The Research Of Fluoxetine’s Mechanism On Promote The Repairation Of Cranial Nerve Function

Objective: To study the relationship between Fluoxetine’s role inimprove brain function and brain tissue nerve stem cells(NSC), in turn explorethe Fluoxetine’s possiblely mechanism in betterring and repair the cranialnerve function.Methods:1Identification and isolated cultivation of neural stem cells in neonatalrats: on the basis of existed rats’ neural stem cells’ isolation and cultureresearch, this experiment adopted the serum-free culture technology, andimproved several operation methods in experiment such as:(1)the use ofmechanical dissociation way during separated and passaged NSC, which canensure part of the primary cells is not affected by pancreaticenzyme.(2)increase the density of stem cells while cultivation in order topromote the growth and proliferation of them), isolated and cultured cellswhich has the ability of division and proliferation from neonatal rats’hippocampus tissue.2The influence of Fluoxetine stimulated neural stem cells (grouped theexperimental animals and samples detection): inoculate the subculture neuralstem cells in the trumpet petri dish respectively, the cell’s concentration is (4~5) x105/ml, while3ml each petri dish and three dish each group, totallyinoculate15trumpet petri dishes, they all applied serum-free conditionedmedium and grouped according to the clinical homeostasis blood drugconcentration of Fluoxetine (500nmol/L) then added into different finalconcentration of Fluoxetine respectively. Grouped into:(1) blank controlgroup (which mix into same volume of PBS)(2) low-dose Fluoxetine group(concentration of100nmol/L)(3) Medium-dose Fluoxetine group(500nmol/L)(the clinical homeostasis group)(4) high-dose Fluoxetine group(1000 nmol/L)(5) ultrahigh-dose Fluoxetine group (5000nmol/L). Apply Rt-pcrand western blo to detect each group’s difference mRNA and proteinexpression level between neural stem cells glia-derived neurotrophic factor(GDNF) and nerve nutrition factor (BDNF).3Apply Flow cytometry to detect the diversity of each group’s positivecell percentage of neural stem cells glial fibrillary acidic protein (GFAP),adopt SPSS18.0software to make statistical analysis of all results’ differences,P value <0.05.Results:1Under the condition of in vitro cultivation,separated and cultivated cellswith the division and proliferation ability which from neonatal ratshippocampus tissue, the pathological nestin immunohistochemical stainingshowed that the nestin antigen’s positive cells was beyond96%, and could beused in the subsequent experiment.2After Fluoxetine stimulate neural stem cells, each neural stem cells’BDNF’s mRNA, GDNF mRNA, BDNF protein expression, GDNF proteinexpression quantity all adopt the folds of control between groups and blankcontrol group by the statistical analysis， the experimental results arerespectively:(1)the basic trend of BDNF’s mRNA is:④＞③＞⑤≥②≥①;(2)basic trend of GDNF’s mRNA is:④≥③＞②≥⑤＞①;(3) basic trend ofBDNF’s protein expression quantity is:④＞③＞⑤≥②≥①;(4) the basictrend of GDNF’s protein expression quantity is：④≥③＞②＞⑤≥①.(PS:≥means P value is0.05or higher, the expression quantity between twogroups that have no significant difference;> means P value’s<0.05, theexpression quantity between two groups has significant difference).3After Fluoxetine stimulate neural stem cells, test result of thepercentage that each group of neural stem cells’ glial fibers acidic protein(GFAP) positive cell taken showed:(1): the blank control group：(12.52+3.83)%;(2) the Fluoxetine low-dose group:(21.73+3.37)%;(3) theFluoxetine medium-dose group:(25.68+4.21)%;(4) the Fluoxetinehigh-dose group:(35.34+4.39)%;(5) the Fluoxetine ultrahigh-dose group: (15.14+3.16)%;Basically trend is:(4)>(3)≥(2)>(5)≥(1).Conclusion:1Nestin immunohistochemical staining results showed that throughexisting experiment that improved and separated rats’ neural stem cells,obtained separation and purification rats’ neural stem cells successfully,andthen be used in the subsequent experiment.2Within a certain dosage range (In this experiment Fluoxetine’s dosageis less than1000nmol/L) Fluoxetine can stimulate neural stem cells toincrease neurotrophic factors’ transcription and translation, and raised someexpression of neurotrophic factors such as the brain derived neurotrophicfactor (BDNF)and glial derived neurotrophic factor(GDNF). But when withina ultrahigh dosage (the dosage of Fluoxetine is higher than1000nmol/L inthis experiment)may cause toxic injury to neural stem cells, and then affect thetranscription and translation of neurotrophic factors. Which explained that incertain dosage, Fluoxetine can promote the restoration and regeneration ofdamaged nerve indirectly by promote neural stem cells raised some expressionof neurotrophic factors.3Within a certain dosage range (In this experiment Fluoxetine’s dosageis less than1000nmol/L.) Fluoxetine can make Glial acidic protein fiber at ahigh expression level in neural stem cells, then increased the proportion ofneural stem cells’ GFAP positive cells, prompted some neural stem cellactivated and proliferated to astrocyte, it suggested that Fluoxetine maythrough the method that promote the astrocyte’s differentiation andproliferation in turn to promote damaged brain tissue’s restoration andstructure reconstruction.