Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells
We examined what effects are exerted by expression of the bcl-2 gene and by treatment
 with nerve growth factor (NGF) on the intensity of apoptosis in cultured pheochromocytoma
 cells (PC12 cells). Half of these cells were transduced with the bcl-2 gene using lentiviral
 plas...
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| Date: | 2014 |
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Інститут фізіології ім. О.О. Богомольця НАН України
2014
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| Cite this: | Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells / G. Han, W. Wei, X. Zhang, Zh. Lai, Ch. Chen // Нейрофизиология. — 2014. — Т. 46, № 4. — С. 375-380. — Бібліогр.: 11 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860245371933949952 |
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| author | Han, G. Wei, W. Zhang, X. Lai, Zh. Chen, Ch. |
| author_facet | Han, G. Wei, W. Zhang, X. Lai, Zh. Chen, Ch. |
| citation_txt | Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells / G. Han, W. Wei, X. Zhang, Zh. Lai, Ch. Chen // Нейрофизиология. — 2014. — Т. 46, № 4. — С. 375-380. — Бібліогр.: 11 назв. — англ. |
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| description | We examined what effects are exerted by expression of the bcl-2 gene and by treatment
with nerve growth factor (NGF) on the intensity of apoptosis in cultured pheochromocytoma
cells (PC12 cells). Half of these cells were transduced with the bcl-2 gene using lentiviral
plasmids, and the respective two groups were denoted as bcl-2-PC12 and control (c) PC12.
Then the cells were incubated in a serum-free medium in six different modes. One group of
c-PC12 cells was incubated in this medium with no additional agents added, another group
was incubated with 1.0 mM H₂O₂
, and the third group was incubated with both 1 mM H₂O₂
and 20 ng/ml NGF (groups 1-3). Cells of the another triad were incubated under the same
conditions, respectively, but these were bcl-2-PC12 cells (groups 4-6). The apoptosis rate
in each group after 1-h-long incubation was measured using a flow cytometry method. A 
bicinchoninic acid (BCA) technique was used for estimation of expression of Bcl-2 protein
in the cultures. As was observed, the action of H₂O₂ significantly increased the apoptosis rate
in both c-PC12 and bcl-2-PC12 samplings, while simultaneous action of NGF considerably
attenuated such increases. At the same time, values of the apoptosis rate for bcl-2-PC12 cells
were much smaller than the respective values for c-PC12 cells under all the three modes of
incubation. In H₂O₂
-treated cultures, the amount of Bcl-2 protein dropped, while the treatment
with NGF counteracted such shifts. The content of this protein in the bcl-2-PC12 groups was
much higher than in the c-PC12 groups. Thus, transduction with the bcl-2 gene significantly
inhibits apoptosis in cultured PC12 cells, and a combined influence of expression of this gene
and treatment with NGF produces a synergistic effect.
Ми досліджували впливи експресії гена bcl-2 та дії нервового фактора росту (NGF) на інтенсивність апоптозу
культивованих клітин феохромоцитоми (PC12). У половину
таких клітин був трансдукований ген bcl-2; відповідні дві
групи зразків були позначені як bcl-2-PC12 та контрольні
(с-PC12). Потім шість груп клітин інкубували в безсироватковому середовищі в різних умовах. Перша група
клітин с-PC12 інкубувалася без дії будь-яких додаткових
агентів, друга група – з додаванням 1 мМ H₂O₂

, а третя – 
в присутності як 1 мМ H₂O₂

, так і 20 нг/мл NGF. Клітини
трьох інших груп (4–6) інкубували в тих самих умовах, але це були клітини bcl-2-PC12. Ступінь апоптозу в кожній
групі після одногодинної інкубації вимірювали з використанням методу флоуцитометрії. Методику з використанням
біцинхонінової кислоти застосовували для оцінки експресії
білка Bcl-2 в культурах. Як було виявлено, дія H₂O₂
істотно
збільшувала ступінь апоптозу в зразках як c-PC12, так і 
bcl-2-PC12, але одночасна дія NGF помітно зменшувала
таке зростання. В той же час інтенсивності апоптозу клітин
bcl-2-PC12 були значно меншими, ніж відповідні значення
у клітин c-PC12 при всіх трьох режимах інкубації. В культурах, підданих впливу H₂O₂

, кількість протеїну Bcl-2 була
зменшеною, тоді як вплив NGF протидіяв таким зрушенням.
