


Vol 7, No 3 (2012)
- Year: 2012
- Articles: 42
- URL: https://genescells.ru/2313-1829/issue/view/6169
Articles
Privetstvennoe slovo professora A.P. Kiyasova
Genes & Cells. 2012;7(3):10-11



Gene and cell therapy of retinal diseases
Abstract
Gene and cell therapy are tightly associated and quickly
developing areas of biomedicine, which purpose is development
of methods to cure diseases caused by genetic defects and/or
death of certain cell types. Current state of methods of retinal
diseases gene and cell therapy is analyzed in this review. We
reviewed development of gene therapeutic approaches to
treatment of the 2-nd form of Lebers congenital amaurosis
and other retinal diseases. We also compared therapeutic
potential of pluripotent stem cells and human adult retinal
stem cells. Therapeutic potential of pluripotent stem cells
seems to be better due to their ability for unlimited expansion
and organogenesis.
Genes & Cells. 2012;7(3):12-20



Can pancreas be the source of hepatocytes?
Abstract
The possibility of hepatocytes differentiation from
pancreatic cells is discussed in the review. Both liver
and pancreas develop from endoderm so there could be a
common stem cell giving rise to both pancreatic and liver
cells. Different experimental models are used to study
the possibility of hepatocytes development in pancreas
such as influence of peroxisomes proliferation stimulators,
hyperexpression of keratinocyte growth factor in pancreatic
islets, Cu depletion-repletion model, etc.). There is no
enough data to confirm which particular pancreatic cell type
can be the source of hepatocytes.
Literature data allow supposing that hepatocytes can
arise in pancreas from three different sources: acinar,
endocrine or ductular cells.
Genes & Cells. 2012;7(3):21-24



Formation of the recombinant adenovirus encoding codon-optimized dysferlin gene and analysisof the recombinant protein expression in cell culture in vitro
Abstract
Dysferlinopathies belong to neuromuscular diseases
associated with aberrant expression and/or function of
dysferlin protein in skeletal muscle, which is caused by
mutations in the dysf (dystrophy-associated fer-1-like, DYSF)
gene. Because of the large size of the codon-optimized dysf
coding region (6243 bp), adenoviral vectors are suitable for
the creation of genetic constructs, which are capable of
delivering a large amount of recombinant genetic information
into both dividing and non-dividing cells, as well as provide a
high level of transgene expression.
We generated a recombinant adenovirus serotype
5 encoding a codon-optimized gene for human dysferlin (Ad5-
Dysf) and analysed recombinant protein expression in vitro in
HEK-293T cell line.
Genes & Cells. 2012;7(3):25-28



Gene transfer to mice organs using non-viral systems for targeted delivery with differenthydrophobicity and with lactose addressing group
Abstract
Biodistribution of lipoplexes formed of cholenim substances
I-III, containing one, two or three cholesterol moieties, and
eukaryotic 14С-DNA and(or) reporter gene into mice organs
using a variety of administration routes (intraperitoneally,
i.p.; portal vein or left renal artery) is studied in this paper. It
is shown that biodistribution doesnt depend on lipoplex lipid
composition under i.p. administration, and depends on lipid
nature under vein and artery administration. Effective in vivo
transfection and reporter gene expression are demonstrated
under portal vein administration of lipoplex formed of
dicholenim II and lactosylated lipid IV (1 to 1 mass ratio). In the
case, the β-Gal gene expression (above 0,3 mcg / g of tissue)
is demonstrated in lungs, liver and spleen histochemically
and spectrophotometrically. Introduction of cholesterol
moieties into oligoethylenimine structure results in optimal
hydrophilicity/hydrophobicity ratio, their stabilization, and
optimal value of critical constant of micelle formation. There
are certain outlooks due to usage of the lipoplexes described
for targeted gene delivery.
Genes & Cells. 2012;7(3):29-33



Changes of the inflammatory activity and fibrosis in patients with alcoholic liver cirrhosis after autologoushematopoietic stem cell transplantation
Abstract
Evaluation of treatment results of chronic liver diseases
should be made on the basis of morphological analysis of
liver biopsies. The aim of our study was to investigate the
effect of autologous hematopoietic stem cell transplantation
on histology activity index and grade of fibrosis in alcoholic
liver cirrhosis patients. The study was performed on liver
biopsies of 11 patients with alcoholic liver cirrhosis. Biopsies
were taken before the injection of autologous peripheral
blood stem cells into celiac trunk, 3 and 12 months after
the procedure. Liver biopsy specimens were stained with
hematoxylin-eosin and Van Gieson's. Results showed
improvement of liver structure and decrease in histology
activity index in liver biopsies performed 3 and 12 months
after transplantation. Our data suggest that autologous
transplantation of hematopoietic stem cell in patients with
alcoholic liver cirrhosis is effective method that is capable
to reduce inflammation activity in the liver, improve its
structure and decrease liver fibrogenesis.
Genes & Cells. 2012;7(3):34-36



