Biomedical Engineering Reference
In-Depth Information
I
NTRODUCTION
It is by this time established that nitric oxide (NO), produced by vascular endothelium,
possesses a great number of activities: is a major factor influencing a tone of vessels, takes
part in inflammatory process and, besides, possesses antipathogenic action concerning a wide
spectrum of infectious agents [3-5, 9, 18, 22]. NO is synthesized by endothelial cells of blood
vessels (ECs) constitutively (eNOS) and at activation (iNOS) by physiological stimulators
and vascular active constituents (6, 8, 10. Infringement of NO production by ECs lead to their
dysfunction that is one of the basic pathogenesis steps of the most widespread diseases of
cardiovascular system, including an atherosclerosis [11, 12, 14].
One of the first steps of atherogenesis is development of inflammatory process in a
vascular wall which can express as a result of the influence of a great number of pathogens,
including and herpesviruses [14]. Herpes simplex virus type 1 (HSV-1), the most widespread
in human population a virus of herpes group, has been found out in biopsies of various bodies
and atherosclerotic plaques [7, 12, 16]. Besides it is known that HSV-1 causes dysfunction in
ECs in experiments in vitro [9, 18]. In clinics practice interferon alpha (IFN - α) is widely
used at treatment of relapses of herpetic diseases [2]. The purpose of the study was in
investigation of NO production by human EC culture under influence of human IFN - α, at an
infection of EC culture with HSV-1 and joint influence on culture with IFN - α and HSV-1.
M
ATERIALS AND
M
ETHODS
Endothelial cells (ECs) were allocated from an umbilical cord, received from healthy
women after normal delivery according to standard protocols, described earlier [17]. Shortly:
umbilical veins were filled by a solution of dispase (0,15 %, MP Biomedicals) and incubated
within 30 minutes at temperature 37ºC. Then veins have been washed out by a physiological
solution, cells have been collected from perfusion solution by centrifugation at 800 g within 5
minutes, resuspended in 199 medium (Gibco) with addition of 10 % fetal calf serum,
endothelial growth factor, heparin (100 mcg/ml) and gentamycin (50 mcg/ml) and are
transferred to plastic dishes. After formation of monolayer cells were resuspended in tripsin-
EDTA (Gibco) and seeded with density 105 cells/sm
2
on 24 well plated . Only 4 days
monolayer of ECs was used.
ECs were infected with herpes simplex virus type I (HSV-1), strain R39, received from
WHO. Multipliciry of infection (MOI) was 0,1-0,0001 TCID
50
/cell. Recombinant interferon
IFN α2b was used ("Immunofarm", Russia). ECs were cultivated with IFN α in different
concentration within 24 hours, cells were washed and infected with HSV-1, in dynamics
samples of cultural medium were selected and samples before research were stored at-20
°
C.
Levels of infectious virus in samples were tested by titration in monolayer of continuous cell
line VERO seeded on 96 wells plates.
NO concentration was determined by the content of nitrite, which is the stable oxidation
product of NO. The content of nitrite in the culture medium reflects the amount of
synthesized NO. Quantity of nitrite was determined by the method of a standard Griess [10].
Griess reagent was prepared by mixing equal volumes of sulfanilamide (1.5% in 1N HCl) and
N-(1 - naphthyl) ethylenediamine dihydrochloride (NEDA) (0,15% in H
2
O). 1 ml sample
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