Вміст згаданого протеїну в групах bcl-2-PC12 був значно
вищим, ніж у групах c-PC12. Отже, трансдукція гена bcl2 істотно гальмує апоптоз культивованих клітин PC12, а 
комбінований вплив експресії цього гена та аплікації NGF
забезпечує сінергічні ефекти.
|
| first_indexed | 2025-12-07T18:36:00Z |
| format | Article |
| fulltext |
Article
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4 375
UDC 612.014.3+576.385.4
G. HAN,1 W. WEI,1 X. ZHANG,2 ZH. LAI,1 and CH. CHEN1
EFFECTS OF TRANSDUCTION OF THE bcl-2 GENE AND OF NERVE GROWTH
FACTOR ON APOPTOSIS OF CULTURED PC12 CELLS
Received 22.08.2013
We examined what effects are exerted by expression of the bcl-2 gene and by treatment
with nerve growth factor (NGF) on the intensity of apoptosis in cultured pheochromocytoma
cells (PC12 cells). Half of these cells were transduced with the bcl-2 gene using lentiviral
plasmids, and the respective two groups were denoted as bcl-2-PC12 and control (c) PC12.
Then the cells were incubated in a serum-free medium in six different modes. One group of
c-PC12 cells was incubated in this medium with no additional agents added, another group
was incubated with 1.0 mM H2O2, and the third group was incubated with both 1 mM H2O2
and 20 ng/ml NGF (groups 1-3). Cells of the another triad were incubated under the same
conditions, respectively, but these were bcl-2-PC12 cells (groups 4-6). The apoptosis rate
in each group after 1-h-long incubation was measured using a flow cytometry method. A
bicinchoninic acid (BCA) technique was used for estimation of expression of Bcl-2 protein
in the cultures. As was observed, the action of H2O2 significantly increased the apoptosis rate
in both c-PC12 and bcl-2-PC12 samplings, while simultaneous action of NGF considerably
attenuated such increases. At the same time, values of the apoptosis rate for bcl-2-PC12 cells
were much smaller than the respective values for c-PC12 cells under all the three modes of
incubation. In H2O2-treated cultures, the amount of Bcl-2 protein dropped, while the treatment
with NGF counteracted such shifts. The content of this protein in the bcl-2-PC12 groups was
much higher than in the c-PC12 groups. Thus, transduction with the bcl-2 gene significantly
inhibits apoptosis in cultured PC12 cells, and a combined influence of expression of this gene
and treatment with NGF produces a synergistic effect.
KEYWORDS: pheochromocytoma PC12 cells, bcl-2 gene, transduction, lentiviral
plasmids, nerve growth factor (NGF), apoptosis.
1 Hangzhou Red Cross Hospital, Hangzhou, China.
2 Department of Cell Biology and Medical Genetics, National Education Base
for Basic Medical Sciences, Institute of Cell Biology, Zhejiang University
School of Medicine, Hangzhou, China.
Correspondence should be addressed to Ch. Chen
(e-mail: zhitree@hotmail.com).
INTRODUCTION
Spinal cord injury (SCI) in most cases causes dramatic
sensory, motor, and autonomic deficits. The patients
may get paraplegia or quadriplegia depending on the
region of spinal lesion [1]. Because there is limited
or no neurological recovery once SCI occurred, the
latter creates great socioeconomic problems related
to serious and stable disability of SCI patients and to
efforts and costs involved in the diagnostics, treatment,
and rehabilitation of this contingent [2].
