高渗盐水通过抑制钠、钾、氯、同向转运蛋白1的表达减轻脑水肿的体外研究
发布时间：2018-09-18 14:40
【摘要】：高渗盐水(Hypertonic saline, HS)常在临床上用于治疗各种原因导致的脑水肿,与甘露醇相比,其疗效更持久、降颅压幅度更大,但尿量却更少。有研究发现其减轻脑水肿的机制并不仅仅是通过传统上认为的渗透性脱水机制,还有非渗透性机制在参与,它可以通过减少脑缺血灶周边组织水通道蛋白AQP4的表达减少对水的转运,和/或抑制脑微血管周边星形胶质细胞上血管内皮生长因子VEGF及其受体VEGFR2的表达,减小血脑屏障对水的通透性从而减轻脑水肿。另一个与脑水肿密切相关的蛋白,钠、钾、氯同向转运蛋白1(Na+-K+-Cl-cotransporterl, NKCC1)受到广泛关注,研究表明利用其特异性抑制剂可以明显减轻脑水肿,并且可改善脑神经功能预后。前期动物实验证实HS可以通过减少脑缺血灶周边NKCC1的表达而减轻脑水肿,还可以抑制脑内小胶质细胞释放炎症因子,但其机制尚不明确。本实验通过体外培养小胶质细胞及星形胶质细胞,探讨HS对小胶质细胞上炎症因子表达及释放的影响、炎症因子对星形胶质细胞上NKCC1的影响,以及HS是否与星形胶质细胞上NKCC1R表达存在直接的关系,以阐明HS与减少炎症因子释放及下调NKCC1表达的潜在机制。 第一章高渗盐水对原代小胶质细胞释放炎症因子的影响 目的：从混合培养的胶质细胞中纯化分离得到小胶质细胞,在缺氧状态下利用HS进行干预,探讨HS是否会影响小胶质细胞释放炎症子如肿瘤坏死因子(tumor necrosis factor,TNF-α)、白介素-1β(interleukin-1beta, IL-1β)及单核细胞趋化蛋白1(monocyte chemoattractant protein-1,MCP-1)。 方法：取出生0-24h的SD大鼠乳鼠的大脑皮层,胰酶消化后进行混合胶质细胞培养,培养7-10天后利用摇床震摇法纯化分离得到小胶质细胞,利用免疫荧光的方法检测小胶质细胞纯度,如果纯度大于95%则可进行下一步实验。 首先检测不同浓度的HS对小胶质细胞活性的影响,将纯化后的细胞分为三组：对照组、缺氧+无糖培养基(缩写为：缺氧组)及缺氧+无糖培养基+HS(缩写为：HS组),其中HS组又根据HS的浓度分为7个亚组：40mM、60nM、80mM、100mM、120mM、140mM及160mM组,缺氧组及HS各组在3%02,5%C02,92%N2条件下进行缺氧。各组进行相应处理4小时后利用CCK-8的方法检测各组细胞活性。 为确定最佳的缺氧时间,将纯化的小胶质细胞分为对照组及缺氧组,缺氧组又根据不同的缺氧时间分为1h组、2h组及4h组。各组处理至相应时间后提取细胞培养基上清,利用ELISA的方法检测培养基中TNF-α含量。 为筛选最佳HS浓度,将小胶质细胞分为对照组、缺氧组、HS组(浓度由前面细胞活性结果决定),缺氧时间由前面最佳缺氧时间决定。各组在相应处理后提取细胞培养基上清进行ELISA实验,检测其中炎症因子TNF-a的含量。 在确定最佳缺氧时间及最佳HS浓度后,探索HS是否可以抑制小胶质细胞释放炎症因子,小胶质细胞被分为3组：对照组、缺氧组及HS组,缺氧组及HS组于3%O2,5%CO2,92%N2的条件下缺氧,在进行相应处理后利用ELISA的方法检测小胶质细胞培养基中炎症因子TNF-α、IL-1β及MCP-1的含量。 结果：混合培养小胶质细胞在培养两天后大多数细胞均可良好贴壁,可较清楚分辨出形态,多呈多边形或棒状；培养至第五天时,混合胶质细胞已铺满瓶底70%,培养基上清中可见少量圆形、折光性强的细胞；第七天胶质细胞已铺满瓶底,折光性强的细胞逐渐增多；第九天时,细胞上清中折光强的细胞大量增多。利用摇床震摇法纯化的小胶质细胞,免疫荧光方法检测纯化后的小胶质细胞的纯度为97.16±0.983%。 细胞活性检测发现HS120mM、140mM、160mM组的小胶质细胞活性明显下降(P0.05),而40mM、60mM、80mM、100mM组的小胶质细胞与对照组相比活性无明显差异(P0.05)；小胶质细胞在缺氧后1,2,4小时均释放大量的炎症介质,以4小时TNF-a含量最高(P0.05);40mM HS.60mM HS、80mM HS、100mM HS组在缺氧4小时后,TNF-a含量均明显减少(P0.05),其中以100mM组减少最为明显。根据上述筛选结果,缺氧组小胶质细胞在缺氧4小时后释放的炎症因子TNF-α、IL-1β及MCP-1明显增加(P0.05)；而使用100mM HS处理的HS组与缺氧组相比,小胶质细胞释放炎症因子明显减少(P0.05)。 