| Astrocytes are the most widely distributed cells in the central nervous system (CNS) of mammals with the biggest size. In the CNS of high vertebrates, various injuries such as trauma, ischemia, infection, intoxation and experimental autoimmune encephalomyelitis (EAE) lead to the proliferation of astrocytes with rapid synthesis of glial fibrillary acidic protein (GFAP), accompanied by phenotype changes such as cellular hypertrophy and migration, which eventually resulting in astrogliosis. GFAP, a member of the classâ…¢intermediate filament proteins, is thought to be specific for astrocytes in CNS. Therefore, GFAP is a reliable and sensitive marker of astrogliosis in CNS.Spinal cord injury (SCI) results in the suspension of neural pathway, neuronal damage and secondary SCI. In high mammals, cell regeneration and functional rehabilitation is hard for injured spinal cord due to the special structure of nerve and the series of pathological changes. Neuroscientists of the world are exerting untiring efforts to surmount this difficult medical problem to benefit mankind. At present, there are still many problems need to be solved on the repairing of SCI. Reactive astrogliosis, which leads to scar formation, is a manner of healing for CNS injury. The astrocytes in scar tissues are connected by intercellular tight junction and gap junction to form networks which inhibit axonal regeneration.In low vertebrates such as gecko, spinal cord can regenerate following SCI. To deeply explore the regeneration mechanism of spinal cord in low vertebrates is a way to study the feasibility of spinal cord regeneration in high vertebrates. Reported researches have revealed that astrocytes are scarce in gecko. It provides a clew that the reactive astrogliosis in gecko may be weaker. This hypothesis is an interesting scientific problem and has been never studied yet. Many reported researches indicated that there were intense reactive astrogliosis after SCI in mammals, which were thought to be the reason of spinal cord regeneration failure. It could provide a inspiration for the spinal cord regeneration of mammals even humans to explore the mechanism of spinal cord regeneration after spinal cord transection in gecko. It is essential to build a reliable and repeatable animal model of SCI in gecko.To clarify the relationship between astrocytes and regeneration of spinal cord in gecko, molecular cloning techniques, real-time quantitative PCR, Western Blotting and fluorescent immunohistochemistry were used to study the astrocyte reaction after spinal cord transection. This study will provide experimental evidence of spinal cord regeneration after transection and will inspire a train of thought on the treatment of human SCI.There is no commercialized gecko GFAP polyclonal antibody, so this experiment was aimed to clone GFAP gene in gecko and to prepare GFAP polyclonal antibody for the following study. First, GFAP mRNA was selected from the cDNA library in Nantong medical college which is lack of 5'end sequence. So we acquired the full sequence of GFAP mRNA by connecting the 5'end sequence synthesized by RACE with the sequence from cDNA library. The expression of GFAP in gecko was verified by Northern Blot. GFAP polyclonal antibody was prepared by immune GFAP antigen in rabbit and the titer was assessed by ELISA. Results showed that the full length of GFAP mRNA was 1770bp, encoded a protein of 446 amino acids and the molecule weight was 52kD. Bioinformatics analysis showed its high homology with gallus and result of Northern Blot showed its high expression in brain and spinal cord. GFAP expression plasmid pGEX-4T1-gGFAP was successfully established. GFAP polyclonal antibody was successfully prepared with a titre of 1:500. So in this part, gecko GFAP gene was successfully cloned and gecko GFAP polyclonal antibody was prepared for the following study.To investigate the changes of GFAP expression in the spinal cord of adult gecko following transection for the purpose of studying the relationship between astrocytes and spinal cord regeneration, real-time quantitative PCR and Western Blotting were applied to detect the GFAP expression at mRNA and protein level respectively. Fluorescent immunohistochemistry was performed to locate the changes of astrocyte morphology. The results of real-time quantitative PCR and Western Blotting similarly showed that the expression of GFAP in the gecko spinal cord close to the injury site increased at 1 day and achieved the highest level at 3 days after transection, and gradually decreased until 2 weeks. The results of fluorescent immunohistochemistry showed that the increase in the labeling of GFAP after SCI appeared in the white matter, especially in the ventral and lateral regions of the white matter. But the number of star-shaped astrocyte was not significantly increased after SCI. The results indicated that the astrocyte reaction had spacial and time limitation after gecko spinal cord transection, completely contrary to that in mammals after SCI. This limitation of astrocyte reaction may be advantageous for spinal cord regeneration after gecko SCI.Summary:The full-length of gecko GFAP mRNA was successfully cloned and characterized by bio-informatic analysis. Prokaryotic expression of gecko GFAP by applying GST fusion system was successfully used to prepare the polyclonal gecko GFAP antiserum. The mRNA and protein expression of GFAP in the gecko spinal cord close to the injury site increased at 1 day and achieved the highest level at 3 days after transection, and gradually decreased until 2 weeks. Fluorescent immunohistochemistry revealed the increase of reactive astrocyte processes but not the amount of cells after SCI. The results validated that the astrocyte reaction after SCI in geckos exhibited spacial and time limitation, completely contrary to that in mammals after SCI. This limitation of astrocyte reaction may be one of the reasons why gecko spinal cord can regenerate after injury.
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