ri^i- vIET NAM THANG 4 • SO 2/2014
Vu Du'cfng Quy, Pham Manh HiJng( 2006):
•>1ien dich ghep". Mien dich hoc. Nha Xuat Ban Y Hgc, tr'277-290.
Colleen R. Balsly, Andrew T. Cotter, Lisa A.
Williams, Barton D. Gaskins, Mark A. Moore and Lloyd Wolfinbarger Jr(2008): "Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts". Cell Tissue Banking (2008) 9:289-298
Grassman S.R.M, D.B. McDonald, G.M.
Thornton, N.G. Shrive, CB. Frank(2002):
"Early healing processes of free tendon grafts within bone tunnels is bone-specific: a morphological study in a rabbit model". The Knee 9(2002):21-26
Hideo Kawakami, Konsei Shine, Masayuki Hamada, Ken Nakata, Shigeto Nakagawa, Norimasa Nakamura, Yukiyoshi Toritsuka, Hideki Yoshikawa,Takahiro Ochi(2004):
"Graft healing in a bone tunne bone-attached graft with screw fixationi versus bone-free graft with extra-articular suture fixation". Knee Surg Sports Traumatol Arthrosc(2004) 12 : pp 384-390
NGHIEN Ciru TAC DUNG KHANG UNG THU* CUA TOI LY SCTN TREN DONG TE B A O UNG THU DAI T R A N G
Nguyen Van Long*, Nguyen Trong Tai**, Nguyen Viet Trung***
T6M TAT
Cac nghien ciru gan day cho thay r5ng toi co tac dyng iTc che tang sinh chong lai nhieu loai ung thu.
Trong nghien cifu nay chiing toi danh gia tac dung cita dich chiet toi Ly Son tren dong te bao ung thu dai trang ngU6i in vitro. Ket qua cho thay, dich chiet to! co kha nSng irc che t3ng sinh ro ret doi vdi te bao ung thu dai trang ngudi dong HT-29 so vdi nhom chirng.
Tat cac thdi diem sau 24, 48 va 72 gid te bao dupc bo sung dich chiet toi Ly Son toan phan d nong do 5 mg/ml va 10 mg/ml cho thay toi co tac dung irc che ro r?t Sl/ tSng smh te bao ciia dong te bao HT-29 (p<0,01).Them vao do, dich chiet toi Ly Son toan phan d nong dp 10 mg/ml cd tac dung irc che ro ret su tSng sinh te bao so vdi nhom dieu tn bSng vincnstine 0,05 pg/ml t?i cac thdi diem sau 48 va 72 gicJ {p<0,05). Phat hien nay da cung cap cac bang chimg ro r^t ve tac dung khang ung thU dai trang ngiTdi ciia toi Ly son.
SUMMARY
I N V E S T I G A T I O N O F ANTI-CANCER EFFECTS OF LYSON GARLIC ON HUMAN COLON CANCER CELLS Previous studies In recent years suggest that garlic has anti-prollferative effects against vanous types of cancer. In this study, we investigated anti-cancer
• Hpc vi$n Quin y
*'D^t H<?c Y Vmh ' " B$nh vten 103
Phan bi^n khoa hgc: PGS.TS. Nguyen Thj Ty
activities of Ly Son's garlic extract on HT-29 human colon cancer cells by using the trypan blue and MTT assays. Results showed that Ly Son's garlic extract solution was significant inhibition of HT-29 cells replication in companson to control group. At the time point of 24, 48, and 72 hours after treated with 5 mg/ml and 10 mg/m! of Ly Son's garlic extracts have been shown significant inhibition the HT-29 cell proliferation (p<0.01). Additionally, at concentration of 10 mg/ml, the Ly Son's garlic extract was significant inhibition of the HT-29 cells replication in comparison to the cells treated with 0,05 pg/ml of vincnstine at 48 and 72 hours following up {p<0.05). This finding indicates anti-replication of Ly Son's garlic on HT-29 human colon cancer cells.
Keywords: Garlic, colon cancer, HT-29 I. DAT VAN OE
Ung thu" la mdt nhdm benh lien quan den roi loan phan ehia te bao mat kiem soat va di can.