Liver pathomorphology of Mus musculus C57BL6 on atherogenic diet
Abstract
Atherosclerosis is one of the leading causes of disability
and death worldwide. Liver plays a huge role in pathogenesis
of atherogenic dislipidemia, development and progression of
atherosclerotic lesions. We studied the effect of atherogenic
diet on liver morphology in animal model of diet-induced
atherosclerosis in mice Mus musculus C57BI6. This strain
has a natural ability to develop atherosclerosis, while some
other mouse stains has not. After 14 weeks on atherogenic
diet a severe hepathomegaly (9% of body mass) and lobular
structure deformation was found. We also observed signs of
micro- and macrovesicular steatosis, cell apoptosis, fibrosis
and inflammatory leukocyte infiltration. So, liver not only plays
an important role in dislipidemia, but it is also a target-organ
in lipid metabolism imbalance.
Genes & Cells. 2012;7(3):37-40



Direct observation of «cholesterol - model of biological membrane» complex by NMR spectroscopy
Abstract
Interaction and aggregation of cholesterol and sodium
dodecyl sulfate molecules were studied in this paper.
Sodium dodecyl sulfate was taken as a model for biological
membranes. Cholesterol-sodium dodecyl sulfate complex was
described by modern methods of nuclear magnetic resonance
spectroscopy.
Nuclear magnetic resonance spectra were recorded on
«Avance-500» spectrometer (Bruker).
To assign 1Н signals of cholesterol, sodium dodecyl
sulfate and cholesterol+sodium dodecyl sulfate mixture
in nuclear magnetic resonance spectra literature data
was used, and 2D homo- end hetero-correlation nuclear
magnetic resonance spectra were recorded. To study the
formation of sodium dodecyl sulfate micelles and complex
of cholesterol-sodium dodecyl sulfate micelles selective
nuclear Overhauser effect spectroscopy experiments were
carried out.
The formation of sodium dodecyl sulfate micelles in dimethyl
sulfoxide solution was confirmed by nuclear Overhauser effect
spectroscopy data. The presence of a complex between sodium
dodecyl sulfate micelles and cholesterol molecules has been
proven by selective nuclear Overhauser effect spectroscopy
experiments. Nuclear Overhauser effect between OHgroup
of cholesterol and «tail» groups of sodium dodecyl
sulfate hydrophobic part was observed in the experiment.
This observation corresponds to close spatial arrangement
of these parts of different molecules and the presence of
a complex between cholesterol and sodium dodecyl sulfate
micelles.
On the basis of the nuclear magnetic resonance
experiments was established that molecules of sodium
dodecyl sulfate form micelles in dimethyl sulfoxide solution
at concentrations above the critical micelle concentration.
Cholesterol molecules form an intermolecular complex with
sodium dodecyl sulfate micelles by interaction of the OH group
of cholesterol and СН3-1 and СН2-2 «tail» aliphatic groups
of sodium dodecyl sulfate. This interaction is similar to the
behavior of cholesterol in phospholipid bilayer membranes in
which cholesterol enters its cyclic part in the hydrophobic
tails of phospholipid molecules oriented primarily across the
bilayers.
Genes & Cells. 2012;7(3):41-48



Tromboliticheskaya i fibrinoliticheskaya aktivnost'bakterial'nykh proteaz
Abstract
In medical practice is increasingly use proteolytic enzymes
of microorganisms. Particular attention of researchers is
attracted proteases, which have fibrinolytic properties,
and can lyse clots. Previously had been isolated and purified
to homogeneity glutamyl endopeptidase, subtilisin-like
proteinase and metalloendopeptidase of Bacillus pumilis
3-19, secreted by B. subtilis JB 2036 recombinant strain.
The analysis of thrombolytic, fibrinolytic, and anticoagulant
properties of the recombinant enzymes was conducted.
It is shown that all investigated proteinases are able
to efficiently lyse the clot. In an in vitro subtilisin and
glutamyl endopeptidase of B.subtilis recombinant strain
have anticoagulant activity. Metalloproteinase is not
able to influence the process clot formation. Subtilisinlike
proteinase and glutamyl endopeptidase possess
fibrinolytic activity and the ability of the activator relative
to plasminogen. Metalloproteinase shows no fibrinolytic
properties. Due to the high incidence of cardiovascular
diseases is urgent search for new enzymes with high
biological activity, specificity and low toxicity.
Genes & Cells. 2012;7(3):49-51



Polymer-stabilised magnetic nanoparticles do not affect the viability of magnetically-functionalised cells
Abstract
Here we report the synthesis of (poly)allylamine-coated
superparamagnetic iron oxide nanoparticles for the surface
modification of living cells. Magnetic functionalisation of cow
embryonic lung cells did not affect the viability of the coated
cells, confluent monolayer formation and proliferation, as
demonstrated using fluorescence and white light microscopy,
flow cytometry and MTT assay. Functionalised cells were
magnetically responsive. We believe that the single-step
approach described here is a novel and potentially promising
way to functionalise mammal cells with magnetic nanoparticles
for the subsequent applications in cell therapy, directed cells
delivery and spatial positioning in tissue engineering.
Genes & Cells. 2012;7(3):52-56



