| Objective:This experiment through cultured SD rat retinal Müller glia cells in vitro under high glucose to establish a model of DR pathological environment,pentazocine is a a selective ?R1 agonist,then it was gived,to observe the influence of pentazocine on producing and secretion of the GDNF and BDNF,and investigated the effects of penzazocine on GDNF and BDNF in cultured Müller cells under high glucose circumstance,to provide a new theoretical basis for pentazocine on the mechanism of retinal Müller glia cells function in DR.Methods: The SD rat were neonatal 3-5 days,we isolated and cultured retinal cells of SD rat in vitro with tissue culturing method,after the 8-12 days,retinal Müller glia cells were about 90% basic confluence state.The cells were went down to the future generation according to the proportion of 1:2.When went down to the 3-4 generation,the cells size and morphology were more uniform.The cells were regarded as retinal Müller glia cells by immune cytochemistry.After the cells cultured 24 hour with free serum culture,then gived high glucose and pentazocine intervention.The experiments consisted of four groups: one group of explants was cultured in normal glucose(concentration of glucose was 5 mmol/L,NC);one group in high glucose medium(concentration of glucose was 30mmol/L,G);pentazocine were added to the normal glucose(concentration of pentazocine was 3 mmol/L,P);pentazocine were added to the high glucose medium(concentration of glucose was 30mmol/L,concentration of pentazocine was 3 mmol/L,GP).After giving pentazocine intervention 24 h,72h and 120 h,determination of intracellular and extracellular GDNF and BDNF protein expression level of retinal Müller glia cells in cultured under high glucose circumstance by westen blot assay and enzyme linked immunosorbent assay(ELISA).Results:1 Culture and identification of retinal Müller glia cells: The cells were regarded as retinal Müller glia cells by immune cytochemistry;2 The expression levels of GDNF and BDNF protein in retinal Müller glia cells were measured by Westen Blot assay:2.1 The expression levels of GDNF after giving pentazocine intervention 24 h,72h and 120 h.1)After giving pentazocine intervention 24h: the expression levels of GDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine grop were: 0.792±0.010,0.956±0.007,1.073±0.022,1.082±0.022,the high glucose grop and normal grop to compare,the expression of GDNF increased obviously(P <0.05);compared pentazocine grop with normal grop,the expression of GDNF increased obviously(P <0.05),whereas,compared high glucose + pentazocine grop with high glucose,there the expression of GDNF was no obvious changing and no statistical difference(P>0.05),compared high glucose + pentazocine grop with normal grop,the expression of GDNF increased obviously(P <0.05).2)After giving pentazocine intervention 72h: the expression levels of GDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine grop were: 0.507±0.007,0.729±0.002,0.851±0.003,1.094±0.016;after giving pentazocine intervention120h: the expression levels of GDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine groups were: 0.638±0.003,0.915±0.003,1.127±0.001,1.206±0.027,the high glucose grop,pentazocine grop,high glucose + pentazocine grop and normal grop to compare,high glucose + pentazocine grop and high glucose to compare,the expression of GDNF increased obviously(P <0.05).2.2 The expression levels of BDNF after giving pentazocine intervention 24 h,72h and 120h: 1)After giving pentazocine intervention 24h: The expression levels of BDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine groups were : 0.792±0.010,0.956±0.007,1.073±0.022,1.082±0.022,the high glucose grop and normal grop to compare,the expression of BDNF decreased(P <0.05);compared pentazocine grop with normal grop and compared high glucose + pentazocine grop with high glucose,there the expression of BDNF was no obvious changing and no statistical difference(P>0.05),compared high glucose + pentazocine grop with normal grop,the expression of BDNF decreased(P <0.05).2)After giving pentazocine intervention72h: the expression levels of BDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine groups were: 0.948±0.029,0.795±0.007,1.111±0.025,0.999±0.014 the high glucose grop and normal grop to compare,the expression of BDNF reduced obviously in high glucose(P<0.05),however,at the same time,compared pentazocine grop,high glucose + pentazocine grop with normal grop and compared high glucose + pentazocine grop with high glucose,the expression of BDNF increased obviously(P<0.05);after giving pentazocine intervention120h: the expression levels of BDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine groups were : 0.774±0.002,0.629±0.004,0.867±0.001,0.755±0.006,the high glucose grop and normal grop to compare,the expression of BDNF reduced obviously in high glucose(P<0.05),however,at the same time,compared pentazocine grop,high glucose + pentazocine grop with normal grop and compared high glucose + pentazocine grop with high glucose,the expression of BDNF increased obviously(P<0.05).3 enzyme-linked immunosorbent adsorption assay(ELISA)to determinate the GDNF and BDNF expression in retinal Müller glia cells culture supernatant: 1)The comparison of expression level of GDNF in the supernatant fluid under the condition of different stimulation after 24 h,72h and 120 h : after giving pentazocine intervention 24 h intervention,the expression levels of GDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine grop were: 2.767±0.279,1.966±0.315,3.096±0.232,2.599±0.305;after giving pentazocine intervention 72 h intervention,the expression levels of GDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine groups were: 2.334±0.261,1.674±0.341,2.852±0.419,2.228±0.240;after giving pentazocine intervention120h: the expression levels of GDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine groups were: 2.334±0.261,1.674±0.341,2.852±0.419,2.228±0.240,high glucose grop compared with normal group found that the expression level of GDNF in the supernatant fluid decreased significantly at 24 h,72h and 120h(P<0.05);in high glucose intervention at the same time giving pentazocine intervention,the expression level of GDNF increased significantly(P<0.01),there the expression level of GDNF was no obvious changing and no statistical difference(P>0.05).2)The comparison of expression level of BDNF in the supernatant fluid under the condition of different stimulation after 24 h,72h and 120h: after giving pentazocine intervention 24 h intervention,the expression levels of BDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine grop were: 6.670±0.224,5.114±0.260,6.278±0.137,4.689±0.251;after giving pentazocine intervention 72 h intervention,the expression levels of BDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine groups were: 6.172±0.260,4.637±0.256,3.574±0.219,2.597±0.139;after giving pentazocine intervention120h: the expression levels of BDNF in normal grop,high glucose grop,pentazocine grop,high glucose + pentazocine groups were: 5.433±0.254,3.939±0.250,2.903±0.213,1.951±0.136,in high glucose intervention,found that high glucose grop compared with normal group,the expression level of BDNF in the supernatant fluid decreased significantly at 24 h,72h and 120h(P<0.01);at the same time giving pentazocine,the high glucose grop,high glucose + pentazocine grop and normal grop to compare,high glucose + pentazocine grop and high glucose grop to compare,the expression level of BDNF decreased significantly(P<0.01).Conclusion:1 The expression level of GDNF increased and secretion level of GDNF decreased in cultured retinal Müller glia cells under high glucose circumstance,after giving pentazocine intervention,the expression level and secretion level of GDNF further up raised.2 The expression and secretion level of BDNF decreased significantly in cultured retinal Müller glia cells under high glucose circumstance,the application of pentazocine could reverse the phenomenon that the expression level of BDNF decreased,however,it could not reverse secretion level of BDNF decreased. |
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