Effect Of Endothelial Progenitor Cells On The Differentiation Of Neural Stem Cells And Its Mechanism

Spine and spinal cord injury (SCI),a common severe trauma often happens in the traffic, labor and sport accidents. Accompanied by the rapid development of modern society, which facilitates the people's life , on the other hand it brings many traumatic accidents. Trauma is also called "developed social sickness". SCI is a kind of extremely severe damage that has high disability rate and is one of the tough problems in the medical. It can result in severe impairment of sensory, motor and autonomic function below the level of injury. More than 11,000 SCI patients are estimated to occur in the United States each year and it has about 253,000 patients with SCI, These patients need 60 billion dollars for the medical cost each year. The number of patients with SCI in China is about 400 thousand and it will increase 10 thousand persons each year. This misfortune brings a lot of trouble and heavy burden to the patients themselves, their family and the whole society. Unfortunately, aside from good medical management, no generally accepted interventional therapies are available. Therefore it is important for the neurologic scientists to put more emphasis on the research for repair of SCI and rehabilitation of paraplegia patients.However, the treatment by tissue engineering on the SCI has brought a ray of hope to the patients with SCI. Tissue engineering is a new branch of biomedical engineering. It is the application of life sciences and engineering techniques to construct different tissues to repair the structure and function of damaged tissues and organs. The study of tissue engineering involves three aspects: seeding cells, scaffold materials and constructing tissues in vitro. Embryonic stem cells(ESCs), neural stem cells(NSCs) and mesenchymal stem cells(MSCs), olfactory ensheathing cells(OECs),schwann cells and fibroblasts are usually choosed as the seeding cells in tissue-engineered spinal cord. The main mechanisms of SCI treatment with cellular transplantation are as follows: 1) The transplanted cells can replace the injured or necrotic neurons; 2) The transplanted cells may filled with defect of injured spinal cord, which offers place and clues for adherence of neurons and axonal elongation; 3) The transplanted cells will provide various neurotrophic factors to promote axonal regeneration; 4) The transplanted cells will play an indirect role for the regeneration: such as promoting angiogenesis and providing basis for regenerative and endogenous cells.NSCs are considered to be the best choice for constructing tissue-engineered spinal cord. NSCs are a kind of stem cells with self-renewal and multi-directional differentiation potentials in the mammal embryonic neural tissue and the adult animal CNS. NSCs can also differentiate into three different lineages: neurons, astrocytes and oligodendrocytes under some certain conditions. It is easy to obtain, culture and identification, meanwhile, with a characteristic of great proliferation and differentiation potential in vitro, and so on. NSCs can be used as the candidate in transplantation. Although the function of NSCs is theoretically perfect, however, researchers have found out that there are many problems in application of NSCs individually for the treatment of adult SCI: the most of NSCs differentiated into glial cells, however few into neurons, and the transplanted cells could not form the effective synaptic connections. Due to the injured partial of spinal cord's blood supply can not be promptly restored and necrotic tissue and toxic cytokines can not be quickly removed, so avascular necrosis frequently occurs to the neural cells in the graft, leading to failure of repair. Therefore, how to promote the differentiation of transplanted NSCs in the injured spinal cord into neurons and promote the vascularization in injured partial of spinal cord is the key problems, which limited the application of tissue engineering in repairing of SCI.Endothelial progenitor cells(EPCs), firstly reported by Asahara in 1997, are isolated from adult peripheral blood and are a group of precursor cells of endothelial cells. EPCs are a group cells with migrating characteristics, which can proliferate and differentiate into endothelial cells. EPCs not only contribute to embryonic angiogenesis, but also play an important role in postnatal neovascularization. These cells can home to the site of ischemia, proliferate and differentiate into mature