摘要
目的：
在动物实验水平观察丙泊酚麻醉对大鼠脑内星形胶质细胞及神经元功能标志蛋白的影响,进而在体外细胞培养实验中明确丙泊酚对星形胶质细胞THBS-1 mRNA及蛋白表达水平的调节作用,观察丙泊酚对单纯培养神经元及与星形胶质细胞共培养神经元突触密度及突起状态的影响。
方法：
①建立大鼠丙泊酚全身麻醉模型,制备大鼠脑组织石蜡切片,应用免疫组织化学法检测星形胶质细胞及神经元标志蛋白；②分离、纯化培养大鼠大脑皮层星形胶质细胞,设置不同浓度和不同时间的丙泊酚处理组,应用免疫荧光化学技术、RT-PCR及Western blotting检测丙泊酚对星形胶质细胞THBS-1 mRNA及蛋白表达的影响；③使用单纯培养及与星形胶质细胞共培养两种方式,培养大鼠大脑皮层神经元,以不同浓度丙泊酚对其进行处理,应用免疫荧光化学技术观察丙泊酚对不同方式培养的神经元突触密度及神经突起状态的影响。
结果：
①动物实验显示丙泊酚处理后大鼠大脑皮层内THBS-1蛋白的表达水平增加,而GFAP、MAP2及Synapsin I蛋白表达水平及分布无明显改变；②丙泊酚可浓度和时间依赖性地上调体外培养大鼠大脑皮层星形胶质细胞的THBS-1 mRNA及蛋白表达水平平,尤以终浓度为30μM和处理时间12h影响最为明显；③与星形胶质细胞共培养的神经元突触密度和突起密集程度均显著高于单纯培养的神经元,300μM丙泊酚可引起单纯培养神经元的突起形态改变,而30μM丙泊酚可一定程度地增加共培养组神经元的突触密度。
结论：
丙泊酚可时间和浓度依赖性地增加大鼠大脑皮层星形胶质细胞分泌THBS-1的水平。与星形胶质细胞共培养可改善体外培养神经元的生长状态,且丙泊酚可影响与星形胶质细胞共培养神经元的突触密度。
Objective:
①To observe the effects of propofol anesthesia on the functional protein of astrocytes and neurons in rat cortex.②To detect the THBS-1 mRNA and protein level of the cultured rat cortical astrocytes in different propofol treat groups.③To compare the influence of propofol on synapses and neurite outgrowth between the stand-alone cultured neuron and co-cultured neuron with astrocytes.
Methods:
①The general anesthesia model of rat using propofol was established firstly. The rat brain tissue slices were used to detect the functional proteins of astrocytes and neurons by immunohistochemisty.②Astrocytes were isolated from pooled rat cortex and grown in culture, then exposed to propofol with different concentrations or different time. The mRNA level of thrombospondin 1 (THBS-1) was detected by RT-PCR, and the protein level of THBS-1 by immunofluorescence cytochemistry and Western blotting.③The rat cortical neurons were cultured in two different ways, co-coltured with astrocytes or stand-alone cultured. Then the cells were treated with different concentrations of propofol. Immunofluorescence cytochemistry was applied to display the state of neuron synapses and neurites.
Results:
①The THBS-1 level was increased in rat cortex after intraperitoneal administration of propofol.②The mRNA and protein level of THBS-1 in cultured rat cortical astrocytes was upregulated by propofol in a time and concentration dependent way. Exposure to 30μM propofol for 12 h may lead to the extreme changes in THBS-1 level.③The synapse density and neurite net intensity of the neurons that were co-cultured with astrocytes surpassed those of the stand-alone cultured ones. The shape change of the neurite was observed in the 300μM propofol exposed group. Even higher synapse density was detected in the co-cultured neurons treated with 30μM propofol.
Conclusions:
The level of THBS-1 secreted by asrtocytes in the rat cortex could be upregulated in a time and dose dependent way when exposed to propofol. The growth state of cultured neuron can be improved by co-culturing with astrocytes. Propofol can influence the synapse density of the co-cultured neurons.
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