Gen fibrin chứa kháng sinh ức chế sự phát triển của vi khuẩn có khả năng hình thành biofilm

Tap chi Cong nghe Sinh hpc 12(2): 229-233, 2014

GEL FIBRIN CH(JA KHANG SINH (JC CHE Str PHAT TRIEN CUA VI KHUAN CO ICHA
NANG HINTH THANH BIOFILM
Doan Nguyen VD, Train Le Ban Ha
Trufmg Doi hpc Khoa hpc Tic nhien, Dai hpc Qudc gia Thanh phd Ha Chi Minh
Ngay nhan bai: 10.5.2014
Ngay nhan Clang: 18.6.2014
TOM TAT
Nhient truing la nguyen nhan gay tin vong tucmg dining tai nan giao th8ng, AIDS va ung thu vii. Khi cac to
bao vi khuan CO ca hei dinh cu tren be mat vat lieu ghep, chung se thay doi kieu hinh de hinh thanh nen clang
mai &roc goi la biofilm, bao ve chung cluing lai he mien dich caa co the va tic dung dm kiting sinh. Gel fibrin
la loai vat lieu co nhieu dac tinh ly trong cho he thong phan Oat khang sinh co kiem sofit via ming dich de
ngan chan su hinh thanh biofilm. Trong nghien dm nay, chung tai tien hanh tap gel fibrin tmc tiep tin huyet
tucmg nguai; sau do, dinh gia gel tao thanh bang quan sat dual kinh hien vi dien tin pet (SEM) va six phan huY
in vitro. Sau do, gel fibrin chira khang sinh duqc (faith gia thong qua khi nang phan phat khang sinh in vitro,
khi nang Lac dung len eking vi khan hinh thinh biofilm, khao sat tinh dac ten to bao nguai in vitro., Ket qua
cho thay, gel fibrin chCra khang sinh
gay d6c cho to bac) nguai in vitro va co khi nang Crc che stn phat
trien dm cluing vi khuan c6 khi nang hinh thanh biofilm in vitro. De tai nay nhu buac khai dau dat nen mang
cho viec Oat trien phuang phap dieu tri hieu qua trong nhiem truing va ngan chan stn hinh thanh biofilm.
Tir khogi: Gel fibrin, biofilm, nhiem trimg,

he thong phan phdi thuac, huyat mong nguai

M O DA U
Hang nam co den 103.000 benh nhan 61 nhiem
thing benh vien tin vong. Theo To chirc giam sat
nhiem thing benh vien quilt gia cua My (NNIS),
38% nhiem truing benh vien la do nhiem truing vet
me). anh hue:mg tai khoing 1.500.000 benh nhan.

V ien phi dm nhiing benh nhan 1)1 mac loai nhiem
khan nay cao han 39.000 USD so v6i benh nhan
binh thuerng va th6i gran nam vien ding keo dai
them 7,5 ngay. Nhang hao ton cua nhiem tiling vet
m6 chiem tai 15 ti USD mill nam 6 My (Smith,
2008; Mandavyapuram, 2010).

Trong cac guy trinh phau thuat, benh nhan
thuemg dirge cung cap kiting sinh; dac biet, trong
phltu thuat chinh hinh vi vet mei rOng va di kern veri
viec cay ghep cac vat lieu sinh hoc. Ngoai ra, khi
hien Wong nhiem truing b nhring vi tri xa (mat de
mach man that)), doi hoi lieu khang sinh cao va dieu
tri trong thin gian keo dai, dieu nay tang nguy co gay
dee . cho ca the benh nhan va khang thuoc
vi
khuan. Vi khuan con san xuat ra ming nhay pi la
biofilm bao ve chung. thong lai he mien dich cila co
the, lam giam ding ke tic dung cua khang sinh
to
bio vi khuan va gay ra nhiem truing man tinh khi vat
lieu con nam trong co the (Pye, 2009; Archer, 2011).

