The Expression Of JARID1B During The Development Of RGCs In Rats And A Study On Its Effect On The Survival Of RGCs In Vitro

ObjectiveThe primary culture pattern of retinal ganglion cells(RGCs)was built up by extracting the retina of primary SD rats,and the purity of RGCs was identified.The expression of JARID1 B in the development of RGCs after birth were detected to study the change of JARID1 B in the development process.The RGCs cell model of JARID1 B overexpression was established to explore the effect of JARID1 B on the survival of RGCs in vitro.It will provide an experimental system in vitro for the basic research of visual function recovery and optic nerve related diseases,and put forward an evidence for the important role of JARID1 B in RGCs development and regeneration.Method1.First,the supernatant containing Thy1.1 antibody was extracted by cultivating OX-7hybridoma cells for positive screening of RGCs;then 12 female and 6 male adult SD rats were selected to mate with each other at 2:1,and the mice within 7 days(P7)after birth were selected to be operated.We digested their retinal tissue with papain to prepare the RGCs suspension,which were screened by macrophage polyclonal antibody,then purified negatively and positively by Goat anti-rabbit IgG(H + L)and Goat anti mouse IgG(H + L)+ supernatant containing Thy-1.1 antibody in turn.The RGCs were suspended and then laid in the 24-well cell culture plates(pretreated with laminin and poly-D-lysine in advance).The purity of the cells was identified by immunocytochemistry with Thy-1.1 antibody 72 h later during the culture.2.Western blots were used to detect the expression of H3K4me3 protein in P6(6 days after birth),P14(14 days after birth)and adult retina.RGCs of P3,P6,P14,10W(10weeks after birth)and 20W(20 weeks after birth)rats were extracted respectively.qRT-PCR and immunofluorescence were used to detect the mRNA and protein expression of JARID1 B and its related molecules.3.The overexpression of JARID1 B in P6 RGCs cultured for 3 days was made by using Cumate-pLenti-JARID1B-SV40-GFP lentivirus plasmid.To observe the effect of JARID1 B on the survival of RGCs with natural apoptosis(or necrosis)in vitro.4.Statistical analysis was done by SPSS software in this paper.All of them were tested by homogeneity of variance.Paired T test was used for paired data.Independent sample T test was used for pairwise comparison between different groups.The test level was 0.05,and the difference was statistically significant if P < 0.05.Result1.The culture results of OX-7 hybridoma cells were good.The concentration of the supernatant containing Thy-1.1 antibody was high.It had a good effect on positivescreening of RGCs in rats,with a yield of 15000 cells / retina.Continuous observation of RGCs culture showed that there is no pollution,and it can be stored for a long time,which is easy to promote.2.The RGCs cultured by this antibody screening method grew well with high purity,and could survive for 9 days in vitro;P3 RGCs had a longer survival time,and the RGCs inoculated with high density formed more intercellular process connections and slower death rate.3.There was no significant difference in H3K4me3 protein expression in different stages of retina development(P6,P14,adult rats)by WB detection;JARID1B mRNA expression increased gradually with development in primary rats,reaching the peak at a certain time point after P14,and decreased at 20W(old age)by qRT-PCR detection;the protein expression confirmed the consistency of transcription level and translation level by immunofluorescence detection.4.Using lentivirus as a vector to overexpress JARID1 B of RGCs cultured in vitro,which was observed that the survival time of RGCs with natural apoptosis(or necrosis)was prolonged in vitro;there were more surviving cells than that of the control group,and the morphology of RGCs was more stable at the same time point.Conclusion1.The antibody supernatant containing Thy-1.1 extracted from the culture of ox-7hybridoma cells had a good effect on RGCs of rats positive screening and is easy to be popularized.The RGCs cultured grew well and had high purity,the survival time of P6 RGCs in vitro could reach 9 days,which can be used for further basic research.2.JARID1 B might be involved in the differentiation,maturation and apoptosis of RGCs and plays different roles in different stages of RGCs development;The interaction between H3K4me3 and JARID1 B in the development of RGCs needs further study.3.Overexpression of JARID1 B can prolong the survival time of RGCs,and maintain morphology and axon connection of RGCs,which shows the potential of JARID1 B in maintaining the activity,inhibiting the apoptosis and promoting the regeneration of RGCs.