Y HOC VIET NAM T H A N G 8 - SO 1/2011 T A I LIEU THAM KHAO
1. Bozzette SA, Ake CF, Tam HK, et al.
Cardiovascular and cerebrovascular
2. events in patients treated for human immunodeficiency virus infection. N Engl ] Med.
2003;348:702-710.
3. J. Selhub. Homocysteine metabolism. Annu. Rev.
Nutr. 1999. 19:217-46.
4. Phillips AN, Carr A, Neuhaus J, et al.
Interruption of antiretroviral therapy and risk of cardiovascular disease in persons with HIV-1
infection:exploratory analyses of the SMART trial.
Antivir Ther. 2008; 13:177-187
Sandra G. Heil, Karin 3. A. Lievers, Godfried H. Boers, Petra Verhoef, Martin den Heijer, Frans 3. M. Trijbels, and Henk J. Blom, Betaine-Homocysteine Methyltransferase (BHMT):
Genomic Sequencing ' and Relevance to Hyperhomocysteinemia and Vascular Disease in Humans. Molecular Genetics and Metabolism.
2000. 71, 511-519.
THIET KE VECTOR BIEU HIEN KHANG THE TAI TO HOP DAC HIEU KHANG NGUYEN CD20 TRONG VI KHUAN E. COLI
Pham Thu T h u y \ Le Th| Hanh^ La Thj Huyen^
Ninh Thj Tuyet Lan^ Le Quang Huan' TOM TAT
Sir dung cac khang the ddn dong dac hieu cac khang nguyen dfch dang dUpc cac nha khoa hpc quan tam nhSm tao Kit chan doan sdm va che pham dieu trj cac benh hi^m ngheo, dac biet la cac benh ung thU c6 y nghTa rat Idn trong viec cham soc va bao ve sire khoe cong dong. Mot trong cac khang the co lien quan tdi ung thU mau la khang the khang CD20. CD20 la khang nguyen be mat chi bieu hien tren te bao B, miTc bieu hien cao (khoang 100.000 phan tir/te bao) trong b?nh ung thU mau dang non-Hodgkin va xuat hien tren be mSt ciia tren 95% te bao lympho trong benh ung thu' nay. Trong nghien ciru nay thiTc hien nhSm thiet ke vector bieu hien khang the tai to hpp dac hieu khang nguyen CD20 va bUdc dau kiem tra siT bieu hien ciia gen antlCDZO. Do vay, chiing toi tien hanh PCR nhan ban doan gen ma hoa cho doan gen khang CD20, Vector bieu hien pEr-22h{+)lantiCD20 dUpc thiet ke va budc dau kiem tra siT bieu hien gen antlCD20 d chiing bieu hien BL21(DE3) vdi dieu kien nuoi cay trong moi trUdng LB d 37°C khi ODeoo dat 0,8, cac te bao dupe cam iTng vdi IPTG nong dp cuoi ciing 0,lmM, 0,5mM, ImM va thu mau sau 3h va 5h cam iTng. Ket qua thu du'dc dUpe siT dung cho cac nghien cull xa hdn.
Tii'khoa-. CD20, £ coli, pEr-22b(+), tai td hpp, ung thu mau.
SUMMARY
DESIGNING EXPRESSION VECTOR FOR GENE CODING RECOMBINANT ANTIBODY CD20
SPECIFIC I N E.COLI
Using specific-antigen recorgnized antibodies in prognosis, eariy diagnosis as well as treatments for 1. Benh vien 198
severe diseases especially cancer has a great meaning in every aspects of life. One of the concerned antibodies of blood cancer is anti-CD20 antibody. CD20 is the surface-antigen only appear in B cells with a high amount (100.000 molecules per cell approximately) and most B cells in non-Hodgkin's (95%) and multiple myeloma. Our study aimed at mass production of recombinant CD20 antibod. Thus, the antiCD20 gene was amplified by PCR. Vector pET- 22b(+)/antiCD20 has been constructed and expressed sucessfully the recombinant CD20 antibody in E. coll strain BL21(DE3) under conditions inducing with 0,1 mM, 0,5mM, 1 mM IPTG at 37°C in 3 and 5 hours when ODeoo of culture is 0,8. Results obtained can be used for next experiments.