fle khic phut nhring nhtroc diem ming nhu ngan
chan str hinh thinh biofilm can co met he thong phan
phat khang sinh có kiern,soat va truing dich. Trong he
thong nay, khang sinh can duqc bao b9c beri cac vat
lieu c6 khi nang to phan huS/; tuy nhien, viec sir dung
nhang loai vat lieu kitting to than se gay ra phan Ong
viem va thai !coal cna co the benh nhan. Fibrin la met

trong nhang foal vat lieu tu than dau tien duqc su
citing trong he thong phan phat khang sinh nhan' kiem
soat so nhiem truing. Fibrin co nhieu dac tinh 15, tuemg
de tr6 thanh Ong vien tiem nang cho mtic dich nay
nhu tinh tucmg hem sinh hoc, khi nang to phan hujr
trong mai tru6ng, co the, tinh chat barn dinh va cau
truc xop tao dieu kien thuan Iqi cho viec phan phat
&roc pham. Tuy nhien, 6 nu6c ta, van de nay con rat
mai; hau nhu khang co cling trinh nghien ciru khoa
hoc nao lien quan doge cong bo. Do do, chint tai tien
hanh nghien dm nay nhu buoy kheri dau dat nen m6ng
cho viec Oat trier' phrrang phau diet' tri hien qua
trong nhiem truing hatt phau (Tsourvakas, 1995;
Mosesson et al., 2001; Canonico, 2003).

VAT Lieu VA PHUONG PHAP
tuvng nghien aim
Mau ngoai vi nguai dai xet nghiem am tinh vii

229


Doan Nguyen Vu & Trail Le Bin Ha
cac bEnh truytn nhiem &roc cung cap to Trung tam
truyen miu huyet hoc bEnh vier' Cher Ray thinh phi)
H6 Chi Minh.
Phirang phi') tao gel fibrin
Mau ngoai vi nguai duqc ly tam 3000 vong/phit
trong 10 phut, thu nhan phan dich .n8i, loai b6 can.
Sau do, mkt bo sung CaCl2 6 cac nong LIE Ichfic nhau

(0,012M; 0,013M; 0,014M), 0 6 37°C trong mot
khoing thai gian dE khio sat thai gian tao gel.
Dinh gia mat so dac tinh can gel fibrin tao thanh
Danh gia eau trac be mat
Ngam gel fibrin tao thinh qua dem trong dung
djch co djnh Mau glutaraldehyde 2,5% Er 4°C; sau do,
khu nu& trong con C; cac nong,d6 giim dan. Mau
gel sau khi khir nuac duqc dua den Vin khoa hoc va
c8ng nghE thanh ph6 HO Chi Minh tit tiEn hanh chop
SEM (scanning electron microscope).
Dcinh gici kha nang phcin hay
Gel fibrin tao thanh Er 3 nling do CaCl 2 khitc
nhau duqc lam kh8 6 nhiEt dO phong qua dem, can
de xac djnh khoi luqng ban dau. Ngam gel trong
huyet twang ngueri, lac 100 vong/phOt 6 37°C. Can
khOi luqng, gel 6 cac thai diem 1 ngiy, 2 ngay va 3
ngiy. Huyet twang &rem thay mai hang ngiy trong
suOt qua trinh thi nghiEm.
Dcinh gici khci ming thai khang sinh caa gel fibrin in vitro
Gel fibrin china 1 mg kiting sinh vancomycin
&roc ngatn trong huyet tuong ngtplii, lac 100

yang/phut 6 37°C. Huy& tucmg &roc thu nhan 11161
-20°C; sau do, b6 sung huytt tuong
ngay va luu
nguai moi. N6ng d6 vancomycin duqc gel fibrin giai
ph6ng vio huyet tuang 6 mai ngiy &roc do bang
phuong phip Kirby-Bauer. Day la phuang phip xac
djnh nong dE kiting sinh trong dung djch thong qua
duErng chuan ttrcmg quan gift duling kinh yang

