Experimental Study Of Building Genetically Engineered Cells Of OECs-VEGF165

Background(?) High incidence and lagging treatment of leukoaraiosisWith the growing aging of population, the reinforcement of people’s awareness about health and the development of imaging technology, leukoaraiosis (LA) has been attracting much attention. It is one of the most common cause of cognitive disorders of the elderly, in comparison with the incidence rate of1.7-8.6%of healthy elderly,78%-93%of the elderly patients with hypertension,30%-31%of the elderly patients with Alzheimer’s disease and36%-100%of the elderly patients with stroke. The LADIS (Leukoaraiosis and Disability in the Elderly) study found that the body’s ability level was related with the extent of white matter damage and the degree of white matter lesions was closely correlated with dementia disability assessment scores in the elderly not suffering from disability. Currently, most scholars believe that LA is due to the brain artery structural changes induced by age, hypertension and other vascular risk factors, which eventually leads to the brain ischemic injury. Hypertension and aging are the most important risk factors for LA.LA is a chronic ischemic disease. After cerebral ischemia neurogenesis and angiogenesis are triggered as two major pathophysiological processes, which have an important role on promoting nerve function recovery after ischemic injuries. To improve the local blood supply is a fundamental solution to LA development, preventing the progression of the disease to the more serious (dementia, cerebral infarction).(?) Research progress and analysis of vascular endothelial growth factorTherapeutic angiogenesis is a new concept in recent years. It is the first to study on myocardial ischemia, aiming to stimulate the growth of the small blood vessels and to promote collateral circulation of the myocardium ischemic area, namely "self-bypass"."Currently, vascular endothelial growth factor (VEGF) is one of the most effective cytokines to stimulate the growth of blood vessels. The biological functions and characteristics of VEGF have attracted more and more attention. VEGF gene therapy is viewed as "molecular bypass surgery", which is expected to partially replace surgical bypass grafting and angioplasty. Study found that VEGF and its receptors upregulated after cerebral ischemia, intracerebral injection of VEGF led to the formation of new blood vessels in the brain and VEGFR-1expression in astrocytes and that VEGF also directly played a neuroprotective effect without relying angiogenesis, which contributed to the damage of cortical neurons from hypoxia and low sugar and stimulated the growth of axons.There are rare reports about VEGF changes in the lesions and treatment of LA at home and abroad. The success of VEGF in the heart cerebrovascular experiments lay a solid foundation for the study of LA.It needs the further explorations about how to apply exogenous VEGF protein and VEGF gene therapy on cerebrovascular disease, improve the blood supply to the damaged brain tissue and reduce the extent of their ischemic, LA pathological changes mainly are ischemic demyelination and the decline in myelin content. Partial remyelination can control the progress of the LA and improve the clinical symptoms.(?) Biological characteristics of olfactory ensheathing cellsOlfactory ensheathing cells (OECs) are cells of the dual characteristics of the astrocytes and Schwann cells and retain many of the characteristics of the developmental stages and plasticity. The olfactory system is the only renewable hub so far and its biological functions are extremely wide, exerting distinct facilitation on the growth, development and survival of neuron as well as promoting neurite outgrowth. Ramon-Cueto, studies have shown that rodents OECs can accelerate axonal regeneration and the remyelination of the demyelinated nerve fibers in rats with spinal cord injury. Therefore, OECs has opened up a new hope for cell transplantation in the treatment of CNS disorders.Based on the biological role of OECs, many studies have focused on the application of olfactory ensheathing cell transplantation to treat demyelination of nerve tissue. Based on the study about the relationship between OECs and apoptosis, it can enhance the understanding of the therapeutic effect and mechanism of OECs and expand its range of treatment and improve their treatment.(?) Composition and significance of the establishment of the genetically engineered cells OECs-VEGFWith the deepening of the study of the mechanism of the molecular level, gene therapy has become the new entry point for cerebrovascular disease prevention and treatment. Gene transfection technology enables the use of both treatment possible and the idea of composing genetically engineered cells is consequently established.Based on the above analysis, it is envisaged that OECs are cultured. VEGF target gene is transferred to the recipient cell and builds OECs-VEGF gene engineering cells of rats with high expression of VEGF. The transplanted cells are injected into the brain. They not only can exert the synergic effect, but also resolve the low expression and poor continuity of VEGF which is the bottleneck problem to provide effective solution. LA cell transplantation and gene therapy to explore a new kind of practical breakthrough. Expected OECs are passaged and amplified in large