Key words. Blood cancer, CD2, £ coll, pET- 22b(+), recombinant.
I. Ma DAU
Non-Hodgkin lymphoma (NHL) la mot trong hai loai cua ung thu' mau lymphoma (NHL va Hodgkin lymphoma). NHL la ung thu" pho bien thii' 7 d nu'dc Mi va cac nu'dc khac tren the gidi.
Tren 9 5 % NHL cd nguon gdc do te bao B va bieu hien cac chi thj khang nguyen be mat te bao B [3]
CD20 la mot phan tiT dfch tiem nang trong y hpc bdi khang nguyen nay hau het bieu hien tren t ^ bao B; miTc bleu hien cao (khoang 100.000 phan tir/te bao); khdng bien dang hay dong hda khi gan vdi khang the va thu'dng khdng phat hien du'dc d dang hda tan [5]
Hien nay, cd nhieu phu'dng phap dieu trj
Y HQC VigT NAM THANG 8 - s 6 1/2011 ung thu' mau dang Non-Hodgkin lymphoma nhu":
xa trj, hda chat, ghep tiiy, dac biet la cac lieu phap sinh hpc. Cac lieu phap sinh hoc nhu' siT dung khang the ddn ddng (khang the tai to hdp) dang du'dc ddng dao cac nha khoa hoe nghien ciru va cung da thu du'dc nhti'ng ket qua nhat djnh. Trong nghien ciTu nay, chiing tdi tien hanh thiet ke vector va bu'dc dau kiem tra siT bieu hien khang the khang CD20 nham iTng dung chuiig trong nghien ciTu chan doan va dieu trj cac benh lien quan tdi khang nguyen be mat GD20.
n. VAT LIEU - PHUbNG PHAP NGHIEN CUU Vat lieu: Chung vi khuan £. coli DH5a, BL21, Vector bieu hien pET22-b(+) (Novagen), Doan gen ma hda cho khang the tai to hdp khang GD20 (antlCDZO) gan trong vector PCR2.1 (du'dc iu'u giCT tai phdng Cong nghe te bao dpng vat, Vien Gong nghe sinh hpc); Cap moi antiCD26F/antiGD20R du'dc thiet ke va tong hdp chu'a trinh tiT nhan biet ciia cac enzyme han che ed trinh tu' cu the nhu' sau:
antiCD20F: 5'- ATGGGCGGGTGAATTCTGTATTATCTG- 3.
£coRI
antiGD20R: 5'- ATrTAGGTTGCTCGAGTGTTACTGTTG-3'.
XhdL
. Mdi tru'dng nudi cay te bao £. coli la mdi tru'dng LB ( 1 % tryptone, 0,8% NaCI, 0,5% eao nam men va 1,5% agar ddi vdi mdi tru'dng th?ch); Cac hda chat va enzyme sir dung trong thi nghiem deu la nhu'ng hda chat ciia cac Hang cd uy tin nhu': enzyme £coRI va XhO., T4-llgase (New England Biolabs), Kit tinh sach DNA tiT gel agarose (Fermemtas), va cac hda chat khac deu la nhu'ng hoa chat tinh khiet du'dc siT dung trong sinh hpc phan tiT va cong nghe gen.
Phu'dng phap: Phu'dng phap PCR du'dc thi/c hien vdi cap moi dac hieu cd gSn cac vj tri nhan biet cua cac enzyme han che EccRl va XhdL.
Cac ky thuat phan tiT diing cho tach ddng gen: tach chiet va tinh sach DNA plasmid, yai ly DNA plasmid vdi cac enzyme han che, gan ndi cac doan DNA vao vector, dien di tren gel agarose.
Xac djnh trinh tiT nucleotide cua gen theo phu'dng phap ciia Sanger.
