Construction And Identification Of Enhanced VEGF Expression Bone Marrow Stromal Cell Line With VEGF Gene-modification

Background:Bone marrow derived mesenchymal stem cell (BMSCs) is a kind of multipotent adult stem cellsderived from bone marrow, the main advantage of its application in cell therapy is autologous transplantation, not transplantation immune rejection, not ethics and induced tumor limitations,compared withembryonic stem celltransplantation; andBMSCs is relatively simple, unlimitedandeasy to obtain,compared with neural stem cells, which were mainly located in the subventricular zone, hippocampus and other structures. Clinical study of BMSCs transplantation has been used in cerebral ischemia and some other diseases.There are many studies in the treatment with brain ischemia throughBMSCs transplantation. The BMSCs transplanted treatment with rat cerebral MCAO by vein, artery and stereotactic local injection had found that transplanted cells could directe to repair the focal ischemic area, andon the other handindirect to repair the focal ischemic area inducing glial cells and neural stem cells walking to the injured area or differentiation and proliferation of the local neural stem cells, through secreting chemokines. It is an important way to reconstruct neural circuit with BMSCs transplantation becauseBMSCs have the ability to differentiate into neural cells. However, most studies found that BMSCs transplantation only hadfew expression of neural or glial cell surface markers, and even a few expression of nerve tissue marker cells, there is no electrophysiological or other evidence shows they have nerve cell function, but can not determine whether they are with other nerve cells established contact.The present studies showed that, BMSCs plays the effect mechanism in many aspects, such as reducing apoptosis of ischemic penumbra cells, promoting proliferation of endogenous neural stem cells, promoting angiogenesis in ischemic region, inhibiting scar of glial proliferation and promoting axon regeneration. In sum, BMSCs are not replaced the missing neurons in the treatment with cerebral ischemia, but played a " molecular factory " role to mediate the above treatment process by secreting various neurotrophic factorsin brain microenvironment.BMSCs are scarce in bone marrow, which should be cell amplification in vitro in order to obtain enough cells in cell transplantation therapy. However, BMSCs will slow death the same as most somatic cell, mainly in two aspects:①With the increase of age, the quantity and quality of BMSCs decreased obviously (the elder is higher risk population inbrain ischemia, is subject to cell transplantation therapy);②In cell amplification processin vitro, BMSCs cell morphology will graduallychange (from fine spindle to wide flat), and gradually lose the ability to cell proliferation, differentiation potential, and homing to focus area.The senescence mechanism of BMSCs is complex and not yet clarified in research. It is well known that BMSCs did not express telomerase. Human telomerase is a ribonucleoprotein, which two most important componentsare the human telomerase catalytic subunit (hTERT) and RNA (hTR) template, and hTR sustained expression, while hTERT is not expressed in most adult cells. Telomerase is different from the general DNA polymerase, which is a specialized reverse transcriptase, using the complementary sequence RNA component itself as a template, catalytic TTAGGG sequences to chromosome ends, so as to maintain telomere length. The telomere structure and composition of chromosome end is closely related to cell in the maintenance and proliferation. Most adult somatic cells’telomere length decreases with cell replication, and limites cell proliferation. In contrast, tumor cells and embryonic stem cells sustain telomere functionthrough the activation of telomerase.BMSCs are not express telomerase, telomere length is gradually shortened with cell division. If it reaches about10Kb, cells will stop dividing and growth arrest. Study on the Baxter MA showed that BMSCs’mean telomere length decreased with the growth of the age (17bp/year), and in vitro amplification of7～10PDs (Population Doublings, population doubling times) the average loss of telomere length is about1Kb. The loss rate is amazing, even in young BMSCs, their life experience telomere reduce the length of the highest can only reach2.5-3.5Kb. Therefore, if you do not take the special processing method, in vitro fully amplified BMSCs back to the patient in the process, actually has greatly shortened the life of BMSCs.In order to