quantities in the the LA model, and eventually differentiated to the nervous system, which has a role to promote axonal regeneration and repair of myelin. Meanwhile, after transfection the OECs have stronger biological activity and continually secret more VEGF to promote vascular endothelial regeneration, thereby improving the LA nerve tissue microcirculation, promoting angiogenesis, and reducing ischemic injury of the nerve tissue.The purpose of this study was to separate, culture and purify OECs from the olfactory bulb. In addition, the lentivirus carrying human-VEGF165gene is construct and transfected OECs. Then OECs-VEGF gene engineered cells are established.Objectives1. To explore the method of introducing human-VEGF165gene into OECs and analysis the feasibility of building the OECs-VEGF genetically engineered cells.2. To research the technique of purifying OECs from the olfactory bulb isolated rat.3. To construct the lentivirus vector carrying the human-VEGF165gene4. To building the OECs-VEGF165genetically engineered cells by human-VEGF165-LV transfecting OECsMethods1. Sampling, separation, cultivation, purification and identification of OECs from olfactory bulbMale SD rats were decapitated. The olfactory nerve layer and granular layer of the olfactory bulb were taken, cut into pieces and digested in trypsin in the incubator. DF12containing FBS terminated the digestion. It was blown and beaten. Draw the upper suspension to be inoculated after standing a while and repeat three times. The OECs were mainly purified by differential adherence method. Cell suspension was joined with dual antibody and cultivated for2d so ld.The cultivated cells were adjusted, planted and adhered. Medium was changed every3days. Take the primary cells, prepare a cell suspension which was inoculated in24-well plates, count the total number of cells, average for the following nine days and make the growth curve.The purity of primary OECs is identified by immunofluorescence. Remove the culture medium, rinse repeatedly, fix and incubate twice. The nuclear was stained with Hoechst33342. Alkaline glycerol was dropped on glass slides. After p75staining, sealed and observed with fluorescence microscopy. Counted the number of nuclear and positive cells in high magnification view and calculated the ratio of the positive cells, which is the purity of OECs.2. Construct the lentivirus carrying the human-VEGF165geneA lentiviral vector carrying the desired gene was constructed by three plasmid co-transfecting of293T cells with the lentivirus packaging kit.1) Cloning Construction:①The carrier preparation which overexpressed objective gene:catching the target gene by PCR, digesting the vector and exchange and transforming its product into competent cells of bacteria.②Making the information about target gene and vector clear.③DNA agarose gel electrophoresis.④Preparing fresh E. coli competent cells by using the CaCl2.⑤Transferring the transformed competent cells to the agar medium. Grown clones were identified by subsequent PCR.2) Plasmid expression detection:①Preparation of the purpose cells.②Target cell plasmid transfection:preparing a cell suspension, seeding in culture plates and transfecting according to the Lipofectamine2000transfection reagent manual operation. In order to determine the efficiency of infection was judged by fluorescently labeled plasmid gene expression.③Proteins were extracted from the collected cells and were detected by western blotting, Meanwhile,7860cells and empty plasmid-transfected293T cells were used as blank control and the negative control.3) Viral packaging, collecting and concentrating:the state of cells was essential for viral packaging, so we needed ensure a good state of cells and less number of passages. After collecting and concentrating, viral biological titer was determinated.4) Viral titer detection:the titer was detected by fluorescence real-time quantitative PCR and determined by comparing the Ct value differences between the control group and the test group.3. Building the OECs-VEGF165genetically engineered cells by human-VEGF165-LV transfecting OECs1) Pre-experiment:four groups, each of which had four different gradients MOI (Multiply of Infection), a total of16holes, there were three wells for every experimental condition. MOI value in each group had a different gradient.2) Immunofluorescence detection:After inoculation and passage untransfected and transfected OECs, discard the culture medium, rinse the cells twice, fix twice at room temperature, wash3times with PBS and incubate after addition of normal goat serum. Then wash, stain nucleus in climbing slices with Hoechst33342, seal with alkaline glycerol and observe under fluorescence microscopy. At the same time, the slides without adding antibody in the dyeing process were used as the negative control slides.3) Real time-PCR:①Extraction of total RNA:blow the cells in6-well plates until they were clear then transfer them to EP tubes. Firmly shake and centrifuge the test tubes. By pipette transfer upper aqueous phase to another EP tube, add an equal volume of isopropanol to precipitate the RNA, centrifuge, and detectRNA concentration with ultraviolet degrees.②RT-PCR:Total RNA of the untransfected and transfected OECs was transcribed reversely after transfection. After thawing the reagents of RT kit, shake up the test tube, slightly centrifugate and perform on ice.