Y HQC VIET NAM THANG 8 - SO 2/2011
7.
prevalence of anemia in anemic Vietnamese women. Am J Clin Nutr 2003;78:284-90.
UNICEF/UNU/WHO (2001). Iron deficiency anemia: assessment, prevention, and control. WHO/ NHD/01.3 Geneva, Switzerland.
http://www.who.int/nut/documents/
ida_assessment_prevention_control.pdf United Nations Administrative Committee
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on Coordination Sub-Committee on Nutrition (ACC/SCN) (2000). Fourth Report on the World Nutrition Situation: Nutrition Throughout the Life Cycle. Geneva: ACC/
SCN in collaboration with the International Food Policy Research Institute.
World Health Organization (2006).
Guidelines on food fortification with micronutrients. Geneva, Switzerland.
KHAO SAT Ml)C DO BIEU HIEN CAC GLYCOPROTEIN HUYET THANH BENH NHAN LEUKEMIA LYMPHO CAP
Nguyen Tien Dung', Do Hiru Chi', Do Thj Vinh An^
Nguyen Bich Nhi', Phan Van Chi'
TOM TAT
Leukemia lympho cap (Acute Lymphoblastic Leukemia, ALL) la dang ung thu' mau du'dc dac truhg bdi su" tang sinh qua miic cua cac nguyen bao lympho. Cac trieu chuhg cua benh ALL thu'dng khong rd rang va de gay nham lan vdi die dang benh khac. Do dd, viec tim kiem cac chi thj sinh hgc cho viec chan doan benh can du'dc quan tam. Cac glycoprotein cd vai trd quan trgng trong te bao va la mdt trong nhu'ng ddi tu'dng nghien culi chii yeu cua khoa hgc proteomics.
Trong nghien ciiu nay, chung tdi phan tich su"
thay ddi miic do bieu hien cua cac glycoprotein huyet thanh benh nhan ALL bang ky thuat sac ky ai lire vdi chat gia conA ket hdp vdi dien di 2 chieu va nhan dien protein hen khdi phd. Ket qua nghien ciili cho thay, 3 protein cd miic do bieu hien thay ddi d mau benh so vdi ddi chuhg, bong dd 1 protein cd miic do bieu hien tang (Alpha-2-HS-glycoprotein), 2 protein cd miic do bieu hien giam (Alphal antibypsin, HapbDglobin a 2 chain).
TCt khoa: Proteomics, ALL, glycoprotein, NanoLC-MS/MS, dien dl2-DE
SUMMARY
Evaluated the expression of glycoproteins from Acute lymphoblastic leukemia patients
Acute lymphoblastic leukemia (ALL), is a form of leukemia, characterized by excess lymphoblasts. The ALL symptoms are so general and may be caused by other diseases. Thus, discovering biomarkers for detecting ALL is a necessary requirement. Glycoproteins play an important role in the cell and have become one of the most attractive targets for proteomic analysises. In this study, the expression of serum glycoproteins from ALL patients was analysed using ConA affinity combined with two- dimensional electrophoresis (2-DE) and Nano- liquid chromatography-electrospray ionization mass spectrometry (NanoLC-ESI-MS/MS). By comparison of serum samples between ALL patients and the healthy, we found that, one protein (Alpha-2-HS-glycoprotein) were up regulated and two glycoproteins were down
regulated (Alphal antitrypsin. Haptoglobin a 2 chain) in ALL serum.
Key words: Proteomics, ALL, glycoprotein, NanoLC-MS/MS, 2-DE
I.
OAT VANof
Benh leukemia lympho cap (ALL) xay ra khi cac tl bao tao mau sa cap (nguyen bao lympho) tang sinh ma khdng phat trien thanh cac tl bao mau binh thudng. Nhimg te bao ac tinh nay dugc nhan len khdng kiem soat, lan at cac tl bao bach ciu trudmg thanh trong tiiy xuomg va he bach huylt, gay tdn thucmg nghiem trgng cho ngudi benh va cd the dan din tu vong. Ty le sdng sdt ddi vdi benh biln thien theo liia tudi, 85% d tre em va 50% d ngudi Idn [2].
