The Study On The Effect Of LEDGFp52 On The Rat RGCs

BackgroundsThe study on enhancing survival and regeneration of injured RGCs has been a common hot spot and focus in ophthalmological research as well as neurobiological investigation. Neuroprotective factor is a prerequisite and substance foundation of gene therapy and transplantation implement in CNS restoration of visual system. However, in the past a few years, the study on neuroprotective factor just focused on the factor itself, up to now, we haven't attained a certain factor which could provide persistent nutritional support for injured RGCs to surmount their regeneration inhibited circumstance and promote axonal outgrowth. If we can find a special factor which could play an important role on transcription or pre-transcription level, perhaps the factor could initiate the growth-chain-reaction of RGCs, the tough problem on axonal regeneration will be solved. LEDGF is a novel growth, adhering, differentiation, anti-apoptosis and survival factor, which was isolated from the cDNA Lib in 2000. The gene of LEDGF was located in 9p21, and it can be alternative splicing produced two isoforms—LEDGFp52 and LEDGFp75. LEDGFp75 was just a lepto-transcription coactivator compared with LEDGFp52, and previous investigation has identified that LEDGFp75 had neuroprotective effect, it can elevate growth and adhering abilities of most cells, such as LEC, RPE and RPR of ocular cells, promote their differentiation and prolong their survival. Sequential researches revealed that LEDGF was regionally expressed in developing brain, and it was involved in the process of neuroepithelia stem cell differentiation and neurogenesis. Therefore, some specialists have suggested LEDGFp75 should be a candidate for remedial protein and neuronutritional transfection factor of retinal desease. Based on the research of neuroprotective effect of LEDGFp75 on some ocular cells, in accordance with the bioinformatics inference that LEDGFp52 is maybe much more competent than LEDGFp75, cohered to the reality that study on LEDGFp52 is very rare in the world, and coped with the requirement of neuroprotective and growth-promoting nutritional factor for RGCs axonal regeneration, we chose LEDGFp52 as our research target. Thus, to be a relatively novel revelation, does LEDGFp52 have a growth-promoting effect on RGCs? Can LEDGFp52 regulate the expression of RGCs neuronal specificity associated gene & protein? An empirical study is necessary to identifying these issues.PurposesTo positively and negatively regulate LEDGFp52 both on gene level and protein level on the base of primary RGCs culturing; to observe their impact on the prominence numbers & axonal length of rat RGCs, to investigate their influence to RGC neuronal specificity associated gene & protein; to understand the effect of LEDGFp52 gene &protein on rat RGCs growth, to lay a foundation for seeking more competent growth and nutrition factor of RGC,to furnish exploration on new path of injured optic nerve recovery with experimental proof .MethodsNeurobasal TM Media serum-free system was used in culturing RGCs, RT-PCR and CMIFC was applied to detect the expression of LEDGFp52 gene & protein on RGCs; rhLEDGFp52 prokaryotic expression vector was constructed, rhLEDGFp52 protein expression was induced and purified; LEDGFp52 adenovirus vector was constructed by homologous recombination in bacteria, in vitro expression was executed and virus was prepared; siRNA-LEDGFp52 eukaryotic expression vector was constructed and identified, and the inhibition ratio on LEDGFp52 protein was examinated.1. Observation of LEDGFp52 gene & protein effect on prominence numbers & axonal length of rat RGCs: experimental groups and control groups were divided. In experiment groups, LEDGFp52(2×10-4g/L) and Ab-LEDGF(2.5×10-4g/L) were added into the RGCs serum-free media, Ad-LEDGFp52(2.5×10-4g/L)(3×10-4pfu/L) and siRNA-LEDGFp52(6×10-4g/L) were transfected into RGCs by Lipofectamine TM 2000 while RGCs culturing 36hours. In treatment 12h, 24h, 36h, 48h, 72h and 96h, observe the alteration under the contrast phase microscope. Positive control group CNTF(10-4g/L) and