Gene transfer using new complexes between cardiolipin-like dicationic lipids and plasmid DNA to tumor cells
Abstract
The lipid vesicles of bisamphiphiles cardiolipin-like dicationic
lipids (CDL) I-IV were studied for creation of lipoplexes with
plasmid DNA of different sizes to obtain stable lipoplexes for
gene transfer to gene therapy. Lipoplexes sizes (300±100 nm)
and stablity (> 2 hrs) of CDL were sufficient to be used in
gene transfer against monolayer and suspension cell cultures.
The CDL total cytotoxicity determined by MTT-test was lower
compare to lipofectin as a control. Transfection conditions
against tumor cells lines were optimized by lipoplexes of CDL
and plasmid DNA. The most efficient transfection for lipoplexes
CDL-plasmid DNA was at the lipid-DNA (L/D) ratio equal to 5
(for lipofectin, it was 2). For monolayer cell cultures, lipoplexes
CDL-I are comparable in terms of transfection efficacy with
lipofectin; in the case of suspension culture, their efficiency
was lower by one order of magnitude. It permits a usage
of lipoplexes suggested as mediators for gene transfer and
delivery to human tumor cells.
Genes & Cells. 2012;7(3):57-61



The cytofluorimetric characteristics of RNAse influence towards pro- and eucariotic cells
Abstract
Cytotoxic ribonucleases (RNAses) are known to be
perspective drugs for cancer therapy. The extension of model
objects range will give possibility to assess the cytotoxic
RNAses selectivity.
We estimated the effect of Bacillus intermedius
ribonuclease (binase) and bovine RNAse A to E. coli K 12 and
lung epithelia of cow embryo (LEC) cells. LEC cells apoptosis
was characterized with a double staining with annexin-FITC and
propidium iodide (PI). E. coli K12 cells vitality was estimate
via PI staning.
Binase and RNAse A were not cytotoxic to LEC and
E. coli K12 cells in investigated concentrations (100 and
300 g/ml).
Low RNAses toxicity for E. coli allows to suppose the
binase and RNAse A in concentrations, capable to display
antitumor activity, will not to effect the tissues microbial
flora during in vivo tests. The lack of apoptosis inducing
activity of binase for LEC cells confirms this RNAse selectivity
for tumor cells.
Genes & Cells. 2012;7(3):62-65



Influence of serum antibodies to DNA on theMadin-Darby сanine kidney epithelial cells in vitroin depend of immunoreactive regions of the IgG molecule
Abstract
The data obtained on cell lines MDCK showed that for
implementation of the biological function of antibodies to DNA
both normal and pathological need the participation as Fab- and
Fc-fragments of molecules of IgG. However, antigen-binding
region is responsible for the manifestation of the biological
function of SLE antibodies to DNA more immunoreactive
than antibodies in normal and constant region is probably
responsible for the active conformation of antibody for pathobiological
function of antibodies to DNA in the cell.
Genes & Cells. 2012;7(3):66-68



Participation of the mononuclear cells of the cord blood in the physiologic regeneration of the rat kidney
Abstract
Nowadays ability to use hematopoietic stem cells for
treatment of various diseases, including kidney pathology,
is widely investigated. Umbilical cord blood as the source of
hematopoietic stem cells becomes more and more promising.
The purpose of our investigation was to study homing and
ways of human umbilical cord blood mononuclear cells
differentiation in an intact rat kidney. We transplanted human
umbilical cord blood mononuclear cells fraction, enrich with
hematopoietic stem cells, into the tail vein of rats. On 2, 5,
7 and 14 days after transplantation paraffin kidney slices
were immunohistochemically stained with antibodies against
human leukocyte antigen (HLA-ABC) to study migration and
differentiation of transplanted cells. Results: HLA-ABC+-
cells were revealed in the epithelium of the distal tubule at all
experimental dates. But HLA-ABC was expressed not in each
distal tubule and not by all tubular cells. We concluded that
human umbilical cord blood mononuclear cells transplanted into
systemic circulation of rat migrate into the intact kidney and
built in the distal tubule epithelium. This data allow to suggest
that distal tubule are stem cell «niche» in the kidney.
Genes & Cells. 2012;7(3):69-71



Antitumor RNase (binase) induces the alteration of cellular permeability
Abstract
Some RNases including ones of microbial origin possess
antitumor activity, which mechanisms remains unclear. Here
we investigated the first step of RNase action towards
eukaryotic cells which is connected with increase of cell
permeability for ions and macromolecules.
Using radiological analysis of 45Са2+uptake by Candida
yeast and fluorescence imaging of human embryo kidney
cells HEK stained by Ca2+-specific Fura-2/АМ day the level
of intracellular Ca2+ under treatment with the RNase of
Bacillus intermedius (binase) was studied. Viability of lung
carcinoma epithelial cells A549 treated by binase was
measured by WST proliferation kit, stability of erythrocytes
was tested by lysis assay.
We have shown that binase induces the permeability
increase of lower and higher eukaryotic cells for Ca2+as
well as the increase of protein permeability of A549 cells.
Binase treatment protects erythrocytes from osmotic
shock.
The protective or cytotoxic binase effect followed by
increase of cellular permeability is realized depending on the
dell type, where the expression of КСa channels and of certain
oncogens, particularly of ras family, is crucial. The obtained
data supports the significance of the cell permeability increase
as a primary step in the mechanisms of binase-induced
biological effects.
Genes & Cells. 2012;7(3):72-76