结论：小胶质细胞在缺氧后各个时间均迅速激活并释放大量炎症因子,而不同浓度的HS均可一定程度上抑制缺氧后小胶质细胞的激活并减少其释放炎症因子的含量,具有减轻炎症反应的作用。 第二章高渗盐水影响小胶质细胞释放炎症因子的机制 目的：探讨100mM HS影响小胶质细胞激活及释放炎症介质的潜在机制 方法：利用p38及JNK信号通路的特异性抑制剂SB203580及SP600125探讨小胶质细胞是否是通过这两条信号通路释放炎症因子。原代培养混合胶质细胞后进行纯化,将纯化的细胞分为五组：对照组、缺氧组、HS组、缺氧+无糖培养基+SB203580组(缩写为：SB203580组)及缺氧+无糖培养基+SP600125组(缩写为：SP600125组),然后在3%O2,5%CO2,92%N2的条件下缺氧4小时,利用ELISA的方法检测各组培养基中炎症因子TNF-α、IL-1β及MCP-1的含量。为检测HS是否会影响小胶质细胞中的p38及JNK两条信号通路,将纯化后的细胞分为对照组、缺氧组及HS组,缺氧组和HS组又根据缺氧时间不同分为30min,1h,2h,4h组,缺氧至相应时间点后,提取各组细胞的总蛋白进行western blot实验,检测Phos-p38及Phos-JNK蛋白水平。 结果：与对照组相比,小胶质细胞在缺氧4小时后释放大量的炎症因子TNF-α、IL-1β及MCP-1(P0.05),而与缺氧组相比,HS组、SB203580组及SP600125组中炎症因子TNF-α、IL-1β及MCP-1含量均明显减少(P0.05); western blot实验显示缺氧组小胶质细胞在缺氧30分钟后即可观察到Phos-p38及Phos-JNK蛋白表达明显增加并持续到4小时(P0.05),而HS组小胶质细胞中,缺氧各个时间点表达的Phos-p38及Phos-JNK蛋白与缺氧组相比均明显减少(P0.05)。 结论：HS可能通过抑制小胶质细胞上的p38及JNK这两条信号通路激活而减少了炎症因子的释放,减轻了缺血缺氧后脑内的炎症反应,防止炎症反应的进一步扩大,从而保护了血脑屏障,有利于减轻脑水肿。 第三章炎症因子TNF-及IL-1β对星形胶质细胞表达NKCC1的影响 目的：探讨炎症因子TNF-α及IL-1β对星形胶质细胞上NKCC1表达的影响,以及TNF-a及IL-1β浓度的变化是否会影响NKCC1的表达。 方法：原代培养星形胶质细胞,利用震摇的方法纯化后将星形胶质细胞分为6组,分别是：对照组、TNF-α (10ng/ml)+无糖培养基组(缩写为：TNF-α (10ng/ml)组)、TNF-α(5ng/ml)+无糖培养基组(缩写为：TNF-α (5ng/ml)组)、IL-1β(10ng/ml)+无糖培养基组(缩写为：IL-1β(10ng/ml)组)、IL-1β(5ng/ml)+无糖培养基组(缩写为：IL-1β(5ng/ml)组)、TNF-α(10ng/ml)+TNFR特异拮抗剂(2μM)(TNFR antagonist组)+无糖培养基组、IL-1β(10ng/ml)+IL-1R拮抗剂(2μM)(IL-1R antagonist组)+无糖培养基组及无糖培养基组(缩写为：Glucose-free medium组)。实验前加入相应浓度的细胞因子,处理4小时后进行相应的检测。然后利用免疫荧光双标、western blot及RT-PCR的方法进行检测星形胶质细胞上NKCC1的表达。 结果：免疫荧光实验表明TNF-α及IL-1β可明显增加星形胶质细胞上NKCC1表达,而TNF-α及IL-1β相应的受体特异拮抗剂则可以明显下调NKCC1的表达；RT-PCR及Western blot结果显示,5ng/ml或10ng/ml的TNF-α或IL-1β均可以明显上调NKCC1mRNA及蛋白的表达,并且随着炎症因子浓度升高,NKCC1mRNA及蛋白的表达也随之增高(P0.05),在应用TNF-α及IL-1β相应的受体特异拮抗剂后NKCC1mRNA及蛋白的表达明显降低(P0.05)；另外,NKCC1的表达不受无糖培养基的影响。 结论：炎症因子TNF-α、IL-1β可以明显上调星形胶质细胞上NKCC1的表达,引起脑水肿;并且随着炎症因子TNF-α及IL-1p浓度的增加,星形胶质细胞上NKCC1的表达也随之增加,从而加重脑水肿。而HS可以明显抑制小胶质细胞释放炎症因子TNF-α及IL-1β,从而下调星形胶质细胞上NKCC1的表达,达到减轻脑水肿的效果。 第四章HS对星形胶质细胞上NKCC1的直接影响 目的：探讨HS是否会直接影响缺氧状态下星形胶质细胞上NKCC1的表达 方法：将原代培养的星形胶质细胞分为对照组(完全正常培养)、缺氧组、HS组及缺氧+无糖培养基+100mM Bumetanide(缩写为Bumetanide组),缺氧组、HS组及Bumetanide组均在3%O2,5%CO2,92%N2的环境中缺氧4小时。