發(fā)布時(shí)間：2018-09-18 14:40

【摘要】：高滲鹽水(Hypertonic saline, HS)常在臨床上用于治療各種原因?qū)е碌哪X水腫,與甘露醇相比,其療效更持久、降顱壓幅度更大,但尿量卻更少。有研究發(fā)現(xiàn)其減輕腦水腫的機(jī)制并不僅僅是通過(guò)傳統(tǒng)上認(rèn)為的滲透性脫水機(jī)制,還有非滲透性機(jī)制在參與,它可以通過(guò)減少腦缺血灶周邊組織水通道蛋白AQP4的表達(dá)減少對(duì)水的轉(zhuǎn)運(yùn),和/或抑制腦微血管周邊星形膠質(zhì)細(xì)胞上血管內(nèi)皮生長(zhǎng)因子VEGF及其受體VEGFR2的表達(dá),減小血腦屏障對(duì)水的通透性從而減輕腦水腫。另一個(gè)與腦水腫密切相關(guān)的蛋白,鈉、鉀、氯同向轉(zhuǎn)運(yùn)蛋白1(Na+-K+-Cl-cotransporterl, NKCC1)受到廣泛關(guān)注,研究表明利用其特異性抑制劑可以明顯減輕腦水腫,并且可改善腦神經(jīng)功能預(yù)后。前期動(dòng)物實(shí)驗(yàn)證實(shí)HS可以通過(guò)減少腦缺血灶周邊NKCC1的表達(dá)而減輕腦水腫,還可以抑制腦內(nèi)小膠質(zhì)細(xì)胞釋放炎癥因子,但其機(jī)制尚不明確。本實(shí)驗(yàn)通過(guò)體外培養(yǎng)小膠質(zhì)細(xì)胞及星形膠質(zhì)細(xì)胞,探討HS對(duì)小膠質(zhì)細(xì)胞上炎癥因子表達(dá)及釋放的影響、炎癥因子對(duì)星形膠質(zhì)細(xì)胞上NKCC1的影響,以及HS是否與星形膠質(zhì)細(xì)胞上NKCC1R表達(dá)存在直接的關(guān)系,以闡明HS與減少炎癥因子釋放及下調(diào)NKCC1表達(dá)的潛在機(jī)制。 第一章高滲鹽水對(duì)原代小膠質(zhì)細(xì)胞釋放炎癥因子的影響 目的：從混合培養(yǎng)的膠質(zhì)細(xì)胞中純化分離得到小膠質(zhì)細(xì)胞,在缺氧狀態(tài)下利用HS進(jìn)行干預(yù),探討HS是否會(huì)影響小膠質(zhì)細(xì)胞釋放炎癥子如腫瘤壞死因子(tumor necrosis factor,TNF-α)、白介素-1β(interleukin-1beta, IL-1β)及單核細(xì)胞趨化蛋白1(monocyte chemoattractant protein-1,MCP-1)。 方法：取出生0-24h的SD大鼠乳鼠的大腦皮層,胰酶消化后進(jìn)行混合膠質(zhì)細(xì)胞培養(yǎng),培養(yǎng)7-10天后利用搖床震搖法純化分離得到小膠質(zhì)細(xì)胞,利用免疫熒光的方法檢測(cè)小膠質(zhì)細(xì)胞純度,如果純度大于95%則可進(jìn)行下一步實(shí)驗(yàn)。 首先檢測(cè)不同濃度的HS對(duì)小膠質(zhì)細(xì)胞活性的影響,將純化后的細(xì)胞分為三組：對(duì)照組、缺氧+無(wú)糖培養(yǎng)基(縮寫(xiě)為：缺氧組)及缺氧+無(wú)糖培養(yǎng)基+HS(縮寫(xiě)為：HS組),其中HS組又根據(jù)HS的濃度分為7個(gè)亞組：40mM、60nM、80mM、100mM、120mM、140mM及160mM組,缺氧組及HS各組在3%02,5%C02,92%N2條件下進(jìn)行缺氧。各組進(jìn)行相應(yīng)處理4小時(shí)后利用CCK-8的方法檢測(cè)各組細(xì)胞活性。 為確定最佳的缺氧時(shí)間,將純化的小膠質(zhì)細(xì)胞分為對(duì)照組及缺氧組,缺氧組又根據(jù)不同的缺氧時(shí)間分為1h組、2h組及4h組。