С-kit-positive progenitor cells activation in rats pancreas after partial hepatectomy
Abstract
One of the most common markers of pancreas stem
cells is a stem cell factor receptor C-kit. According to some
authors, this marker is presented in islet cells of normal rat
pancreas. But it is unknown about the behavior of these cells in
disorders of carbohydrate metabolism during liver disease. The
aim of our study was to evaluate C-kit expression in pancreas
after partial hepatectomy in rats. Partial hepatectomy was
performed for 27 white male rats. The expression of C-kit,
insulin and glucagon in rats pancreas was studied. The
expression of C-kit in islets and interstitial cells was shown
in results after 3 days of the experiment, and double staining
showed that these cells can express glucagon. Thus, there
is the activation of C-kit+ progenitor cells in pancreas after
partial hepatectomy and the beginning of there differentiation
to -cells of Langerhance islets.
Genes & Cells. 2012;7(3):77-79



Isolation, culture and differentiation of rat (Rattus norvegicus) and hamster (Mesocricetus auratus)adipose derived multipotent mesenchymal stromal cells
Abstract
Cell therapy of various diseases is one of the most
perspective fields in modern medicine. Adipose derived
autological stem cells can be obtained for therapeutic
purposes. Animal model of human diseases are essential for
cell therapy research. However, the most frequently used
laboratory animals, such as rats and mice, cant suffer the
whole rate of common diseases of modern society. At the
same time Syrian hamsters can provide scientists with an
appropriate animal models of these diseases. Nevertheless,
we couldnt find any data on hamsters stem cells isolation
and their characteristics. In this study we first isolated Syrian
hamsters adipose derived stem cells, characterized their
morphology, features and differential potential in several
ways. These cells are much alike multipotent mesenchymal
stromal cells and can go through osteogenic and, adipogenic
differentiation. We have also shown that these cells can
differentiate in neurogenic way.
Genes & Cells. 2012;7(3):82-87



The action of bacterial serine proteases on cultured animal cell lines
Abstract
Our research is direct to determination of serine
proteases (subtilisin and glutamyl endopeptidase) action at
establish cell lines. We determined that the serine proteases
of bacillus are capable cytotoxic action at establish cell lines
of animals. The cell lines are differentiating by sensitivity to
proteins. The cell lines LEK and NGUK were more sensitive
to subtilisin, and line Vero - glutamyl endopeptidase.
Genes & Cells. 2012;7(3):88-91



Human umbilical cord blood mononuclear cells transfected with dual cassette plasmids(vegf + neurotrophic factor) for the treatment of amyotrophic lateral sclerosis
Abstract
To increase the viability of neural cells in neurodegenerative
diseases, after neurotraumas and ischemic strokes the
most important neurotrophic and neuroprotective factors,
which can be used as therapeutic agents were identified
in long-term studies in vitro and in vivo. These include
brain-derived neurotrophic factor (BDNF), glial-derived
neurotrophic factor (GDNF), insulin-like growth factor (IGF)
and vascular endothelial growth factor (VEGF). One of the
promising ways of the delivery of supporting neuron survival
factors is considered to be transplantation of genetically
modified cells overexpressing recombinant therapeutic
genes. This article describes generation of cellular delivery
vectors of therapeutic genes - human umbilical cord blood
mononuclear cells genetically modified by dual cassette
plasmids, expressing two therapeutic genes. Efficiency of
transgene expression was confirmed in vitro using RT-PCR.
Analysis of survival, migration, and phenotype of genetically
modified cells was performed 2 weeks after transplantation
into transgenic mice with amyotrophic lateral sclerosis
phenotype.
Genes & Cells. 2012;7(3):92-97



Cellular and receptor mechanisms of impairment of myocardium and aorta contractilityat Alzheimers disease model
Abstract
Introduction. Recent studies certify the existence of link
between Alzheimers disease and cardiovascular pathology,
however the mechanisms of this phenomenon is unclear. Here
we studied the influence of Alzheimers β-amyloid peptide
(βAP) on the contractility of rat myocardium and aorta.
Material and methods. Contractility of myocardium ventricle
strips and transverse fragments of abdominal aorta was
measured at Power Lab setup using conventional myographic
technique. Contractile responses of aorta strips were evoked
by application of receptor agonists, contractile responses of
myocardium - by electrical stimulation. Contractile responses
of aorta strips after application of carbachol (10-6-10-4 М),
histamine (10-6-10-4 М), norepinephrine (10-5-10-3 М) and
ATP (10-6-10-4 М) were measured.
Results and discussion. We found the impairment of
carbachol- and histamine-induced contractility of aorta,
appearing as perverse contractile reactions (relaxation instead
of contraction) under the action of βAP (10-6 М). Next, we
found βAP-induced impairments of ventricle myocardium
contractility, appearing as decrease of relaxation phase
duration and increase of relaxation speed (positive lusitropic
effect). Also, own positive lusitropic effect of norepinephrine
was absent in presence of βAP (10-6М).
Thus, βAP(25-35) significantly impairs the contractility
of rat myocardium and aorta, as well as processes of its
regulation. Obtained data significantly broad our understanding
of mechanisms of Alzheimers disease pathogenesis and
pathophysiology of cardiovascular system.
Genes & Cells. 2012;7(3):98-100



