| This subject took hippocampal neurons of newborn rats as the research o-bject.Study the suitable conditions of primary hippocampcal neurons cultured invitro objectively.The hippocampal nerve cells were cultured with 10-HDA of d-ifferent concentration.The neurual survival was detected by MTT assay.Nissl's staining and NSE immunohistochemistry were used to evaluate neurons prolifer-ation.BrdU immunofluorescence was used to detecte cell proliferation.And,the r-esearch results indicate:1.The newborn hippocampus were reacted by 0.125% parenzyme in a 37°C water bath for 10 min.Add DMEM/F12 (10% calt serum) to stop parenzyme digestion for 5min. Dissociated by pipetting up and down with a pipette for 20times.Centrifuge cells at 100g for 5min,twice.Filter cells with 200 mesh screen,then we could get good hippocampal nerve cells.2.Adjusted the cells concentration to 1×105cells/mL and incubate to the pe-tri dish coated with poly-ornithine in 5% CO2 incubator of 37℃. Planting me-dium were replaced by DMEM/F12 maintaining medium(without FCB,with 2% B27 supplement after 24h.Add AraC solution(final concentration of 5μg/mL)after48h to inhibit growth of glial cells.Replaced new DMEM/F12 maintaining medi-um after 48h of AraC effect.Then half-medium was changed three times a we-ek. Neurons could grow well.3.The best condition for Nissl's staining:color cells with cresyl violet at 25℃for 30 minutes,destain with 75% alcohol for 60 seconds.Then neurons and glial cells could be distinguished obviously.4.The best condition for NSE immunohistochemistry:incubate cells at 37℃with primary antibody for 1h and incubate cells at 37℃with secondary antib-ody for 20min.Then,neurons and glial cells could be distinguished obviously.5. The results of neurons survial with MTT indicated that 10-HDA of 1.0μmol/L could promote the survial and proliferation of neurons(p<0.05), the surv-ial and proliferation rate was(17.25±0.074)%.6. The detection results of BrdU immunofluorescence indicated that 10-HDA of 1.0μmol/L was positive to neuron's proliferation(p<0.05),the proliferation rate was(19.4±2.354)%.Compared with the control group,the number of neurons of the low concentration(0.1μmol/L)and high concentration(10μmol/L) didn't ch-ange obviously.7.The results of Nissl's staining indicated that 10-HDA of 1.0μmol/L was positive to neuron's proliferation(p<0.05), proliferation rate was(18.6±2.354)%. Compared with the control group,the number of neurons of the low concentrat-ion(0.1μmol/L)and high concentration(10μmol/L)didn't change obviously.8.The results of NSE indicated immunohistochemistry that 10-HDA of 1.0μmol/L was positive to neuron's proliferation(p<0.05),proliferation rate was (18.6±2.354)%.Compared with the control group,the number of neurons of the low concentration(0.1μmol/L)and high concentration(10μmol/L)didn't change obviou-sly.The results indicated that 10-HDA of appropriate concentration could stimulate the proliferation of rat hippocampal nerve cells in vitro. |