在缺氧前30分钟,HS组及Bumetanide组分别加入终浓度为100mM的HS和100μM的Bumetanide。各组相应处理4小时后,利用免疫荧光双标、western blot及RT-PCR的方法检测星形胶质细胞上NKCC1的表达。 结果：缺氧组、HS组及Bumetanide组星形胶质细胞在缺氧之后,NKCC1mRNA的表达较对照组明显上调；但HS及bumetanide组与缺氧组相比,NKCC1mRNA的表达量明显减少。免疫荧光实验结果显示,与对照组相比,缺氧4小时后缺氧组、HS组及Bumetanide组中星形胶质细胞上NKCC1的表达均明显上升；而HS组及Bumetanide组与缺氧组相比,NKCC1蛋白表达则明显下降(P0.05)。 Western blot结果表明缺氧组、HS组及Bumetanide组在缺氧4小时后,NKCC1蛋白表达均明显上升(P0.05)；而HS组及Bumetanide组与缺氧组相比,NKCC1蛋白表达量明显下降(P0.05)。 结论：缺氧以后星形胶质细胞上NKCC1的表达明显增加,而HS可以在缺氧条件下直接抑制星形胶质细胞上NKCC1的表达,从而减轻脑水肿,但HS直接抑制NKCC1表达的确切机制仍有待进一步研究。
[Abstract]:Hypertonic saline (HS) is often used clinically to treat brain edema caused by various causes. Compared with mannitol, HS has a more lasting effect, a greater reduction in intracranial pressure, but less urine volume. Some studies have found that its mechanism of reducing brain edema is not only through the traditional osmotic dehydration mechanism, but also through the non-osmotic mechanism. Involved, it can reduce water transport by reducing the expression of aquaporin AQP4 in peripheral tissues of cerebral ischemic foci, and/or inhibit the expression of vascular endothelial growth factor-VEGF and its receptor-VEGFR2 on astrocytes around cerebral microvasculature, thereby reducing the permeability of blood-brain barrier to water and thus reducing brain edema. Relevant proteins, Na + - K + - Cl - cotransporter 1 (NKCC1) have attracted much attention. Studies have shown that specific inhibitors can significantly reduce brain edema and improve the prognosis of neurological function. In this study, we investigated the effect of HS on the expression and release of inflammatory factors on microglia, the effect of inflammatory factors on NKCC1 on astrocytes, and the relationship between HS and astrocytes by culturing microglia and astrocytes in vitro. There is a direct relationship between NKCC1R expression and HS to elucidate the underlying mechanism of reducing inflammatory factor release and NKCC1 expression.