Phu'dng phap bien nap vector mang gen antlCDZO vao chiing bleu hien £. coli BL21(DE3) du'dc thiTc hien bang each tao te bao kha bien va soc nhiet. Chpn lpc cac ddng mang gen tren mdi tru'dng LB thach chu'a Ampieillin (100 ng/ml).
Bleu hien gen ma hoa antiCD2(>. Khuan lac xuat hien tren dia chpn lpc LB thach du'dc nudi qua dem trong mdi tru'dng LB Idng cd b6 sung
Ampieillin. Khi ODeoo ciia djch nudi cay dat 0,6 0,8, cac te bao du'dc cam iTng vdi IPTG va du'dc nudi lac vdi tdc dp 200 vdng/phut d nhiet dp thfeh hdp. Mau doi chiTng la te bao khdng cam iTng IPTG. Thu te bao d thdi diem 3, 5 va 16 h de kiem tra kha nang bieu hien ciia gen antlCD20 bang dien di bien tfnh tren gel polyacrylamide.
I I I . KET QUA VA BAN LUAN
Tong hdp doan gen ma hoa cho vung hoat dong cua anti CD20 bIng kl thuat PCR: De cd the thu du'dc lu'dng gen ma hoa khang the khang GD20 dii Idn, chiing tdi thiTc hien phan iTng nhan gen bang k9 thuat PCR vdi 2 moi dac hieu antiCD20F/R cho doan gen antlCD20 vdi hai vj tri nhan biet ciia hai enzyme han che du'dc thiet ke (trinh tiT du'dc neu trong phan phu'dng phap). Doan gen dich du'dc nhan ban diTa tren trinh tiT doan gen ma hda cho khang the tai to hdp khang CD20 gan trong vector PCR 2.1, va tien hanh theo chu trinh nhiet sau: giai doan bien tfnh 94°G: 3 phut, gen du'dc khuech dai theo ehu'dng trinh: 94°C: 45 giay;
55°C: 45 giay; 72°C: 1 phiit; lap lai 30 chu ky va san pham du'dc bao quan d 4°G. San pham PCR la mdt bang dac hieu, rd net, kfch thu'dc khoang 727 bp dung nhu' tfnh toan ly thuyet (Hinh 1).
Y HOC VIET NAM THANG 8 - SO 1/2011
7Sftp. . <•» Plum PCR
Hinh 1 . Ket qua dien di san pham PCR nhan gen antiCD20
Du'dng chay M: Maker DNA Ikb Du'dng chay 1: gen antiCD20
Nhu' vay cd the khang djnh chiing tdi da nhan ban tiianh edng gen antlCD20, gen thu du'dc mang hai vj trf eat ciia cac enzyme gidi han
£coRI va XhdL dam bao eho cac thf nghiem tiep theo.
Thiet ke vector bieu hien pET- 22b(-H)/antiCD20: TiT nguon PGR d tren chung tdi tien hanh tinh sach tiT gel agarose. Do cd vj trf cat ciia enzyme han che £coRI va XhdL d 2 dau cua gen nen sau khi tien hanh xOr ly san pham PCR triTc tiep vdi hai enzyme tren, se thu du'dc
1 M 2 3 4 5 e M
Kiem tra kit qua gSn doan gen antiCD20 vao vector bilu hiSn dugc tiSn hanh bang each su dung tr^c tiSp 4 khuin lac chpn nglu nhien tir dia nuoi cdy tiSn h^nh PCR vai cap m6i T7F/R cua vector pET- 22b(+), a tdt ca cac duomg ch^y cho thay san pham PCR xudt hi?n mot bang dJic hifu co kich thuac khoang 900 bp. Kich thudc nay la hoan toan phu hop vdi tinh todn v6 t6ng kich thudc ciia do£in gen
doan gen antiCD20 vdi cac dau dinh can thiet eho viec gan vao vector bieu hien. Vector pET- 22b(+j cung du'dc cat md vdng bang hai enzyme han che tren. Doan gen antiCD20 da Y.ii ly vdi enzyme sau khi du'dc tinh sach bang bd kit tinh sach se du'dc gan vao vector pET-22b(+) nhd enzyme T4-ligase. San pham bien nap se du'dc bien nap vao chiing DHSor de chpn ddng, ket qua chpn ddng bang phu'dng phap PCR vdi cap moi T7F/R the hien d hinh 2.
