發(fā)布時間：2018-04-17 08:10

  本文選題：神經(jīng)元 + 細胞　； 參考：《中國組織工程研究》2016年51期

【摘要】：背景:神經(jīng)元的體外原代培養(yǎng)在研究神經(jīng)系統(tǒng)的發(fā)育、再生、信號轉(zhuǎn)導(dǎo)機制、神經(jīng)藥理學以及基因表達方面具有極其重要的地位。目的:建立一種操作更加簡單且能夠得到較高純度新生SD大鼠皮質(zhì)神經(jīng)元原代培養(yǎng)的方法。方法:取1 d新生SD大鼠皮質(zhì)。傳統(tǒng)方法實驗組:取整個皮質(zhì);改良方法實驗組:取SD大鼠表面2.0-3.0 mm處皮質(zhì)。木瓜蛋白酶消化后離心,制備成單細胞懸液,以1×105/孔的濃度接種至含神經(jīng)元培養(yǎng)液的24孔培養(yǎng)板進行原代培養(yǎng)。培養(yǎng)3 d時采用免疫細胞化學染色方法,使用神經(jīng)元特異性標志物Tuj1與MAP-2雙標記法鑒定所培養(yǎng)的細胞;采用倒置相差顯微鏡觀察6,24,48,72 h及5,7 d細胞數(shù)和突起長度并記錄。結(jié)果與結(jié)論:1所培養(yǎng)的細胞可表達神經(jīng)元特異性標志物Tuj1與MAP-2,因此所培養(yǎng)細胞為神經(jīng)元,可用于之后實驗;2實驗組培養(yǎng)至第3天時神經(jīng)元的純度已達到峰值92%,而普通實驗組神經(jīng)元的純度為51%;32組細胞培養(yǎng)至6 h均已貼壁且長處小突起,培養(yǎng)至第3天時,細胞已初步形成神經(jīng)網(wǎng)絡(luò),培養(yǎng)至5 d時神經(jīng)網(wǎng)絡(luò)密集;4研究所用方法簡便能夠穩(wěn)定的培養(yǎng)出純度較高的神經(jīng)元,可以用于SD大鼠皮質(zhì)源性神經(jīng)元的相關(guān)實驗研究。
[Abstract]:Background: primary culture of neurons in vitro plays an important role in studying the development, regeneration, signal transduction mechanism, neuropharmacology and gene expression of nervous system.Aim: to establish a simple method for primary culture of cortical neurons in neonatal SD rats.Methods: the cortex of 1 day newborn SD rats was taken.The whole cortex was taken from the experimental group and the cortex from 2.0 mm to 3.0 mm on the surface of SD rats.After digesting papain, a single cell suspension was prepared and inoculated into a 24 well culture plate containing neuron culture medium at a concentration of 1 脳 10 5 / well for primary culture.The cultured cells were identified by immunocytochemical staining with neuron specific markers Tuj1 and MAP-2 at 3 days, and the number of cells and the length of processes were observed and recorded by inverted phase contrast microscope.Results and conclusion the cells cultured at 1: 1 could express the neuron-specific markers Tuj1 and MAP-2.Therefore, the cultured cells were neurons.The purity of neurons in the experimental group reached the peak value at the 3rd day, while the purity of the neurons in the ordinary experimental group was 51kW. The cells in the experimental group were all adherent to the wall and had small protrusions until 6 h, and at the third day of culture, the purity of the neurons in the experimental group reached the peak, and the purity of the neurons in the experimental group reached the peak at the third day.The neural network has been formed preliminarily, and the method of dense neural network is simple and stable to produce high-purity neurons, which can be used to study cortical neurons in SD rats.
【作者單位】： 昆明醫(yī)科大學神經(jīng)科學研究所;云南省第一人民醫(yī)院普外二科;昆明理工大學醫(yī)學院;
【基金】：國家自然科學基金(31560295,31260253,81260075) 云南省應(yīng)用基礎(chǔ)研究昆醫(yī)聯(lián)合專項(2015FB098,2014FZ066) 云南省教育廳科學研究基金項目(2014C020Y) 2014年省級臨床重點專科建設(shè)項目-普外科(云南省第一人民醫(yī)院)~~
【分類號】：R338

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