endothelial cells and contribute to new blood vessel formation. These features make EPCs have a broad clinical application and EPCs can be used as seeding cells in the tissue engineering research. In recent years, the studies on functions of EPCs mainly focus on repairing of ischemic myocardium, ischemic limbs, injured skin and cornea,etc. They have not been used for SCI treatment. Thus in this research, NSCs and EPCs were choosed as seeding cells to construct tissue-engineered spinal cord to repair SCI, which can meet the need of "nerve nutrition " and "blood vessel nutrition" , this two main problems. However, NSCs and EPCs are originated from different germ layers and they require different growing environment in vivo and in vitro. Thus, it is necessary to co-cultured these two seeding cells in vitro before transplanting into host, which can provide experimental basis and theory support to build vascularized tissue engineering to repairing SCI and central nervous system diseases. So, in this experiment, EPCs were isolated and cultured from rat peripheral blood mononuclear cells at first, then co-cultured with NSCs in vitro. The effects of EPCs on inducing NSCs proliferation and differentiation was examined and finally the mechanism of which was investigated .Main Methods and TechniquesThe experiment was divided into three parts1. NSCs were isolated from E14 cortex of Sprague-Dawley rats and induced to differentiation by serum. The indentification of NSCs were examinated by immunocyto- chemistry method with antibodies of Nestin,β-tubulin-Ⅲand GFAP to identify NSCs respectively. Mononuclear cells were separated from rat peripheral blood by density gradient centrifugation combined with different rate of adhering characteristics. EPCs were identified by morphology observation and immunofluorescent examination.2. The NSCs and EPCs were co-cultured in 3 different media: NB, DMEM and NB + DMEM medium. Then the growing behaviors of these two kinds of cells in different group were observe to screening the suitable conditioned medium. The regulation of EPCs on inducing NSCs proliferation and differentiation was detected and the differentiating trend of NSCs co-culture with EPCs at different rate was primarily discussed.3. The effect mechanism of NSCs and EPCs co-cultured in vitro was investigated. The content of VEGF and BDNF in EPCs, NSCs and EPCs co-culture media was examined by ELISA. The affect of VEGF combined with bFGF on NSCs differentiation was observed. At the same time, whether VEGF plays main roles in inducing NSCs differentiating into neurons when co-cultured with EPCs was proved through interfering with antibody of VEGF.Main Results and Conclusion are as follow:1. It is easy to obtain, culture and identification NSCs. NSCs also can differentiate into three main different neural lineages: neurons, astrocytes and oligodendrocytes under some certain conditions. The rat peripheral blood is a rich source for EPCs and EPCs can be induced to differentiate into endothelial cells directly under the conditional culture system. These suggested that NSCs and EPCs are successfully cultured as seeding cells in vitro under this condition。2. The suitable condition for co-culturing NSCs and EPCs is NB+DMEM media . EPCs could promote NSCs proliferation and the percentage of neurons in EPCs group(68.40%) was much more than those in fetal bovine serum group(28.70%). In certain ranges(the ratio of EPCs and NSCs from 0.1:1 to 10:1), the more the EPCs, the high rate of neurons is. These results suggested that the co-culture of EPCs and NSCs is successful in this study and EPCs can promote the differetiation of NSCs to neurons.3. The content of VEGF and BDNF in EPCs, EPCs and NSCs supernatant solution was 813.13pg/ml, 917.78pg/ml and 46.03pg/ml, 88.53pg/ml respectively by ELISA examination, which are remarkably more than these of NSCs. It suggested that EPCs mainly express and secrete the VEGF.4. The ratio of neuron differentiated from NSCs with VEGF and bFGF medium is 60.3%, 60.4% respectively, and up to 80.3% with both VEGF + bFGF. It suggested that VEGF can promote NSCs, differentiating into neurons significantly and present synergistic effect on NSCs, differentiation into neuron with bFGF.5. The effect of EPCs inducing NSCs differentiating into neurons in co-culture and then the action can be inhibited by VEGF antibody, but the role can be of inversion by added into equal VEGF. These results demonstrated that EPCs can promote the differentiaton of NSCs onto neuron through secreting VEGF.