Protective Effects Of ADSCs Exosomes On Stress-induced RGCs Damage In Rats

Objective : To study the protective effect of ADSCs exosomes on damaged RGCs by establishing a rat RGCS stress damage model in vitro.Methods:(1)Cultivate ADSCs to collect supernatant extract and identify exosomes.(2)Cultivate and identify rat RGCs in vitro,establish models of rat RGCs damage caused by stress,and group them:(1)control group: RGCs cultured normally;(2)model group: sealed the RGCs and applied different pressures(40 mm Hg,80 mm Hg,120 mm Hg)for 8 hours;(3)treatment group: add exosome culture to RGCs with different pressure damage.(3)CCK8 method was used to detect the proliferation activity of RGCs cells in each group.(4)The q PCR method detected the expression levels of BDNF and Caspase-3 m RNA in each group of RGCs.(5)Western Blot detects expression levels of BDNF and Caspase-3 proteins in each group of RGCs.Results:(1)The morphology of the exosome was observed to match the characteristics through transmission electron microscopy;Western Blot detected positive expression of the marker proteins CD9 、 CD63 and CD81.(2)Immunofluorescence identification of RGCS showed that its specific antibody,THy-1.1,was expressed positively fluorescently in green.(3)The CCK-8 test found that in the control group,compared with cell proliferative activity at 24 h,cell proliferative activity increased at 48 h,and the difference was statistically significant(P<0.05).At 48 h,compared with pressurized 40 mm Hg,80 mm Hg,and 120 mm Hg model groups,cell activity increased after adding exosomes,and the difference was statistically significant(P<0.05).(4)The q PCR test found that compared with the control group,the m RNA expression of BDNF decreased in the 40 mm Hg group.The difference was not statistically significant(P>0.05),and the m RNA expression of BDNF decreased in the 80 mm Hg and 120 mm Hg groups.The difference was statistically significant(P<0.05,P=0.001).The m RNA expression of BDNF in RGCS increased after pressurized 40 mm Hg and 80 mm Hg groups added exosomes.The difference was statistically significant(P<0.05).The difference was statistically significant(P<0.05).After pressurizing the 120 mm Hg group added the exosome,the m RNA expression of BDNF in RGCS increased,but the difference was not statistically significant(P > 0.05).Compared with the control group,the m RNA expression of Caspase-3 increased in the pressurized 40 mm Hg group.The difference was not statistically significant(P > 0.05),and the m RNA expression of caspase-3 in the pressurized 80 mm Hg and 120 mm Hg groups both increased.The difference was statistically significant(P<0.05,P=0.001).The m RNA expression of caspase-3 of RGCS decreased after the pressurized 40 mm Hg and 80 mm Hg groups were added to exosome treatment.The difference was statistically significant(P<0.05).After the pressurized 120 mm Hg group added exosome treatment,the m RNA expression of RGCS decreased,but the difference was not statistically significant(P > 0.05)(5)Western Blot’s test found that compared with the control group,the protein expression of BDNF decreased in the pressurized 40 mm Hg group.The difference was not statistically significant(P>0.05).The protein expression of BDNF decreased in the pressurized 80 mm Hg and 120 mm Hg groups,and the difference was statistically significant(P<0.001).Compared with the model group,BDNF protein expression increased after exosome treatment was added,and the difference was statistically significant(P<0.05).Compared with the control group,the protein expression of Caspase-3 increased in each model group after pressurization,and the difference was statistically significant(P<0.05);compared with the model group,the protein expression of each group of Caspase-3 decreased after exosome treatment was added,and the difference was statistically significant(P<0.05).Conclusions: Exosomes derived from ADSCs can increase the cell proliferation activity of rat RGCs damaged by different pressures,increase the m RNA and protein expression levels of BDNF,and reduce the m RNA and protein expression levels of Caspase-3,indicating that exosomes derived from ADSCS have a protective effect on pressure-damaged rat RGCs.