各組處理至相應(yīng)時(shí)間后提取細(xì)胞培養(yǎng)基上清,利用ELISA的方法檢測(cè)培養(yǎng)基中TNF-α含量。 為篩選最佳HS濃度,將小膠質(zhì)細(xì)胞分為對(duì)照組、缺氧組、HS組(濃度由前面細(xì)胞活性結(jié)果決定),缺氧時(shí)間由前面最佳缺氧時(shí)間決定。各組在相應(yīng)處理后提取細(xì)胞培養(yǎng)基上清進(jìn)行ELISA實(shí)驗(yàn),檢測(cè)其中炎癥因子TNF-a的含量。 在確定最佳缺氧時(shí)間及最佳HS濃度后,探索HS是否可以抑制小膠質(zhì)細(xì)胞釋放炎癥因子,小膠質(zhì)細(xì)胞被分為3組：對(duì)照組、缺氧組及HS組,缺氧組及HS組于3%O2,5%CO2,92%N2的條件下缺氧,在進(jìn)行相應(yīng)處理后利用ELISA的方法檢測(cè)小膠質(zhì)細(xì)胞培養(yǎng)基中炎癥因子TNF-α、IL-1β及MCP-1的含量。 結(jié)果：混合培養(yǎng)小膠質(zhì)細(xì)胞在培養(yǎng)兩天后大多數(shù)細(xì)胞均可良好貼壁,可較清楚分辨出形態(tài),多呈多邊形或棒狀；培養(yǎng)至第五天時(shí),混合膠質(zhì)細(xì)胞已鋪滿(mǎn)瓶底70%,培養(yǎng)基上清中可見(jiàn)少量圓形、折光性強(qiáng)的細(xì)胞；第七天膠質(zhì)細(xì)胞已鋪滿(mǎn)瓶底,折光性強(qiáng)的細(xì)胞逐漸增多；第九天時(shí),細(xì)胞上清中折光強(qiáng)的細(xì)胞大量增多。利用搖床震搖法純化的小膠質(zhì)細(xì)胞,免疫熒光方法檢測(cè)純化后的小膠質(zhì)細(xì)胞的純度為97.16±0.983%。 細(xì)胞活性檢測(cè)發(fā)現(xiàn)HS120mM、140mM、160mM組的小膠質(zhì)細(xì)胞活性明顯下降(P0.05),而40mM、60mM、80mM、100mM組的小膠質(zhì)細(xì)胞與對(duì)照組相比活性無(wú)明顯差異(P0.05)；小膠質(zhì)細(xì)胞在缺氧后1,2,4小時(shí)均釋放大量的炎癥介質(zhì),以4小時(shí)TNF-a含量最高(P0.05);40mM HS.60mM HS、80mM HS、100mM HS組在缺氧4小時(shí)后,TNF-a含量均明顯減少(P0.05),其中以100mM組減少最為明顯。根據(jù)上述篩選結(jié)果,缺氧組小膠質(zhì)細(xì)胞在缺氧4小時(shí)后釋放的炎癥因子TNF-α、IL-1β及MCP-1明顯增加(P0.05)；而使用100mM HS處理的HS組與缺氧組相比,小膠質(zhì)細(xì)胞釋放炎癥因子明顯減少(P0.05)。 結(jié)論：小膠質(zhì)細(xì)胞在缺氧后各個(gè)時(shí)間均迅速激活并釋放大量炎癥因子,而不同濃度的HS均可一定程度上抑制缺氧后小膠質(zhì)細(xì)胞的激活并減少其釋放炎癥因子的含量,具有減輕炎癥反應(yīng)的作用。 第二章高滲鹽水影響小膠質(zhì)細(xì)胞釋放炎癥因子的機(jī)制 目的：探討100mM HS影響小膠質(zhì)細(xì)胞激活及釋放炎癥介質(zhì)的潛在機(jī)制 方法：利用p38及JNK信號(hào)通路的特異性抑制劑SB203580及SP600125探討小膠質(zhì)細(xì)胞是否是通過(guò)這兩條信號(hào)通路釋放炎癥因子。