Uluchshenie dostavki kompleksov plazmidnoy DNKs polikationom v kletki chelovekav prisutstvii bloksopolimerov etilen-i propilenoksida
Abstract
The work is aimed at the study of the effect of novel
block copolymers of ethylene oxide and propylene oxide
on the delivery of plasmid DNA and its complexes with
cationic polymers into human cells. Tri-functional amphiphilic
block copolymers on the basis of glycerol (LaprolsTM),
polyethyleneimine (25 kDa) and commercial transfection
reagent TurboFectTM were tested as delivery systems.
TurboFect was found to form more compact and positively
charged polyplexes with plasmid DNA (pEGFP-N2) and
provided higher expression of GFP in HEK 293 cells
compared to polyethyleneimine. Laprols weakly interacted
with plasmid DNA and did not improve its intracellular
delivery. However they markedly promoted cell transfection
by DNA-polyethyleneimine complexes. Results show that
Laprols exhibit mild cytotoxicity and produce the interest for
gene therapeutics delivery into cells.
Genes & Cells. 2012;7(3):101-104



Biosafety model of adenovirus infection: effects of bacterial proteases for infection of human cells in vitro
Abstract
To determine the antiviral activity of various biologically
active compounds, the model of adenovirus infection on the
basis of cell cultures of human HEK293A and recombinant
adenovirus Ad-EGFP, expressing green fluorescent protein
EGFP. Adenoviruses have a capsid size of 70-90 nm and are
able to infect dividing and nondividing cells in vitro and in vivo.
Recombinant adenoviruses are the replicative defect in the
cells of humans and animals. The developed model allowed
us to determine the effect of bacterial proteases in the
infected cell cultures with adenovirus. This model can also
be used for screening drugs with potential protivivovirusnoy
activity.
Genes & Cells. 2012;7(3):105-107



Effect of binase on phorbol myristate acetate-induced apoptosis of human peripheral bloodgranulocytes and monocytes
Abstract
Effect of binase (RNAse of Bacillus intermedius) on
phorbol myristate acetate-(PMA)-induced apoptosis of human
peripheral blood granulocytes and monocytes was studied in
vitro by flow cytometry. Both toxic (400 μg/ml) and nontoxic
(40 μg/ml) binase concentrations were tested. The binase
end-point effect was dependent on the target cell population
and the binase concentration. In a granulocyte subset, the
400 μg/ml concentration resulted in strongly pronounced
stimulation of PMA-induced apoptosis. In a monocyte subset,
the 40 μg/ml concentration developed a protective effect as
judged by an increase in a percantage of viable cell subset and
by slowing-down cells transtion from an early to late PMAinduced
apoptotic phase.
Genes & Cells. 2012;7(3):108-111



Isolation and cultivation of myofibroblasts from rats liver using explantation method
Abstract
During liver fibrosis development connective tissue
is produced by myofibroblasts that could originate from
two hepatic populations: hepatic stellate cells and portal
fibroblasts. A marker of myofibroblasts is the expression
of -smooth muscle actin (-SMA). Distinctive feature of
myofibroblasts, derived from hepatic stellate cells, is the
preservation of the hepatic stellate cells marker expression -
desmin. The processes of activation, proliferation and cells
trans-differentiation into myofibroblasts are closely related
to the activity of transcription factor NF-kB and its inhibitor
IkB. The aim of our work was to obtain a culture of hepatic
myofibrobasts, to study their origin, phenotype, relations
between NF-kB and IkB expression and the processes of
activation and cells trans-differentiation into myofibroblasts.
For this purpose we isolated heterogeneous population of
cells from rat liver by the method of explantation. Almost
all the cells had desmin and -SMA expression. On this
basis, we suppose that these myofibroblasts were hepatic
stellate cells derivatives, and singular desmin-negative cells
originated from portal fibroblasts. Thus, hepatic stellate cells
have major potential to activation, growth, proliferation and
transdifferentiation into myofibroblasts in comparison to
portal fibroblasts. Activated state of the cells was confirmed
by stable expression of NF-kB and its inhibitor IkB in all the
cells throughout the whole experiment.
Genes & Cells. 2012;7(3):112-115



Efficiency and mechanism of antitumor activity of cardiolipin-like lipid/thymidine kinase gene HSV-tklipoplexes in the presence of gancyclovir
Abstract
The herpes simplex virus thymidine kinase/gancyclovir
(HSV-tk/GCV) system is studied as cytotoxic lipoplex based
on cardiolipin-like dicationic lipid CDL-I. It is proposed to be
used as nonviral gene transfer system in cancer gene therapy
protocols. Аn efficient transfection of MCF7 and HEC293 cell
lines with this lipoplex was earlier demonstrated. Non-viral
system based on the CDL-I/HSV-tk lipoplex and ganciclovir
treatment causes efficiently death of tumor cells with an
involvement of apoptosis key stages. It was proved that
depolarization of mitochondrial membrane and increased level
of NF-kB transcription factor take place as a response to CDL-I
/ HSV-tk lipoplex action followed by gancyclovir treatment. It
suggests an early involvement of apoptosis mitochondrial way
to an action of this certain «suicide» system, delivered using
dicationic lipid.
Genes & Cells. 2012;7(3):116-120



