The Effect Of Different Sources Of Endothelial Progenitor Cells On Proliferation And Differentiation To The Neural Stem Cells

A Comparative Study on the Characteristics between the Different Gestational Ages of Spinal Neural Stem Cells from SD Fetal Rats.Part 1Objective To study and discuss the differentiation and proliferation ability of the spinal neural stem cells at different gestational age in SD fetal rats.Methods Three different gestational age of SD fetal rats were randomly divided into three groups.Marked group A(12 days of pregnancy)、group B(14 days of pregnancy)and group C(16 days of pregnancy).Neural stem cells were separated by the method of enzyme-assisted microdis-section.The diameter and each number of neural stem cells were measured at different times.The cell growth curve were drew with CCK8 colorimeter. Neural stem cells were identified with 5-Brdu BrdU/Nestin immuno-histochemical staining.We induced neural stem cells differentiate into nerve cells and astrocytes with 10% fetal bovine serum.Ten days later,immunocytochemistry was used to examine the expression of the neuronal specific marker β-tubulinⅢ and glial fibrillary acidic protein.Finally,We analyzed the differentiation proportion of neural stem cells among different groups by the software of SPSS statistics.Results It was indicated by immunofluorescence staining that neural stem cells and the differentiated cells were positive for Brdu/Nestin/β-tubulinⅢ/GFAP staining.There was a significant difference in the rates of the proliferation anddifferentiation(22.74±0.79%) of neural stem cells between the group B and other two groups(P<0.05).However, no significant difference in differentiation was seen between group B and group C. But the cell vitality in the 5th day of third-generation neural stem cells in group C has a notable difference compared with group B(OD0.5107±0.0272)(P < 0.05),but the differentiation of the protoplasmic astrocytes in group C with the highest proportion(53.19±2.42%)among the whole groups.Conclusion The method of enzyme-assisted microdissection gave neural stem cells intact morphology, high quantity, vigorous growth and maintained a lot of biologic and morphologic characteristics in vitro.Isolate the neural stem cells from 14 days of pregnancy pregnant rats will get a higher percentage of neuronal differentiation(22.74±0.79%),but separate out neural stem cells from 16 days of pregnancy pregnant rats will more easily and will get a higher percentage of protoplasmic astrocytes which will promote the differentiation of neurons.Exploring the difference in biological characteristics between spinal neural stem cells and brain-derived neural stem cells Part 2Objective To isolate and culture spinal neural stem cells and brain-derived neural stem cells from gestational age 14 days of SD fetal rats and to study the cultivation condition in vitro and differentiation of neural stem cells from the brain and spinal cord and to explore the difference in biological characteristics between two sources of neural stem cells.Methods Neural stem cells were isolated from gestational age 14 days of SD fetal rats by the method of enzyme-assisted microdis-section.The serum-free culturing technology was used to isolate and culture neural stem cells. we detected expressions of Nestin,Brd U by using indirect immunofluorescent staining.Proliferation was evaluated by direct cell counting and CCK-8 wad used to examine the cells viability.Neural stem cells were differentiated into nerve cells and astrocytes by 10%fetal bovine serum and immunocytochemistry was used to examine the expression ofβ-tubulinⅢ and GFAP.In the end we analyzed the proportion of neural stem cells between spinal neural stem cells and brain-derived neural stem cells by the software of SPSS statistics.Results Neural stem cells from the pallium and spinal cord of SD fetal rat at age of gestational 14 days can be cultured, passaged and differentiated,and immunohistochemistry demonstrated that the different origin cells were positive for Nestin, Brdu,β-tubulinⅢ and GFAP.There were significant differences in the rates of the neural differentiation and the cell viability between spinal neural stem cells and brain-derived neural stem cells.(P<0.05). The differentiation rate of neurons from brain-derived neural stem cells(27.56±0.79)% is higher than spinal neural stem cells(25.58±0.72)%. In addition, the spinal neural stem cells has a notable difference on protoplasmic astrocytes differentiation rate( 53.09±2.88) % compared with brain-derived neural stem cells(50.60±2.88)%.Conclusion Neural stem cells from the fetal rat cerebral cortex and spinal cord tissue can be cultured, passaged and differentiated. Brain-derived neural stem cells have a higher differentiation rate of neurons than spinal neural stem cells.The biological effect of different sources of endothelial progenitor cells to the spinal cord derived neural stem cells.Part3Objective To explore the peripheral blood derived endothelial progenitor cells and bone marrow derived endothelial progenitor cells affect the proliferation and differentiation ability of spinal cord derived neural stem cells.Methods Endothelial progenitor were isolated and cultured with the method of density gradient centrifugation from Peripheral blood and bone marrow. EPCs were cultured in high glucose DMEM + 20% FBS and 50 ng/m L of VEGF163.Immunocytochemistry including detection of markers CD34 and v WF and double-staining for Dil-Ac-LDL and FITC-UEA-1 verified the purity of the cultured EPCs. Spinal neural stem cells were separated by the method of enzyme-assisted microdis-section from 14 days of pregnancy pregnant rats and were identified with Nestin immunohistochemical staining.We induced spinal neural stem cells differentiate into neuron and astrocytes with neurbasal medium +10% fetal bovine serum. Cells cultured in Transwell were divided into three groups: control group(C):cultured with free DMEM medium+NSCs; experimental group A:peripheral blood derived endothelial progenitor cells+NSCs and experimental group B:bone marrow derived endothelial progenitor cells+NSCs. We measured and compared diameter and each number of neural stem cells at different times,and specific marker β-tubulinⅢand glial fibrillary acidic protein were used to examine and compare the expression of the neuronal and astrocyte among the three groups.Results As shown in Figure 1, the peripheral blood derived endothelial progenitor cells and bone marrow derived endothelial progenitor cells were positive for markers CD34 and v WF and double-staining for Dil-Ac-LDL and FITC-UEA-1,but there were apparent differences in the morphological changes between the two different kinds of EPCs as shown in figure 4,as well, the spinal neural stem cells and the differentiated cells were positive for Nestin,β-tubulinⅢ and GFAP staining. The results showed that the two experimental groups have a greater proliferation activity than the control group(P<0.05), in addition,group B has a notable difference on neuronal differentiation rate(27.37±0.80)%and protoplasmic astrocytes differentiation rate(53.78±2.37)% compared with other two groups.Conclusion The experiment showed the bone marrow derived endothelial progenitor cells have a greater ability to promote pinal neural stem cells differentiate into neuron than peripheral blood derived endothelial progenitor cells.