khing lchuan vi dung djch co !tong do kiting sinh da
Wet.
Mir nghiem lac clang caa gel fibrin chaa khang sinh
len to bao vi khucin Staphylococcus aureus
flat gel fibrin chira lmg kiting sinh vancomycin
vat) cac eppendorf china vi khuan S. aureus yeti mat
to bao/ml. U eppendorf a 37°C trong 24 h, sau
dO
do loai moi trinling, them dung djch crystal violet
1%, a 20 phid 6 nhiEt di) phong. Loai bo dung Bich
crystal violet, hob tan thuoc nhuEm con lai trong to
bio vi khuan bang con curet diii. Do mat (V quang
hoc 6 bulic song 570nm.
Khoo sat dac tinh caa gel fibrin chart khang sinh dal
vai nguyen bao sal ngwai
NuOi cay nguyen bio sqi ngtrai dEn khi phil kin
80-90% be met dia nu8i cay. Dar gel fibrin chira
khing sinh vio trong tam cila dia nu8i, bo sung moi
tnrang nuoi cay, net) tuc 0 6 37°C, 5% CO2. Sau
24h, quan sat inh hulling dm gel fibrin china kiting
sinh len hinh thai, mat dia, cub to bao ducti kinh hien
vi dito nguqc. Sir dung cao su thien nhien (latex) lam
nhom chimg ducmg. Tien chuan so sfinh theo bang 1
(Theo tieu chuan cub Hip hEi thir nghiEm va vat liEu
M9-ASTM).

Bang 1. Chi so dQc t6 cap tinh.
Chi s8
0
1

2
3
4
5

Me to
Kheng c6 vung to bac) chat nhan biet xung quanh hoec ben duel vet lieu
VOng to bac chat giai han & khu v rc ben &raj vet lieu
Viing to bac chat Ian ra ngoai it nhet han 0,5cm so vai vat lieu
Vbrig to bao chat Ian ra ngoai tin 0,5 — 1cm so vai vet lieu
Khu vtyc ti bao chi& Ian ra ngoai leen han 1cm so vai vet lieu nhung khOng Ian ra het dia
Khu vir to bao chat Ian ra toan be dia

KET QUA VA THAO LUAN

nhau. biting do CaCl2 cang cao thi thai gian to gel
cang nhanh (Bing 2).

To gel fibrin

Kin tang nting 80 CaCl2 them vio huyEt twang,
lung ion Ca' theo do ding tang len. Dieu nay anh
huling den hai buErc trong qui trinh tao gel fibrin:

Theo ktt qui,khio sit, thai gian tao gel to huyEt
tucrng vai cac nong di) CaCl2 khic nhau Ia khic
230


Tgp chi Cong nghe Sinh hoc 12(2): 229-233, 2014

thilc day su hoat him thrombin, qua do day nhanh
qua trinh chuyen tibrinogen thank fibrin, cling nhu
thac day qui trinh hoat hoa nhan to &Ong mau XIII,
tao lien ket cheo giira cac sqi fibrin thanh mang luOi
sqi, day nhanh qua trinh tao gel.
Dinh gii ciu trtic be mit
Cac sqi fibrin dan chang chit v6i nhau tao thanh
mat mang 1u6i,vai cac nhanh va cac lien ket ,cheo,
tao nen nhieu
tren be mat gel (Hinh 1). Ket qua
nay phis hop vai nhieu ding trinh ding bo tnr6c day
(Mosesson et al., 2001; Woolverton, 2001;
Canonico, 2003).
Dinh gia kha nang phan hay
Cac Mau gel fibrin tao ra ter ba n6ng do CaCl2
khac nhau c6 so phan huy khac nhau dtra teen mix
dO giam khoi luong theo phin tram (Hinh 2). Kh6i

luong gel fibrin giam gin 50% trong ngay &Au nen,
nhfing ngay sau la str giam tit tit cua khoi Itrong gel.
Trong do, gel fibrin tao ra to ,nong da CaC12 0,014
M c6 so phan hiry nhanh Mat trong ngay dau tien
(mit 42,89% khoi Wong ban diu), gel tao ra tit n6ng
dia CaCl2 0,013 M giam it nhat (mat 23,47% khoi
luong ban diu). Sau 3 ngay thi nghigm, khoi Itrong
con lai ctia gel fibrin tao ra bang cach them 0,013 M
CaC12 la nhieu nhat (68%), trong khi do gel tao ra to
nong da CaCl2 0,014 M co khoi lutmg con lai sau 3
ngity thap Mit (49%).
Theo (Woolverton, 2001, Janmey et al., 2009),

gel fibrin tao ra v6i thin gian cang cham thi mat da
16 cang day va kich thuerc 11) cang nh6, gel tucrng dOi
chic chin hon so v6i gel fibrin tao thanh voi th6i
gian nhanh hon. Do d6, gel v6i, nong d6 CaCl2
0,0I4M c6 cau tnic it chac chan nhat, nen
bi phan
htiy nhit.

a

Bang 2. Th61 gian tao gel fibrin theo n6ng dO CaCI,
Nang dO CaCl2 (M)

0,012

0,013

0,014

Th&I gian (phtit)

20 ± 0,471

15 ± 0,428

13 ± 0,340

Hinh 1. Cau trtic dm gel fibrin khi chop bang kinh hien vi dian tit (pet (SEM).

