| There are two main methods in experimental study of using neural stem cells (NSCs) to repair spinal cord injury (SCI). One way is through transplantation of NSCs into the spinal cord injury site, by exogenous NSCs proliferation and differentiation, alternativing the damage neurons, reconstructing the neural pathways to repair the injured spinal cord; the other way is through all kinds means inducing the proliferation and differentiation of endogenous NSCs in spinal cord injury site, alternativing the damage neurons, reconstructing the neural pathways to repair the injured spinal cord. Current researchs indicate that exogenous NSCs transplanted into spinal cord injury site is essentially improved the local environment of the site of injury after spinal cord injury, and ultimately by improving the environment to induce the of endogenous NSCs’ neurogenesis to promote the recovery of the injured spinal cord.Neurogenesis is often accompanied by blood vessels, suggesting that vascular factors play an important role in nerve repair process, effective vascular germinal should be able to bring significant improvement in spinal microenvironment, thereby promoting neurogenesis in injury site. Endothelial progenitor cells (EPCs) are a kind of stem cells which can get isolated and cultured from peripheral blood, and can effectively promote vascular germinal. This experiment is designed to isolate EPCs from peripheral blood of Sprague-Dawley SD rats and transplant into the spinal cord of SCI rats by promoting vascular germinal to improve inner environment of SCI and neurogenesis of NSCs, which rebuild neural pathways to repair spinal cord injury. At first, study isolated and cultured rat peripheral blood-derived EPCs and spinal cord derived NSCs. Then co-cultured peripheral blood-derived EPCs and spinal cord derived NSCs in vitro. The study identified the impact of peripheral blood derived EPCs to spinal cord derived NSCs’proliferation and differentiation by inverted microscope and immunohistochemical methods, and assayed the supernatant of VEGF (Vascular endothelial growth factor) content changes, study its mechanism. Next, the study transplanted the peripheral blood-derived EPCs into injury site of SCI rat model. Through BBB ratings and Rivlin oblique pull test we observated the recovery of SCI. By histological observation of neuronal regeneration and immunofluorescence staining we observated the proliferation and differentiation of spinal cord derived NSCs. Identify its role in promoting neurogenesis, identify expression of VEGF to reflect the mechanism of peripheral blood-derived EPCs repairing spinal cord nerve function. To lay foundation of achieve autologous EPCs promote endogenous NSCs germinal effective clinical application in spinal cord injury research.The study is divided into four parts. In the first experiment,5-7 days SPF neonatal SD rats were sacrificed by cervical dislocation, remove the thoracolumbar spinal cord tissue under sterile conditions, removal of excess blood vessels and soft meninges, cut, filtration, centrifugation, separation, and the neural stem cells balls in suspension grown were successfully isolated and cultured, by immunohistochemistry to identify which can express neural stem cells specific antigens nestin. When serum was added to cultured it can differentiate into cells with axons and dendrites which were identified by immunohistochemistry expressing neuron-specific antigen β-tubulin-III, namely neurons, ELISA measured that the 7 days’culture supernatant was visible VEGF and brain-derived neurotrophic factor (BDNF) expression, indicating that the isolated and cultured cells may be determined to be rat spinal cord derived NSCs, and can secrete VEGF and BDNF.In the second experiment, the male SPF SD rats in weight of 90～120 g were intraperitoneal anesthesiaed with 10% chloral hydrate (0.3 ml/100 g), after anesthesia satisfaction taking the neck incision under sterile conditions, cut skin and subcutaneous tissue open to the sides and fixed, carefully separate the muscles along the gap to expose the double carotid artery, and then use a sterile medical blood collection tube take blood from bilateral carotid arteries, about 10 ml, transferred over net workbench immediately after the completion of blood collection, isolated mononuclear cell layer with lymphocyte separation medium according to kit instructions, the cells were washed by centrifugation, the cell pellet obtained suspended in EBM-2 complete medium and weight to 5×105/ml cells, then plated in the 24-well plates which contained pre-FN-coated cover slips,37℃, constant temperature incubator with 5% CO2. About 9 d around, cell fusion reached about 90% and passaged. Cell morphology observed was cobblestone arrangement which consistent with reported in the literature. Identified by immunohistochemistry expressing cell surface antigen CD133 and VEGFR-2, Dil-Ac-LDL uptake and binding FITC-UEA-1 double positive, indicating success isolated and cultured EPCs from peripheral blood.ELISA measured culture supernatant of 7 days seen a lot of expression