| Background:Cerebrovascular disease is one of three lethal diseases that can do serious harm to people’s health. It has a high incidence of morbidity, disability rate, mortality and recurrence rate, which brings a great burden to family and society. Among cerebrovascular diseases, ischemic cerebrovascular disease accounted for80%. However, only one drug-recombinant tissue plasminogen activator (rt-PA) has got the FDA approval for the treatment of acute cerebral ischemia so far. But rt-PA treatment has strict time limit:must be applied in within3hours after stroke occurred. In addition, rt-PA therapy increased risk of cerebral hemorrhage. So how to promote the effective tissue and function restoration after cerebral ischemia is both a hot point and difficult point in recent research. To this end, stem cell transplantation has spring up as a favorable method to treat cerebral ischemia with good clinical application prospects recently.Bone marrow derived mesenchymal stem cell (BMSCs) is a kind of multipotent adult stem cells derived from bone marrow, the main advantage of its application in cell therapy is autologous transplantation, no transplantation immune rejection, no ethics problem and induced tumor limitations, compared with embryonic stem cell transplantation; and BMSCs is relatively simple, unlimited and easy to obtain, compared with neural stem cells, which were mainly located in the sub-ventricular 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 through BMSCs transplantation. The BMSCs transplanted treatment with rat cerebral MCAO by vein, artery and stereotactic local injection had found that transplanted cells could directly repair the focal ischemic area, and on the other hand indirectly repair the focal ischemic area by 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 because BMSCs have the ability to differentiate into neural cells. However, most studies found that BMSCs transplantation only had few 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 cannot 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 factors in 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 in brain ischemia, is subject to cell transplantation therapy);②In cell amplification process in vitro, BMSCs cell morphology will gradually change (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 components are 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 limits cell proliferation. In contrast, tumor cells and embryonic stem cells sustain telomere function through the activation of telomerase.BMSCs cannot 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 labs have in biochemical research with BMSCs immortalization. There were able to successfully establish BMSCs’ immortalization cell 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, and sustain 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 be immortalization by transfecting hTERT gene. In the previous studies, there were not found significant malignant cells in using the 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 PDGFR and 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 apply human telomerase catalytic subunit h.TERT gene lentiviral expression vector and the expression of VEGF gene lentiviral expression vector of BMSCs gene to modify the elderly hBMSCs, 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. Human bone marrow stromal cells (hBMSC) isolation, culture and identification: The human BMSCs were harvested from three healthy male donors, aged64,67and71respectively. The study was approved by the local institutional review board of Southern Medical University affiliated Zhujiang Hospital. hBMSCs were harvested and cultured as described previously. Briefly,5-10ml bone marrow was diluted to20ml with the culture medium (Dulbecco’s-modified Eagle’s Medium-Low Glucose supplemented with GlutaMAX [DMEM-LG Gibco Invitrogen, Carlsbad, CA] with10%fetal bovine serum[Gibco Invitrogen, Carlsbad, CA],1%penicillin/streptomycin [Gibco Invitrogen]),and cultured in two10cm petri dishes in a humidified incubator at37℃and5%CO2. After48hours, the non-adherent cells were removed by replacing the medium. The medium was changed subsequently every4days. About2-3weeks later the culture reached90%confluence. Then the cells from every dish were lifted by0.05%trypsin and0.02%EDTA (Hyclone) and replated in two new dishes as passage1(P1) cells. Since P1cells were not enough for the follow-up experiments, the passage2(P2) cells were used for subsequent transfection. WhenP2cells reached50-60%confluence, transfection of hBMSCs was carried out with hTERT lentiviral (MOI:5, PU/cell) or VEGF165(MOI:5, PU/cell) or both two constructs (MOI:5, PU/cell) in the presence of8μg/mL polybrene (sc-134220, Santa Cruz, CA95060USA). Then the cells were divided into four groups:hBMSC (untreated), hBMSC-V, hBMSC-T and hBMSC-V&T. Successfully transfected cells