Nhihig nghien ciiu proteomics ddi vdi leukemia hien nay nham muc dich lam sang td vl phan loai leukemia, xac dinh cac khang nguyen ung thu, chan doan sdm va danh gia qua trinh dilu tri benh. Nghien ciiu cua mdt sd tac gia cho thiy, miic do bieu hign qua mire cua mdt sd protein lai cimg nhu mdt sd protein cua te bao tien than cd anh hudmg rd ret tdi su bieu hien cua toan bd protein trong te bao dd va la nguyen nhan quan trgng gay ra benh Leukemia [7].
Cac protein bi glycosyl hda chiem hom 50% trong cac bien ddi sau dich ma. Sir ' glycosyl hda bat thudng cua cac protein cd the la tiln de cua nhieu benh ly khac nhau [5]. Trong nghien ciiu nay, chung tdi khao sat mdt sd bien ddi miic do glycosyl hda ciia cac protein huylt thanh bgnh nhan ALL vdi muc dich tim kiem cac glycoprotein tiem nang cho qua trinh chan doan va dieu hi benh.
II. VAT UEU VA PHUONG PHAP NGHIEN COU
2.1. Vat lieu
Cac mlu huylt thanh ngudi binh thudng va benh nhan Leukemia do Benh vien Bach Mai cung cip. Cac boa chit dugc su dung bao gdm: Concanavalin A (Amersham Bioscience), hoa chit dien di SDS-PAGE va dien di 2-DE (Bio-Rad) va cac hoa chit ca ban khac.
2.2. Phu-otig phap
Sdc ky di luc v&i chdt gid ConA
ConA dugc hda vao trong dem can bang (Tris-HCl 20mM NaCl 0.5M, pH 7.4) va nhdi len cdt. Huylt thanh nguyen dugc hda trong 10 lin thi tich dem can bing va dugc dua len cdt (0,lml/phiit). Phin khdng bam cdt dugc rua bing 8ml dem can bang; cac glycoprotein bam cdt dugc thdi ra bing 8 ml dung dich dudng methyl-a-D- glucopyranoside 0.5 mM.
Di^n di2DE vaphan tich muv dp bieu bi^n Hon hgp glycoprotein da thu nh?ui dugc tiia bing acetone rdi phan tich bang dien di 2DE vdi thanh Strip 7cm, pH4-7 theo quy h-inh va he thdng thilt bi cua hang Bio-Rad (My). Kit qua dign di 2DE dugc phan tich dl xac dinh miic do bilu hign cua cac protein giiia mlu benh va ddi chiing bing phin mIm PD Quest V 7.1 (Bio-Rad).
Nhan dien protein bdng sdc ky long kit ndi khii phd (NanoLC-MS/MS)
Cac dilm glycoprotein quan tam dugc cit va thuy phan vdi 0,2 pg trypsin. Hon hop peptide sau dd dugc phan tich tren he thdng IDnanoLC-MS/MS. Tir phd nanoLC- MS/MS thu dugc, chiing tdi nhan dign cac protein bing phan mem Mascot vl.8 ket ndi vdi ca sd dir lieu NCBI cap nhat.
Y HQC VI$T NAM THANG 8 - SO 2/2011
III. KET QUA
3.1. Thu nhan glycoprotein bing sic ky ai lire ConA
Trong nghien ciiu nay, chiing tdi su dung phuang phap sic ky ai lire vdi chit gia ConA dl thu nh$n cac glycoprotein huylt thanh benh nhan ALL. Kit qua qua trinh thu nhan dugc kilm tra bing dien di SDS-PAGE, 12,6% (Hinh 1).
kDa M
^ ', 'A'- ? '•A
14.4
Hinh 1. Dien di dd SDS- PAGE ket qua thu nhan glycoprotein bang sac ky ai luc vdi ConA. M: Marker, Dudng chay 1: Huyit thanh nguyen; Dudng chay 2-6:
Phdn protein bdm cot.