Nerve growth factor (NGF), a prototypic member of
the neurotrophin family, was initially recognized as a
pro-survival and pro-differentiation factor for sensory
and sympathetic neurons [3]. It can play a positive
role in the recovery of nerve functions after injury
[4]. Among pathological changes in the injured spinal
cord, strong intensification of apoptosis occupies one
of the crucial positions. An anti-apoptotic protein from
B-cell CLL/lymphoma 2, Bcl-2, was shown to be a
powerful inhibitor of apoptotic and necrotic cell death
[5]. Chen et al [6] found that the proto-oncogene bcl-2
plays a critical role in neuronal morphogenesis in the
CNS of mammals.
Our work was to examing figure out of how
expression of the bcl-2 gene and treatment with NGF
affect apoptotic cell death in a model system, cultured
pheochromocytoma (PC12) cells.
METHODS
In our study, a rat pheochromocytoma cloned cell line
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4376
G. HAN, W. WEI, X. ZHANG
(PC12 cells) was used. Samples of these cells were
purchased from the Shanghai Institute of Cell Bank
(China). Cultured PC12 cells have been recognized
to be a suitable model for certain directions of
neuroscience research [7]. These cells are known to be
rather close to the nerve cells in a few properties; under
definite conditions, such cells can be differentiated
into neuron-like units.
Lentiviral plasmids carrying the bcl-2 gene and the
analogous plasmids that did not carry this gene were
purchased from the Shanghai Jima Biotech Company
(China).
PC12 Cell Culturing and Lentiviral Plasmid
Transduction. PC12 cells were conserved in a liquid
nitrogen tank. Cells were recovered and incubated at
37 °C in an atmosphere of air enriched with СO2 to
5%. A Dulbecco’s modified Eagle’s medium (DMEM;
Gibco BRL, USA) supplemented with 10% heat-
inactivated fetal bovine serum (FBS), 5% heat-
inactivated horse serum (HS, both from Hyclone,
USA), and penicillin-streptomycin was used. Cells
were reseeded for two or three generations to be fully
restored.
The PC12 cells were seeded in the above DMEM
complete medium for transduction with bcl-2 lentiviral
plasmids and lentiviral plasmids with no bcl-2 at
a multiplicity of infection (MOI) of 100. The cells
successfully transduced with bcl-2 lentiviral plasmids
are denoted below as bcl-2-PC12, while those
transduced with no bcl-2 lentiviral plasmids are denoted
as control, c-PC12 (Figs. 1 and 2). Then, the cells were
cultured on collagen-coated 6-well plates (105 cells
per well) and incubated for 1 h at 37°C in a serum-free
medium (Invitrogen, USA) in six different ways denoted
as groups 1-6. Group 1 served as an intact control;
these were c-PC12 cells (with no induced expression
of the bcl-2 gene) that were not subjected to the action
of any additional factor. In group 2, c-PC12 cells
were incubated in a medium with 1 mM H2O2 added.
Hydrogen peroxide (H2O2) is a key signaling molecule
that intensifies apoptosis by inducing oxidative stress
via free oxygen radicals. In group 3, c-PC12 cells were
subjected to a combined action of 1 mM H2O2 and NGF
(final concentration 20 ng/ml). Another triad, groups
4-6, was incubated under conditions similar to those
for groups 1-3, respectively, but these were transfected
PC12 cells (group 4, bcl-2-PC12; group 5, bcl-2-PC12+
+ H2O2, and group 6, bcl-2-PC12+H2O2+NGF).
Apoptosis Assessment by Flow Cytometry. The
cells were trypsinized, centrifuged at 1200 min–1 for
5 min, and collected. Apoptosis was evaluated using
the Alexa Fluor® 488 annexin V/Dead Cell Apoptosis
Kit (Invitrogen; USA).The cells were resuspended in
100 μl of annexin-binding buffer after washed twice with
cold (4°C) phosphate-buffered saline (PBS). According
to the apoptosis detection kit reference, 5 μl of annexin
V-fluorescein isothiocyanate (FITC) and 1 μl of a
propidium iodide (PI) solution (100 μg/ml) were added to
each sample. After incubated for 15 min at 20°C, 400 μl
20 μm
20 μm
F i g. 1. PC12 cells successfully transduced by bcl-2-carrying lenti viral
plasmids and observed under an inverted fluorescence microscope.
