Transplantion Of Human Umbilical Cord Blood Stem Cell Improves Neurological Function Recovery And Microenvironment Factors Mechanisms After Spinal Cord Injury In Rats

Part 1. Analysis of influencing factors on spinal cord injury animal model in rats Objective: To compare the device and effect with four kinds of spinal cord injury (SCI) animal models and establish a easy and reliable one. Methods: Adopting the method of occluding abdominal arteries, clipping, weight-dropping and weight-pressing, their virtues, defects and neurological function were assessed. Results: The method of occluding abdominal arteries was instability, which the mortality was higher and the rats, preoperative anal temperature was obviously different(P<0.05)to the postoperative one .The exposed extent of spinal cord was larger and made the more bleeding in the clipping device.The operation was complicated in weight-dropping .Finally, in weight-pressing ,the operation was easy and the mortality was lower . Conclusion: The weight-pressing device is a easy and reliable animal model,which is the easier operation, lower mortality and better stability . Part 2. The collection, culture, differentiation and identification of human umbilical cord stem cell in vitro Objective: To explore the collection, culture, induce, differentiation and identification of human umbilical cord stem cell in vitro. Methods: Human cord blood samples were obtained sterilely preservative-free heparin. Mononuclear cells were isolated by lymphocyte separation medium and suspended in RPMI 1640 with 10%FBS. Then the mononuclear cells were cultured and purified by the different sensitivity to Trypsine. The surface antigen expression of mesenchymal stem cells (MSCs) was detected by flow cytometry. The passage 2-3 of the expanded MSCs were induced to differentiate into neuron-like cells using "Inducing Medium"containing all-trans-retinoic acid(RA)and human NGF. Specific markers of the induced cells were further detected by immunocytochemistry methods. Results: MSCs isolated from human umbilical cord blood show as the osteoclast-like cell, epithelial-like cell and MSCs respectively in the primary cultures. Flow cytometry results showed that MSCs did not express CD34, but expressed CD14,CD25,CD29,CD105,CD106,HLA-ABC,HLA-DR. NGF+RA may induce and differentiate MSCs to neuron-like and glia-like cell. Immunohistochemistry staining showed that the induced cell expressing NF200,NSE and GFAP. Conclusion: MSCs isolated from human umbilical cord blood can be cultured and passaged in vitro and differentiated into non-mesenchymal cells after induction. Part 3. Transplantion of human umbilical cord blood stem cell improves neurological function recovery after spinal cord injury in rats Objective: To observe the translated human umbilical cord blood stem cells infiltrating into the spinal cord injury region and explore the role of that improving neurological function recovery in rats. Methods: Adopting the animal model of weight-pressing, the human umbilical cord blood stem cells which were marked by Brdu were translated via vein into rats. The damaged region of spinal cord was removed at 1 and 3 weeks, localized and made the pathological observation. The neurological function was assessed using BBB score and inclined plane test. Results: The spinal cord injury region can be observateb the human umbilical cordblood stem cell marked by Brdu. Neurological function recovery determined by BBB score and inclined plane test was significantly improved in the group receiving human cord blood stem cells compared with the control group(P<0.05). Conclusion: Via vein administered human cord blood stem cells can infiltrate into the spinal cord injury region and survive and improve neurological function in rats.Transplantion of human cord blood cells may provide a novel strategy. Part 4. The change of microenvironment factors following the translated human umbilical cord blood stem cells improving neurological function recovery in spinal cord injury rats. Objective: To investigate the change of NGF, BDNF, IL-8 level in spinal cord injury region and explore the mechanisms of the translated human umbilical cord blood stem cells improving neurological function recovery in rats. Methods: Adopting the animal model of weight-pressing ,the damaged region of spinal cord was removed at 3 weeks after human umbilical cord blood stem cells translated and conserveed under -70℃. Spinal cord tissue was made homogenate. The NGF, BDNF, IL-8 level in supernatant was determined. Results: The NGF, BDNF level in spinal cord injury region was significantly raiseed in the group receiving human cord blood stem cells compared with the control group(P<0.05). Conclusion: The translated human umbilical cord blood stem cells can promote to secrete the NGF, BDNF in spinal cord injury region and help repair injury tissue.