Ket qua dien di cho thay, trong hdn hgp glycoprotein tir huylt thanh benh nhan ALL da loai bd dugc phin ldm Albumin, tao dieu kien cho qua trinh phan tach bing dien di 2-DE.
3.2. Phan tich mire do bieu hien bing dien di 2-DE va phan mem PD Quest v7.1 Lugmg glycoprotein huyet thanh ciia mau benh Leukemia va mau ddi chiing dugc tinh toan tuong duomg nhau bang phuomg phap Bradford. Miic do bieu hien cvia cac glycoprotein huylt thanh miu benh va binh thudng dugc so sanh tren ban dien di 2-DE vdi sir ho trg ciia phan mem PD Quest v7.1. Ket qua phan tach cac glycoprotein huyet thanh bang dien di 2-DE dugc the hien d Hinh 2.
Dudi su ho trg ciia phin mem PD Quest v7.1, 3 viing diem cd miic do bieu hien thay doi dang kl giiia hai mau da dugc phat hien trong do: 1 vung diem cd miic do bieu hien tang (viing diem 1- Hinh 2) va 2 vung dilm cd miic do bilu hien giam (vimg diem 2,3 Hinh 2) d mau bgnh so vdi ddi chiing.
m-
* •
«
BT05 L2.1 Hinh 2. Ket qua phan tach 2-DE cac glycoprotein huylt thanh ngudi benh ALL (L2.1) va
ngudi binh thudng (BT05)
•n. IP .^^
V^"'
• 3 1
#%
1
3.3. Nhan dien cac protein cd muc do bieu hien thay doi
Cac vvmg dilm glycoprotein cd sir thay ddi khac biet giua miu thudng va mau bgnh dugc cit, thuy phan va phan tich tren he thong sic ky ldng nano 1 chiiu kit ndi khoi phd, nhan dien bang phan mem Mascot vl.8 (Hinh 3).
,.i.l.l;!,'J- rll. . . „ . J ; , Lil.. .. 1,
W,^™FJ Mascot Search Results
Protein View
Match t o : gil296653; Score: 78 hpH alpha t^ono aapi-cno]
Found in search of
C:\KIK^^:\?roflle3\AD^5IKI-:.\LOC»LS-l\TelBp\l^a3i33.t
Hinh 3. Minh hga ket qua nhan dien viing diem 3
Tir kit qua nhan dien bing phin mIm Mascot vl.8, 3 glycoprotein da dugc xac dinh (Bang 1).
Viing diem
1 2 3
Sd dang ky
ail 120095 011177831 0114826762
Ten protein
Alpha-2-HS-glycoprotein Alphal- antitrypsin Haptoglobin-a2-chain
Khdi lu'dng
49 kDa 46,85 kDa
16.9 kDa
Mure do bieu hien
Tang Giam Giam
Bang 1. Kit qua nhan dign cac protein cd miic do bieu hign thay ddi.
IV. BAN LUAN
Alphal-antitrypsin (AIAT) ddng vai trd quan trgng trong cac ca che kiem soat viem, ddng mau va sira chiia cua ca the. Sir bieu hien ciia AIAT cd lien quan den qua trinh thuc hien chiic nang ciia nhieu protein khac, do nd lie che manh me su hoat ddng ciia cac protease phan cit cac protein trong mau va md. Khao sat trudc day cua chiing tdi phat hien su bieu hien giam ciia AIAT trong huyet thanh bgnh Leukemia man ddng tiiy (CML) [I]. Trong nghien ciiu nay, su giam dang ke ham lugng ciia AIAT trong huyet thanh benh nhan ALL da dugc ghi nhan.
Hien nay, da cd cac md ta ve sir nhay cam ciia Haptoglobin (Hp) va Alpha-2-HS- glycoprotein (AHSG) din mdt sd trang thai
benh ly [4, 6]. Nghien ciiu ciia chiing tdi budc diu cho thiy sir thay ddi dang ke cua ndng do cua 2 protein nay trong huylt thanh benh nhan ALL. Cac kit qua nay cho thay tilm nang cua cac nghien ciiu proteomics cho qua trinh tim kilm cac biomarker cho chan doan va dieu tri bgnh.
