| Spinal cord injury (spinal cord injury, SCI) is one of the serious diseases, in recent years, the incidence has increased year by year. Once it occurred, the event of subsequent neurological deficits is very difficult to recover. it bring a heavy financial burden and psychological burden to society, families and patients,for most patients have to rely on a wheelchair or bed-ridden life. Therefore, how to reconstruct the continuity of the structure of spinal nerve and restore nerve function of the spinal cord is the hot spot and problem that the world's scientists are working on. In recent years, with modern medicine,molecular biology and gene technology developing, through cell transplantation to treat spinal cord injury has become a hot topic, especially Schwann cells on the repairment of nerve injury, causing more and more people'attention. Schwann cells (Schwann cells, SCs) is the peripheral nervous system glial cells .In physiological circumstances, it nutrient, support, protect, insulat the peripheral nerve; in the central or peripheral nerve injury and disease ,it play an important role in the repairment of peripheral nerve, through its own proliferation, migration, adhesion, and secreting extracellular matrix, and secreting a variety of neural factors,to promot neurons and glial cells survival and promote proliferation, inhibit apoptosis, repair of central nervous system . Neuregulin1 (NRG1) is a signaling protein found in recent years, secreted mainly by neurons and glial cells. It forms homologous or heterologous ErbB Dimerization formation, through the extracellular binding structure of the receptor tyrosine kinase ErbB3 and ErbB4 to activ intracellular signaling pathways resulting in cell biological effects, including stimulating cell proliferation, apoptosis, migration, differentiation and adhesion, etc.It also can stimulate the neurons and glial cells, inhibit apoptosis, promote nerve injury repair and regeneration.In view of the unique biological effects of Schwann cells and NRG1 in the nervous system ,in the experiment,we will Transfect NRG1 gene into Schwann cells vector ,then transplant it into semi-amputated rat model of spinal cord to observe the treatment of spinal cord injury and to explore its relevant mechanisms in repairment of the spinal cord injury, and to provid a theoretical basis for clinical treatment of spinal cord injury.the content and the main results are as follows: 一,Schwann cells primary culture and purificationUsing mputation cut the sciatic nerve, making pre-nerve degeneration in vivo, 7 days latter,getting the sciatic nerve from Wistar rats, by transplantation, changing the concentration of fetal bovine serum in BS medium, and low concentration enzyme double digestion methods, without any division factor to promot proliferation , the adult Schwann cells can rapid proliferation in a short period ; without any cell division inhibitor to prevent contamination of fibroblasts, at last , The density of Schwann cells is about 2.74×106/mm2 and the purity is about 96.1%.二,NRG1 transfected Schwann cells and NRG1, ErbB2, ErbB4 expressionThe recombinant plasmid pcDNA3.1-NRG1 was transfected into Schwann cells through Fugene6 (for control group), transfected cells with a clear division and proliferation. Using Immunofluorescence method to detect cells'transfection rate was 57.83%, using immunocytochemical to detect the expression NRG1 in Schwann cells.After transfection 24h, 48h, 72h, collect the cells'culture supernatant fluid respectively in each group , using ELISA to detect the expression of NRG1 ,results showed that: the control group 1.581±0.051 ng / ml, transfected 24h group 1.537±0.110 ng / ml, transfected with 48h group 2.022±0.059 ng / ml, transfected 72h group 2.250±0.165 ng / ml, NRG1 expression was time-dependent increased, 48h, 72h group compared with the control group ,the difference is significant (p <0.05); After transfection 24h, 48h, 72h,cells were harvested in each group and lysis, using ELISA to detect the expression of NRG1and ErbB2, ErbB4 expression in the cells, results showed that: NRG1 expression was time-dependent increased in all groups, compared with the control group, there is a significant difference between groups, the 72h group is the most significant (p <0.05); ErbB2, ErbB4 was time-dependent increased in each group, compared with the control group ,there is a significant difference between groups, the 72h group is the most significant (p <0.05). These results suggest that: After NRG1 transfected Schwann cells, it could promote cells division and proliferation ,meanwhile,it could enable cells to express the NRG1 protein real-timely, high-leveland stablely, and to promote its specific receptor ErbB2, ErbB4 expression.三,transplantation genetically modified Schwann cells to treat the spinal cord injury in rats Using the speacial needle to make rat models of spinal cord injury(Hemi-sectde Spinal Cord Injury, HSCI) ,after operation the rats were scored by BBB (Basso, Beattie, Bresnahan locomotor rating scale) ,the qualified rats were divided randomly into three groups , each group 24 : A group for transplant DMEM control group, B group, Schwann cell transplant group, C group of genetically modified Schwann cells transplantation group, the normal control group for the D group; after cells transplantation , from the beginning to the end of the experiment in the eighth week,to assess the functionalof the experimental animals every week (BBB score);After Cells transplantation, 4 animals were sacrificed which were randomly selected from each group every 2 weeks, to observe the survival of cells through spinal cord frozen section and observed the number and changes of neurons and oligodendrocyte morphology in injuried spinal cord by immunohistochemistry,at the same time , detect and compare NRG1 and its receptor ErbB2, ErbB4 expression and distribution in the spinal cord; after cell transplantation, four animals were sacrificed which were randomly selected from each group every 4 weeks,then injuried spinal cord was fixed for TEM detection and HE staining to observe morphological changes in spinal cord after MRI testing. After 4 weeks ,four animals were selected randomly in each group for SEP detection and observe the electrophysiological recovery in injuried limb . The results showed that: transplanted cell can be survived in the region of spinal cord in each group , the genetically modified cells may secrete a large number of NRG1, not only to promot receptor ErbB2, 4 expression but also promot gliosis proliferation in injuried pinal cord; BBB, SEP, HE, TEM, MRI Testing showed that each experimental group had varying degrees recovery, but the transgenic cells group is the most significant.From the above study,we get the following conclusions :一,Transfected the NRG1 gene into Schwann cells, exogenous NRG1 gene can enable cells to express the NRG1 protein immediately, high-level and stablely, and to promote its specific receptor ErbB2, ErbB4 expression.二,Transgenic cells can be continuously survived in the region of injuried spinal cord, not only does it secrete a large number of NRG1 protein to promote glial cells proliferation in the injuried spinal cord , but also to promote its specific receptor ErbB2, ErbB4 expression time-dependent increased in in neurons and glial cells to protect damaged neurons. 三,After the NRG1 gene transfection into Schwann cells and situ implantation in rat model of Hemi-sectde Spinal Cord Injury, it can improve the motor function in rat hindlimb obviously, effectively promote the recovery of neurological function of spinal cord injury in a significant therapeutic effect,and opened up a new way for clinical treatment of spinal cord injury .
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