extend life cycle of BMSCs, or some other scientific purposes, many labshave in biochemical research with BMSCsimmortalization. There were able to successfully establish BMSCs’immortalizationcell lines by modified BMSCs with the human papilloma virus (HPV) E6/E7gene, simian virus40(SV40) large T antigen gene or Bmi-1gene. These cancer gene could activate expression of human telomerase catalytic subunit gene hTERT, andsustain cells’telomere length in the amplification process, however, the major problems of BMSCs cell lines using a cancer gene modified was chromosome instability, often caused cells malignant, and not for transplantation.In recent years, there are many laboratories using hTERT transfection and successfully obtained BMSCs immortalization cell lines. The established cell lines were not significantly reduced telomere length in vitro amplification, and maintain the same primary BMSCs similar or even better proliferation and multi-directional differentiation potential. More importantly, BMSCs cell line was established by using hTERT transfected in vitro maintained normal karyotype, cell growth inhibition in vitro and in vivo, contact, experiments were not obvious tumorigenicity.In addition to BMSCs, other somatic cells skin fibroblasts, vascular endothelial cells, skin cells, mammary epithelial cells, skin melanoma cells, lymphocytes, and by BMSCs induced differentiation of the osteoblast, can beimmortalizationby transfecting hTERT gene. In the previous studies, there were not found significant malignant cellsin usingthe hTERT gene modified.Whether BMSCs express vascular endothelial growth factor receptor (VEGFR), different experiments have different reports.BMSCs are not expressed in adult VEGFR, but expressed PDGFR-A, PDGFR-B and NRP-1. VEGF play important role through the VEGFR conjugate of PDGFRand enhance the effect by NRP1. Cerebral ischemia due to cerebral vascular atherosclerosis or cerebral vascular occlusion cause nerve dysfunction, VEGF can promote ischemic vascular endothelial cell chemotaxis and aggregation, induced the formation of new blood vessels, thus increasing the nutrient supply to the ischemic area, reduce ischemia caused by functional disorder of God, therefore, will be of great value in clinical application construction of VEGF gene overexpression of BMSCs in the elderly.This experiment will construct the hTERT lentiviral vector and VEGF gene lentiviral vector, to study the effect of hTERT lentiviral vector and VEGF gene lentiviral vector combined modification in elderly patients with BMSCs, establish the feasibility of enhancing cell lines expressing VEGF, and the hTERT lentiviral vector and VEGF gene slow virus vector modified BMSCs studies phenotype identification and function. Because the genetic information of BMSCs containing autologous, research results will be helpful to explore the preparation of individual (patient-specific), for the treatment of cerebral ischemia (disease-specific) stem cell products, but also a solid foundation for further experimental study.Objectives:BMSCs transplantation is an effective strategy for the treatment of cerebral ischemia, however, BMSCs will slow death with aging and amplification process in vitro, which hinders the treatment application. This study intends to human telomerase catalytic subunit hTERTgene lentiviral expression vector and the expression of VEGF gene lentiviral expression vector of BMSCs gene modified the elderly, explore the construction of enhanced expression of VEGF permanent biochemical BMSCs, detection of its biological characteristics. Because the genetic information of BMSCs containing autologous, research results will be helpful to explore the preparation of individual (patient-specific), for the treatment of cerebral ischemia (disease-specific) stem cell products, but also a solid foundation for further experimental study.Methods:1.Construction of lentivirus vector containing hTERT gene and VEGF gene.①The amplification of hTERT and VEGF gene fragments by PCR in vitro.②Construction of pENTR11-hTERT-IRES-EGFP and pENTR11-VEGF-IRES-EGFP entry vector.③Construction of pLenti6/V5-DEST-hTERT-IRES-EGFP和pLenti6/V5-DEST-VEGF-IRES-EGFPexpression vector.④Packaging, expression and analysis of lentiviral vectors pLenti6/V5-DEST-hTERT-IRES-EGFP and pLenti6/V5-DEST-VEGF-IRES-EGFP.2. To study the effect of hTERT gene lentiviral vector and VEGF gene lentiviral vector combined modification in elderly patients with BMSCs, establish the feasibility of enhancing cell lines expressing VEGF.