blank control group were set up and observation timing point were same as above. In each control groups and experimental groups 12 wells cells were observed and each well was repeatedly observed 3 times. The observation indexes were prominence numbers & axonal length of rat RGCs and the analyses software was IPP image analytical system.2. Investigation of LEDGFp52 gene & protein regulating RGC neuronal specificity associated gene & protein: the treatment factors and subgroups were same as above. The detection indexes were gene expression changes & protein expression variations of GAP-43, NF-L and MAP-2, the gene changing was studied by RT-PCR and the protein varying was detected by CMIFC. The analyses softwares were Quantity One & LSM510-Meta-Expert, the former was used in semiquantitative analysis relative amount of each gene in mRNA level and the latter was applied in semiquantitative analysis mean fluorescence intensity of each protein.Results1. The serum-free Neurobasal TM Media is a better culturing system for RGCs, both gene and protein of LEDGFp52 was expressed in RGCs.2. LEDGFp52 gene was inserted into prokaryotic expression vector by gene recombination technique, the construction was identified by enzyme digesting and sequencing, and the target protein dissoluble expression was induced by IPTG, the expression amount of rhLEDGFp52 was accounted to 34.63% of total bacteria protein. The Westernblotting result revealed that rhLEDGFp52 protein could be specifically integrated with LEDGF monoclone antibody. rhLEDGFp52 was purified by Ni-NTA His.Bind.Resin Method, and the final concentration was reached to 520μg.mL-1, its purity was 87.93%.3. LEDGFp52 gene fragment was successfully inserted into the adenoviral shuttle plasmid pAdTrack-CMV and pAdTrack-CMV-LEDGFp52 was obtained. Then, it was cotransfected BJ5183 bacteria with TyV adenoviral backbone plasmid pAdeasy-1, the recombinant adenoviral vector pAd-LEDGFp52 was produced by homologous recombination in bacteria. And, pAd-LEDGFp52 was transformed into 293cells with lipofectamine TM 2000, the recombinant adenovirus Ad-LEDGFp52 was bred, and the titre was upto 5×1012 pfu.L-1.At last, 293cells were transfected by Ad-LEDGFp52 in vitro, the expression of LEDGFp52 was confirmed by CPE and Westblotting.4. LEDGFp52 gene RNA interference eukaryotic expression vector was successfully constructed, the alteration of LEDGFp52 protein expression was detected by Westblotting in 48h once recombinant plasmid transfected HeLa cells, and the expression was significantly downregulated about 70%.5. The RGCs growth was regulated by LEDGFp52 gene as well as protein, and manifestation was as follows: the axonal length was increased by up-regulation, and decreased by down-regulation; the peak potency appeared in positive regulation.6. LEDGFp52 was an arborisation factor as well as an axonal elongation factor of RGC.7. The RGCs neuronal specificity growth associated genes & proteins—GAP-43 , NF-L and MAP-2 were noticeably regulated by LEDGFp52 gene & protein, and manifestation was as follows: all genes & proteins expression were heightened by up-regulation and depressed by down-regulation.Conclusions1. The expression of LEDGFp52 on RGCs was first established.2. rhLEDGFp52 protein was successfully obtained, LEDGFp52 recombinant adenovirus was prepared and small interference RNA recombinant was constructed which could be used in suppressing expression of LEDGFp52. All the protein, virus and recombinant had provided material prerequisites for further studying on biological effect of LEDGFp52 on ocular region.3.The process numbers & axonal length of RGCs were significantly impacted by LEDGFp52 gene & protein .And LEDGFp52 is much more competent than CNTF.4. LEDGFp52 was an arborisation factor of RGCs as well as axonal elongation factor .5. LEDGFp52 gene & protein noticeably regulated the RGCs neuronal specificity growth associated gene &proteins—GAP-43, NF-L and MAP-2 regulating RGCs growth. And this is the main mechanism of LEDGFp52 regulating RGCs growth.6. There were some other arborisation factors participating in controlling RGC arborisation by LEDGFp52 gene.