③Real-time PCR. Melting curve analysis.4) ELISA detection:enzyme-linked pro and material were added to the standard holes and test sample hole. After microplate coated and drained after incubation, the substrate was added to each well in accordance with the order respectively. After kept in the dark, the liquid to terminate the reaction was added. OD value was shown with a microplate reader. Calculated with Logit-Log linear regression and draw a standard graph.Results1. Sampling, separation, cultivation, purification and identification of OECs from olfactory bulb1) The primary cells cultivated by differential adherent method approximately accounted for98%of all cells. More the times of passage were, lower the purity of OECs was. In the second generation the purity of OECs declined to78%and the third was58%.2) Cellular morphology and growth characteristics:the cellular morphology after purification was consistent, mainly spindle-shaped, accounting for about90%of the total cells. There were other common forms, multiple swelling and irregular. Cellular morphology changed, tight junctions disappeared and cells changed from spindle-shaped to multiple swelling after passage. In the case of culture liquid nutrients fully and completely, cells with poor activity died gradually and seperated from the bottom. But the most of the cells could be transformed from multiple projections to spindle-shaped form with stronger proliferation ability and continued to proliferate.3) The proliferative capacity of primary OECs was strong. But with the times of passages increased, the purity of OECs decreased. The cell shape diversified and the proliferation slowed.2. To construct the lentivirus carrying the human-VEGF165gene1) Construction of plasmid cloning vector. The target gene fragment was amplified from the plasmid containing humanVEGF165gene, and re-linked to the plasmid used as lentivirus packaging vectors. By sequencing the positive transformants and comparing with the NCBI on the target gene sequences, the target gene sequence was clear.2) Detection of plasmid expressing the gene. The plasmids labeled with the EGFP protein gene were transfected into293T cells by liposome. Significant fluorescence the cells could be observed after24hours, which indicated that the expression of target gene and EGFP was normal. The predicted size of the target gene protein was22KD. By Western Blot analysis, the protein extracted from plasmid transfected293T cells could be detected22kD near bands, which was consistent with the protein synthesized by target. This indicates that the connection was successful.3) Synthesis of viral titer. Ct value appeared2circulating difference between one ten thousandth group (i.e. gradient group the10-4ul dilution) and Con group by real-time PCR. It was considered that there were virus particles in the samples10-4ul and virus titer of2×108/ml was obtained.3. Building the OECs-VEGF165genetically engineered cells by human-VEGF165-LV transfecting OECs1) From different concentrations of the virus and the comparison of different transfection conditions, the best infection conditions of lentiviral gene vector of OECs were as follows:for lentiviral synthetic, at MOI=10to obtain greater than80%of the efficiency of infectionat; relatived to the normal medium, Eni.s medium did not significantly improve the infection efficiency; Polybrene also little changed in the efficiency of virus infection.2) After transfection, the growth activity of OECs was affected."Telogen" within12hours, after24hours, an increase in the dead cells in the cell culture supernatant which was not visible under fluorescence, and48hours more died cells in transfection group. After72hours the weak fluorescence was visible in a small amount of cells. After96hours, the majority of cells expressed EGFP.3) GFP-positive cells accounted for more than80%in the three transfection sub-groups with different MOI. But after transfection,10and20subgroups cells died more. Cells of MOI5died less and cell function recovered quickly. VEGFA immunofluorescence staining was performed in the transfected OECs and the expression of the exogenous VEGFA was observed. From transfection efficiency and the impact of transfection on the growth of OECs, MOI=5was a more appropriate choice.Conclusion1. OECs from the olfactory bulb which are purified by differential adherent method appear high purity and good activity. That is a simple and economical way for extraction and purification of primary cells in vitro studies, which is suitable for further relevant studies.2. With the times of passages increase, the purity of OECs declines significantly. Thus to make the results more representative, it is important to select the appropriate number of passages during OECs research. 3. Target gene can be transferred into OECs by lentiviral vector carrying the human-VEGF gene. Because of the high infection efficacy of lentivirus in primary cells, higher transfection efficiency can be achieved in the case of the influence of the activity of OECs as small as possible.4. OECs are infected by the lentiviral vector carrying the human-VEGF gene to construct the human-VEGF-OECs genetically engineered cells, which can express of VEGF165gene stably and efficiently. The synthetic protein can be secreted to the extracellular which has the same physiological characteristic as VEGF165protein and can play a biological role in a normal body.