①The old BMSCs was purchased from biotech companies, or from clinical volunteers (aged) bone marrow adherent method to obtain BMSCs, combined with density gradient centrifugation.②Grouping:unmodified BMSCs group, empty lentiviral modification group, VEGF gene modification group, hTERT gene modification group, hTERT/VEGF double gene modification group (each gene modification group stable expression clones were selected by blasticidin).③Fluorescence microscope and transmission electron microscope observecells’ changes in different generation cell morphology; flow cytometry method detectescells’ changes indifferent generation cell surface markers; RT-PCR method detectes cell hTERT and VEGF level of mRNA; fluorescence immune cell chemical method and Western blot method detecteexpression ofcell protein.3. Study on modification of BMSCs phenotype and function in hTERT/VEGF double gene①Grouping:unmodified BMSCs group, empty lentiviral modification group, VEGF gene modification group, hTERT gene modification group, hTERT/VEGF double gene modification group.②Flow cytometry method detectescells’changes indifferent group cell surface markers; transmission electron microscope observescells’ changes in different group cell ultramorphology; confocal microscopy observescells’ changes in different groupcell markers by immunofluorescence; MTT assay and colony assay observe cell growth and proliferation; single cell scratch assay and transwell chamber assay observecell migration and invasion; RHG band karyotypeanalysis observes evaluation of chromosome stability; nude mice inoculated tumor formation experiment of recombinant cell tumorigenicity; the level of VEGF secreting cells telomerase activity and cell was detected by TRAP-ELISA.4.Data are expressed as mean SD. Statistical comparisons were performed using one-way ANOVA (LSD). Statistical significance was set at P<0.05. All statistical analysis was operated with SPSS13.0software.Results:1.Successful construction of hTERT gene lentiviral vector and VEGF gene lentiviral vector.①Successful amplified hTERT gene and VEGF gene by using pEGFP-hTERT, pEGFP-VEGF165plasmid (our laboratory existing) as a template.②Using EcoR I and Not I enzyme cut pENTR11entry vector and pIRES vector, and IRES sequence fragment from pIRES vector cut with T4DNA ligase connected into pENTR11to construct pENTR11-IRES, the recombinant plasmid was transformed into DH5acompetent cell, screening, PCR, enzyme digestion and sequencing, the success of the pENTR11-IRES.③Using Nco I, EcoR I pENTR11-IRES plasmid and hTERT gene or VEGF gene PCR product, hTERT fragment or VEGF fragments with T4DNA ligase cloned into vector pENTR11-hTERT-IRES or pENTR11-VEGF-IRES plasmid, by enzyme digestion, sequencing; finally, Xbo I, Not I restriction enzyme pENTR11-hTERT-IRES or pENTR11-VEGF-IRES plasmid and belt EGFP PCR gene product green fluorescent marker, T4DNA ligase to construct pENTR11-hTERT-IRES-EGFP or pENTR11-VEGF-IRES-EGFP plasmid by restriction enzyme digestion, sequencing, pENTR11-hTERT-IRES-EGFP and pENTR11-VEGF-IRES-EGFP; successful entry vector.④The preparation of pENTR11-hTERT-IRES-EGFP and pENTR11-VEGF-IRES-EGFP entry clone, expression vector pLenti6/V5-DEST and Gateway TM LR ClonaseTM enzyme Ⅱ mix mixed reaction, construction with poison slow disease table with double gene as carrier pLenti6/V5-DEST-hTERT-IRES-EGFP and pLenti6/V5-DEST-VEGF-IRES-EGFP, will connect the product into Stb13TM E.coli cells, screening positive clone identification. Successful expressing pLenti6/V5-DEST-hTERT-IRES-EGFP and pLenti6/V5-DEST-VEGF-IRES-EGFP vector.⑤Lipofectamine2000pLenti6/V5-DEST-hTERT-IRES-EGFP or (and) the transient expression of pLenti6/V5-DEST-VEGF-IRES-EGFP gene plasmid was transfected into COS-7cell line, the expression of recombinant protein, lentivirus vector verification of construction, with satisfactory results.2. Successfully enhanced and identified expression of VEGF immortalization BMSCs cell lines①Using ViraPowerTM Lentiviral Expression System recombinant plasmid and packaging purified mixtures were transfected into293FT cell line, collect the virus supernatant from293FT cell line, for titer determination; in the appropriate MOI with the virus supernatant transfection of bone marrow stromal cells; screening to produce stable expression of pLenti6/V5-DEST-hTERT-IRES-EGFP or by blasticidin (and) pLenti6/V5-DEST-VEGF-IRES-EGFP cell line; the RT-PCR and Western-blot testing to determine the mRNA and protein expression and has higher activity.