Changes In The Expression Of Jarid1b During The Development Of RGCs And The Expression Of Jarid1b And H3K4me3 In Rats After Optic Nerve Injury

Objective:The primary culture model of retinal ganglion cell(RGCs)was established by extracting the retina of 6-day-old SD rats,and its purity was identified by immunocytochemical method,and the expression of Jarid1 b during the development of postnatal RGCs was detected.By establishing a rat model of optic nerve injury,the expression changes of H3K4me3 and Jarid1 b after optic nerve injury were determined whether Jarid1 b plays a certain role in the process of optic nerve injury and subsequent recovery,and to provide some basic basis for clinical diagnosis and treatment after optic nerve injury.Method:Firstly,the 6-day-old(P6)suckling mice were selected,their eyeballs were taken after anesthesia,and the retinal tissues of suckling mice were dissected and digested with papain to prepare RGCs cell suspension.the RGCs was screened and purified by the supernatant of macrophage polyclonal antibody,goat anti-rabbit Ig G and goat anti-mouse Ig G + Thy-1.1 antibody.The suspended RGCs was cultured in 24-well cell,and the purity of RGCs cultured for 3 days was identified by immunocytochemical method with Thy-1.1 antibody.1.The RGCs,of P6(6 days after birth)and adult rats were extracted respectively and the changes of mRNA expression of Jarid1 b were detected by qRT-PCR.2.45 clean adult SD rats of both sexes were selected.Each rat took the left eye as the injured eye and used miniature vascular clamp to clamp the vertical optic nerve at about2 mm behind the eyeball for about 15 s to establish the model of optic nerve injury,and the right eye was used as the control eye,the optic nerve was exposed only for 15 s,and the blank control was carried out.If the pupil of the injured eye is dilated,the direct light reflex disappears,the indirect light reflex exists,and there is no retinal blood vessel bleeding,the complete recovery of blood flow in 5min after injury can be included in the experimental group.After injury or exposure of the optic nerve,pupil examination and FVEP detection were performed in both eyes to ensure the establish of the optic nerve injury model.On the3 rd,7th and 14 th day,10 rats in each group were killed.The retinas of experimental eyes and control eyes were used to extract RGCs,and qRT-PCR was used to detect the changes of Jarid1 b mRNA expression of RGCs in the injured group and the control group.3.After the establishment of optic nerve injury model on the 3rd,7th and 14 th day,in each group 5 rats were randomly killed,and the pathological sections of optic nerve tissue of right eye and left eye were taken as control group and injury group.The sections were stained with immunohistochemistry to observe the expression of H3K4me3 and Jarid1 b in the optic nerve of the two groups,statistical analysis was carried out.Results:1.The in vitro survival time of RGCs,extracted from p6 suckling mice was about 7days under light microscope,which provided a basis for follow-up cell research.By using the supernatant of Thy-1.1-containing antibodies extracted from cultured OX-7 hybridoma cells,the positive yield of RGCs was good and the purity was high,which provided the basis for RGCs-related experimental research.2.qRT-PCR detection of the expression of Jarid1 b in different stages of rat retinal development(P6,adult rats)showed that the mRNA expression of Jarid1 b decreased gradually with the development of rat retina.3.There was no significant change in the expression of Jarid1 b in optic nerve axons in control group.The expression of Jarid1 b in the acute injury group was significantly lower than that in the control group.4.There was no significant change in the expression of H3K4me3 in optic nerve axons in normal control group.The expression of H3K4me3 in the acute injury group was significantly higher than that in the control group.Conclusion:1.The supernatant of antibody containing Thy-1.1 extracted by culturing OX-7hybridoma cells can be used for screening positive RGCs in rats.The cultured RGCs has good growth and high purity.The in vitro survival time of RGCs from 6-day-old suckling mice is up to 7 days,which can be used for further basic research.2.Jarid1 b may be involved in the differentiation,maturation and apoptosis of RGCs,and play different roles in different stages of RGCs development.3.After optic nerve injury,the expression of Jarid1 b and H3K4me3 suggesting that:(1)the expression of H3K4me3 is related to the expression of Jarid1 b in optic nerve injury;(2)after optic nerve injury,the low expression of Jarid1 b suggests that it may be one of the mechanisms of inhibition of RGCs axonal regeneration.