| Over the past five decades, cardiovascular surgical techniques have made great progress, success rate has been greatly improved. But cardiovascular surgery-related complications and mortality resulting still high, especially in some critically ill patients, such as complex congenital heart disease and aortic aneurysm involving the aortic arch, these difficult surgery, operation complex, and during surgery generally require temporary interruption of the brain, the spinal cord of normal blood supply due to blocked aorta caused by spinal cord ischemic injury (such as:lower limb dysfunction, paresis and paraplegia), it is a serious postoperative complications, and prognosis, quality of life and psychological cause serious injury. So, how to prevent the occurrence of neurological complications is a major critical problem faced by clinical medicine and also a major key scientific and technological problems, bottlenecks.At present, the researches on the mechanisms of spinal cord ischemia-reperfusion injury showed that spinal cord ischemia-reperfusion injury in the occurrence and development of a variety of factors are involved, including excitatory amino acids, nitric oxide, free radicals and lipid peroxidation intracellular calcium overload and apoptosis. Effect of various differences in both the loss factor have a synergistic effect.Thus, the mechanism for reperfusion injury in spinal cord ischemia, now that the heart and great vessels surgery means of protecting the spinal cord in addition to improving surgical technique to ensure that the blood supply of the spinal cord and cerebrospinal fluid drainage and other measures, there are the following means: ① Hypothermia:suitable cryogenic It can reduce the body’s metabolic rate, improve spinal cord ischemia and hypoxia tolerance, reducing cerebrospinal fluid secretion and glutamate release. ② Drugs:the spinal cord has a protective effect of drugs: corticosteroids, superoxide dismutase, receptor antagonist, nerve growth factors, and other drugs. ③ Protective autoimmunity:autoantigens on the central nervous system components, such as myelin basic protein in active or passive immunization may be a viable cell therapy, is expected to fully recover after spinal cord injury is not neurological. ④ Ischemic preconditioning:previously given by the organization transient, non-invasive ischemia, organizations have the capacity to ischemia ensued longer tolerate a certain effect, it is an endogenous protective mechanism. ⑤ Neural stem cells (NSCs) transplantation.Wherein the NSCs transplantation is expected to become an important repair central nervous system lesions therapy. We know that the central nervous system damage is limited ability to repair itself, the traditional view is that the brain, neurological dysfunction caused by spinal cord injury is impossible to restore the neuronal cell death would cause irreversible decrease. Therefore, when the trauma and ischemia causes severe central nervous system damage caused by a large number of neuronal loss, the inability to generate new neurons, can not establish new synaptic connections, central nervous system damage will be difficult to reverse. In 1992, Reynolds and Weiss found from adult hippocampal NSCs, and finally updated the central nervous system injury awareness, research suggests that the adult central nervous system tissue has the ability to repair itself. With the deepening of neural stem cell research in recent years, more and more evidence suggests that NSCs not only can promote the regeneration of nerve cells, promote the repair of brain or spinal cord tissue, but also for therapeutic purposes by transplanting NSCs. The results of gene therapy vector more to promote neural stem cell transplantation therapy applications, are expected to be used to treat a number of central nervous system injury so far no effective treatments.Neural stem cell transplantation as a treatment of central nervous system injury advantages:in vitro amplification to obtain a sufficient number of NSCs, can provide adequate donor; NSCs from the central nervous system transplant survival rate of a host of high energy improve the success rate of cell therapy. Because NSCs in the spinal cord ability to repair itself is extremely limited, so through in vitro amplification of NSCs transplanted into spinal cord injury, degeneration and necrosis of the site, a new way to treat severe spinal cord injury disease, with great theoretical and clinical value.In this study, NSCs from fetal rat brain tissue were used as donor cells. By transfection of tyrosine kinase C (TrkC) gene, TrkC overexpressing NSCs were prepared. After establishing a rat model of spinal cord ischemia-reperfusion injury, NSCs and TrkC overexpressing NSCs were transplanted into the spinal cord injury site. Recovery of motor function, pathological changes, and heat shock protein 70 (Hsp70) levels were analyzed, in order to the effect and possible mechanism of TrkC overexpressing NSCs transplantation on spinal cord ischemia-reperfusion injury.Part one:Isolation, culture, and identification of neural stem cells and transfection of TrkC geneObjective:In vitro isolation, culture and identification of rat NSCs, and prepared by transfection TrkC gene transfer TrkC gene NSCs.Methods:Pregnant rats 14d Wistar removed fetal Wistar rat hippocampus, mechanical digestion into single cell suspension. Join NSCs whole culture, suspension culture, passed two generations after use. By observing cell morphological identification NSCs. Design and synthesis of primers TrkC extracted mouse brain total RNA, using reverse transcription PCR kit reversal rate of mRNA into cDNA, recovered fragment, then get pMD18-T-TrkC plasmid by digestion and ligation, transformation, amplification and extraction pMD18 -T-Trkc plasmid, followed by TrkC gene sequencing to construct pECFP-C1-TrkC plasmid using liposome 2000/pMD18-T-TrkC plasmid NSCs, the last expression of TrkC detected by immunofluorescence.Results:(1) primary cultured cells are round bright spherical, the third day adherent differentiation, cell gradually becomes fusiform, extending protrusion; 6th day of a large number of neurons and glial cells, interleaved projection into a mesh. (2) by plasmid transfection TrkC gene NSCs can make a strong expression of TrkC gene.Conclusion:The fetal rat brain tissue can be isolated NSCs, as the transplanted cells. Plasmid transfection method can be prepared transfer TrkC gene NSCs.Part two:Effects of TrkC overexpressing gene neural stem cells transplantation on spinal cord injury and heat shock protein 70Objective:To observe the effects of transplantation of TrkC overexpressing gene neural stem cells (NSCs) on heat shock protein 70 (Hsp70) levels.Methods:Forty-five female or male Wistar rats underwent aortic cross-clamping to build a model of spinal cord ischemia-reperfusion injury. Rats were randomly assigned to three groups, aortic cross-clamping group (Group 1), transplanted NSCs at 1 day after aortic cross-clamping (Group 2), and transplanted TrkC overexpressing gene NSCs at 1 day after aortic cross-clamping (Group 3). At times of 1 d,2 d,3 d,7 d, and 14 d after operation, three rats of each group were evaluated by Tarlov score, then tail vein blood were collected to detect the expressions of Hsp70 by enzyme immunoassay. At last, rats were killed to perform histopathological examination.Results:All 45 rats survived. Hindlimb motor function score showed that at 7 d after transplantation, Group 3’s scores were significantly higher than Group 1 (P <0.05); at 14 d, Group 2’s scores were significantly higher than blocking group, Group 3’s scores were significantly higher than the other two groups (P<0.05). ELISA results showed that levels of Hsp70 in three groups increased after operation and peaked at 3 d. Hsp70 in Group 3 were significantly higher than other groups at all time points (P<0.01). Among 5 time points, cell morphology of three groups showed significant difference at 1 week after operation. Histopathological results showed that neurons in Group 3 were preserved better and showed fewest apoptotic cells compared with the other groups.Conclusion:Transplantation of TrkC overexpressing NSCs may protect spinal cord from ischemia-reperfusion injury, probably through replacing injured neurons and enhancing the synthesis and release of Hsp70 which might inhibit stress. |
PDF Full Text Request