Chapter 1 the effect of hypertonic saline on the release of inflammatory factors from primary microglia
AIM: To isolate microglia from mixed cultured glial cells and to investigate whether HS can affect the release of inflammatory molecules such as tumor necrosis factor (TNF-a), interleukin-1 beta (IL-1beta) and monocyte chemoattractant protein-1 (monocyte c) from microglia under hypoxia. Hemoattractant protein-1, MCP-1).
Methods: Mixed glial cells were cultured in the cerebral cortex of SD rats born 0-24 hours after trypsin digestion. After 7-10 days of culture, microglia were purified and isolated by shaking method. The purity of microglia was detected by immunofluorescence. If the purity was more than 95%, the next experiment could be carried out.
The purified cells were divided into three groups: control group, hypoxia + sugar-free medium (abbreviated as: hypoxia group) and hypoxia + sugar-free medium + HS (abbreviated as: HS group). HS group was divided into seven subgroups according to the concentration of HS: 40mM, 60nM, 80mM, 100mM, 120mM, 140 mM and 160m, respectively. The oxygen group and HS groups were hypoxic at 3% 02, 5% C02, 92% N2. The cell viability of each group was detected by CCK-8 after 4 hours of corresponding treatment.
In order to determine the optimal hypoxia time, the purified microglia were divided into control group and hypoxia group, and the hypoxia group was divided into 1 hour group, 2 hour group and 4 hour group according to different hypoxia time.
In order to select the best HS concentration, microglia were divided into control group, hypoxia group, HS group (the concentration was determined by the results of cell activity) and hypoxia time was determined by the optimal hypoxia time.
After determining the optimal hypoxia time and concentration of HS, to explore whether HS can inhibit the release of inflammatory factors from microglia, microglia were divided into three groups: control group, hypoxia group and HS group, hypoxia group and HS group under the condition of 3% O2, 5% CO2, 92% N2. After corresponding treatment, microglia culture medium was detected by ELISA method. The levels of inflammatory factors TNF-, IL-1 and MCP-1.
RESULTS: Most of the microglia cells adhered well to the wall after two days of culture and could be distinguished clearly, most of them were polygonal or rod-shaped. By the fifth day of culture, the mixed glia cells were covered with 70% of the bottom of the bottle, and a few round and refractive cells were found in the supernatant of the culture medium. On the seventh day, the glia cells were covered with the bottom of the bottle. On the ninth day, the number of highly refractive cells in the supernatant increased. The purity of the purified microglia was 97.16 6550
The activity of microglia in HS120 mM, 140 mM, 160 mM group was significantly decreased (P 0.05), while the activity of microglia in 40 mM, 60 mM, 80 mM, 100 mM group was not significantly different from that in control group (P 0.05); microglia released a large number of inflammatory mediators at 1, 2, and 4 hours after hypoxia, and the content of TNF-a was the highest at 4 hours (P 0.05). TNF-a content in HS, 80mHS and 100mHS groups decreased significantly after 4 hours of hypoxia (P 0.05), especially in 100mHS group. The release of inflammatory factors by glial cells was significantly reduced (P0.05).
CONCLUSION: Microglia can activate and release a large number of inflammatory factors rapidly at various times after hypoxia, and HS at different concentrations can inhibit the activation of microglia and reduce the release of inflammatory factors to a certain extent.
The second chapter is about the mechanism of hypertonic saline affecting microglia to release inflammatory factors.
Objective: To explore the potential mechanism of 100mM HS affecting microglia activation and release of inflammatory mediators.
