| BackgroundHypoxic-ischemic brain damage (HIBD) is a disease caused by all kinds of cerebral hypoxia and reduces or disrupts of your cerebral blood flow, which leads to encephaledema and then reconstruction of brain. Besides, it also results in continuous loss of brain cells and vesiculation of substantial alba in the surrounding of brain ventricle and neighborhood cortex. It can lead to mass loss of nerve cells, which in turn cause certain kinds of functional disturbance of nerve. And it is the primary cause of children's cerebral palsy. The number of children with cerebral palsy is becoming larger and larger as the improvement of salvage of neonate in recent years. In recent years, the therapeutic method with the cerebral palsy was unsatisfied. It is both a new conviction and a new way to cure HIBD children that to find a new way to reconstruction of the cell, and replace the injured neural cell. Transplant of neural stem cells (NSCs) is a new therapeutic method that researchers have been work for. Stem cell can be obtained by umbilical cord blood, umbilical cord, bone marrow, brain of fetus, amnion cell of placenta, and be induced into neural stem cells. It also can proliferate and differentiate into kinds of neural cells, such as:neurons, glial cell, oligodendroglia cell etc. However, as it is limited by a lot of factor, when it is induced in vitro, the success ratio is very low. Besides, the ratio of survival cell is also too low after it was transplanted into the host. So genetic engineering of neural stem cells becomes a major source of neural stem cells. Recent researches show that neural stem cells are rich in embryo brain. Scientists have been study the development regular of embryo brain, they show that it is regulated by several kinds is an essential gene which can regulate and control the development of brain structure. It will lead to cerebral hemisphere dysplasia if it is absence. It has been proved that it is reappearance and continuance of embryo after newborn animal suffer a Hypoxic-ischemic brain damage, and Foxgl plays a vital role during the development of brain. Foxgl gene can be expressed continuously after born in the sub ventricular zone (SVZa) and sub granular zone (SGZ), which is famous as the stem cells assemble zone. It also plays a vital role during the process of proliferation and the differentiation of neural stem cells. It is confirmed that NSCs transplant has got a favorable curative effect in the therapy of brain damage, Parkinson syndrome, demyelinating diseases, Alzheimer disease, cerebral infarction, brain tumor, spinal injury, and epilepsy, which can't be cured with common mean of therapy provides a wide thread and enticeable perspective to the scientist. It is proved that the NSCs is controlled by multiple factors during the differentiation, such as genes, translate factors and the micro-environment of NSCs. Of all the factors, Foxgl is one of them. However, what is the mechanism that the Foxgl control the NSCs? What is its role during the proliferation and differentiation? We don't have good answers. However, overseas'studies have shown that, Foxgl can regulate neural stem cell proliferation by inhibiting the expression of p21 gene. In our study, cord blood derived neural stem cell was transplanted and observed in neonatal rat brain with HIBD in SGZ area. To observe the impact of NSCs transplantation on the two genes. Provide genetic engineering with the theoretical basis of neural stem cells. ObjectiveThis experiment aims to separation and cultivate mesenchymal stem cells umbilical cord blood, directional induced neural stem cells to differentiate into certain direction, transplant them into HIBD newborn rat body through the tail meridians, using in situ hybridization method, determines and records Foxgl and p21 mRNA genes and gene expression changes respectively on different time points in sub granular zone area after HIBD, discusses the change trend of both in the hippocampus express change after HIBD, and the relationship between them at genetic level. So that we can explore neural the regulatory mechanism of stem cell proliferation and differentiation of brain after stem cell transplants in the damaged area, so as to improve with proliferation and differentiation efficiency, which is of important significance for genetic engineering, providing theoretical basis for further application of neural stem cells in the clinical.MethodsTo collect healthy full-term puerperal placenta cord blood, separate and cultivate mesenchymal stem cells, then directional differentiation, immune cells chemical method appraisal.7 day-old SD newborn rats as research object, with total number 150, no limit of male or female. Using random number table method, divides them into HIBD-NSCs group, HIBD transplantation group, and sham group randomly, with 50 rats each group. Using classical Rice method for HIBD model, transplant cultivated NSCs into the HIBD rats after the model were successfully made one day. Take the 3d,14d,7d,21d, and 28d for checkpoint after the model were successfully making. At each observation points, respectively in, take the right hemisphere of the brain of death rats, All the tissues were fixed in 10% neutral formalin and desiccated and embedded in paraffin, HE dyed to appraisal model. in order to in situ hybridization, Determination different time points p21 mRNA and Foxgl mRNA expression changes in the hippocampus, observation the influence of neural stem cell transplantation to the both expression, to explore their immanent connection.Results1 The original generation cultivated mesenchymal stem cells morphological observationThe original generations cultivating MSCs assumes the circular, seven days later, they grow into short spindle, single nuclear, refractivity is stronger. Then gradually stretch out bumps, mixed with osteoclastic-like giant cells, large size, assumes the circular, multiple nuclear. About 28 days cells will be spread bottle.2 The induction and identification of umbilical cord blood derived neural stem cellsThe original generation cells be spread bottle to 80%, then add inducer with EGF and bFGF. Induce them differentiate into neural stem cells. Using immune cell chemical dye, observe and count Nestin, NSE, and GFAP positive cells. The expression increase compared with before induction3 Model identificationConventional HE dyeing examination results shows:the left (ligation side) brain shows significant pathological changes, the neurons in cerebral cortex and the hippocampus apparent necrosis.4 In situ hybridization detection results:(1) Foxgl mRNA genes in the first three days is expressed after surgery in each group, and expressed highest 7 day, then gradually declining.(2) p21 mRNA genes in the first three days is expressed after surgery in each group, and expressed lowest 7 day in HIBD group and HIBD-NSCs group,5 Statistical resultsWhether in HIBD group, HIBD-NSCs transplant groups or sham group, expression of mRNA Foxg1 mRNA and p21 mRNA are closely related to negatively correlated. In HIBD group, its two correlation coefficient is 0.743, in sham group, the correlation coefficient is -0.891 (P<0.05), in HIBD-NSCs group correlation coefficient is -0.841.Conclusions1 HIBD can change the microenvironment in vivo so that Foxg1 mRNA expression increased after injury and can promote the repairof brain structure and function.HIBD can change the microenvironment of brain, thereby rise Foxg1 expression after injury, promote repair of brain structure and function.2 NSCs transplantation can improve the environment of the brain, thus contributing to increased expression of proliferation-related gene Foxg1.3 The expression of p21 mRNA decreased with the increase of Foxg1 expression in SGZ areas brain tissue, the expression of p21 gene in hippocampal tissue regulated by Foxg1. |
PDF Full Text Request