 93,00 ± 29,02 270,8 ± 56.93
Doi chilu tOng cap so lieu cua tung dudi
nhdm t l bdo, ta thay ddp Ong cDa t l bdo lympho tdch tO bgnh nhan mgnh han nhilu so vdi ddp Ong cOa t l bdo taeh tO ngudi binh thudng mpt cdeh cd y nghTa.
Crilin T nong dd cao cho ddp Ong tit han
ndng dd thlp, dap Ong tit han khi t l bdo iympho T d bpnh nhdn da bj suy gidm so vdi t l bao cua ngudi binh thudng.
Sir che tilt IL-2 va TNFa d cac mau nuoi cay lympho
Kit qua dugc thi hipn trong bdng 6.
Bdng 6: IL-2 vi TNFa sau 48 gid nudi ciy cda chi phim Crilin T so v&i chimg dwang
Churg am Levamisol Crilin T LT Crilin T LC
Nguai binh thudng IL-2 (ng/ml)
9,940 ± 0.5 13.04 ±0.9 19.84 ±1.6 21.66 ±2.5
TNFa (ng/ml) 456.7 ± 6.3 485.3 ± 4.9 477.7 ±21.8
510.0 ± 3.2
B$nh nhSn UTVMH lL-2 (ng/ml)
11.63±1,0 27,50 ± 6,3 22.25 ±1.8 27,00 ± 3.5
TNFo (ng/ml) 452,5 ± 9,1 488,1 ±7,8 494,7 ± 6.2 510.9 ±19.2
Sy chl tilt IL-2 va TNFa d cdc mau nuli ely lympho cCia ngudi binh thudng va bgnh nhdn ung thu vdm hpng giua chOng dm vd chOng duang Id cd sy khac bidt cd y nghTa (p<0,05), trong khi so sdnh giOa ede nhdm diu cho kit qud khdng khdc bipt (p>0,05). Sy bilu Id TNFa cao han nhilu so vdi IL-2.
Ban lu$n
s l lugng ede dudi nhdm t l bao lympho T trudc nudi ely trong gidi hgn thudng gap d ngudi binh thudng va ngudi bpnh UTVMH phCi hgp vdi nhilu tac gia chuydn nghidn cOu UTVMH '^'^vd trj so sinh hpc ngudi binh thudng thi kf 20. Ddy eung la mpt edeh kilm tra ky thudt cCia chung tdi trong cdc dgt nghidn cOu khdc nhau. Cdc mau chOng dm cho kit qud rit thlp han mlu chOng duang Id hgp ly.
TJSf CHI DirgtC HQC - 05/2013 (SO 445 NAM 53)
Vi dap Ong vdi ede logi chit tang cudng miln djch thi dap Ong cua t l bdo da bj suy giam mien djeh tit han, mgnh han dap ung cua te bao mien djch binh thudng. Bang 2-5 deu minh hog cho nhdn xet nay vd cung phu hgp vdi nhdn xdt cCia Do Hod Binh, 2004 '"l
Levamisol Id chat kich thieh miln djch da dup'c su dung nhieu trong dieu tri nhung Igi cd han chl do mpt so tae dyng phy ngng n l (ryng tde, tdm thdn...). Crila tnjdc ddy vd nay la Criiin T Id cdc thuoc tu thao duge, ehua thiy tac dyng phy trong Idm sang, ca trdn nghidn cuu dpc tfnh d p , bdn cap tren thye nghidm. D l danh gid Crila, chung tdi da lam tren cdc md hinh thuc nghiem in vitro, va in vivo d chuOt nhat, chugt cong trdng '^'. Ddp Ong d ehu|t d l ding nhit han cd d ngudi vi cdc cd thi ngudi da dgng han. Chua cd nhilu nghidn cOu tren t l bdo
25
• Nghien CIFU - Ky thuat
lympho c h i l t tach t u benh nhdn UTVMH cung nhu b i t ky loai ung t h u nao khdc, do dd khong t h i so sdnh v d i so lieu cua cdc tdc gia nao khdc. Khi da thay Crilin T NDC tdt han NDT nen vdi Crilin T M1 chung tdi cung chi thO v d i NDC do te bdo lympho thu d u g c t u bdnh nhan Id khdng d u g c nhieu. Crilin T, c h l p h i m mdi cua trinh n u Crila, gdm cd cdc phan doan alkaloid v d flavonoid ma Nguydn Thj Nggc Trdm ehua cdng bo cu the, nhung cac phan dogn hgp thdnh Crilin T cung da d u g c chOng mlnh t h y c nghiem v l tac dung tang m i l n djch, chong oxy hda va ngan can s y tang sinh cua mgt so ddng t l bao t h u d n g tryc ung t h u ngudi ^^\
Lan ddu tien Crilin T d u g c thO tren t l bao lympho ngudi binh t h u d n g va benh nhan ung t h u vdm miji hong bj suy giam mien djch cho k i t qud tang sd lugng t l bdo lympho T gdn v d i levamisol hogc mgnh han levamisol khi t h u trdn t l bao lympho bdnh nhdn da cd suy giam m i l n djeh. K i t qua ndy cung phu hap vdi nhung nhgn xdt cua Do Hda Binh, 2004 '"l Phgm Huy C u d n g , thanh vidn nhdm nghidn eOu cilia ehung tdi cung da chung minh Crilin T co xu h u d n g dap u n g tot han levamisol d ca hai cytokin la IL- 2 v d TNFa d thai diem 8h va 4h sau nudi cay, mgt each t u a n g Ong, d ca mOc dp b i l u Ig gen va protein '^. Dap Ong vdi TNFa d i l n ra rat s d m chi sau 4 gid nudi c i y da cho t h i y m u e dp t i l t eao. Gan L. va es '^' cung da thay phuc hp-p glycoprotein cua Lycium barbarum lam tang bieu lp 2 cytokin (cd d gen v d protein) la lL-2 va TNFo d te bao d a n nhdn mdu ngoai vi nudi cay Idm c a s d cho t i l m nang chong ung t h u cua cay thuoc nay.
