The Study Of The Protection And Mechanism Of RhG-CSF On Cultured Rat Retinal Ganglion Cells In Vitro

The common pathophysiological characteristics of Ischemic optic neuropathy,glaucoma, diabetic retinopathy and retinal vascular obstruction are retinal ischemia andhypoxia. This feature will directly damage the retinal ganglion cells, lead to thedegeneration and apoptosis, and ultimately cause the loss of visual function. Therefore, toexplore the mechanism of injury and apoptosis of the retinal ganglion cells, developingnew drugs to promote the survival and axonal regeneration of the retinal ganglion cells, isnot only the key to save the visual function of patients but aiso a major issue in thecontemporary nerve biomedical field.As we all know, granulocyte colony stimulating factor (G-CSF) is named. by the functionof stimulating selectively the differentiation of the hematopoietic precursor cells togranulocyte colony in vitro. It promotes the survival, differentiation and proliferation of cellsof the neutrophilic lineage,the migration of the stem cells, the effect of anti-inflammationand anti-apoptosis by a variety of ways, and therefore it is payed close attention in the fieldof neuroprotection and gradually applied to the clinical study. Now, recombinant humangranulocyte colony-stimulating factor (rhG-CSF) have been produced artificially byrecombinant DNA technology, there is not significant different between the activity of therhG-CSF and of natural G-CSF, and they are also very similar in structure and function. Inthis study, we investigate the protection and mechanism of G-CSF on cultured rat retinalganglion cells in vitro using recombinant human granulocyte colony-stimulating factor(rhG-CSF).1,the study of the effect of G-CSF on activity and proliferation mechanisms Of RGC-5RGC-5cell line is described as derived from postnatal day1rat retinal cells that hasbeen transformed with the Ψ2E1A adenovirus. The advantage of this cell line is the unifiedphenotype, stable traits and unlimited ability to divide.that displayed the samecharacteristics and physiological functions of ganglion cells in addition to the originalelectrophysiological properties. Now,the cell line has become the perfect cell model to study the activity and function of RGCs. In our experiments, We cultured RGC-5cell line in vitrofirstly, observed their shape and characters, detected the change of the activity andproliferation of RGC-5, presumed its mechanism after the intervention of rhG-CSF withMTT assay, flow cytometry, Western Blot. Found that G-CSF can enhance the activity of thecultured RGC-5in vitro, and this effect is dose-dependent, can promote the proliferation ofRGC-5, can be induced STAT3to phosphorylation, In addition,we also found that theG-CSF-R is involved in the activation of STAT3. All of above prompt us that G-CSF canpromote the activity and proliferation of RGC-5by inducting activation of STAT3throughG-CSFR pathway.2The cultivation, purification and identification of the retinal ganglion cells in vitroThe purpose of purification Of RGCs is to prevent other cells in the retina (Müller cells)interfere with the effect of the various factors on RGCs. Purified RGCs is a key to study thedamage mechanisms of RGCs and to evaluate the effect of protective.agent. As we known,Thy1.1antigen only expresses in the surface of the rat retinal ganglion cell, it is a sign ofmaturity of RGCs of mammals.Thy-1.1monoclonal antibody can only purified RGCs, butalso lead to the damage of non-ganglion cells, promote activity and axonal regeneration ofRGCs. Thy-1.1monoclonal antibody for the separation and purification of RGCs not onlywill get a higher purity, but also receive more cultured cells compared with the previousmethods. Culturing RGCs with NEUROBASALTMMedium can eliminate the heterogeneityof the serum, avoid the effect of many unknown ingredients in the serum on experiment, andextend survival time of the nerve cells in vitro. It supports the growth of the department ofthe purified neurons in spite of glial cell feeder layer. More importantly, in view of itsexclusion from the interference of the unknown composition of serum, NEUROBASALTMMedium has a greater advantage in detecting the factors that promote the neuronal survivaland growth. in addition, medium that dedicated to neurons can also reduce the proliferation ofnon-neuronal cells in some certain cultured conditions, so it can make neurons purified.Therefore, in this experiment, we selected specific Thy1.1monoclonal antibody andNEUROBASALTMMedium to purify the rat retinal ganglion cells, and obtained the goodresults, The purity of retinal ganglion cells can be as high as95%. Then we cultured RGCs respectively with NEUROBASALTMMedium and DMEM containing serum, compared themorphological changes, then detected the viability and apoptotic rate of the two groups ofRGCs with MTTand flow cytometry. the result is that NEUROBASALTMMedium cansignificangtly improve survival rate and survival time of RGCs in vitro, reduce the rate ofapoptosis of RGCs in vitro. In sum, the combination of Thy-1.1monoclonal antibody andNEUROBASALTMMedium to purified and cultured RGCs in vitro, can significantlymaintain the activity and prolong survival time of the RGCs in vitro.3,The effect of G-CSF on the growth of purified cultured rat RGC processes and on theoptic nerve protection under hypoxiaGAP-43is used as a molecular marker of nerve plasticity during development andinjury repair. A high level of GAP-43expression in RGCs is critical for RGC axonalregeneration. MAP-2is an important member of the MAP family, distributed in RGC bodiesand dendrites, and is a crucial component for maintaining RGC structure and promoting therepair of RGCs processes. RhoA is a protein in the Rho GTPase family, and has been shownto play an important role in signal conduction of axonal inhibitors. A variety of injuries canactivate RhoA enzymes in RGCs, and activated RhoA conducts signals through Rockactivation to inhibit axonal repair. Caspase3, bcl-2and bax are all apoptosis-related protein,thy play the role of regulation in the process of neuronal apoptosis. There is rare report athome and abroad that G-CSF Can protect the optic nerve by adjusting the above proteins.To this end, in vitro cultured RGCs were treated with G-CSF to investigate the effectson axonal production. In addition, RGCs were exposed to CoCl2to investigate the influenceof G-CSF on injured RGCs,to detect the the amount of the GAP-43, MAP-2, RhoA,Rock,Bcl-2, Caspase3and Bax expression in RGCs with rt-PCR and Western Blot. whiledetect the apoptosis rate of RGCs with flow cytometry. In the our study, GAP-43andMAP-2expression were increased with prolonged periods of hypoxia. GAP-43and MAP-2expression were significantly increased in the G-CSF group compared with the hypoxiagroup at12hours following hypoxia, indicating that G-CSF can repair RGCs axons byincreasing the expression of axon-associated protein. Western blot analysis revealed that Rhoand Rock protein content in RGCs exposed to hypoxia was significantly increased compared with the control group, but G-CSF treatment significantly attenuated this increase. Thisfinding indicates that G-CSF promoted axonal repair and regeneration, possibly through theRhoA/Rho pathway. Then Bcl-2expression were significantly increased in the G-CSF groupcompared with the hypoxia group, on the contrary Caspase3and Bax were significantlyreduced.In summary,these studies detecte the relevant factors using cell model in vitro to provethat G-CSF can increase the activity of RGCs and promote the growth of processes,andprotect the injured RGCs with anti-apoptotic pathways and increase the amount of growthassociated protein to promote the regeneration of processes. Provide a better experimentalbasis and theoretical basis for more in-depth study of hypoxic ischemic optic nerve disease,also provide a new direction and possibilities. for its treatment.