Different Periods Of Filum Terminale And Growth Factor Effect On Neural Stem Cells Of Proliferation And Differentiation

BackgroundCongenital spinal cord lesion is a diseases with serious impact on children central nervous system development.It’s Include tethered cord syndrome(TCS),Split cord malformation(SCM), myelomeningocele.Especially TCS with impact on children growth development.TCS is due to the lesions filum terminale to stretch the end of the conus, result in changes of spinal cord ischemia hypoxia, which led to the bowel and bladder dysfunction, deformity of the lower limbs and sensory dysfunction.At present TCS main treatment is resection the lesion filum terminale, release the end of the spinal cord,gradually returned to normal levels, and then terminate or improvement of clinical symptoms.But this treatment the results are unsatisfactory.In1992Reynolds isolated from mouse striatum in vitro proliferation of pluripotent cells, proposed the concept of neural stem cells (neural stem cells, NSCs).The concept of NSCs changed people in the past for the nerve cell is a permanent cell damage non-renewable understanding.Give a new hope of treatment diseases of the nervous system, as well as a new treatment strategy-neural stem cell transplantation for the treatment of central nervous system diseases. In the past spinal cord NSCs that exists only in the subependymal,there are no NSCs in the filum terminale.In the past the filum terminale is considered just a simple structure of fibrous tissue.Mercy and Varghese respective in2009and2010use of the human filum terminale tissue to culture the NSCs, then prove there is NSCs in human filum terminale tissue.This finding change people to understanding of the NSCs again, currently there is no reported on use of the human filum terminale tissue to culture NSCs in the domestic.In this study we use the fetal filum terminale tissue to culture NSCs preliminary study for isolating and identifying in the NSCs. This study provides some reference for isolation and culture of NSCs.Objective1. By primary cultured the NSCs with filum terminale tissue and identification the cells with Nestin.with positive expression is NSCs.Prove there are NSCs in the filum terminale tissue.2. To compare the characteristics of growth number and differentiation of NSCs in different types of filum terminale tissue.3. Observed the characteristics of proliferation and differentiation to NSCs, analysis epidermal growth factor (EGF), fibroblast growth factor (bFGF),and leukemia inhibitory factor (LIF) impact on NSCs in growth number and differentiation.Materials and methods1. Collect15cases of12to8w abortion Fetus in2011.11-2012.8provided by third affiliated hospital of zhengzhou university department of Obstetrics.Based on the gestational age divided into3groups:group A (12to17W), group B (18to23W), group C (24to28W). Primary cultured NSCs with the filum terminale tissue and periodical observe the growth of NSCs with inverted microscope.Count the number of neurospheres compare the growth characteristics of the three groups of NSCs.Identification the cells with Nestin.positive expression is NSCs.Prove there are NSCs in the filum terminale tissue.2. Collect9cases of12to20w abortion Fetus.Based on additives different factors in NSCs basal medium DMEM/F12to divided into three groups:group A (DMEM/F12+B27+EGF+bFGF), B (DMEM/F12+B27+EGF+bFGF+LIF), C group(DMEM/F12+B27).C group is the control group.Observed three groups of NSCs growth,induced differentiation NSCs with fetal bovine serum.Identify three groups NSCs in NSE and GFAP with immunocytochemistry method,observed proportion of differentiation in NSCs.Analysis EGF,bFGF and LIF affect on growth number and differentiation of NSCs.3. Collect5cases filum terminale tissue of TCS Children in2011.09-2012.05provided by third affiliated hospital of zhengzhou university department of pediatricorthopedics. Communication with parents of children with their agreement and consent hospital ethics committee agreement in preoperative, then to experimental study.Observe the Lesions and normal filum terminale tissue NSCs proliferation and differentiation,Compare characteristics of NSCs growth number and differentiation in different types of filum terminale tissue.4. Statistical analyzes were performed using SPSS17.0statistical package for analysis. The experimental results expressed as(x±s), one-way ANOVA was used to compare the three groups of NSCs growth number,NSE and GFAP percentage.LSD-t test was used to compare the two groupsof NSCs growth number,NSE and GFAP percentage. Significant level being0.05.Result1. Observed cells morphology by inverted microscope, found those cells have NSCs morphological characteristics,those cells expression was positive by nestin identified, indicating that the cells are NSCs.2. The number of NSCs cultured in different gestational age groups with differences, analysis results of the three groups with one-way ANOVA (p＜0.05).Prompt NSCs growth number in different gestational age groups had significant difference, each two comparison the results of three groups,compare group A and B(P＞0.05), no significant difference, compare group A and C,group B and C(p＜0.05), the difference was statistically significant.3. NSCs growth number and differentiation have differences in different NSCs culture medium,analysis results of the three groups with one-way ANOVA(p＜0.05). Prompt used contain different cell factor medium to isolated and cultured NSCs, in growth number, NSE and GFAP-positive cell percentage were significant differences. In number of NSCs, compare three groups(P<0.05), the difference was statistically significant. In the differentiation of NSCs,compare three groups(p＜0.05), the difference was statistically significant.4. NSCs growth number and differentiation have differences in different types of the filum terminale tissue, analysis results of the two groups with one-way ANOVA (p＜0.05), the difference was statistically significant.Prompt using different types of filum terminale tissue to isolated and cultured NSCs,in the growth number and differentiation were significantly different.Conclusion1. The filum terminale tissue under specific conditions can isolation and culture the NSCs,to prove there are NSCs existence in the final filament tissue. Gestational age and the NSCs they are closely related.This may be related to the proliferation potential of NSCs in the Young embryonic group.2. Different growth factors influence the proliferation and differentiation of NSCs in the filum terminale tissue, LIF has obvious effect in promote NSCs proliferation and inhibit differentiation.3. Lesions final filament and normal filum terminale both cultured NSCs,but the number of neurospheres and the NSE proportion in differentiation of neurospheres the normal filum terminale group was significantly higher compared with the lesions filum terminale group.The normal fetal filum terminale tissue is suitable as tissue-derived to isolation and culture NSCs in vitro.