Р и с. 1. Клітини PC12 після успішної трансдукції з викорис-
танням лентівірусних плазмід, що несуть ген bcl-2.
F i g. 2. PC12 cells before transfection.
Р и с. 2. Клітини PC12 перед трансфекцією.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4 377
EFFECTS OF TRANSDUCTION OF THE bcl-2 GENE AND OF NERVE GROWTH FACTOR ON APOPTOSIS OF CULTURED PC12 CELLS
of annexin-binding buffer was added to each suspension,
which was analyzed by flow cytometry (BD Biosciences,
USA). Annexin V-FITC-negative and PI-negative cells
were considered living cells. Cells positive with respect
to annexin V-FITC but negative for PI were classified as
being in the stage of early apoptosis. Cells positive for
both annexin V-FITC and PI were considered to be in the
stage of late apoptosis, while cells positive with respect
to PI were considered to be in necrosis. Normalized
numbers (%) of the cells belonging to the above groups
were calculated. The analysis was performed using
FACSDiva software (BD Biosciences, USA).
Assessment of Expression of Bcl-2 Protein.
All groups of the cells were cultured for 24 h. After
treatment, cells were harvested, washed once with
PBS, and lysed with phenylmethanesulfonyl fluoride
(PMSF) at 4°C for 30 min. The protein produced due
to bcl-2 gene expression was obtained after 10-min-
long centrifugation at 12,000 min–1 at 4°C. The amount
of this protein was determined using the bicinchoninic
acid (BCA) method.
Statistical Analysis. Statistical analysis was
performed using SPSS13.0 (SPSS, USA). The
apoptosis rates in the groups were compared with
the nonparametric Mann–Whitney U test. Protein
concentrations followed a normal distribution, so the
Student’s t-test was used for intergroup comparisons.
Numerical data are presented below as means ± s.e.m.,
unless stated otherwise. The differences with P < 0.05
were considered statistically significant.
RESULTS
Effects of Expression of the bcl-2 Gene and Treatment
with NGF on the Apoptosis rate of Cultured PC12
Cells. As was found, the rate of apoptosis in group
1 (c-PC12 cells incubated under above-described
conditions with no additional influences) was equal
to 15.63% on average; this value was taken as 100%
in comparison with other groups. Under the influence
of 1 mM H2O2, the level of apoptosis in group 2 was
significantly higher than that in group 1 (by 68.8%, on
average). The above index in group 3 (c-PC12 + H2O2 +
+ NGF) noticeably exceeded the respective value in
the intact control group 1 (by 28.2%), but, at the same
Q1
Q1
1
4
2
5
3
6
Q1 Q1
Q1 Q1
Q4
Q4 Q4 Q4
Q4 Q4Q3
Q3 Q3 Q3
Q3 Q3
Q2
Q2 Q2 Q2
Q2 Q2
1.34%
normal H2O2-treated H2O2 + NGF-treated
с-PC12 cells
bcl-12-PC12 cells
3.71% 1.42% 3.10%
1.12% 1.56%
90.4%
81.1% 71.7% 76.2%
86.1% 90.3%7.82%
14.8% 26.6% 20.2%
12.4% 7.61%
0.453%
0.379% 0.348% 0.458%
0.388% 0.520%
F i g. 3. Estimation of the apoptosis rate using flow cytometry. 1-6) Groups of PC12 cells incubated under different conditions.
Р и с. 3. Оцінка ступеню апоптозу з використанням флоуцитометрії.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4378
G. HAN, W. WEI, X. ZHANG
time, it was much lower than that in group 2 (c-PC12 +
+ H2O2). If we take the apoptosis rat in group 2 as
100%, a decrement of this index related to NGF action
in group 3 corresponded to 24.0%.