Methods: The specific inhibitors of p38 and JNK signaling pathway, SB203580 and SP600125, were used to investigate whether microglia release inflammatory factors through these two signaling pathways. The content of inflammatory factors TNF-a, IL-1 beta and MCP-1 in SB203580 group and SP600125 group (abbreviated as SP600125 group) were detected by ELISA after 4 hours of hypoxia in 3% O2, 5% CO2, 92% N2 medium. The purified cells were divided into control group, hypoxia group and HS group, hypoxia group and HS group according to different hypoxia time and divided into 30 min, 1 h, 2 h, 4 h groups. After hypoxia to the corresponding time point, the total protein of each group was extracted and the levels of Phos-p38 and Phos-JNK protein were detected by Western blot.
Results: Compared with the control group, microglia released a large number of inflammatory factors TNF-a, IL-1 beta and MCP-1 after 4 hours of hypoxia (P 0.05). Compared with the hypoxia group, the contents of inflammatory factors TNF-a, IL-1 beta and MCP-1 in HS group, SB203580 group and SP600125 group were significantly decreased (P 0.05); Western blot showed that microglia in hypoxia group were significantly reduced at 30 minutes of hypoxia (P 0.05). The expression of Phos-p38 and Phos-JNK proteins increased significantly and lasted for 4 hours (P 0.05). In HS microglia, the expression of Phos-p38 and Phos-JNK proteins decreased significantly at different time points of hypoxia (P 0.05).
CONCLUSION: HS may reduce the release of inflammatory factors by inhibiting the activation of p38 and JNK signaling pathways on microglia, alleviate the inflammatory reaction in the brain after ischemia and hypoxia, prevent the further expansion of inflammatory reaction, thus protect the blood brain barrier and help to reduce brain edema.
The third chapter is the effect of inflammatory factors TNF- and IL-1 beta on the expression of NKCC1 in astrocytes.
AIM: To investigate the effects of inflammatory factors TNF-a and IL-1beta on the expression of NKCC1 in astrocytes and whether the changes of TNF-a and IL-1beta concentrations affect the expression of NKCC1.
Methods: Primary cultured astrocytes were purified by shaking method and then divided into 6 groups: control group, TNF-a (10ng/ml) + sugar-free medium group (abbreviated as TNF-a (10ng/ml), TNF-a (5ng/ml) + sugar-free medium group (abbreviated as TNF-a (5ng/ml), IL-1 beta (10ng/ml) + sugar-free medium group (abbreviated as TNF-a (5ng/ml), IL-1 beta (10ng/ml) + sugar-free medium group (abbreviated as follows:TNF-a (5ng/ml)). Written as: IL-1 beta (10ng/ml), IL-1 beta (5ng/ml) + sugar-free medium group (abbreviated as: IL-1 beta (5ng/ml), TNF-a (10ng/ml) + TNFR specific antagonist (2ugM) (TNFR antagonist group) + sugar-free medium group, IL-1 beta (10ng/ml) + IL-1R antagonist (2ugM) (IL-1R antagonist group) + sugar-free medium group (abbreviated as: Glucose-free medium group). The expression of NKCC1 on astrocytes was detected by immunofluorescence double labeling, Western blot and RT-PCR.
Results: Immunofluorescence assay showed that TNF-a and IL-1 beta could significantly increase the expression of NKCC1 on astrocytes, while TNF-a and IL-1 beta receptor-specific antagonists could significantly down-regulate the expression of NKCC1. RT-PCR and Western blot showed that 5 ng/ml or 10 ng/ml of TNF-a or IL-1 beta could significantly up-regulate the expression of NKCC1 mRNA and protein. The expression of NKCC 1 mRNA and protein increased with the increase of inflammatory factor concentration (P 0.05). The expression of NKCC 1 mRNA and protein decreased significantly after the application of TNF-a and IL-1 beta receptor-specific antagonists (P 0.05). In addition, the expression of NKCC 1 was not affected by sugar-free medium.
Conclusion: Inflammatory factors TNF-alpha and IL-1beta can significantly up-regulate the expression of NKCC 1 on astrocytes and induce brain edema, and with the increase of the concentrations of inflammatory factors TNF-alpha and IL-1p, the expression of NKCC 1 on astrocytes also increases, thus aggravating brain edema. HS can significantly inhibit the release of inflammatory factors TNF-alpha and IL-1p from microglia. 1 beta, thereby reducing the expression of NKCC1 on astrocytes to reduce the effect of cerebral edema.