Hinh 2. Thiet ke vector bieu hien Du'dng chay M: Marker DNA Ikb
Du'dng chay 1: Vector pET22b(-H) da xii ly vdi enzyme £coRI va Xhd.
Du'dng chay 2: Gen antiCD206a xii ly vdi enzyme
£coRI va XhdL
Du'dng chay 3 6: San pham PCR tiT cac khuan lac vdi cap moi T7F/R
antiCD20 vdi mpt phin cua vector. De kilm tra chieu va khung dpc cua gen, chung toi tiln hanh xic djnh trinh tvr gen trong plasmid tai td hgrp thu dugc. Sau khi xac djnh trinh tu, chiing tdi thu dupe kit qua trinh t\r gen ma hda khing the dom ddng tdi t6 hpp (dang don chuoi) d§c hifu khang nguy6n CD20 trong vector bilu hifn vdi trinh tu nucleotide nhu sau ( Hinh 3).
1 ATGAAATACC TGCTGCCGAC CGCTGCTGCT GGTCTGCTGC TCCTCGCTGC 51 CCAGCCGGCG ATGGCCATGG ATATCGGAAT TAATTCGGAT CCGAATTCTG 101 TATTATCrCA ATCTCCTGCA ATTTTATCTG CATCTCCTGG AGAAAAAGTA 151 ACAATGACAT GTCGTGCATC TTCTTCTGTA TCTTATATGC ATTGGTATCA 201 ACAAAAACCr GGATCTTCTC CTAAACCTTG GATTTATGCA GCTTCTAATT 251 TAGCATCTGG AGTACCTGCA CGI I IIICTG GATCTGGATCTGGAACATCT 301 TATTCTITAA CAATTTCTCG TGTAGAAGCA GAAGATGCAG CAACATATTA
Y HQC VIET NAM THANG 8 - S6" 1/2011
351 TTGTCAACAA T G C T C T T T T A ATCCTGCTAC ATTTGGAGGA GGAACAAAAT 401 TAGAATTAAA ACGTTCTTCT GGAGGAGGAG GATCTGGAGG AGGAGGATGT 451 GGATCTGAAG CATATTTAGA ACAATCTGGA GCAGAATTAG TACGTCCTGG 501 AGGATCTGTA AAAATGTCTT GTAAAGCATC TGGATATAGA TTTACATCTT 551 ATAATATGGA TTGGGTAAAA GAAACACCTC GTCAAGGATT AGAATGGATT 601 GGAGGAATTT ATGCTGGAAA TGGAGATAGA TCTTATAATG AAAAATTTAA 651 AGGAAAAGGA ACATTAAGAG TAGATAAATG TTCTTCTAGA GCATATATGC 701 AATTATCTTC TTTAAGATCT GAAGATTCTG GAGTATATTT TTGTGCACGT 751 GTAGTATATT ATTGTAATTC TTATTGGTAT TTTGATGTAT GGGGAAGAGG 801 AAGAACAGTA ACACTCGAGC ACGACCACCA CGACGACTGA
Trinh tiT amino acid suy dien tir trinh tiT nucleotide cua gen tu'dng iTng la:
1 MKYLLPTAAA GLLLLAAQPA MAMDIGINSD PNSVLSQSPA ILSASPGEKV 51 TMTCRASSSV SYMHWYQQKP GSSPKPWIYA PSNLASGVPA RFSGSGSGTS
101 YSLTISRVEA EDAATYYCQQ WSFNPPTFGA GTKLELKRSS GGGGSGGGGS
151 GSQAYLQQSG AELVRPGASV KMSCKASGYT FTSYNMHWVK QTPRQGLEWI 201 GAIYPGNGDT SYNQKFKGKA TLTVDKSSST AYMQLSSLTS EDSAVYFGAR 251 W Y Y S N S Y W Y FDVWGTGTTV T L E H H H H H H *
Hinh 3. Ket qua xae djnh trinh tiT nucleotide ciia gen ma hda khang the scFv dac hieu CD20 trong vector bieu hien pET 22-b(+)
M i 2 3
» 0
Hinh 4. Ket qua bieu hien protien tai to hdp Du'dng chay M: Thang marker protein chuan Du'dng chay 1: Mau tru'dc cam iTng
Du'dng chay 2-4: Mau sau cam iTng sau 3h vdi cac nong dd IPTG Ian lu'dt la 0,lmM; 0,5mM;
ImM
Qua ket qua d hinh 3, chiing tdi cd the ket luan rSng da gan thanh edng doan gen antiCD20 vao vector pET-22b(+) dung chieu va diing khung dpc. Den day chung tdi ed the khang djnh da thiet ke thanh cong vector bieu hien pET- 22b{^)lantlCD20.