Cac benh ung thu" thu'dng gap nhat tren toan the gidi (2008) la ung thu" phoi (1,61 trieu ca, 12,7%
tong sd), ung thu' vu (1,38 trieu ca, 10,9%) va ung thu- dai trirc trang (1,23 trieu ea, 9,7%) [3].
Ung thu' dang la mdt thach thirc Idn ciia y hpc, nghien ciru de tim ra nhuTig thudc mdi dieu trj ung thu, dac biet la cac hdp chat ed nguon goc thien nhien dang la mpt linh vi/c du'oc u'u tien hang dau. Tdi la mot loai du'oc lieu cd nhieu cdng dung trong chu'a benh nhu' chdng virus, khang vl khuan, nam, kha nang chdng goe tu" do, ho trd dieu tri cac benh li tim mach va chdng ung thU
Y HOC VIET NAM THANG 4 - SO 2/2014
[1,2,4]. O nu'dc ta cd mdt giong tdi dac biet du'de trong tai dao Ly Sdn cd tiem nang trong dieu t n mpt sd benh ly. Trong cong trinh nay chting toi nghien cii'u tac dung li'c che tang sinh te bao cua djch chiet toi Ly Sdn tren te bao ung thuYdai trang ngu'di.
II. €>6l TUONG VA P H U a N G PHAP NGHIEN CUTJ 2 . 1 . Do! tu'dng va v a t lieu nghien cu'u Dieh chiet tdi Ly Sdn (TLS) toan phan du'de bao che tai Hgc vien quan y bang phirdng phap tach chiet hoi luXi.
Vincristine la mot vinca alkaloid tif Catharanthus roseus (eay dira can). Nd la mot chat li'c che phan bao du'de sir dung trong hda tri lieu ung thu'.
Te bao ung thir dai trang ngu'di ddng HT-29 (ATCC, USA) du'de nudi cay, tang sinh trong moi tru'dng McCoy's 5a Medium Modified them 1 % dung dich Penieillin/Streptomycin (lOx) va 10%
dung dich huyet thanh bao thai be (FBS) trong mdi tru'dng 5% CO2 va 37°C. HT-29 la mdt ddng te bao ung thu" bieu md dai trang du'de phan lap tir mpt benh nhan ntT 48 tuoi. Ddng te bao nay bieu hien IgA, CEA, protein gan ket chuyen dang yeu t d tang tru'dng beta; mucin, myc -1-; ras + ; MYB -I-; fos +; sis -1-; p53-i-, ...
Bp kit MTT assay (3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) va dung dich Trypan Blue.
2.2. Pliu'cjng phap nghien cu'u De danh gia kha nang gay dpc triTc tiep va tac dung khang ung thir ciia dieh chiet TLS toan phan chung tdi tien hanh thir nghiem Trypan Blue va thir nghiem MTT tren eac dTa 96 gieng nudi cay ddng te bao ung thu' HT-29. Cae gieng nudi te bao du'de ehia thanh 7 nhdm, du'de danh sd tir 1-7 tu'dng irng nhu" sau:
Nhdm 1; te bao HT-29 du'de nudi vdi cac mdi tru'dng McCoy's 5a Medium Modified them 1 % dung djch Penicillin/Streptomycin (lOx) va 10% FBS Nhdm 2: te bao HT-29 du'de nudi trong mdi tru'dng nudi eay nhu" nhdm 1 va cd bo sung vincristine 0,05pg/ml
Nhdm 3: te bao HT-29 du'de nudi trong mdi tru'dng nudi cay nhu' nhdm 1 va cd bo sung them nong dp djch chiet TLS toan phan lOmg/ml.
Nhdm 4: te bao HT-29 du'de nuoi trong mo tru'dng nudi cay nhu' nhdm 1 va cd bo sung then- ndng dp djch chiet TLS toan phan 5 m g / m l .
Nhdm 5: te bao HT-29 du'de nudi trong mo tru'dng nuoi cay n h u nhdm 1 va cd bo sung then:
nong dd dich chiet TLS toan phan 2,5mg/ml.
Nhdm 6: te bao HT-29 du'de nuoi trong moi tru'dng nudi cay nhu' nhdm 1 va cd bo sung them ndng dp dich chiet TLS toan phan l,25mg/ml.