The fourth chapter is about the direct effect of HS on NKCC1 in astrocytes.
Objective: To investigate whether HS directly affects the expression of NKCC1 on astrocytes in hypoxia state.
Methods: The primary cultured astrocytes were divided into control group (completely normal culture), hypoxia group, HS group and hypoxia + sugar-free medium + 100mM Bumetanide group (abbreviated as Bumetanide group), hypoxia group, HS group and Bumetanide group were hypoxia in 3% O2, 5% CO2, 92% N2 environment for 4 hours. The expression of NKCC1 on astrocytes was detected by immunofluorescence double labeling, Western blot and RT-PCR after treatment for 4 hours.
Results: The expression of NKCC1 mRNA in astrocytes of hypoxia group, HS group and Bumetanide group was significantly higher than that of control group after hypoxia, but the expression of NKCC1 mRNA in HS and bumetanide group was significantly lower than that of hypoxia group. The expression of NKCC1 in glial cells increased significantly, while that in HS group and Bumetanide group decreased significantly compared with hypoxia group (P 0.05).
Western blot showed that the expression of NKCC1 protein in hypoxia group, HS group and Bumetanide group increased significantly after 4 hours of hypoxia (P 0.05), while the expression of NKCC1 protein in HS group and Bumetanide group decreased significantly compared with hypoxia group (P 0.05).
Conclusion: After hypoxia, the expression of NKCC1 on astrocytes is significantly increased, while HS can directly inhibit the expression of NKCC1 on astrocytes under hypoxia, thus reducing brain edema. However, the exact mechanism of direct inhibition of NKCC1 expression by HS remains to be further studied.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
本文编号：2248230
[Abstract]:Hypertonic saline (HS) is often used clinically to treat brain edema caused by various causes. Compared with mannitol, HS has a more lasting effect, a greater reduction in intracranial pressure, but less urine volume. Some studies have found that its mechanism of reducing brain edema is not only through the traditional osmotic dehydration mechanism, but also through the non-osmotic mechanism. Involved, it can reduce water transport by reducing the expression of aquaporin AQP4 in peripheral tissues of cerebral ischemic foci, and/or inhibit the expression of vascular endothelial growth factor-VEGF and its receptor-VEGFR2 on astrocytes around cerebral microvasculature, thereby reducing the permeability of blood-brain barrier to water and thus reducing brain edema. Relevant proteins, Na + - K + - Cl - cotransporter 1 (NKCC1) have attracted much attention. Studies have shown that specific inhibitors can significantly reduce brain edema and improve the prognosis of neurological function. In this study, we investigated the effect of HS on the expression and release of inflammatory factors on microglia, the effect of inflammatory factors on NKCC1 on astrocytes, and the relationship between HS and astrocytes by culturing microglia and astrocytes in vitro. There is a direct relationship between NKCC1R expression and HS to elucidate the underlying mechanism of reducing inflammatory factor release and NKCC1 expression.
Chapter 1 the effect of hypertonic saline on the release of inflammatory factors from primary microglia
AIM: To isolate microglia from mixed cultured glial cells and to investigate whether HS can affect the release of inflammatory molecules such as tumor necrosis factor (TNF-a), interleukin-1 beta (IL-1beta) and monocyte chemoattractant protein-1 (monocyte c) from microglia under hypoxia. Hemoattractant protein-1, MCP-1).
Methods: Mixed glial cells were cultured in the cerebral cortex of SD rats born 0-24 hours after trypsin digestion. After 7-10 days of culture, microglia were purified and isolated by shaking method. The purity of microglia was detected by immunofluorescence. If the purity was more than 95%, the next experiment could be carried out.
The purified cells were divided into three groups: control group, hypoxia + sugar-free medium (abbreviated as: hypoxia group) and hypoxia + sugar-free medium + HS (abbreviated as: HS group). HS group was divided into seven subgroups according to the concentration of HS: 40mM, 60nM, 80mM, 100mM, 120mM, 140 mM and 160m, respectively. The oxygen group and HS groups were hypoxic at 3% 02, 5% C02, 92% N2. The cell viability of each group was detected by CCK-8 after 4 hours of corresponding treatment.