were selected with blasticidin at a final concentration of5μg/mL (Blasticidin, Invitrogen) for7days. Afterwards, the culture medium was changed to normal full medium for further maintenance.2. groups of Experiment:cells were divided into the following five groups:①unmodified BMSCs group;②empty lentiviral modification group;③VEGF gene modification group;④hTERT gene modification group;⑤hTERT/VEGF double gene modification group.3. Flow cytometry method detected cells’changes in different group cell surface markers;4. Matrigel matrix (BD Biosciences) was added to a24-well plate at0.5mL/well and incubated for3h at37℃. hBMSCs (104/well) were added in0.5mL serum-free medium containing10ng/mL VEGF and incubated for6h at37℃. The plate was observed and photographed under an inverted microscope (CKX41, OLYMPUS). Totally12visual fields of3wells were selected and calculated in four groups. Percentage of cells form capillary structures was calculated by capillary structure cells divided by all the cells in a visual field.5. Analysis of capillary formation in vivo was performed as described previously. Briefly,158-week male athymic nude mice weighing25-30g were raised in specific pathogen-free conditions. The DiI labeled PD10cells (5×105) of four groups suspended in0.5mL Matrigel were injected subcutaneously in the back of recipient mice using a23-G needle, carefully positioning the needle between the epidermis and the muscle layer. Cell-free Matrigel plugs with serum-free culture medium of the same size were used as control here. Three weeks later, mice were deeply anaesthetized by sodium pentobarbital and sacrificed. Then the plugs were removed, fixed with formalin for24hrs in room temperature, and subsequently cut into10-μm thick sections on a freezing microtome. The sections were stained with immunofluorescence stained for vWF.9visual fields of each group were selected and photographed under an immunofluorescence microscope. Vessel circumference length was calculated by Image-Pro Plus. Vessel density was calculated by numbers of vessels divided by the area of every visual field.6. Cell transplantation therapy was applied to transient MCAO model rats. Multi-level tests such as behavioral activities, histological analysis, cytology and protein were applied to test the effectiveness of the therapy, and reveal Its possible mechanism.7. MCAO rats with cell transplantation were tested by CatWalk behavioral analysis system, in order to find the most suitable indicator, and evaluate the effect of the treatment.8. Brain tissue of MCAO rats were studied for outcome of transplanted cells in the body and its effect of secretion of cytokines on ischemic brain.7. 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. Previous experiments verified lentivirus on hBMSCs transfection is successful and effective. Cell lifespan had been significantly prolonged. Telomerase and the expression of VEGF were significantly increased. There is no evidence of tumorigenicity.2. On the basis of the preliminary experiment results, our further experiments show that VEGF and hTERT transfection can dramatically enhance angiogenesis ability of hBMSCs in vitro.3. Immunofluorescence showed DiI labeled transplanted cells increased the formation of vascular structure with expression of vWF in Nude mice. VEGF and hTERT transfection can significantly improve the formation of vascular structure in density and perimeter after hBMSC transplanted in nude mice.4. NSS were improved after transplantation, hBMSC-V&T has the most obvious result (P<0.05).5. TTC results revealed the infarction area have decreased in rats, among which hBMSC-V&T and hBMSC-V groups were more obvious than other groups (P<0.05).6. MRI imaging results are basic consistent with TTC staining results.7. Model mouse tissue section showed that transplanted cells mainly distributed in the ischemic penumbra region. There is still a strong fluorescence expression at the end of observation.8. Genetic testing results show that the tissue of model mouse VEGF (human and rat), hTERT (human), synaptophysin p38and growth protein GAP-43expression increased. CD31staining show that cells are integrated into the newborn blood vessels and blood vessel number and density have increased compared with control group (P<0.05).Conclusions:1. The method of transfection hBMSCs with VEGF and hTERT genes by lentivirus is feasible, effective and safe, which has important clinical value.2. The verification of therapeutic effect of modified cells in transient cerebral ischemia model of rats is significantly. Neural functional recovery was obviously improved compared with the control group.3. Possible mechanisms of its treatment effect may be mainly increased secretion of neurotrophic factor around the ischemic zone, and promoted formation of new blood vessels in the local area. |
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