The Research Of17-β Estradiol Down-regulates PTEN To Prevent Apoptosis In H₂O₂-induced Astrocytes Of Rat Spinal Cord

Objectives: To collect primary astrocytes from spinal cord of neonatal rats, and toculture and passage for identification and determination of purity of astrocytes.Methods: The astrocytes were isolated from spinal cord of neonatal rats which wereborn within3days，inoculated in the75cm²bottle by107¹09cells/L，placed in theincubato（r37℃、5%CO₂），cultivated by DMEM media added with15%fetal bovineserum for8⁹days， and depurated by differential velocity adherenttechnique,swinging and passaging.We will observe the morphological change of thecells and identify the astrocytes by immunofluorescence staining.Results: High purity astrocytes were achieved by combination of differentialvelocity adherent technique and shaking，and with the gradual passage.The cells willbecame more purified gradually,and the morphology of astrocytes also changedgradually.The cells turned on clear boundary,and cytoplasmic process long.Conclusion: Primary astrocytes can be collected from spinal cord of newborn ratand and high-purity astrocytes can be obtained by shaking and differential velocityadherent technique. Objectives: To establish the injury model of astrocytes of spinal cord induced byH₂O₂whose concentration was established by the method of MTT.And to observethe morphological change and survival rate after treatment with20nmol/L estrogenfor2hours prior to exposure to400μmol/L H₂O₂for24hours.Methods: The astrocytes were isolated from spinal cord of neonatal rats which wereborn within3days,inoculated in the75cm2bottle by107¹09cells/L，placed in theincubato（r37℃、5%CO₂），cultivated by DMEM media added with15%fetal bovineserum for8⁹days， and depurated by differential velocity adherenttechnique,swinging and passaging. The model of astrocytes injury was made byH₂O₂treating for24h whose concentration was established by the method ofMTT.After treatment with20nmol/L estrogen for2hours prior to exposure to400μmol/L H₂O₂for24hours,we observed the change of morphology and survivalrate which were determined by the optical density of490nm.Results: After H₂O₂treating for24h,part of cells became smaller,cytoplasmicprocess became shorter,a few of cells became round and float in the media. However,after treatment with20nmo/L estrogen for2hours prior to exposure to H₂O₂for24hours， the majority of cell body became larger,the cytoplasmic process becamelonger and interwined,fewer of cells dead. The cell viability in astrocytes oftreatment with H₂O₂was reduced. But exposure to estrogen prior to exposure toH₂O₂provided partial restoration of cell viability.Conclusion: The injury model of astrocytes of spinal cord can be established byH₂O₂.17-βestradiol can increase the cell viability and protect the astrocytes whichinjuried by H₂O₂. Objective: To investigate the mechanism of protective effects of17-β estradiol onthe experimental model of spinal cord injury （SCI） rats.Method: First,the primary astrocytes from spinal cord of SD rat within3days werecultured and identified. When the astrocytes were cultured the third generation,theastrocytes were induced by H₂O₂whose concentration was established by the methodof MTT. The cells were divided into five groups randomly：controlled group; thegroup of treatment with400μmol/L H₂O₂for24hours; the group of treatment with20nmol/L estrogen for2hours prior to exposure to400μmol/L H₂O₂for24hours;the group of treatment with20nmol/L estrogen for26hours; the group of treatmentwith DMSO for26hours. The protein which was extracted from these cells aftertreatment with kinds of facters was detected by western blotting.Results: Exposure of astrocytes to H₂O₂resulted in significantly increased PTENand caspase-3expressions. However,treatment cells with17-βestradiol prior to H₂O₂exposure down-regulate PTEN （F=290.003P=0.001<0.01） and caspase-3（F=46.158P=0.023<0.05） expressions compared with the group of treatment astrocytes withH₂O₂.Treatment of astrocytes with H₂O₂resulted in significantly decreased levels ofp-Akt and Bcl-2.17-β estradiol treatment of cells increased the levels of p-Akt（F=49.173P=0.033<0.05）and Bcl-2（F=115.916P=0.001<0.01）compared withthe group of treatment astrocytes with H₂O₂.Conclusion: These findings suggest that attenuation of PTEN expression mediatedby estrogen is associated with an increase in phosphorylation/activation of the Aktand the Bcl-2expressions. Our studies suggest that the protective effects of 17-βestradiol on the experimental model of SCI rats may depend on the estrogenprotection to the astrocytes which may be mediated through decrease in PTENexpression.