| Objective To explore the changes of gene expression in regeneration of retina gangling cells after neural transplantation and protection of Kallikrein-Binding Protein(KBP) to Retinal Ganglion Cells (RGCs)Methods 39 Listed Hooded rats were researched by the approach of case control, by spliting which into normal control group, neural amputation group and experiment group on rats of which neural amputations were performed and then peripheral nerves were transplanted. Classic peripheral nerve spliced models were adopted, and immunohistochemical technique was performed to detect changes of expression of GFAP, GAP-43, NF-Kb, HPS-27 and rhodopsin protein during different periods in control and experimental group. RT-PCR, Real-time PCR techniques were adopted to detect mRNA concentration changes of 13 genes in Rats' RGCs. NOVA was adopted to compare, and LSD test was used to Post-Hoc test to explore changes of gene expression in regenerating of RGCs after Optic nerve injury. In addition,34 SD rats, splited in to the experimental group, control group and normal group. In the rat model of optic nerve injury by direct pressurization method, immunohistochemical technique was adopted to observe the structure of retinal, measure the thick of retina and count the number of survival RGCs, and data were analyzed by JRuler Software. The rat optic nerves were collected to extraction protein for Western blot analysis to determining expression of GAP-43 and data was analyzed by Software of Band Scan 5.0 to identify whether KBP has protective function to retinal ganglion cells and promote regeneration of axon.Results (1) Amphiblestrodes cut sheets displayed GFAP increased visibly on the 5th and 14th day after neural amputation. Compared with injured amphiblestrodes, on the 5th,14th and 21st day after neural transplantation, GFAP expressed in a low level in transplanted and anageneticed nerves.Tinction in control group and neural amputation group showed negative immune reaction on GCL. On the 5th day after a piece of ischiadic nerve was transplanted, GAP-43 espression inceased visibly on GCL, and GAP-43 immunoreactivity increaseed greatly on the 14th day after transplantion, and after 21 days, only a little GAP-43 expressed on GCL. In anagenetic amphiblestrodes, NF-κb transferred into nucleoloid structures. HPS-27 seemed to redistribute in RGC on the 5th day after optic neural amputation. On the 14th day after optic neural amputation, amphiblestrode RGCs had exhausted, and little HPS-27 could be detected on GCL. In nerve amputated+transplanted group, HSP-27 expressed persistently for 21 days. (2)No marked change was found in these gene's RT-PCR test results. (3) Real-time PCR results indicated 5 days later, amphiblestrode GFAP increased significantly in amputation group, experiment group and control group (p<0.01). But GFAP expressiong was not showed significantly differences at all times. 5 days later, amphiblestrode GAP-43 in nerve amputated+transplanted group increased much more siginificantly than that in control group (146±11%, P<0.05), however, same increase was not found in amputation group(108±31%, P<0.05). At time points after that, GAP-43 expression in both two experiment groups was decreased much more significantly than control group (P<0.05).The degree of decrease in nerve amputated+transplanted group was much lighter, and differences were not statistically significant, compared with the 5th day (14days:64±12% vs.39±4%; 21 days:46±6% vs.42±1%). At all times, amphiblestrode Thy-1 expression in both two experiment groups decreased significantly (P<0.01). There was no marked qualitative difference about Thy-1 expression in both nerve amputated+transplanted group and amputation group. Results:Amphiblestroid ET-1 expression of nerve amputated+ transplanted group increased more significantly than amputation group as time went on(14 days:108±24% vs 148±21%). Instead, differences of amphiblestroid ET-1 expression between the two groups were not statistically significant at all times P.There was significant difference between all groups about amphiblestroid ETB receptors' expression at all timesP. (4)The optic nerve injury rats with HE staining, we observed the structural changes in the all levels of the retina. The double-blind RGCs counting and retinal thickness measurement showed the statistical significance differences between the groups. (5)We measured the expression of the nerve regeneration marker protein GAP-43 by Western-blot. The statistical signify can were showed between the inter-group. Conclusions (1)GFAP is distributed in astrocytes of GCL and NFL of retinal ganglial cells (RGCs) in normal rats; up-regulated expression of GFAP in nerve amputated indicated Muller cells and astrocytes may be inhibited; GFAP's low expression of in nerve amputated+transplanted group indicated RGCs may regenerate. (2)There is no GAP-43 expression in normal and amputated rats'retina, however, GAP-43's up-regulated expression indicated expression of GAP-43 may related to regeneration of RGCs. (3)NF-κB is located in cytoplasm in normal rats'retina, and NF-κB shifted into karyon after nerve amputeated. This indicated activation of NF-κB participates regeneration of RGCs. (4)HSP-27 may expressed in RGCs of normal rats but not in nerve amputated group. HSP-27 expressed persistently in nerve amputated+ transplanted group suggested HSP-27 may have protective effect on RGCs as a reactive protein. (5)Erythropsin didn't express in the three groups and suggested no significant relation between erythropsin and regeneration of RGCs. (6)GFAP mRNA up-regulation expressed in both in nerve amputated+transplanted group and amputated group, showing increased GFAP may provide an eligible microcircumstance for nerve regeneration as a neurotrophic factor. (7)GAP-43 mRNA increased in expression in RGCs of nerve amputated+transplanted group, showing GAP-43 may participate regeneration of RGCs. (8)Thy-1 mRNA decreased in expression in RGCs of nerve amputated+transplanted group, which can be used as a index to assess regeneration capacity of RGCs. (9)ET-1 mRNA increased significantly in expression in retina of nerve amputated+transplanted group. As a neurotropic factor, it can maintain integrality of nerve and vessels and promote regeneration of RGCs. (10)No significant change of ETB mRNA expression in the three groups showed no relationship between ETB and regeneration of RGCs. (11)PBS solution was pured into vitreous chamber after incomplete injury of optic nerves, and nucleolus dye of rats' RGCs was deep mixed. Nuclear fragmentation, vesicular nucleus, reduced RGCs, disorganized cell polarity and thinner inner plexiform layer indicated PBS can't protect injured nerves. (12)KBP protein solution was pured into vitreous chamber after incomplete injury of optic nerves, and every layers was clear in rats' retina. RGCs was arrayed in a monolayer, nucleolus were level dyeing, number of RGCs was reduced, inner plexiform layer was slight thinner and cell polarity was disorganized slightly. These suggested KBP protein may protect injured nerves. (13)KBP protein solution was pured into vitreous chamber after incomplete injury of optic nerves, and average quantity of RGCs, average thickness of retina and GAP-43 expression were higher than rats that were pured into PBS solution in their vitreous chambers. This suggested KBP protein may protect injured nerves. (14)KBP protein solution was pured into vitreous chamber, which can provide theoretical and practical foundations to clinical treatment of optic nerve injury. |
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