②Observation of optical microscope, primary cell Gang inoculation suspended in a DMEM liquid culture medium, the nucleus was round, located in cytoplasm of central. In general,2-4h after inoculation of primary cells became adherent,48h after most primary cell wall. The medium was changed after removal of nonadherent cells adherent cells can be seen in many forms, fusiform or polygonal, nucleus large, oblate, cytoplasmic can extend lamellipodia in different directions. Adherent primary cells to disperse the colony clonal rapid proliferation,1W around each colony contains about hundreds of cells, cell morphology was long fusiform fibroblast-like. Passage cells begin to adherent to1-2h after inoculation,24h completely adherent, passaged cells showed a broad flat polygon,6-7d cells completely confluent, cells become elongated, morphology were fibroblast-like.③There were not obvious structure changed in the ultrastructure of different generation BMSCs cells by transmission electron microscope.④The expression level of flow cytometry assay for the detection of different generations of BMSCs cell surface markers CD29, CD34, CD44, CD45, CD90and CD105, CD44, primary cultured cells showed positive expression of CD90, CD29was weakly positive expression of CD34, CD45, CD133, and almost no expression; cell line (P3, P9) CD44the positive expression of CD29, CD90, weakly positive expression, while CD34, CD45, CD133almost no expression, and primary cells are basically the same.⑤RT-PCR, Western-blot and immunofluorescence assay to determine the different groups of mRNA and protein expression and has higher activity.3. Successful construction of hTERT/VEGF modified BMSCs cell line and studies on the phenotype and function①The expression level of flow cytometry assay for the detection of different groups of BMSCs cell surface markers CD29, CD34, CD44, CD45, CD90and CD105, each group was CD44, the positive expression of CD90, CD29was weakly positive expression of CD34, CD45, CD133, and almost no expression.②Transmission electron microscope was used to observe the ultrastructure of BMSCs cells, and different groups showed that, WP bodies were detected VEGF gene expression in BMSCs cells, plasma membrane vesicles and secretion of vWF, the other groups were not expressed.③the laser confocal microscopy of BMSCs cell protein expression of different groups, each group results show that, BMSCs cells could express CD31and UEA-1, but only the expression of VEGF BMSCs cells to express vWF protein.④RHG banding chromosome analysis BMSCs analysis of karyotype of different groups of changes, results show that, the group BMSCs cell karyotype, no genetic variation.⑤The MTT experiment and plate cloning test in different groups of the BMSCs cell growth and proliferation, results showed that, hTERT gene expression in BMSCs cell growth and proliferation was significantly increased.⑥BMSCs cells of different groups of detecting the single cell scratch assay and Transwell chamber assay invasion and migration, results show that, expression of hTERT gene or VEGF gene of BMSCs cell migration ability enhancement, and BMSCs cell groups were no invasion.⑦Tumorigenicity, different groups of BMSCs cells to nude mice tumor formation experiment test results show that, the group BMSCs cells are not tumorigenic.⑧The level of VEGF, the secretion of TRAP-ELISA method for the detection of different groups of BMSCs cells telomerase activity and cell showed that, hTERT gene expression or enhance telomerase activity of BMSCs cells with VEGF gene, VEGF secretion increased.Conclusions:The experiment will construct the hTERT lentiviral vector and VEGF gene lentiviral vector, in order to study the effect of hTERT lentiviral vector and VEGF gene lentiviral vector combined modified elderly patients with BMSCs, establish the feasibility of enhancing cell lines expressing VEGF, and the hTERT lentiviral vector and VEGF lentiviral vector modified BMSCs studies phenotype identification and function. Therefore, we found that the following conclusions: 1. Establishedenhanced VEGF expression BMSCs cell line with hTERT and VEGF gene lentivirus vector combined modified elderly patients with BMSCs, which has important clinical value.2.Enhanced VEGF expression BMSCs cell line with hTERT and VEGF gene lentivirus vector combined modified elderly patients with BMSCs expresses the characteristics of the surface of BMSCs cells, can be amplified in vitro without limit, and can secrete VEGF protein function.3. Enhanced VEGF expression BMSCs cell line with hTERT and VEGF gene lentivirus vector combined modified elderly patients with BMSCs has fast cell proliferation, strong ability of migration, notinvasiveness and tumorigenicity, and chromosome karyotype stability.