K e t l u a n
Cdc c h l p h i m Crilin T NDT vd NDC (ke cd M l NDC) ddu cd tdc dyng tang c u d n g m i l n djch n h u levamisol hay manh han levamisol.
Crilin T nong dO cao c d tdc dyng tot h a n n i n g dp thdp lam tang ed so l u a n g t l bao va khd nang c h l t i l t cytokin d 48 g i d .
Ddp Ong v d i cdc chat thO cua t l bao lympho bj suy giam m i l n dich mgnh h a n ddp Ong cua te bdo lympho ngudi binh t h u d n g .
lL-2 v d TNFa vdn d u g c c h l t i l t t i t d thdi diem nudi cay 48 g i d .
Tai lieu tham khao 1. Nguyen Th| Ngoc Tram, Tran Cong Khdnh (2012), "Tnnh nu CRILA-Cr/num latifolium L. var.
cnlae Tram & Khanh, var n.: Mot thO mdi cCia lodi trinh nO hoang cung-Crinum latofolium L. (Ho ndng - Amaryllidaceae) d Vidt Nam", T$p chi Sinh hgc, 34(2), tr. 190-193.
2. Nguyen Thi Ngoc Tram (2009), "Nghidn ciiu kha nang kich thich hd mien djch chlng ung thu' cOa cac alkaloid va Flavonoid dugc chiet xuit tO cay trinii nu hoang cung {Crinum latifonium L ) de s d dung lam nguyen lieu san xuat thuoc ho trp dieu tn ung thu", Bao cao ting h<?p kit qua khoa hoc cong nghi nhi^m vi,j hap tac quic te ve khoa hoc vi cong nghi theo nghi dinh thw giQa Viet Nam va Bungari. Bp khoa hcfc vd cong nghe.
3. Nguyin Thi Ngoc Tram, Phan Thj Phi Phi, Phan Thj Thu Anh vd cs (2008), 'Tdc dung phyc hoi ton thuong te bao lympho T va dong tCiy cCia vien nang Crila tnnh nO hodng cung (Crinum latifolium L.)", T^p chi Duac hoc. 5, tr. 12-14
4. DO Hoa Binh (2004), "Mpt so thay d l ! ve s6 lugng va chuc nang cua t l bao lympho va t i n suat bieu lp Protein LMP1, P53, MDIV12 d benh nhSn ung thu vom hong", Luan an Tien sT Y hoc.
5. T N. Dung (2000), "Nghien cuu cac thflng s6 miln dich - sinh hpc giup tien lugng, phat hien sdm tai phat UTVH va kit hgp vien M sau xg tn nhim gidm tai phat", Luin in tien sT Y hoc.
D. Jenny M. et al. (2011), "Crinum latifolium leave extracts suppress immune activation cascades in peripheral blood mononuclear cells and proliferation of prostate tumor cells", Sci Pharm.. 79(2), p. 323- 335
7. P. H. Cudng (2012), "ThO nghi#m tac dyng tang cudng miln dich in vitro cua cdc che pham cfly trinh nu hoang cung", LuSn vin th^c sf. Tru&ng Dgi hoc Khoa hocty nhidn, Ha Noi
8. Gan L. et al. (2003), "A polysaccharidfr- protein complex from Lycium barbarum upregulates cytokine expression in human peripheral blood mononuclear cells", Eur. J.
Phannacol. 471(3): p. 217-22.