In order to determine the optimal hypoxia time, the purified microglia were divided into control group and hypoxia group, and the hypoxia group was divided into 1 hour group, 2 hour group and 4 hour group according to different hypoxia time.
In order to select the best HS concentration, microglia were divided into control group, hypoxia group, HS group (the concentration was determined by the results of cell activity) and hypoxia time was determined by the optimal hypoxia time.
After determining the optimal hypoxia time and concentration of HS, to explore whether HS can inhibit the release of inflammatory factors from microglia, microglia were divided into three groups: control group, hypoxia group and HS group, hypoxia group and HS group under the condition of 3% O2, 5% CO2, 92% N2. After corresponding treatment, microglia culture medium was detected by ELISA method. The levels of inflammatory factors TNF-, IL-1 and MCP-1.
RESULTS: Most of the microglia cells adhered well to the wall after two days of culture and could be distinguished clearly, most of them were polygonal or rod-shaped. By the fifth day of culture, the mixed glia cells were covered with 70% of the bottom of the bottle, and a few round and refractive cells were found in the supernatant of the culture medium. On the seventh day, the glia cells were covered with the bottom of the bottle. On the ninth day, the number of highly refractive cells in the supernatant increased. The purity of the purified microglia was 97.16 6550
The activity of microglia in HS120 mM, 140 mM, 160 mM group was significantly decreased (P 0.05), while the activity of microglia in 40 mM, 60 mM, 80 mM, 100 mM group was not significantly different from that in control group (P 0.05); microglia released a large number of inflammatory mediators at 1, 2, and 4 hours after hypoxia, and the content of TNF-a was the highest at 4 hours (P 0.05). TNF-a content in HS, 80mHS and 100mHS groups decreased significantly after 4 hours of hypoxia (P 0.05), especially in 100mHS group. The release of inflammatory factors by glial cells was significantly reduced (P0.05).
CONCLUSION: Microglia can activate and release a large number of inflammatory factors rapidly at various times after hypoxia, and HS at different concentrations can inhibit the activation of microglia and reduce the release of inflammatory factors to a certain extent.
The second chapter is about the mechanism of hypertonic saline affecting microglia to release inflammatory factors.
Objective: To explore the potential mechanism of 100mM HS affecting microglia activation and release of inflammatory mediators.
Methods: The specific inhibitors of p38 and JNK signaling pathway, SB203580 and SP600125, were used to investigate whether microglia release inflammatory factors through these two signaling pathways. The content of inflammatory factors TNF-a, IL-1 beta and MCP-1 in SB203580 group and SP600125 group (abbreviated as SP600125 group) were detected by ELISA after 4 hours of hypoxia in 3% O2, 5% CO2, 92% N2 medium. The purified cells were divided into control group, hypoxia group and HS group, hypoxia group and HS group according to different hypoxia time and divided into 30 min, 1 h, 2 h, 4 h groups. After hypoxia to the corresponding time point, the total protein of each group was extracted and the levels of Phos-p38 and Phos-JNK protein were detected by Western blot.
Results: Compared with the control group, microglia released a large number of inflammatory factors TNF-a, IL-1 beta and MCP-1 after 4 hours of hypoxia (P 0.05). Compared with the hypoxia group, the contents of inflammatory factors TNF-a, IL-1 beta and MCP-1 in HS group, SB203580 group and SP600125 group were significantly decreased (P 0.05); Western blot showed that microglia in hypoxia group were significantly reduced at 30 minutes of hypoxia (P 0.05). The expression of Phos-p38 and Phos-JNK proteins increased significantly and lasted for 4 hours (P 0.05). In HS microglia, the expression of Phos-p38 and Phos-JNK proteins decreased significantly at different time points of hypoxia (P 0.05).
CONCLUSION: HS may reduce the release of inflammatory factors by inhibiting the activation of p38 and JNK signaling pathways on microglia, alleviate the inflammatory reaction in the brain after ischemia and hypoxia, prevent the further expansion of inflammatory reaction, thus protect the blood brain barrier and help to reduce brain edema.
The third chapter is the effect of inflammatory factors TNF- and IL-1 beta on the expression of NKCC1 in astrocytes.