原代培養(yǎng)混合膠質(zhì)細(xì)胞后進(jìn)行純化,將純化的細(xì)胞分為五組：對(duì)照組、缺氧組、HS組、缺氧+無(wú)糖培養(yǎng)基+SB203580組(縮寫(xiě)為：SB203580組)及缺氧+無(wú)糖培養(yǎng)基+SP600125組(縮寫(xiě)為：SP600125組),然后在3%O2,5%CO2,92%N2的條件下缺氧4小時(shí),利用ELISA的方法檢測(cè)各組培養(yǎng)基中炎癥因子TNF-α、IL-1β及MCP-1的含量。為檢測(cè)HS是否會(huì)影響小膠質(zhì)細(xì)胞中的p38及JNK兩條信號(hào)通路,將純化后的細(xì)胞分為對(duì)照組、缺氧組及HS組,缺氧組和HS組又根據(jù)缺氧時(shí)間不同分為30min,1h,2h,4h組,缺氧至相應(yīng)時(shí)間點(diǎn)后,提取各組細(xì)胞的總蛋白進(jìn)行western blot實(shí)驗(yàn),檢測(cè)Phos-p38及Phos-JNK蛋白水平。 結(jié)果：與對(duì)照組相比,小膠質(zhì)細(xì)胞在缺氧4小時(shí)后釋放大量的炎癥因子TNF-α、IL-1β及MCP-1(P0.05),而與缺氧組相比,HS組、SB203580組及SP600125組中炎癥因子TNF-α、IL-1β及MCP-1含量均明顯減少(P0.05); western blot實(shí)驗(yàn)顯示缺氧組小膠質(zhì)細(xì)胞在缺氧30分鐘后即可觀察到Phos-p38及Phos-JNK蛋白表達(dá)明顯增加并持續(xù)到4小時(shí)(P0.05),而HS組小膠質(zhì)細(xì)胞中,缺氧各個(gè)時(shí)間點(diǎn)表達(dá)的Phos-p38及Phos-JNK蛋白與缺氧組相比均明顯減少(P0.05)。 結(jié)論：HS可能通過(guò)抑制小膠質(zhì)細(xì)胞上的p38及JNK這兩條信號(hào)通路激活而減少了炎癥因子的釋放,減輕了缺血缺氧后腦內(nèi)的炎癥反應(yīng),防止炎癥反應(yīng)的進(jìn)一步擴(kuò)大,從而保護(hù)了血腦屏障,有利于減輕腦水腫。 第三章炎癥因子TNF-及IL-1β對(duì)星形膠質(zhì)細(xì)胞表達(dá)NKCC1的影響 目的：探討炎癥因子TNF-α及IL-1β對(duì)星形膠質(zhì)細(xì)胞上NKCC1表達(dá)的影響,以及TNF-a及IL-1β濃度的變化是否會(huì)影響NKCC1的表達(dá)。 方法：原代培養(yǎng)星形膠質(zhì)細(xì)胞,利用震搖的方法純化后將星形膠質(zhì)細(xì)胞分為6組,分別是：對(duì)照組、TNF-α (10ng/ml)+無(wú)糖培養(yǎng)基組(縮寫(xiě)為：TNF-α (10ng/ml)組)、TNF-α(5ng/ml)+無(wú)糖培養(yǎng)基組(縮寫(xiě)為：TNF-α (5ng/ml)組)、IL-1β(10ng/ml)+無(wú)糖培養(yǎng)基組(縮寫(xiě)為：IL-1β(10ng/ml)組)、IL-1β(5ng/ml)+無(wú)糖培養(yǎng)基組(縮寫(xiě)為：IL-1β(5ng/ml)組)、TNF-α(10ng/ml)+TNFR特異拮抗劑(2μM)(TNFR antagonist組)+無(wú)糖培養(yǎng)基組、IL-1β(10ng/ml)+IL-1R拮抗劑(2μM)(IL-1R antagonist組)+無(wú)糖培養(yǎng)基組及無(wú)糖培養(yǎng)基組(縮寫(xiě)為：Glucose-free medium組)。實(shí)驗(yàn)前加入相應(yīng)濃度的細(xì)胞因子,處理4小時(shí)后進(jìn)行相應(yīng)的檢測(cè)。然后利用免疫熒光雙標(biāo)、western blot及RT-PCR的方法進(jìn)行檢測(cè)星形膠質(zhì)細(xì)胞上NKCC1的表達(dá)。 