Bieu hien protein khang CD20 tai to hdp: Plasmid tai to hdp pET-22b(+)/antiCD20 diTdc dula vao chiing bleu hien BL21(DE3) tao ra dong cd kha nang bieu hien antlCD20 tai to hdp.
Dong te bao cd kha nang cd kha nang san xuat ra antiCD20 du'dc nudi lac trong mdi tru'dng LB chii^ khang sinh Amplcilin 100 pg/ml d 37°C, khi ODfioo = 0,8 hon hdp nudi cay du'dc cam uYig vdi IPTG dat nong dp cudi ciing la 0,1 mM, 0,5 mM va 1 mM, cac mau thu d cac thdi diem 3h va 5h.
De kiem tra kha nang bieu hien ciia ddng te bao thu du'dc, cac mau du'dc dien di kiem tra tren gel polyacrylamide, ket qua dien di the hien tren
hinh 4. Qua hinh anh dien di ta thay, tren cac mau cam iTng vdi IPTG ed xuat hien mot bang protein mdi khac biet so vdi cac mau khdng cam u'ng va tru'dc khi cam iTng, bang protein thu du'dc cd kfch thu'dc khoang 30kD tu'dng du'dng vdi kfch thu'dc theo tfnh toan ly thuyet. Do do, cd the khang djnh sd bp da bieu hien du'dc CD20 va da tao ra du'dc ddng te bao co kha nang tong hdp antiCD20 tai to hdp.
IV. KET UJMi
1. Da thiet ke thanh edng vector bieu hien gen ma hda khang the ddn ddng tai to hdp dac hieu khang nguyen GD20.
2. Da bu'dc dau bieu hien gen anti-CD20 trong vi khuan Ecoll chiing BL21(DE3) vdi cac dieu kien cam iTng khac nhau.
Y HOC VI^T NAM T H A N G 8 - S6' 1/2011 TAI LIEU THAM KHAO
1. La Thi Huyen (2009), Nghien ciru tao khang the tai to hpp khang CD25 de iTng dung trong y hpc.
Luan an tien sT.
2. Claudia De Lorenzo, Angela Arciello, Rosanna Cozzolino, Donald B. Palmer, Paolo Laccetti, Renata Piccoli, and Giuseppe D'Alessio (2004), "A Fully Human Antitumor ImmunoRNase Selective for ErbB-2-Positive Carcinomas". Cancer res 64 4870-4874.
3. Fisher, R. I. (2003), "Overview of non-Hodgkin's lymphoma: biology, staging, and treatment".
Seminars in oncology. 30: 3 - 9.
4. Fusion Protein Production (2001), Stockinger Lab.
5. Ginaldi, L., De Martinis, M., Matutes, E., Farahat, N., Morilla, R., Catovsky, D. (1998),
"Levels of expression of CD19 and CD20 in chronic B cell leukaemias". Journal of clinical pathology 51: 364-369.
