本文關(guān)鍵詞：大腦皮質(zhì)小膠質(zhì)細胞IL-6，IL-1β不對稱分泌及LPS相關(guān)受體的研究　出處：《汕頭大學》2008年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 腦不對稱 小膠質(zhì)細胞 白細胞介素6 白細胞介素1β TLR4/MD-2

【摘要】： 腦不對稱是機體的生物學特性之一,是普遍存在的生物學現(xiàn)象,主要表現(xiàn)在大腦左右兩半球在解剖結(jié)構(gòu)、遞質(zhì)分泌以及功能等方面存在異質(zhì)性。左、右兩側(cè)大腦皮質(zhì)對免疫系統(tǒng)的調(diào)控具有不對稱性,大腦皮層和中樞神經(jīng)許多神經(jīng)核團可影響機體的免疫應答,且不同部位作用不同。正常機體左側(cè)皮層擔負正反饋免疫調(diào)節(jié),右側(cè)皮層則為負反饋免疫調(diào)節(jié),兩側(cè)大腦皮層介導不同的免疫功能。神經(jīng)系統(tǒng)可通過HPA軸調(diào)節(jié)免疫系統(tǒng)的功能,腦不對稱可通過不對稱的神經(jīng)內(nèi)分泌作用于免疫系統(tǒng),使之具有不對稱性。小膠質(zhì)細胞是中樞神經(jīng)系統(tǒng)的巨噬細胞,激活后表現(xiàn)為中樞神經(jīng)系統(tǒng)的免疫效應細胞,是腦內(nèi)細胞因子的重要來源和作用部位;相對星形細胞,小膠質(zhì)細胞對LPS刺激的反應性更強,LPS與小膠質(zhì)細胞膜表面的LPS受體復合物結(jié)合后,激活小膠質(zhì)細胞并使其分泌IL-6,IL-1β等。LPS的受體復合物包括CD14,TLR4/MD2,在小膠質(zhì)細胞上均有表達。我們的實驗結(jié)果表明左、右兩側(cè)大腦皮質(zhì)小膠質(zhì)細胞在LPS刺激24小時后,右側(cè)大腦皮質(zhì)小膠質(zhì)細胞分泌的IL-6、IL-1β顯著高于左側(cè),即兩側(cè)細胞因子分泌不對稱,試想兩側(cè)皮質(zhì)小膠質(zhì)細胞膜表面的LPS受體復合物的表達是否也存在不對稱性呢?而細胞因子的產(chǎn)生與腦不對稱呈明顯的相關(guān)性,因此,本文旨在前期研究的基礎上,通過建立Balb/c小鼠大腦皮質(zhì)小膠質(zhì)細胞體外培養(yǎng)的模型,探討在正常情況及LPS刺激情況下左、右兩側(cè)大腦皮質(zhì)小膠質(zhì)細胞分泌細胞因子水平的變化,以及LPS受體復合物在兩側(cè)皮質(zhì)小膠質(zhì)細胞的表達水平,以期從神經(jīng)小膠質(zhì)細胞的免疫生物學效應上尋找其與腦不對稱性的關(guān)系;為揭示腦不對稱影響免疫功能的物質(zhì)基礎提供理論依據(jù)。我們做了以下工作: 1.探討B(tài)alb/c小鼠兩側(cè)大腦皮質(zhì)中小膠質(zhì)細胞接受LPS刺激前后IL-6的分泌情況及左、右兩側(cè)分泌情況的比較,以及其與腦不對稱影響免疫功能間的關(guān)系; 2.探討腦不對稱小鼠大腦皮質(zhì)中小膠質(zhì)細胞接受LPS刺激前后IL-1β的分泌情況及左、右兩側(cè)分泌情況的比較,以及其與腦不對稱影響免疫功能間的關(guān)系; 3.初步探討LPS受體復合物的在左、右腦兩側(cè)大腦皮質(zhì)小膠質(zhì)細胞上的表達,了解受體表達是否也存在不對稱性,以及這種異質(zhì)性是否為引起細胞因子分泌不對稱的原因之一。 材料和方法 新生(1d)的BALB/C小鼠,常規(guī)皮膚消毒,斷頭殺鼠,在無菌條件下迅速剝除硬腦膜,鈍性分離大腦皮層,將左、右兩側(cè)大腦皮層分別制成混合細胞懸液。培養(yǎng)20-25天,用溫和胰酶消化的方法得到純的小膠質(zhì)細胞懸液,細胞計數(shù),調(diào)整細胞濃度5×105/ml。轉(zhuǎn)入6孔板培養(yǎng)24小時(含10%FCS),24小時后用無FCS的培養(yǎng)液輕輕清洗小膠質(zhì)細胞3次,加入無FCS的培養(yǎng)液以及LPS,使LPS的終濃度為10μg/ml。培養(yǎng)24小時收集上清,凍存于-80℃?zhèn)溆�。用雙抗夾心法測上清中IL-6和IL-1β的含量。6孔板中的細胞的用途:提取RNA,在RNA水平檢測LPS受體復合物的表達情況。 結(jié)果 1.小鼠大腦皮質(zhì)小膠質(zhì)細胞分泌IL-6、IL-1β水平: ①正常對照組左、右腦大腦皮質(zhì)小膠質(zhì)細胞均分泌少量IL-6,左、右腦無明顯差別(P=0.28);用LPS刺激24小時后,IL-6濃度明顯增加,且右腦濃度高于左腦(P0.05)。 ②正常對照組左、右腦大腦皮質(zhì)小膠質(zhì)細胞均分泌少量IL-1β,且右腦較左腦高(P0.05);用LPS刺激24小時后,IL-1β的濃度增加,右腦濃度高于左腦(P0.05)。 2.小鼠大腦皮質(zhì)小膠質(zhì)細胞LPS受體表達: ①半定量PCR分析顯示,小膠質(zhì)細胞表面有CD14表達,且左、右腦大腦皮質(zhì)小膠質(zhì)細胞表達有差別,右側(cè)高于左側(cè),在LPS刺激后CD14表達上調(diào)(P0.05)。 ②半定量PCR分析顯示,小膠質(zhì)細胞表面有TLR4有表達,但MD-2少量表達,LPS刺激后左、右腦大腦皮質(zhì)小膠質(zhì)細胞有差別,右側(cè)高于左側(cè),TLR4表達下調(diào)(P0.05)。 結(jié)論 1.用溫和胰酶消化的方法得到大量高純度的小膠質(zhì)細胞。 2.左、右兩側(cè)大腦皮質(zhì)的小膠質(zhì)細胞對LPS刺激的反應性上存在差別,右側(cè)顯著高于左側(cè),提示當機體遭受革蘭陰性細菌感染時,右側(cè)大腦皮質(zhì)的小膠質(zhì)細胞較左側(cè)分泌較多的IL-6。 3.左、右兩側(cè)大腦皮質(zhì)的小膠質(zhì)細胞在LPS刺激下釋放IL-1β水平具有顯著差異,右側(cè)大腦皮質(zhì)的小膠質(zhì)細胞釋放IL-1β明顯高于左側(cè)。 4.左、右兩側(cè)大腦皮質(zhì)小膠質(zhì)細胞膜表面LPS受體復合物表達有明顯差異,右側(cè)高于左側(cè),在LPS刺激后CD14表達上調(diào),TLR4表達下調(diào)。 本實驗研究結(jié)果提示大腦巨噬細胞-小膠質(zhì)細胞在腦不對稱中發(fā)揮重要作用。
[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.

【學位授予單位】：汕頭大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

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