Gel 0.012

.••••• Gel 0,013
Gel 0.014
0

1

2

3

4

thiri gran (ngay)
Hinh 2. Phan tram khoi krong gel fibrin phan huji trong huyit twang
231


Doan Nguyen WI & Tran Le Bao HA
Ket qua danh gia kha nang thai khang Binh cua
gel fibrin in vitro
Trong ngay dau fib thai thu6c, gel fibrin too boi
nang de CaCl2 0,012M va 0,0I3M cho kat qua thai
thuric gan giang nhau (khoang 30%), trong khi luong
thuoc thai cfm gel too boi the tich CaC12 0,014 M rat
thap (khoang 14%) (Bang 3).
Tsourvakas (1995) cling cid chimg minh kha
nang thai cila gel fibrin &A veri nhieu loai khang sinh
khac nhau la khac nhau; tuy nhien, khoang 30% 60% khang sinh se duuc thai ra trong 48h dau tien va
lucmg con Iai se duric thai cham o cac ngay sau do.

Tu nhOng kat qua tren, c6 the lila), gel fibrin too
thanh vei ming do, CaCl2 0,0I3M co do, ben va kha
nang thai khang sinh tot nhat;Do d6, cluing Col quyet
dinh chon ming do nay di tien hinh cac thin nghiim
flap theo.
Tic di:Ing ciia gel fibrin cilia khang sinh len to
bac) vi khuin S. aureus

Kat qua kiem tra tac dens clia gel fibrin chin
khang sinh len ti bao vi khufin S. aureus cho thay
sau 24 h, chi s6 OD dm mei trueng vi khufin cao
hcm rat nhieu so voi 2 nh6m con lai (Bang 4). Ben
canh do, chi se OD cua mai trutmg vi khuan co gel
chtra khang sinh va moi truong vi khuan chi co
khang sinh tying dung nhau (khang khac nhau vi
mat thong ki). Chfrng to gel fibrin china khang Binh
vi khuan S
co tac dung frc chi so phat trim
aureus nuei cay trong mai trutrng giau dinh du6ng.
D6c tinh ciia gel fibrin chum khing sinh dei veri
nguyen bao soi ngittri
Ket qua 6 hinh 3 cho thiy mit do, hinh thai cita
to bao phat trien xung quanh va ngay ca ben dual
gel deu binh thizong, khong quan sat thay to bao
chat hay bi bong troc. Ngtrqc lai, vat lieu cao su
thien nhien lam cho toan,bt) to bao trong &fa nuoi cay.
chat va bong tric kh6i be mit. TO kit qua tren c6 the
ket luin gel fibrin chda kiting sinh khong gay dee
tinh cap tinh tren nguyen bao soi ngutri.

lulu

Bing 3. Phan tram khoi luang vancomycin gel fibrin thai ra trong ngay day tien.
Nang d6 CaCl2 (M)

Phan tram kh6i krcrng vancomycin thai (%)

0,012 ,

34,022 t 0,026

0,013

30,264 t 0,019

0,014

14,195 t 0,024

Bang 4. Chi s6 OD clia cac nhorn thi nghiem sau 24 glee.