Nhdm 7; te bao HT-29 du'de nuoi trong moi tru'dng nudi cay nhu' nhdm 1 va cd bo sung them nong dp djch chiet TLS toan phan 0,625mg/ml.
Thiy nghiem Trypan Blue:le bao sau khi thu hoach tir dTa nudi cay du'dng kinh 10 cm, du'de hda deu trong moi tru'dng nudi cay va du'de dieu chinh de mat dp te bao dat 10^ te bao/ml. Sir dung 100 \i\ dung djch chiTa te bao nay cho vao tirng gieng tren eac dTa nuoi cay 96 gieng va sir dung mdi tru'dng nudi eay cd chira cac thuoc thii"
theo 7 nhdm nhu" tren. Cho cac dTa nuoi cay vao til am CO2 il trong vdng 1 gid. Sau dd bo sung 100 pi dung djch Trypan Blue 0,4% vao tirng gieng chira te bao. Hiit mdi tru'dng chiTa te bao trong tiTng gieng cho vao budng dem Neubauer, ehd 1-2 phut d dieu kien nhiet dp phdng. Sau do, sir dung kinh hien vi de dem so lu'dng te bao chet va tdng sd te bao cd trong buong dem co dien tich I m m ^ Te bao chet se bat mau xanh dac tru'ng ciia Trypan Blue, te bao sdng khong bat mau. Sau d d , so sanh siT khae biet ve ti le te bao chet tren tong sd te bao trong mpt gieng giu'a cac nhdm nghien ciTu sir dung cac moi tru'dng chira djch chiet TLS toan phan d cac nong dp khac nhau so vdi nhdm sir dung vincristine va nhdm chirng khdng su'dung thudc.
Thiy nghiem MTT [3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide] du'de thut hien theo qui trinh md ta dau tien bdi Mosmann (1983). Nguyen li cua phan irng la sir dung chifc nang chuyen hda cita ti the d te bao song cd kha nang chuyen hda chat MTT mau vang thanh mot san pham cd mau tim khdng tan trong niTdc nhu'ng tan trong dung dieh chuyen dyng. Khi te bao chet thi ti the khong cdn kha nang chuyen hda MTT thanh ehat mau. San pham tao ra tir phan irng MTT cd mau dac tru'ng va co the djnh lu'dng du'de bang each do mat dp quang vdi bu'c!:
sdng hap thu dac trung bang may dpc ELISA 6
T H y C VIET NAM THANG 4 • SO 2/2014
kinh lex: 570nm. Mat dp quang do diTde tiT thir nghiem MTT ti le thuan vdi sd lu'dng cac te bao sdng cd mat trong cac gieng nudi cay. Thir nghiem MTT diTdc thi/e hien d eac thdi diem 24, 48 va 72 gid. Ooi vdi moi nhdm lam thi nghiem tien hanh tren 3 gieng nudi cay te bao. Sau do, moi quy trinh thi nghiem du'de lap lai 3 lan.
2.3. X u r i i s o l i e u
Sd lieu diTdc xif \'\ bang phan mem thdng ke Statvlew 5.0.
III. K ^ QUA NGHIEN CU'U
3.1. Ket qua thit nghiem doc tinh bang Trypan Blue tren dong te bao ung thU dai trang
Ket qua thir nghiem Trypan blue eho thay:
djeh chiet TLS toan phan d nhCTng nong dp khac nhau (tir 0,625 mg/ml, 1,25 mg/ml, 2,5 mg/ml, 5 mg/ml, 10 mg/ml) khdng gay chet te bao HT-29.
Bieu hien, ty le te bao chet va te bao sdng giCTa nhuTig gieng nudi cay sir dung djeh chiet TLS toan phan tiT nong dp thap (0,625 mg/ml) den nong dp cao (10 mg/ml) so vdi nhdm chirng khong sir dung djch chiet TLS toan phan la khdng cd sir khac biet cd y nghTa thong ke.