In groups 4-6, the apoptosis rates for PC12 cells
successfully transfected with bcl-2 gene were
significantly lower than those in the corresponding
groups 1-3 (PC12 cells with no transfected bcl-2 gene).
This index in group 4 was about two times smaller
than that in group 1 (by 48.1%, u = 2.16, P < 0.05). In
group 5 (bcl-2-PC12 cells subjected to isolated action
of 1 mM H2O2), the apoptosis rate was noticeably
greater than in group 4. At the same time, this index
was significantly (more than two times) smaller than
that in the corresponding group 2 (c-PC12+ H2O2,
u = 2.38, P < 0.05). The analyzed index in group
6 (bcl-2-PC12+ H2O2+NGF) was much lower than that
in group 5; this value was nearly equal to that in group
4 and 2.5 times smaller than the respective index in
group 3 (c-PC12 + H2O2 + NGF; u = 2.83, P < 0.05).
Effects of Transduction with the bcl-2 Gene
and Treatment with NGF on the Amount of Bcl-2
Protein in Cultured PC12 Cells. The mean amount
of the mentioned protein in group 1 (c-PC12 cells
with no additional influence) was 0.32 ± 0.02 µg/l.
The addition of 1 mM H2O2 to the cultured medium
resulted in a dramatic drop in the concentration of this
protein (by 43.7% as compared with group 1). At the
same time, when H2O2 and NGF influenced cultured
c-PC12 cells in group 3 simultaneously, the mean
amount of Bcl-2 protein was much greater than that in
group 2 and even exceeded the respective index in the
“intact” group 1 (by 37.5%, on average).
In cultured PC12 cells transfected with the bcl-2
gene (groups 4-6), the amounts of Bcl-2 protein
significantly exceeded the corresponding indices in
the respective groups of c-PC12 cell. In group 4, the
mean value was, on average, 175% of that in group
1. In group 5, the value was 256% of that in group
2 (t = 2.75, P < 0.05, both groups were treated with
H2O2). In group 6, the Bcl-2 content was 134% of the
value in group 3 (t = 2.23, P < 0.05; as was mentioned,
these groups were simultaneously influenced by H2O2
and NGF). Thus, transduction with the bcl-2 gene
significantly increases the amount of the respective
protein, and a combined influence of such transduction
and treatment with NGF produces a synergistic effect.
DISCUSSION
At present, major treatment of SCI includes
application of neuroprotective agents used to prevent
damage development within the acute phase. This
therapy includes the improvement of the oxygenation
ability, scavenging of free radicals, and correction of
biochemical disorders. Within the sub-acute and late
phases, neurotrophic factor (NTF) is used to prevent
nutritional deficiency in neurons. This agent can also
promote axonal growth. There are other methods to
intensify long-distance regeneration ability of the
axons, such as neuronal transplantation associated
with genetic-engineering cell import and applications
of antibody proteins associated with the myelination
process. Unfortunately, all these techniques are at
present characterized by insufficient efficacies.
I t has been reported that NGF, the f i rs t
representative of typical neurotrophic factors that was
described and studied, exerts certain positive effects
on the development of the nervous system, functional
0
0.2
0.4
0.6
0.8
0 1 2 3 4 5 6
5
10 8.11 7.85
12.27
15.63
26.38
20.04
15
20
25
30
% c-PC12
bcl-2-PC12
F i g. 4. Apoptosis rates (%) in the groups of PC12 cells (1-6)
incubated under different conditions.
Р и с. 4. Ступінь апоптозу (%) у групах клітин PC12, інкубованих
у різних умовах (1–6).
1 2 3 4 5 6
0.56 ± 0.02
0.46 ± 0.02
0.59 ± 0.02
0.32 ± 0.02
0.18 ± 0.02
0.44 ± 0.02
F i g. 5. Concentrations (µg/l) of Bcl-2 protein expressed in the
groups of PC12 cells (1-6) incubated under different conditions.