6. The leukemia and lymphoma Society Fighting blood cancer. Facts 2010-2011.
HIEU QUA DIEU TRj U ONG TUYEN MO HOI BANG LASER COj
Dao Hoang Thien K i m * , N g u y i n HCu Siiu*
TOM TAT
MiJC tieu: Danh gia hieu qua dieu trj benh u ong tuyen mo hoi (UOTMH) bang laser CO2. Ool tu'dng va phu'dng phap nghien cu'u: ThiT nghiem lam sang tren 38 benh nhan UOTMH dUpc dieu tri bang laser CO2 vdi cong suat phat 2-2,5W. Ket qua dUpc danh gia diTa tren siT cai thien ve lam sang (so lUpng, mire dp pliSng, mau sac ton thUPng) va cac bien chirng sau 2 tuan, 1 thang, 3 thang dieu trj. Ket qua: Sau 3 thang dieu trj, 97,4 % benh nhan BN dat ket qua tot va kha, 81,6 % BN hai long vdi ket qua dieu tri. 06 da la tac dung khong mong muon gap trong 52,4 % trUdng hdp sau dieu trj 2 tuan va 0% sau 3 thang. Co 28,9 % co bieu hien tSng sac td sau 1 thang va 15,8% sau 3 thang dieu trj, chu yeu d nhuYig benh nhan co nu'dc da tham mau. Khong co trUdng hpp nao bj giam sac td hay seo xfu sau dieu tri: Ket Juan: Dieu trj UOTMH bSng laser CO2 la 1 phUPng phap co hieu qua, va it tac dung phu.
TCtkhoar. u ong tuyen mo hoi, laser CO2 SUMMARY
EVALUATION OF THE EFFICACY OF LASER CO2 IN TREATMENT OF SYRINGOMAS
Objective: to evaluate the efficacy of laser CO2 in the treatment of syringoma Patients and Methods: Clinical trials on 38 patients suffering from with syringoma treated by laser CO2 with single pulse 0,1 sec duration at powwer 2-2.5W. The results was assessed by clinical improvements(number of lesions, surface, erythema) and complications after 2 weeks, 1 and 3 months of treatment. Results: After 3 months of treatment 97,4% of patients had good and
* Tn/dng Dai hgc YHa Ngl
Phan bi$n khoa hgc: PGS.TS. Nguyen Van Thu'dng
excellent results, 86,1% of patients satisfied themselves with the treatment. Erythema was observed in 52,4% of patients after 2 weeks of treatment and no patients had this complication after 3 months. Hyperpigmentation post- laser after 1 and 3 months of treatment were 28,9% and 15,8% of patients. Scarring or hypochromia did not occur in this series. Conclusions: Laser CO2 appeared as an effective technique and less side-effects for the treatment of syringomas.
Key words: syringoma, carbon dioxide laser
I . OAT VAN DE
U ong tuyen mo hdi (UOTMH Syringoma) la u da lanh tfnh do siT ph^t trien qua mufc ciia cac te bao ong dan tuyen mo hdi [6]. Benh xuat hien tren mpi chiing tdc va thu'dng gap d nu"
nhieu hdn nam. Benh tien trien cham, khong thuyen giam neu khdng dieu trj. Dii khdng gay nguy hiem hhutig UOTMH lai lam anh hu'dng den tham my va anh hu'dng den chat lu'dng cupc song khien ngudi benh cam thay thieu b/ tin khi giao tiqj.
Nhieu phu'dng phap dieu trj UOTMH da du'c^c ap dung nhu' dot dien, phau thuat eat bd, mai mdn da hoac laser CO2 ... Moi each thufc cd nhijrig u'u, khuyet diem rieng va ket qua cdn tuy thupc vad bac sT dieu tri. 6 Viet nam, hau het cac cd sd y te siT dung laser CO2 de dieu trj U 6 T M H . Tuy nhien, cho den nay chu'a cd nghien cii\j nao danh gia hieu qua dieu trj cua phu'dng phap n^y.