Hinh 1: Te bao HT-29 sau thir nghiem Trypan Blue
3 . 2 . Tac dung cua dich chiet TLS toan phan tai thdi diem sau 2 4 gid tren dong t e bao ung thu' dai trang
Ket qua thir nghiem MTT sau 24 gid cho thay, mat dp quang d cac gieng sir dung djch chiet TLS toan phan nong do 5 - 10 mg/ml va vincristine 0,05 pg/ml thap hdn ed y nghTa thdng ke so vdi cac gieng chirng khdng su" dung thudc vdi p<0,001. Mat khac, d cae gieng sir dung djch chiet TLS toan phan cd nong dp thap hdn 5 mg/ml khdng cd siT khac biet so vdi nhdm chirng vdi p>0,05 (hinh 2).
Hinh 2: Ket qua thir nghiem MTT sau 24 gid tren dong te bao HT-29
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0.0055 0.0004 0.0086 0.5368 0.8346 0.7184 0.9875 0.4270 0.0001 0.0002 0.0006
3.3. T^c dung cua djch chiet TLS toan phan tai thdi diem sau 4 8 gid tren d6ng t e b^o ung thu* d^i tr^ng
Y HOC VIET NAM THANG 4 - SO 2/2014
Hinh 3: Ket qua thiT nghiem MTT sau 48 gid tren ddng te bao HT-29
^ tt^
Pl-2 Pl-3 Pw Pl-S Pl-6 Pl-7 P2-3 P2-4 P2-5 P2-6 P2-7
<0.0001
<0.0001
<0.0001 0.6659 0.0553 0.0869 0.0473 0.5572
<0.0001
<0.0001
<0.0001
Ket qua do mat do quang d cac gieng thi nghiem eho thay vincristine 0,05 pg/ml, djch chiet TLS toan phan ndng dp 5 va 10 mg/ml deu CO tac dung iSc che si/ tang sinh cua cac te bao ung thu' HT-29 vdi p<0,0001. Oac biet la SLT ire che te bao ung thu' dal trang ciia dich chiet TLS toan phan 10 mg/ml manh hdn so vdi dung dich vincristine 0,05 pg/ml vtifi p=0.0473 (hinh 3).
3.4. Tac dung ciia djch chiet TLS toan phan tai thdi diem sau 72 gid tren dong te bao ung thu' dai trang
Ket qua thi nghiem sau 72 gid, eho thay dung djch vincristine 0,05 pg/ml, djch chiet TLS toan phan ndng dp tir 2,5-lOmg deu cd tac dung ire che siT tang sinh ciia ddng te bao ung thu' dai trang HT-29 (p<0,01). Trong dd, mat dp quang trong eae gieng sir dung djeh chiet TLS toan phan 10 mg/ml thap hdn cd y nghia thdng ke so vdi vincristine 0,05 pg/ml vdi p<0,0001 (hinh 4).
Hinh *?.' Ket qua thir nghiem MTT sau 72 gid tren ddng te bao HT-29
M^
Pl-2 Pl-3 Pl-4 Pl-5 Pl-6 Pl-7 P2-3 P24 P2-5 P2-6 P2-7
<0.0001
<0.0001
<0.0001 0.0051 0.0560 0.1188
<0.0001 0.0175
<0.0001
<0.0001
<0.0001
Z ; VIET NAM THANG 4 - SO 2/2014
IV. BAN LUAN
Nghien ciru eiia ehiing tdi cho thay djch chiet TLS toan phan cd tac dung irc che si/ tSng sinh ciia ddng te bao HT-29 in vitro. Cac nghien ciru khac cung cho thay tdi cd tiem nang trong iic ehe SLT phat trien eiia ung thu' ndi chung va ung thu' dai trang ndi rieng. Tae dyng iic che ung thu' dai trang ciia tdi thong qua a5 che gay apoptosis, tu'dng tac vdi mdt sd protein lien quan den chu trinh te bao...[4,5,6].
Nghien cu'u ciia Seki T. va CS (2008) danh gia hieu qua cua diallyl trisulfide (DATS), mdt hdp chat sulfur chiet xuat tir tdi [5]. Khi te bao ung thu* dai trang du'de cam irng vdi DATS, ehu trinh te bao bj thay doi va dirng d pha G2/M va cac te bao vdi caspase-3 hoat dpng da du'de tang len dang ke khi dieu tri vdi DATS [ 5 ] . DATS gian doan hinh thanh mang lu'di vi ong ciia te bao [5].