AIM: To investigate the effects of inflammatory factors TNF-a and IL-1beta on the expression of NKCC1 in astrocytes and whether the changes of TNF-a and IL-1beta concentrations affect the expression of NKCC1.
Methods: Primary cultured astrocytes were purified by shaking method and then divided into 6 groups: control group, TNF-a (10ng/ml) + sugar-free medium group (abbreviated as TNF-a (10ng/ml), TNF-a (5ng/ml) + sugar-free medium group (abbreviated as TNF-a (5ng/ml), IL-1 beta (10ng/ml) + sugar-free medium group (abbreviated as TNF-a (5ng/ml), IL-1 beta (10ng/ml) + sugar-free medium group (abbreviated as follows:TNF-a (5ng/ml)). Written as: IL-1 beta (10ng/ml), IL-1 beta (5ng/ml) + sugar-free medium group (abbreviated as: IL-1 beta (5ng/ml), TNF-a (10ng/ml) + TNFR specific antagonist (2ugM) (TNFR antagonist group) + sugar-free medium group, IL-1 beta (10ng/ml) + IL-1R antagonist (2ugM) (IL-1R antagonist group) + sugar-free medium group (abbreviated as: Glucose-free medium group). The expression of NKCC1 on astrocytes was detected by immunofluorescence double labeling, Western blot and RT-PCR.
Results: Immunofluorescence assay showed that TNF-a and IL-1 beta could significantly increase the expression of NKCC1 on astrocytes, while TNF-a and IL-1 beta receptor-specific antagonists could significantly down-regulate the expression of NKCC1. RT-PCR and Western blot showed that 5 ng/ml or 10 ng/ml of TNF-a or IL-1 beta could significantly up-regulate the expression of NKCC1 mRNA and protein. The expression of NKCC 1 mRNA and protein increased with the increase of inflammatory factor concentration (P 0.05). The expression of NKCC 1 mRNA and protein decreased significantly after the application of TNF-a and IL-1 beta receptor-specific antagonists (P 0.05). In addition, the expression of NKCC 1 was not affected by sugar-free medium.
Conclusion: Inflammatory factors TNF-alpha and IL-1beta can significantly up-regulate the expression of NKCC 1 on astrocytes and induce brain edema, and with the increase of the concentrations of inflammatory factors TNF-alpha and IL-1p, the expression of NKCC 1 on astrocytes also increases, thus aggravating brain edema. HS can significantly inhibit the release of inflammatory factors TNF-alpha and IL-1p from microglia. 1 beta, thereby reducing the expression of NKCC1 on astrocytes to reduce the effect of cerebral edema.
The fourth chapter is about the direct effect of HS on NKCC1 in astrocytes.
Objective: To investigate whether HS directly affects the expression of NKCC1 on astrocytes in hypoxia state.
Methods: The primary cultured astrocytes were divided into control group (completely normal culture), hypoxia group, HS group and hypoxia + sugar-free medium + 100mM Bumetanide group (abbreviated as Bumetanide group), hypoxia group, HS group and Bumetanide group were hypoxia in 3% O2, 5% CO2, 92% N2 environment for 4 hours. The expression of NKCC1 on astrocytes was detected by immunofluorescence double labeling, Western blot and RT-PCR after treatment for 4 hours.
Results: The expression of NKCC1 mRNA in astrocytes of hypoxia group, HS group and Bumetanide group was significantly higher than that of control group after hypoxia, but the expression of NKCC1 mRNA in HS and bumetanide group was significantly lower than that of hypoxia group. The expression of NKCC1 in glial cells increased significantly, while that in HS group and Bumetanide group decreased significantly compared with hypoxia group (P 0.05).
Western blot showed that the expression of NKCC1 protein in hypoxia group, HS group and Bumetanide group increased significantly after 4 hours of hypoxia (P 0.05), while the expression of NKCC1 protein in HS group and Bumetanide group decreased significantly compared with hypoxia group (P 0.05).
Conclusion: After hypoxia, the expression of NKCC1 on astrocytes is significantly increased, while HS can directly inhibit the expression of NKCC1 on astrocytes under hypoxia, thus reducing brain edema. However, the exact mechanism of direct inhibition of NKCC1 expression by HS remains to be further studied.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
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