Preparation and reversible aggregation of human cells encased in biocompatible polysaccharide shell
Abstract
A deposition of cationic (chitosan) and anionic (alginic
acid) polysaccharides onto the surface of normal and cancer
human cells was studied with the use of dynamic light
scattering. A method for preparation of multilayer polymeric
shell by means of electrostatic adsorption of polysaccharides
onto cell plasma membrane has been proposed. According to
confocal microscopy, the polymeric shell evenly covers the cell
and is 1-5 μm in thickness. Under experimental conditions,
the modification with polysaccharides inhibits human skin
fibroblasts growth but does not exhibit cytotoxicity to
HeLa cells. We developed an approach to controllable and
reversible aggregation of modified cells by their cross-linking
in the presence of calcium ion. Resulting aggregates have
spherical shape and the size of 100-500 μm. Proposed
approaches and methods represent an alternative to cell
microencapsulation technique and are of interest for the
development of three-dimensional cell models and their
delivery in vivo.
Genes & Cells. 2012;7(3):121-124



Survival and differentiation of endogenous Schwann cells migrating into spinal cordunder the influence of neurotrophic factors
Abstract
Schwann cells are a major figure in the process of
regeneration in the peripheral nervous system. They migrate
into the injury region of spinal cord, which are involved in
remyelination and are regarded as the source of numerous
molecular signals that could potentially support the growth
of axons in the central nervous system. In the present work
we describe the behavior of migrating into the injury dosed
region spinal cord Schwann cells under the influence of
neurotrophic factors - vascular endothelial growth factor
(VEGF) and fibroblast growth factor 2 (FGF2), delivered
by direct introduction of «naked» plasmid DNA and by
transplantation of genetically modified human umbilical cord
blood mononuclear cells.
Using immunohistochemical detection of markers of S100,
GFAP, Krox20 and HSP25 identified different phenotypes
of migrating into the spinal cord of endogenous Schwann
cells. Found that greatest influence on their numbers in the
injury region provides local delivery of genes vegf and fgf2
by human umbilical cord blood mononuclear cells. However,
the direct introduction of the same plasmid may also be
promising in the case of synthetic platforms that enhance
its transfection activity.
Genes & Cells. 2012;7(3):125-129



Analysis of recombinant vegf gene expression by genetically modified umbilical cord bloodmononuclear cells in experiment in vivo
Abstract
To obtain a significant therapeutic effect transplanted
genetically modified cells should have an enhanced ability
to survive and active expression of the therapeutic
gene. In this paper, by using immunofluorescent staining
we investigated the functional activity of the gene-cell
formulation designed to deliver a therapeutic gene into the
area of regeneration. As a model we used transgenic SOD1-
G93A mice with amyotrophic lateral sclerosis phenotype
which received xenotransplantation of human umbilical cord
blood mononuclear cells, genetically modified with adenoviral
expression vector encoding vascular endothelial growth
factor (VEGF) and the reporter green fluorescent protein
(EGFP).
Results of the study allowed to establish not only the
duration of survival of transplanted cells, but also the
efficiency of expression of recombinant genes in genetically
modified cells in vivo. Double immunofluorescent staining
with antibodies against human nuclear antigen HNA and
VEGF detected HNA+/VEGF+ cells in the terminal stage of
disease 15 weeks after transplantation. These data suggest
that genetically modified umbilical cord blood mononuclear
cells, transplanted into SOD1-G93A transgenic mice, are
able to penetrate the blood-brain barrier and migrate into
the area of degeneration of nerve tissue and survive from
the time of transplantation until the death of animals at the
terminal stage of disease. At that time adenoviral expression
vector encoding therapeutic gene is functionally active in
transplanted cells, and secretory products of recombinant
gene act on target cells by a paracrine mechanism.
Genes & Cells. 2012;7(3):130-134



The mechanism of -amyloid peptide influence on the retrograde axon transport
Abstract
Impairment of axon transport is widespread and early
event in a number of neurodegenerative diseases. The goal
of study is to investigate the mechanisms of retrograde axon
transport impairment in mouse spinal motoneurons after
application of -amyloid peptide (AP) (25-35) on the central
stump of transected sciatic nerve.
Retrograde fluorescent tracer Fluorogold (5%), AP
(25-35) (10-6 М), or mix was applied to the proximal stump
of the transected sciatic nerve of mouse under the general
anesthesia. At 24 hours after surgery lumbar spinal cord
was processed for morphometric and immunohistochemical
analysis.
The amount of Fluorogold-positive motoneurons at control
was 1223,7162,7 (n = 7), whereas after application of
AP(25-35) - 393,285,3 (n = 5, p < 0,01), which certifies
pronounced inhibition of retrograde axonal transport. Staining
with polyclonal antibodies against caspase-3 did not reveal
motoneurons in apoptotic state. Staining with monoclonal
antibodies against the AP (25-35) was negative both at
operated and intact sides of spinal cord.
Thus, revealed inhibitory action of AP (25-35) on the
retrograde axon transport is not related to apoptotic death of
neurons or accumulation of AP (25-35) inside the neuronal
soma, but, evidently, is mediated by intraaxonal effects.
Obtained data has great importance for understanding of
mechanisms of Alzheimers disease pathogenesis.
Genes & Cells. 2012;7(3):135-137



