Vol 7, No 3 (2012)

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.

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.

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.

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.

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.

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 ƒg/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.

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.

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 ƒ-cells of Langerhance islets.

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.

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.

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.

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 ƒ-smooth muscle actin (ƒ-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 IkBƒ. The aim of our work was to obtain a culture of hepatic myofibrobasts, to study their origin, phenotype, relations between NF-kB and IkBƒ 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 ƒ-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 IkBƒ in all the cells throughout the whole experiment.

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.

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.

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 ƒ-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.

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.

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.

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.

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.

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.

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».