Nhtrng ket qua nay cho thay rang DATS ed ddng gdp vai trd quan trong trong tae dung chong ung thu-ciiatoKS].
Nghien cim Sujatha G. va CS (1996) da eho thay tae dung chdng tang sinh ciia diallyl disulfide (DADS), mot ehat sulfur hiTu cd tir djch chiet tdi, tren tde cip tang tru'dng ciia ddng te bao ung thu' dai trang ngu'di HCT-15 [ 6 ] . Chuot thieu hut mien dieh mang khoi ung thu' dai trang ngu'di du'de tiem mang bung I m g DADS ba lan mol tuan eho ket qua klioi lu'dng khoi u glam^
69% (p<0,05) ma khdng cd hau qua dang ke nao ve toan than [6].
Nhu" vay, cac dCr lieu thu du'de tir eae nghien cull tac dung ciia djch chiet tdi cung nhu mot so hdp chat cu the dirde tach chiet tir tdi ed tae dung irc che siT tang sinh ciia ung t h u dai trang in vitro dJnq nhu' in vivo.
V. KET LUAN
Djch chiet TLS toan phan ndng dp 5 mg/ml va 10 mg/ml cd tac dyng ixc che rd ret siT tang smh ciia ddng ung t h u dai trang HT-29 so vdi nhdm chu'ng sau eac thdi diem 24, 48 va 72 gid vdi p < 0 , 0 1 . Dich chiet TLS toan phan nong dp 10 mg/ml cd tae dung ifz che rd ret siT tang sinli ciia ddng ung thu' dai trang HT-29 so vdi te bao dUdc dieu tri vdi vincristine 0,05 pg/ml tai cac thd'i diem 48 va 72 gid (p<0,05).
TAI LIEU T H A M KHAO
1. Nguyen Minh Chinh, Nguyen Van Long, Dao Van Don, N g u y i n Duy ThtTc (20U), "Nghien ciru thanh phan hoa hgc cua toi Ly sdn {Allium saltivum L)", Tap chi YDu'de hoc Quan sW, 21(2), tr. 91-95.
2. Bui Khac Cu'dng, N g u y i n Linh l o a n (2011),
"Anh hu'dng ciia dich chiet toi tren te bao ung thU vu ngudi". Tap chi -y hoc thwc hanh, 771(6), tr.
43-49.
3. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. (2010), "Estimates of worldwide burden of cancer in 2008: GLOSOCAN 2008", Int3 Cancer.. 127(12), pp. 2893-917.
4. Marta Corzo-Marti 'nez, Nieves Corzo and Mar Villamiel (2007), "Biological properties of onions and garlic". Trends tn food Science &
Technology, 18, pp. 609-625.
3. Seki T, Hosono T, Hosono-Fukao T, Inada K, Tanaka R, Ogihara J, Ariga T. (2008), "Anbcancer effects of diallyl trisulfide denved from garlic", Asia PacJOin /l/i/&-.,17(Suppl 1), pp. 249-52.
6. Sujatha G. Sundaram and John A. Milner (1996), "Diallyl Disulfide Suppresses the Growth of Human Colon Tumor Cell Xenografts in Athymic Nude Mice", J. Nutr., 126, pp. 1355-1361.
DAC DIEM LAM SANG, CAN LAM SANG KEM PHAT TRIEN CHIEU NGANG XiraNG H A M TREN
6" TRfi SAU PHAU THUAT KHE HCT MOI VOM MIENG
T 6 M TAT
Mvc tieu nghien c i h i : Nghien ciTu thUc hien tren 28 tre 6 - 15 tuoi, da phau thuat tao hinh khe h6
' Vl4n DTRSng Ham MSt
Phan bl$n khoa hgc: PGS.TS Mai Dinh HuTig
Nf!u>en Thi Thu Phu'ong*, Tran Huirng Tr^*
mol vom mieng, duoc chan doan kem phat tnen chieu ngang xi/ong ham tren nham muc tieu mo ta mot so dac diem lam sang, can lam sang ciia nhiing benh nhan nay. Phu'dng phap nghien ciTu: mo ta cat ngang, tham kham lam sang, mau thach cao va phim xquang mat thing, Ket qua: Sai khdp c3n loai III (theo Angle) chiem ti le Icin nhat (50% 6 cung ham
