| Objects and significancesNeuroblastoma (SHSY5Y) is a kind of cell lines in reseach of neurological diseases. The SHSY5Y extract prepared in experiment is the proteins in SHSY5Y cells which can express neurotrophic factors. Stem cell therapy is an important method used in the research of tissue repair after injury. Umbilical cord mesenchymal stem cells (UC-MSCs) can secrete many kinds of cytokines, including nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). The nerve repair can be promoted by increasing the content of neurotrophic factors related to injuries portion.This research adopted the SHSY5Y extract disposing umbilical cord mesenchymal stem cells, aimed to detect the secretion changes of neurotrophic factor and observe the repair of sciatic nerve injury by the disposed stem cells. In conclusion, we hope to provide a useful idea for the treatment of peripheral nerve injury.Materials and methodsThe human neuroblastoma cells (SHSY5Y) extract was obtained by ultrasonic crushing method and the protein stability of cells extract was detected by BCA protein quantitative. UC-MSCs of the third passage prepared by our laboratory was recovered, and cultured by serum free medium in batches. After passage, the cells were seeded in a6-orifice at a density of105cells per hole, and then ware passaged with medium exchange. In the process of culturing, after passage or medium exchange, the SHSY5Y cells extract was added in the experimental group in a final concentration of50mL/L for induction of7days. After7days of culturing, the cell surface markers of control group and experimental group were detected respectively. Everyday cells of the control group and experimental group were collected respectively. The expression of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in cells was tested with the normalization of beta actin. Finally the animal model of rat sciatic nerve compression injury was set up with36SD female rats. All the rats were divided into6groups (A～F) randomly as saline group, neuroblastoma cell extract group, stem cell group, extract and stem cell group, induced stem cell group and normal control group. Body condition of each group was observed postoperative:gait changes, toe contractures, muscle atrophy and recovery. Footprint analysis was consulted to Shen JiangNing, sciatic nerve function index (SFI) was tested by Brain formula. Leg triceps atrophy recovery and muscle wet weight recovery rate (lateral crus triceps wet weight/normal controls side x100%) were detected after three weeks. The muscle was cut in leg triceps load-point of rats with blood and connective tissue removing. Statistical analysis was conducted after precise weighing for the evaluation of the regenerated nerve’s dominance in the target tissue. The sciatic nerve function index, lower limb triceps wet weight ratio and pathology HE staining tissue biopsies were analyzed to compare the recovery of each group after different treatment.Results1. Umbilical cord mesenchymal stem cells were obtained by collagenase digestion technique and purified with differential adherent method.2. The cell phenotype of two groups was identified and no change was found in the experimental group. Immunophenotypic analysis showed that MSCs are positive for CD29, CD44, CD105and HLA-ABC.3. Total RNA of the two groups was extracted respectively for polymerase chain reaction and DNA gel electrophoresis experiment. The results demonstrated that in the experimental group, the expression of NGF and BDNF has a certain increase.4. Sciatic nerve transection was used as the model of sciatic nerve compression injury. The rat muscle spasm level degree of the experimental group is better than that of normal saline control group. Among them, especially the neuroblastoma cells extract induced stem cell group recovered best. 5. God sciatic function index determination results showed that there had a significant difference between the treatment group and normal saline groups (P<0.05).6. Leg triceps wet weight recovery rate determination results showed that there had a significant difference between the treatment group and normal saline groups (P<0.05).7. Pathology detection, the crosscutting HE staining of side leg triceps and injury of sciatic nerve was processed3weeks after operation which showed that the induced stem cell group has a very small amount of neutrophils, and its myelin recovery is better compared to other groups.Conclusions1Neuroblastoma extract can increase the expression of nucleic acid of certain neurotrophic factor in umbilical cord mesenchymal stem cells.2Umbilical cord mesenchymal stem cells induced by neuroblastoma cells extract had a better curative effect on repair of nerve injury. |
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