結(jié)果：免疫熒光實(shí)驗(yàn)表明TNF-α及IL-1β可明顯增加星形膠質(zhì)細(xì)胞上NKCC1表達(dá),而TNF-α及IL-1β相應(yīng)的受體特異拮抗劑則可以明顯下調(diào)NKCC1的表達(dá)；RT-PCR及Western blot結(jié)果顯示,5ng/ml或10ng/ml的TNF-α或IL-1β均可以明顯上調(diào)NKCC1mRNA及蛋白的表達(dá),并且隨著炎癥因子濃度升高,NKCC1mRNA及蛋白的表達(dá)也隨之增高(P0.05),在應(yīng)用TNF-α及IL-1β相應(yīng)的受體特異拮抗劑后NKCC1mRNA及蛋白的表達(dá)明顯降低(P0.05)；另外,NKCC1的表達(dá)不受無(wú)糖培養(yǎng)基的影響。 結(jié)論：炎癥因子TNF-α、IL-1β可以明顯上調(diào)星形膠質(zhì)細(xì)胞上NKCC1的表達(dá),引起腦水腫;并且隨著炎癥因子TNF-α及IL-1p濃度的增加,星形膠質(zhì)細(xì)胞上NKCC1的表達(dá)也隨之增加,從而加重腦水腫。而HS可以明顯抑制小膠質(zhì)細(xì)胞釋放炎癥因子TNF-α及IL-1β,從而下調(diào)星形膠質(zhì)細(xì)胞上NKCC1的表達(dá),達(dá)到減輕腦水腫的效果。 第四章HS對(duì)星形膠質(zhì)細(xì)胞上NKCC1的直接影響 目的：探討HS是否會(huì)直接影響缺氧狀態(tài)下星形膠質(zhì)細(xì)胞上NKCC1的表達(dá) 方法：將原代培養(yǎng)的星形膠質(zhì)細(xì)胞分為對(duì)照組(完全正常培養(yǎng))、缺氧組、HS組及缺氧+無(wú)糖培養(yǎng)基+100mM Bumetanide(縮寫(xiě)為Bumetanide組),缺氧組、HS組及Bumetanide組均在3%O2,5%CO2,92%N2的環(huán)境中缺氧4小時(shí)。在缺氧前30分鐘,HS組及Bumetanide組分別加入終濃度為100mM的HS和100μM的Bumetanide。各組相應(yīng)處理4小時(shí)后,利用免疫熒光雙標(biāo)、western blot及RT-PCR的方法檢測(cè)星形膠質(zhì)細(xì)胞上NKCC1的表達(dá)。 結(jié)果：缺氧組、HS組及Bumetanide組星形膠質(zhì)細(xì)胞在缺氧之后,NKCC1mRNA的表達(dá)較對(duì)照組明顯上調(diào)；但HS及bumetanide組與缺氧組相比,NKCC1mRNA的表達(dá)量明顯減少。免疫熒光實(shí)驗(yàn)結(jié)果顯示,與對(duì)照組相比,缺氧4小時(shí)后缺氧組、HS組及Bumetanide組中星形膠質(zhì)細(xì)胞上NKCC1的表達(dá)均明顯上升；而HS組及Bumetanide組與缺氧組相比,NKCC1蛋白表達(dá)則明顯下降(P0.05)。 Western blot結(jié)果表明缺氧組、HS組及Bumetanide組在缺氧4小時(shí)后,NKCC1蛋白表達(dá)均明顯上升(P0.05)；而HS組及Bumetanide組與缺氧組相比,NKCC1蛋白表達(dá)量明顯下降(P0.05)。 結(jié)論：缺氧以后星形膠質(zhì)細(xì)胞上NKCC1的表達(dá)明顯增加,而HS可以在缺氧條件下直接抑制星形膠質(zhì)細(xì)胞上NKCC1的表達(dá),從而減輕腦水腫,但HS直接抑制NKCC1表達(dá)的確切機(jī)制仍有待進(jìn)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R741

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