V. KET LUijlN
Da phat hign 3 protein cd miic do bieu hien thay doi d mau huylt thanh benh nhan ALL so vdi ddi chiing, trong dd I protein cd miic do bilu hign tang (Alpha-2-HS- glycoprotein), 2 protein cd miic do bieu hign giam (Alphal-antitrypsin, Haptoglobin-a2- chain).
Y HQC VI$T NAM THANG 8 - S6 2/2011
TAI UEU THAM KHAO
1. Nguyen Tien Dung, Vu Khic Nggc, Pham Due Dan, Do Thj Vinh An, Nguyen Bich Nhi, Phan Van Chi, (2010). "Sir thay doi miic do bieu hien hg glycoprotein huylt thanh benh nhan leukemia kinh dong bach ciu hat". Ky yen Hoi nghi Sinh hgc phdn tir va Hoa sinh Y hgc toan qudc lan 2, Ha noi,
18-19/9/2010: 132-137.
2. Nguyen Anh Tri, Nguyen Hiru Toan (1995). "Tir tiin leukemia din leukemia cd/7",NXBYhgc, HaNoi.
3. Bradford MM., (1976). "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding". 'Anal.
Biochem 11: 248-254.
4. Epelbaum R, Shaltin C, Segal R, Valansi C, Arselan I, Faraggi D, Leviov M, Ben-
Shahar M, Haim N (1998). "Haptoglobin - relate Protein as a Serum Marker in Malignant Lymphoma". Pathology Oncology Research 4(4): n\-216.
5. Jakob B, Bartosz JP, Alexandre VP, Jacek RW (2004), "Screening for N-glycosylated proteins by liquid chromatography mass spectrometry". Proteomics 4: 454-465.
6. Kwak JY, Ma TZ, Yoo MJ, Choi BH, Kim HG, Kim SR, Yim CY, Kwak YG (2004).
"The comparative analysis of serum proteomes for the discovery of biomarkers for acute myeloid leukemia" Experimental hematology 32(9): 836-842.
7. Melnick A, Licht JD (1999).
"Deconstructing a Disease: RAR{alpha}, Its Fusion Partners, and Their Roles in the Pathogenesis of Acute Promyelocytic Leukemia". Blood 93: 3167-3 215.
NGHIEN ClJfU KNOCK-DOWN GEN ST3GAL-I BANG SIRNA TREN MO HINH TE BAO UNG THlT VU IVICF7
Do Thi Phuong', Do Thi Thao', Nguyen Thj Cue', Nguyen Thi Nga', Nguyen Thi Trang'
TOM T A T
Enzyme sialyltransferase ST3Gal-I du'dc ma hoa bdi gen ST3Gal-I cd tac dung xuc tac de chuyen CMP-sialic acid tdi gan ket vao nhanh ciia loi galactose 1 (Galpl,3GalNAc) 0-glycan va thu'dng bieu hien manh d nhu'ng benh nhan ung thu* dang trong giai doan di can. De bu'dc dau tim hieu vai trd cua gen ST3Gal-I, chiing tdi da chuyen nhiem siRNA vao te bao ung thu' vu MCF7 de knock-down gen ST3Gal-I. Hieu qua chuyen nhiem len tdi gan 92% sau khi du'dc kiem chiing
' Vi^n Cong ngh? sinh h(>c, Vi?n Khoa hgc va Cong ngh? Vi$t Nam
bang phan ling RT-PCR. Hinh thai te bao sau khi bj knock-down gen nay cd nhu'ng thay ddi nhat djnh. Thong qua thii nghiem vdi bg kit almar blue, te bao sau khi chuyen nhiem siRNA cho thay kha nang sdng sdt va phat trien tu'dng t i i nhu" cac te bao khong bj chuyen nhiem. Nhu" vay qui trinh knock-down gen ST3Gal-I bang siRNA da thanh cdng tren md hinh te bao MCF7.
Tu'khoa: Glycosyl, sialyltransferase, knock- down, siRNA, ST3Gal-I.
