大脑皮质小胶质细胞IL-6，IL-1β不对称分泌及LPS相关受体的研究

发布时间：2018-01-07 09:07

本文关键词：大脑皮质小胶质细胞IL-6，IL-1β不对称分泌及LPS相关受体的研究　出处：《汕头大学》2008年硕士论文　论文类型：学位论文

【摘要】： 脑不对称是机体的生物学特性之一,是普遍存在的生物学现象,主要表现在大脑左右两半球在解剖结构、递质分泌以及功能等方面存在异质性。左、右两侧大脑皮质对免疫系统的调控具有不对称性,大脑皮层和中枢神经许多神经核团可影响机体的免疫应答,且不同部位作用不同。正常机体左侧皮层担负正反馈免疫调节,右侧皮层则为负反馈免疫调节,两侧大脑皮层介导不同的免疫功能。神经系统可通过HPA轴调节免疫系统的功能,脑不对称可通过不对称的神经内分泌作用于免疫系统,使之具有不对称性。小胶质细胞是中枢神经系统的巨噬细胞,激活后表现为中枢神经系统的免疫效应细胞,是脑内细胞因子的重要来源和作用部位;相对星形细胞,小胶质细胞对LPS刺激的反应性更强,LPS与小胶质细胞膜表面的LPS受体复合物结合后,激活小胶质细胞并使其分泌IL-6,IL-1β等。LPS的受体复合物包括CD14,TLR4/MD2,在小胶质细胞上均有表达。我们的实验结果表明左、右两侧大脑皮质小胶质细胞在LPS刺激24小时后,右侧大脑皮质小胶质细胞分泌的IL-6、IL-1β显著高于左侧,即两侧细胞因子分泌不对称,试想两侧皮质小胶质细胞膜表面的LPS受体复合物的表达是否也存在不对称性呢?而细胞因子的产生与脑不对称呈明显的相关性,因此,本文旨在前期研究的基础上,通过建立Balb/c小鼠大脑皮质小胶质细胞体外培养的模型,探讨在正常情况及LPS刺激情况下左、右两侧大脑皮质小胶质细胞分泌细胞因子水平的变化,以及LPS受体复合物在两侧皮质小胶质细胞的表达水平,以期从神经小胶质细胞的免疫生物学效应上寻找其与脑不对称性的关系;为揭示脑不对称影响免疫功能的物质基础提供理论依据。我们做了以下工作: 1.探讨Balb/c小鼠两侧大脑皮质中小胶质细胞接受LPS刺激前后IL-6的分泌情况及左、右两侧分泌情况的比较,以及其与脑不对称影响免疫功能间的关系; 2.探讨脑不对称小鼠大脑皮质中小胶质细胞接受LPS刺激前后IL-1β的分泌情况及左、右两侧分泌情况的比较,以及其与脑不对称影响免疫功能间的关系; 3.初步探讨LPS受体复合物的在左、右脑两侧大脑皮质小胶质细胞上的表达,了解受体表达是否也存在不对称性,以及这种异质性是否为引起细胞因子分泌不对称的原因之一。材料和方法新生(1d)的BALB/C小鼠,常规皮肤消毒,断头杀鼠,在无菌条件下迅速剥除硬脑膜,钝性分离大脑皮层,将左、右两侧大脑皮层分别制成混合细胞悬液。培养20-25天,用温和胰酶消化的方法得到纯的小胶质细胞悬液,细胞计数,调整细胞浓度5×105/ml。转入6孔板培养24小时(含10%FCS),24小时后用无FCS的培养液轻轻清洗小胶质细胞3次,加入无FCS的培养液以及LPS,使LPS的终浓度为10μg/ml。培养24小时收集上清,冻存于-80℃备用。用双抗夹心法测上清中IL-6和IL-1β的含量。6孔板中的细胞的用途:提取RNA,在RNA水平检测LPS受体复合物的表达情况。结果 1.小鼠大脑皮质小胶质细胞分泌IL-6、IL-1β水平: ①正常对照组左、右脑大脑皮质小胶质细胞均分泌少量IL-6,左、右脑无明显差别(P=0.28);用LPS刺激24小时后,IL-6浓度明显增加,且右脑浓度高于左脑(P0.05)。 ②正常对照组左、右脑大脑皮质小胶质细胞均分泌少量IL-1β,且右脑较左脑高(P0.05);用LPS刺激24小时后,IL-1β的浓度增加,右脑浓度高于左脑(P0.05)。 2.小鼠大脑皮质小胶质细胞LPS受体表达: ①半定量PCR分析显示,小胶质细胞表面有CD14表达,且左、右脑大脑皮质小胶质细胞表达有差别,右侧高于左侧,在LPS刺激后CD14表达上调(P0.05)。 ②半定量PCR分析显示,小胶质细胞表面有TLR4有表达,但MD-2少量表达,LPS刺激后左、右脑大脑皮质小胶质细胞有差别,右侧高于左侧,TLR4表达下调(P0.05)。结论 1.用温和胰酶消化的方法得到大量高纯度的小胶质细胞。 2.左、右两侧大脑皮质的小胶质细胞对LPS刺激的反应性上存在差别,右侧显著高于左侧,提示当机体遭受革兰阴性细菌感染时,右侧大脑皮质的小胶质细胞较左侧分泌较多的IL-6。 3.左、右两侧大脑皮质的小胶质细胞在LPS刺激下释放IL-1β水平具有显著差异,右侧大脑皮质的小胶质细胞释放IL-1β明显高于左侧。 4.左、右两侧大脑皮质小胶质细胞膜表面LPS受体复合物表达有明显差异,右侧高于左侧,在LPS刺激后CD14表达上调,TLR4表达下调。本实验研究结果提示大脑巨噬细胞-小胶质细胞在脑不对称中发挥重要作用。
[Abstract]:Brain asymmetry is one of the biological characteristics of the body, is a biological phenomenon, mainly in the two hemispheres in the anatomical structure, the heterogeneity of neurochemical and functional aspects. Left with asymmetric regulation on the immune system of the right sides of the brain cortex and many central nervous nuclei can affect the immune response of the body, and different parts of different effects. The normal body left neocortex enhances immune feedback, right cortex is negative feedback regulation of immunity, on both sides of the cerebral cortex mediated immune function. Different nervous system can regulate the immune system through the function of HPA axis, brain asymmetry through neuroendocrine role asymmetry in immune system. Which is asymmetric. Microglia are central nervous system macrophages after activation showed immune effect of the central nervous system of fine The cell, is an important source and site of action of cytokines in the brain; relative to more reactive astrocytes, LPS stimulated microglia, LPS receptor complex LPS and microglial cell membrane binding, activation of microglia and the secretion of IL-6 receptor complexes including CD14, TLR4/MD2, IL-1 beta.LPS, expressed in microglial cells. Our experimental results show that the left and right sides of the brain microglia stimulated 24 hours after LPS, the right side of the brain cortex secretion of microglia IL-6, IL-1 beta was significantly higher than the left, is on both sides of the cytokine secretion asymmetry, expression of the LPS receptor complex on both sides think cortical microglia of the membrane surface is there asymmetry? And the production of cytokines and brain asymmetry was significantly correlated. Therefore, this paper based on the previous studies, through the establishment of Balb/c mouse brain cortex Quality of microglial cells in vitro model of normal and LPS stimulation of the left and right sides cytokines of cortical microglia, and LPS receptor complexes in the expression level of cortical microglia, in order to find the relationship between cerebral asymmetry from immune nerve biology effect microglia on; to provide a theoretical basis for the material basis of influence of brain asymmetry immune function. We do the following work:
1. to investigate the secretion of IL-6 and the secretion of left and right sides of small glial cells in Balb/c mice after LPS stimulation, and the relationship between them and the effect of brain asymmetry on immune function.
2. to investigate the secretion of IL-1 beta and the secretion of left and right sides of small glial cells in the brain cortex of mice with asymmetric brain stimulation, and the relationship between LPS and brain asymmetry and immune function.
3., we preliminarily investigated the expression of LPS receptor complex in the microglia on the left and right sides of the cerebral cortex, to know whether the receptor expression is also asymmetric, and whether this heterogeneity is one of the reasons causing cytokine secretion asymmetry.
Materials and methods
The new (1D) BALB/C mice, normal skin disinfection, beheaded kill the rats under aseptic conditions, rapid stripping of the dura mater, blunt dissection of the cerebral cortex, the left and right sides of the cerebral cortex were made from mixed cell suspension. After cultured 20-25 days, pure microglial cell suspension obtained by mild trypsinization cell count, adjust cell concentration of 5 * 105/ml. into 6 well culture plates for 24 hours (including 10%FCS), after 24 hours in culture medium without FCS gently cleaning microglial cells 3 times, adding liquid and LPS medium without FCS, the final concentration of LPS is 10 g/ml. 24 hour culture supernatant was collected, frozen to use the content of.6. -80 C spare orifice plate with sandwich method to measure IL-6 and IL-1 beta in the supernatant of cells: extraction of RNA, the RNA expression level of LPS receptor complex was detected.
Result
1. the microglia of the cerebral cortex secretes IL-6 and IL-1 beta in the cerebral cortex of mice.
(1) in normal control group, microglia in left and right cerebral cortex secreted a small amount of IL-6, but there was no significant difference between left and right brain (P=0.28). After stimulation with LPS for 24 hours, the concentration of IL-6 increased significantly, and the concentration of right brain was higher than that of left brain (P0.05).
(2) in normal control group, microglia in left and right cerebral cortex secrete a small amount of IL-1 beta, and the right brain is higher than the left brain (P0.05). After stimulation with LPS for 24 hours, the concentration of IL-1 beta increases and the concentration of right brain is higher than that of left brain (P0.05).
2. the expression of LPS receptor in microglia in cerebral cortex of mice:
(1) semi quantitative PCR analysis showed that there was CD14 expression on the surface of microglia, and the expression of microglia in the left and right cerebral cortex was different, the right side was higher than the left side, and the expression of CD14 was up-regulated (P0.05) after LPS stimulation.
(2) semi quantitative PCR analysis showed that TLR4 was expressed on the surface of microglia, but MD-2 was expressed in small amounts. After LPS stimulation, there were differences in microglia between left and right cerebral cortex, right side was higher than left side, and TLR4 expression was down regulated (P0.05).
conclusion
1. a large number of high purity microglia were obtained by mild trypsin digestion.
2., there were differences in the responsiveness of microglia on the left and right sides of the cerebral cortex to LPS stimulation. The right side was significantly higher than the left side. It indicated that when the body suffered from gram negative bacterial infection, the microglia in the right cerebral cortex secrete more IL-6. than the left side.
3. there was significant difference in the release of IL-1 beta between microglia in the left and right bilateral cerebral cortex stimulated by LPS, and the release of IL-1 beta from microglia in the right cerebral cortex was significantly higher than that on the left side.
4. there was significant difference in the expression of LPS receptor complex on the surface of microglia on the left and right sides of the brain. The right side was higher than the left side. After LPS stimulation, the CD14 expression was up-regulated and the TLR4 expression was down regulated.
The results of this study suggest that the brain macrophages - microglia play an important role in brain asymmetry.

【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【相似文献】