Р и с. 5. Концентрації білка Bcl-2 (мкг/л), експресованого в
групах клітин PC12, котрі інкубувалися в різних умовах.
µg/l
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4 379
EFFECTS OF TRANSDUCTION OF THE bcl-2 GENE AND OF NERVE GROWTH FACTOR ON APOPTOSIS OF CULTURED PC12 CELLS
maintenance, and reparation [1-3]. It was shown to
facilitate regeneration and reparation of impaired
neurons. Major mechanisms of the NGF function are
as follows: (i) inhibition of the release of toxic amino
acids; (ii) inhibition of calcium overloading; (iii)
inhibition of the release of superoxide radicals, and
(iv) inhibition of the apoptosis process.
Gene therapy is one of the rapidly developing
approaches in the field of biomedical research.
Expression of the proto-oncogene bcl-2 was shown
to be a powerful anti-apoptotic factor. Chen et al.
[6] found that Bcl-2 protein, the product of the bcl-2
gene can promote regeneration in the severed
mammalian CNS, in particular regeneration of axons
after SCI. Such findings provide a theoretical basis
for introduction of gene therapy in pathophysiological
situations needing neural regeneration [6]. Takahashi
et al. [8] found that injection of DNA plasmids
carrying the bcl-2 gene can reduce neuronal apoptosis
after SCI [8]. There are also other observations that
the bcl-2 gene can exert a significant protective effect
with respect to neuronal apoptosis caused by injury
under both in vivo and in vitro conditions [9-11].
We tested the effects provided by transduction with
the bcl-2 gene and by application of NGF in an in
vitro model system, cultured rat pheochromocytoma
PC12 cells. These cells are not neural units in a strict
meaning of the term, but this cell line possesses
certain properties rather similar to those of neurons
(see above). These cells can be cultured for rather
long time intervals, the technology of culturing is
standardized, and the influences exerted by different
factors on the viability of such cells can be estimated
much more easily and accurately than in the in vivo
experiments.
Our measurements showed that transfection with
the bcl-2 gene provides significant intensification of
the synthesis of protein Bcl-2 in cultured PC12 cells.
Under conditions of our experiments, the amounts
of this protein in groups 4-6 exceeded the control
index in group 1 of c-PC12 cells (sham-transfected)
by 44-84% (Fig. 5). As is known, Bcl-2 is a powerful
inhibitory factor limiting apoptotic and necrotic cell
death [5]. So, increased levels of Bcl-2 in cultured
groups 4-6 clearly correlate with significantly lower,
than in the corresponding groups 1-3 (c-PC12 cells),
rates of apoptosis.
On the other side, NGF also demonstrated in our
experiments clear anto-apoptotic properties in our
experiments. In c-PC12 cells of group 2, the addition
of 1 mM H2O2 to the incubation medium increased
the apoptosis rate by nearly 70%. At the same time,
when NGF was added to the above medium (group 3),
the respective increment shortened to less than 30%.
Even more impressive effects of the NGF addition to
the medium were found in bcl-2-PC12 samplings. The
action of 1 mM H2O2 increased the apoptosis rate in
group 5 by nearly 80%. In group 6, NGF practically
completely eliminated the negative effect of H2O2.
The mean apoptosis rate in this group was found to be
even somewhat lower than in the intact control group
1 (Fig. 4). Thus, we conclude that a combined action
of Bcl-2 protein and NGF provide a clear synergistic
effect.
Of course, at present it is too early to believe that
a combination of gene therapy and action of the
agents intensifying recovery and growth processes
in nerve elements is ready to be directly applied in
clinics. Nonetheless, our findings together with the
results of other researchers allow us to think that
such combinations look as promising approaches in
the treatment of the consequences of CNS traumas
and of SCI in particular. This, of course, implies the
need of detailed studies directed toward adequate
interpretation of the mechanisms of such combined
effects and development of the most expedient modes
of the action of the respective agents.