C-kit-positive pancreas islets cell of rats pancreas as a endocrine cells progenitor during alloxan diabetes
Abstract
One of the most common markers for stem cells in
pancreas is the stem cell factor receptor C-kit (CD117) that
plays a main role in differentiation of progenitor endocrine
cells of pancreas islets in prenatal development and persists
after birth. But still the role of C-kit positive cells in islet
-cells regeneration during the diabetes mellitus type I has
not been studied. Thats why the aim of our work was to
study the dynamic of C-kit expression in the pancreas islets
during the experimental alloxan diabetes in rats. The work
was made on 33 rats with the experimental diabetes. Blood
glucose levels, levels of insulin and glucagon were measured.
And also we studied the expression of C-kit, insulin and
glucagon in rat pancreas. The results of the study showed
the C-kit expression after one day of the experimental
hyperglycemia. These cells were also expressed insulin and
glucagon. We suppose that C-kit+-cells, which produce
insulin, were enable to correct disrupted carbohydrate
metabolism during alloxan diabetes.
Genes & Cells. 2012;7(3):138-141



Chromatographic purification of plasmid DNA for clinical applications (gene therapy)
Abstract
For the successful application of plasmid vectors in gene
therapy protocols it is necessary to develop methods for
purification of highly homogeneous preparations of recombinant
DNA that do not contain contaminants, primarily chromosomal
DNA, bacterial proteins, RNA and endotoxins. In the course
of our study we performed optimization of the purification of
plasmid supercoiled DNA by three chromatographic steps from
an alkaline lysate of bacterial strain of E. coli. We determined
an optimal conditions for alkaline lysis step in order to increase
the yield and minimize the duration of the plasmid purification
by gel filtration.
Genes & Cells. 2012;7(3):142-145



Autophagy induction in peripheral blood T-lymphocytes of atopic asthma patients
Abstract
Autophagy is a fundamental process that ensures
the regulation of T-cell homeostasis. In case of apoptosis
induction disruption in the cell it could be single mechanism of
the cell death. Previously was shown inhibition of lymphocyte
apoptosis in patients with bronchial asthma, so the main study
of this work has focused on the study development process
of autophagy in T-lymphocytes of patients with bronchial
asthma. The article presents the main morphological changes
in cells associated with activation of autophagy (formation
autophagosome). In addition to morphological changes in
lymphocytes, we have shown the expression of autophagy
marker protein (LC3B). We found that in T-lymphocytes of
patients with severe asthma are simultaneous activation of
both autophagy and apoptosis, and autophagy is a stimulus
to cell death.
Genes & Cells. 2012;7(3):146-150



Influence of recombinant histone H1.3 on the efficiency of lentiviral transduction of human cells in vitro
Abstract
Lentiviral vectors are widely used in genetic modification
of human and animal cells (lentiviral transduction) to enhance
their therapeutic potential by expression of recombinant
protective and trophic factors. Genetic modification of cells in
vitro or ex vivo achieves the specificity of viral transduction,
as modified are just cells that have been manipulated in
the laboratory. In addition, the introduction of genetically
modified cells, but not pure virus, helps to avoid introduction
of viral particles into the body of the recipient. This approach
allows us to control the expression of therapeutic genes, the
immunogenicity of viral vectors and viral transduction. To
date, different approaches are used to improve the lentiviral
transduction (polycations, protamine sulfate, etc.), but these
methods suffer from limited efficacy or high toxicity. For the
first time we demonstrated that the recombinant histone
N1.3 increases the efficiency of lentiviral transduction by
more than 2 times and has no toxic effect on target cells in
a wide range of concentrations studied.
Genes & Cells. 2012;7(3):151-154



Endogenous secretion of vascular endothelial growth factor by multipotent mesenchymal stromal cellsderived from human third molar dental follicles
Abstract
Human stem cells secretome is currently a very hot area
of research. We report that multipotent mesenchymal stromal
cells isolated from human third molar dental follicles (MMSCTMDF),
are able to secrete high levels of vascular endothelial
growth factor (VEGF) when cultured in vitro. Due to the fact
that VEGF is a well known angiogenic and neuroprotective
factor, the use of MMSC-TMDF is promising for the
development of stem cell therapy of various degenerative
human diseases.
Genes & Cells. 2012;7(3):155-158



Influence of Trichoderma asperellum metabolites on tissue regeneration against pyrene
Abstract
We studied the influence of the culture fluid of fungi of
the genus Trichoderma on Swiss Webster CFW mice after
exposure to pyrene - polycyclic aromatic hydrocarbons, which
can cause pathological changes in the body. Beneficial effect
of Trichoderma metabolites on haematological parameters,
the functioning of liver and nephros was shown, the trend
toward regeneration of the structure of skin and liver after
the damages, caused by the introduction of pyrene, was
identified.
Genes & Cells. 2012;7(3):159-163



Generation of recombinant adenoviruses and lentiviruses expressing angiogenic and neuroprotectivefactors using Gateway cloning technology
Abstract
The critical aspect in gene and gene-cell therapy is to
find an optimal vector - a carrier of genetic information.
Viruses represent a natural biological system for gene
transfer into eukaryotic cells. One of the most effective
and proven vectors for delivery of recombinant nucleic acids
into mammalian cells are adenoviruses and lentiviruses. In
this study using the Gateway cloning we have constructed
adenoviral and lentiviral vectors encoding angiogenic and
neuroprotective factors: various isoforms of vascular
endothelial growth factor vegf121, vegf165, vegf189; basic
fibroblast growth factor fgf2; glial cell-derived neurotrophic
factor gdnf. The efficiency of transduction of HEK293A cell
line with generated recombinant viruses and expression of
recombinant proteins were confirmed by immunofluorescent
analysis.
Genes & Cells. 2012;7(3):164-168