VEGF, BDNF little expression, indicating that EPCs can secrete a large number of VEGF.In the third experiment, subjects were randomly divided into three groups:EPCs and NSCs co-cultured group, anti-VEGF group and the control group. The control group: collect the 3rd generation of NSCs after purified culture, percussion into a single cell suspension and inculated in 24 holes plates, incubated for 7 days to observe the growth, then with 5% serum induced culture NSCs 7 d, observed differentiation. EPCs and NSCs co-cultured group:on the basis of the control group,24-well plates Transwell were placed in the hole, taking cover glass containing adherent good growth primary EPCs, EPCs on the Transwell membrane, NSCs located under the Transwell membrane, culture 7 days to observe the growth of NSCs, then remove EPCs and Transwell membrane, plus 5% serum induced cultured NSCs 7 d, observed differentiation. Anti-VEGF group:on the basis of EPCs and NSCs co-cultured group,24-well plates Transwell were placed in the hole, taking cover glass containing adherent good growth primary EPCs, EPCs on the Transwell membrane, NSCs located under the Transwell membrane, culture 7 days to observe the growth of NSCs, then remove EPCs and Transwell membrane, plus 5% serum induced cultured NSCs 7 d, observed differentiation.The results can be seen under an inverted microscope on day 7, the number of neurons ball of co-culture group significantly more than the control group, and significantly increased the ball diameter,, the difference of the co-culture group and the control group was statistically significant (P<0.05); after 5% serum induced culture NSCs 7 d, (3-tubulin-Ⅲ immunofluorescence staining, calculated percentage of β-tubulin-Ⅲ+/total cells under the microscope, co-culture group was significantly higher (P<0.05). ELISA assay supernatant VEGF levels of three groups cultured of 7 days and found in anti-VEGF group VEGF levels were lower than EPCs and NSCs co-culture group (P<0.05), while the ability to induce proliferation and differentiation of NSCs also significantly lower (P<0.05). EPCs in vitro can significantly promote the proliferation and differentiation of spinal cord derived NSCs which relevant with VEGF secreting.In the fourth experiment,4-month-old SPF SD rats were randomly divided into EPCs transplantation group, NSCs transplantation group and control group,24 in every group. All rats were given 50μg/g BrdU per day by intraperitoneal injection from three days before surgery until a week after surgery. After using modified Allen’s method to prepare spinal cord injury model in rats, in EPCs transplantation group, we injected EPCs 5μl into spinal cord about 0.5～1.0mm in the proximalion of the lesion with Hamilton micro-syringe, the total number of cells was 5×105/ml, in NSCs transplantation, we injected NSCs 5μl into spinal cord about 0.5～1.0mm in the proximalion of the lesion with Hamilton micro-syringe, the total number of cells was 5 x 105/ml, in the control group, we injected saline 5μl into spinal cord about 0.5～ 1.0mm in the proximalion of the lesion with Hamilton micro-syringe. In 1,2,4, and 6 weeks after surgery using the BBB motor function scoring system, Rivlin swash plate motion test for detection of motor function recovery in rats; histological observation and double immunofluorescence staining for pathological changes of spinal cord tissue and EPCs’Effect of endogenous NSCs proliferation and differentiation, ELISA test detected the changes of expression of VEGF in the spinal cord content. It was found that after the 2th week BBB motor function score of EPCs transplantation group was significantly higher than NSCs transplantation group and control group, the difference was statistically significant (P<0.05). Histological observation showed that EPCs transplantation group structures and morphologies of neurons in the spinal cord recover better and becoming more normal, double immunofluorescence staining showed that spinal cord tissue that transplanted EPCs BrdU+/β-tubulin-III+cells compared with NSCs transplantation group and control group increased, the difference was statistically significant (P<0.05), ELISA test found that EPCs transplanted spinal cord tissue VEGF level was higher than the other two groups (P<0.05). These results suggest that EPCs can so as to promote the recovery of neurological function in rats with spinal cord injury in vivo environment by promoting endogenous NSCs proliferation and differentiation, and may be associated with VEGF secretion of EPCs.In summary, in the rat spinal cord tissue can be successfully extracted endogenous NSCs, EPCs can be successfully isolated from peripheral blood, peripheral blood-derived EPCs in vitro could promote the proliferation and differentiation of endogenous NSCs. Spinal cord injury in rats transplanted EPCs can promote the proliferation and differentiation of endogenous NSCs and promote the recovery of neurological function in rats with spinal cord injury and may be associated with VEGF secretion of EPCs. |
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