Г. Хан1, У. Уей,1 Кс. Жанг,2 Ж. Лай,1 Ч. Чен1
ВПЛИВИ ТРАНСДУКЦІЇ ГЕНА bcl-2 ТА ДІЇ НЕРВОВО-
ГО ФАКТОРА РОСТУ НА АПОПТОЗ КУЛЬТИВОВАНИХ
КЛІТИН PC12
1 Лікарня Червоного Хеста, Ханчжоу (Китай).
2 Національна освітня база фундаментальних медичних
наук Інституту клітинної біології Чжеджанського Медич-
ного Університету, Ханчжоу (Китай).
Р е з ю м е
Ми досліджували впливи експресії гена bcl-2 та дії нер-
вового фактора росту (NGF) на інтенсивність апоптозу
культивованих клітин феохромоцитоми (PC12). У половину
таких клітин був трансдукований ген bcl-2; відповідні дві
групи зразків були позначені як bcl-2-PC12 та контрольні
(с-PC12). Потім шість груп клітин інкубували в безси-
роватковому середовищі в різних умовах. Перша група
клітин с-PC12 інкубувалася без дії будь-яких додаткових
агентів, друга група – з додаванням 1 мМ H2O2, а третя –
в присутності як 1 мМ H2O2, так і 20 нг/мл NGF. Клітини
трьох інших груп (4–6) інкубували в тих самих умовах,
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4380
G. HAN, W. WEI, X. ZHANG
але це були клітини bcl-2-PC12. Ступінь апоптозу в кожній
групі після одногодинної інкубації вимірювали з викори-
станням методу флоуцитометрії. Методику з використанням
біцинхонінової кислоти застосовували для оцінки експресії
білка Bcl-2 в культурах. Як було виявлено, дія H2O2 істотно
збільшувала ступінь апоптозу в зразках як c-PC12, так і
bcl-2-PC12, але одночасна дія NGF помітно зменшувала
таке зростання. В той же час інтенсивності апоптозу клітин
bcl-2-PC12 були значно меншими, ніж відповідні значення
у клітин c-PC12 при всіх трьох режимах інкубації. В куль-
турах, підданих впливу H2O2, кількість протеїну Bcl-2 була
зменшеною, тоді як вплив NGF протидіяв таким зрушенням.
Вміст згаданого протеїну в групах bcl-2-PC12 був значно
вищим, ніж у групах c-PC12. Отже, трансдукція гена bcl-
2 істотно гальмує апоптоз культивованих клітин PC12, а
комбінований вплив експресії цього гена та аплікації NGF
забезпечує сінергічні ефекти.
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|
| id | nasplib_isofts_kiev_ua-123456789-148307 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0028-2561 |
| language | English |
| last_indexed | 2025-12-07T18:36:00Z |
| publishDate | 2014 |
| publisher | Інститут фізіології ім. О.О. Богомольця НАН України |
| record_format | dspace |
| spelling | Han, G. Wei, W. Zhang, X. Lai, Zh. Chen, Ch. 2019-02-17T20:12:45Z 2019-02-17T20:12:45Z 2014 Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells / G. Han, W. Wei, X. Zhang, Zh. Lai, Ch. Chen // Нейрофизиология. — 2014. — Т. 46, № 4. — С. 375-380. — Бібліогр.: 11 назв. — англ. 0028-2561 https://nasplib.isofts.kiev.ua/handle/123456789/148307 612.014.3+576.385.4 We examined what effects are exerted by expression of the bcl-2 gene and by treatment
 with nerve growth factor (NGF) on the intensity of apoptosis in cultured pheochromocytoma
 cells (PC12 cells). Half of these cells were transduced with the bcl-2 gene using lentiviral
 plasmids, and the respective two groups were denoted as bcl-2-PC12 and control (c) PC12.