Transplanted Hepatic Stellate Cells participate in liver regeneration after partial hepatectomywithout risk of hepatic fibrosis
Abstract
Hepatic stellate cells are considered as one of the potential
stem cells candidates in the liver. The aim of our work was to
study the probability of hepatic stellate cells transplantation
to rats after partial hepatectomy, their further homing, the
ways of differentiation and hepatocytes repopulation in the
recipient liver. For this reason fresh isolated rat`s hepatic
stellate cells were transplanted into portal vein of intact rats
(control group) and rats immediately after partial hepatectomy
(experimental group). Before transplantation cells were
labeled by adenovirus expressing green fluorescent protein.
Our results showed that it was possible to detect 2 types of
donor cells in the recipient liver of control and experimental
groups: 1) hepatocyte-like cells in liver parenchyma; 2) small,
spindle-shaped, rounded and triangular cells in liver sinusoids
and portal areas. Transplantation after partial hepatectomy
leads to significant increase of transplanted cells homing and
stimulation of their differentiation into hepatocytes. Over
the whole experiment there was no hepatic stellate cells
transdifferentiation into myofibroblasts, thus there is no risk of
liver fibrosis development after this cell type transplantation.
In summary hepatic stellate cells after being transplanted are
able to differentiate into hepatocytes and do not induce liver
fibrosis, that confirms their role in organ regeneration and
probable belonging to hepatic progenitor cells.
Genes & Cells. 2012;7(3):169-172



Comparative study of an activity of rat spinal ganglion cells and PC12 cells on the surfacesmodified with bioadhesive polymers
Abstract
We studied the adsorption of bioadhesive polymers
(polyornithine, gelatin, laminin) on polystyrene surface
by the use of dynamic light scattering. The contribution
of biopolymers to resulting zeta potential of the modified
surface was assessed. PC12 cells do not exhibit selective
adhesion in the presence of foetal bovine serum. Polystyrene
with adsorbed polyornithine promotes primary adhesion
of PC12 cells cultured in serum-free medium with nerve
growth factor. Subsequently adsorbed laminin induces
spreading and differentiation of the cells into neuronal
direction. Primary neurons isolated from rat spinal ganglion
adhere preferentially on the polyornithine-modified surface.
On the polyornithine-laminin surface neurons intensively
form neuritis that correlates with proliferation of glial
cells positive for S100 protein. The results show that
PC12 cells and primary neurons exhibit similar response to
surface material with the latter cells being more sensitive
to this factor. Isolated cell culture can be used to study the
relationship between neurite outgrowth and Schwann cells
proliferation on different biomaterials.
Genes & Cells. 2012;7(3):173-176



Correction of the face soft tissue defect using autologous fat tissue enriched by cellsof stromal-vascular fraction
Abstract
The article presents a clinical case of successful surgical
treatment of a patient with idiopathic progressive hemifacial
atrophy. We have used lipofilling with autologous fat tissue
enriched by stromal-vascular fraction cells.
Observation of the patient within two years suggests that
this method of treatment is safe and highly effective method
of face soft tissue defect substitution of such pathology.
Genes & Cells. 2012;7(3):177-179



The first clinical experience of direct gene therapy using VEGF and bFGF in treatmentpatients with critical lower limb ischemia
Abstract
In this paper we present the clinical observation of
successful treatment of distal form of peripheral artery
disease of the lower extremity with symptoms of critical
ischemia in a 60 years old patient.
Intramuscular injection of dual expression plasmid, encoding
vascular endothelial growth factor VEGF and basic fibroblast
growth factor FGF2, was performed to the affected lower
extremity. The effect of treatment was evaluated by functional
tests: measurement of ankle-brachial index, treadmill test, the
recovery time, shoulder-ankle index after strain and temporary
occlusion. Performed immunohistochemical examination of
biopsy samples of the affected lower extremity muscles.
Genes & Cells. 2012;7(3):180-184



Life without water: cryptobiosis of invertebrates as a model for next generation techmologyof biomaterials preservation
Abstract
To date, advances in the field of tissue engineering,
cell transplantation and genetic engineering have made
the biological materials of different origin an important
therapeutic tool in clinical medicine. Currently, cells
preservation is achieved by freezing at -80°С or in liquid
nitrogen. Cryopreservation technology is expensive and
has considerable limits during transportation. Preservation
of viable biological material in dry state under ambient
temperature is considered as attractive, but yet fully
achieved alternative. There are organisms which are able to
survive complete water loss. Understanding of mechanisms
underlying dehydration tolerance will allow the development
of dry preservation technology for molecules, cells and
organs, and further use of these methods in medicine,
pharmacology and biotechnology.
Genes & Cells. 2012;7(3):185-189



Yubiley
Abstract
В июне этого года отметила свой юбилей профессор Нина Андреевна Онищенко - заведующая лабораторией биотехнологии стволовых клеток ФГУ «ФНЦ
Трансплантологии и искусственных органов им. В.И. Шумакова» МЗ СР, участник редакционного совета журнала «Клеточная трансплантология и тканевая инженерия».
Genes & Cells. 2012;7(3):190-191