Chi at:, OD

Mei trtrong vi khuin

Moi trutmg vi khuan ce gel china
vancomycin

Moi truirng vi khuin co
vancomycin

0,204 t 0,02

0,063 t 0,03

0,07 t 0,02

b. Gel fibrin china khang s•
Hinh 3. Ket qui khio sat dec tinh hen nguyen baO scri ngutri a. Cao su thien nhien (latex)

232


Tap chi Gong nghe Sin!: hoc 12(2): 229-233, 2014

KET LUAN
Chang Vol da tao thanh cong gel fibrin co kha
rang phan phat khang sink. Ngoai ra, gel fibrin chira
khang sinh, co kha rang tic the six tang sinh
to
bao vi khufin S. aureus in vitro. Nghien dm nay ma
ra met huerng di mai, ,met phucmg phap dieu tri hieu
qua ham
yeti van de nhibn thing hat: phau.
Uri cam an: Nghien ctiu nay &roc tai tro. poi BO
Khoa hoc va Gong nghe trong khuon kW de tai ma
so DTDL. 2012-G34.
TAI LIEU THAM KHAO
Archer NK, Mazaitis JM, Costerton JW, Leid JG, Powers
ME, Shirtliff ME (2011) Staphylococcus aureus biofilms:

properties, regulation, and roles in human disease.
Virulence 2: 445-459.
Canonico S (2003) The use of human fibrin glue in the
surgical operations. Acta Biomed 74: 21-25.
Janmey PA, Winer JP, Weisel JW (2009) Fibrin gels and
their clinical and bioengineering applications. J R Sac
Interface 6: 1-10.

Mandavyapuram HB (2010) Antibiotic delivery system for
surgical site infection prevention in spinal implant surgery.
Disertation of Master of Science. Case Western Reserve
University.
Mosesson MW, Siebenlist KR, Meh DA (2001) The
structure and biological features of fibrinogen and fibrin.
Ann N Y Acad Sci 936: 11-30.
Pye AD, Lockhart DE, Dawson MP, Murray CA, Smith AJ
(2009) A review of dental implants and infection. J Hosp
Infect 72: 104-110.
Smith PW, Bennett G, Bradley S, Drinka P, Lautenbach E,
Marx J, Mody L, Nicolle L, Stevenson K (2008)
SHEA/APIC Guideline: infection prevention and control
in the long-term care facility. Am J Infect Control 36: 504507.
Tsourvakas S, Hatzigrigoris P, Tsibinos A,
Kanellakopoulou K, Giamarellou H, Dounis E (1995)
Pharmacokinetic study of fibrin clot-ciprofloxacin
complex: an in vitro and in vivo experimental
investigation. Arch Orthop Trauma Surg 114: 295-297.
Woolverton CJ, Fulton JA, Salstrom Si, Hayslip 1, Haller
NA, Wildroudt ML, MacPhee M (2001) Tetracycline
delivery from fibrin controls peritoneal infection without

measurable systemic antibiotic. J Antimicrob Chemother
48: 861-867.

FIBRIN GEL CONTAINING ANTIBIOTIC PREVENTS GROWTH OF BIOFILM
BACTERIA
Doan Nguyen VII', Tran Le Bao Ha
University of Science, Vietnam National University Ho Chi Minh City

SUMMARY
Infeztion diseases cause of death for patients at the same number of patients died by traffic accidents, AIDS
and breast cancer. When the bacterial cells have a chance to settle on the surface of graft materials, they will
change the phenotype and produce mucus to form a new form called biofilm to protect themselveg against the
immune system of the body and the effects of antibiotics. The fibrin gel has many useful characteristics as
biocompatibility, biodegradation in the human body environment, adhesion and spongy structure for development
of antibiotic delivery system to prevent the formation of biofilm. In this study, human plasma was collected form
human peripheral blood. After that we created a fibrin gel directly from human plasma by supplement ion Cat*,
then gel was evaluated gel formed by scanning electron microscope (SEM) and degradation in vitro. Fibrin gel
containing antibiotics was assessed through ability to deliver antibiotics in vitro, effect on biofilm bacteria, and
poison for human fibroblast cells in vitro. Of the investigated concentration of CaCl2 applied for the fibrin gel
formation, the gel created by higher concentration of CaCl2 was degraded in vitro lower than the gel generated by
lower concentration of CaCl2. Most important, fibrin gels containing antibiotic were not toxic for human fibroblast
cells in vitro and inhibited the growth of biofilm bacteria in vitro. In Viet Nam, this issue is still very new;
virtually no scientific studies published concerning. This research as the first step for the development of antibiotic
delivery for effective treatments for infection diseases.
Keywords: Fibrin gel, biofilm, infection, drug delivery, human plasma

Author for correspondence: Tel: +84-1648064416; E-mail: dnyua,hcmus.edu.vn

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