 Then the cells were incubated in a serum-free medium in six different modes. One group of
 c-PC12 cells was incubated in this medium with no additional agents added, another group
 was incubated with 1.0 mM H₂O₂
 , and the third group was incubated with both 1 mM H₂O₂
 and 20 ng/ml NGF (groups 1-3). Cells of the another triad were incubated under the same
 conditions, respectively, but these were bcl-2-PC12 cells (groups 4-6). The apoptosis rate
 in each group after 1-h-long incubation was measured using a flow cytometry method. A 
 bicinchoninic acid (BCA) technique was used for estimation of expression of Bcl-2 protein
 in the cultures. As was observed, the action of H₂O₂ significantly increased the apoptosis rate
 in both c-PC12 and bcl-2-PC12 samplings, while simultaneous action of NGF considerably
 attenuated such increases. At the same time, values of the apoptosis rate for bcl-2-PC12 cells
 were much smaller than the respective values for c-PC12 cells under all the three modes of
 incubation. In H₂O₂
 -treated cultures, the amount of Bcl-2 protein dropped, while the treatment
 with NGF counteracted such shifts. The content of this protein in the bcl-2-PC12 groups was
 much higher than in the c-PC12 groups. Thus, transduction with the bcl-2 gene significantly
 inhibits apoptosis in cultured PC12 cells, and a combined influence of expression of this gene
 and treatment with NGF produces a synergistic effect. Ми досліджували впливи експресії гена bcl-2 та дії нервового фактора росту (NGF) на інтенсивність апоптозу
 культивованих клітин феохромоцитоми (PC12). У половину
 таких клітин був трансдукований ген bcl-2; відповідні дві
 групи зразків були позначені як bcl-2-PC12 та контрольні
 (с-PC12). Потім шість груп клітин інкубували в безсироватковому середовищі в різних умовах. Перша група
 клітин с-PC12 інкубувалася без дії будь-яких додаткових
 агентів, друга група – з додаванням 1 мМ H₂O₂
 
 , а третя – 
 в присутності як 1 мМ H₂O₂
 
 , так і 20 нг/мл NGF. Клітини
 трьох інших груп (4–6) інкубували в тих самих умовах, але це були клітини bcl-2-PC12. Ступінь апоптозу в кожній
 групі після одногодинної інкубації вимірювали з використанням методу флоуцитометрії. Методику з використанням
 біцинхонінової кислоти застосовували для оцінки експресії
 білка Bcl-2 в культурах. Як було виявлено, дія H₂O₂
 істотно
 збільшувала ступінь апоптозу в зразках як c-PC12, так і 
 bcl-2-PC12, але одночасна дія NGF помітно зменшувала
 таке зростання. В той же час інтенсивності апоптозу клітин
 bcl-2-PC12 були значно меншими, ніж відповідні значення
 у клітин c-PC12 при всіх трьох режимах інкубації. В культурах, підданих впливу H₂O₂
 
 , кількість протеїну Bcl-2 була
 зменшеною, тоді як вплив NGF протидіяв таким зрушенням.
 Вміст згаданого протеїну в групах bcl-2-PC12 був значно
 вищим, ніж у групах c-PC12. Отже, трансдукція гена bcl2 істотно гальмує апоптоз культивованих клітин PC12, а 
 комбінований вплив експресії цього гена та аплікації NGF
 забезпечує сінергічні ефекти. en Інститут фізіології ім. О.О. Богомольця НАН України Нейрофизиология Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells Впливи трансдукції гена bcl-2 та дії нервового фактора росту на апоптоз культивованих клітин pc12 Article published earlier |
| spellingShingle | Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells Han, G. Wei, W. Zhang, X. Lai, Zh. Chen, Ch. |
| title | Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells |
| title_alt | Впливи трансдукції гена bcl-2 та дії нервового фактора росту на апоптоз культивованих клітин pc12 |
| title_full | Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells |
| title_fullStr | Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells |
| title_full_unstemmed | Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells |
| title_short | Effects of Transduction of the bcl-2 Gene and of Nerve Growth Factor on Apoptosis of Cultured PC12 Cells |
| title_sort | effects of transduction of the bcl-2 gene and of nerve growth factor on apoptosis of cultured pc12 cells |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/148307 |
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