本文關(guān)鍵詞： 神經(jīng)節(jié)苷脂鈉 內(nèi)皮細(xì)胞 PI3K GSK-3 NF-κb　出處：《吉林大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：目的 大腦中動(dòng)脈栓塞（middle cerebral artery oclusion,MCAO）法復(fù)制大鼠局灶性腦缺血模型，探討單唾液酸神經(jīng)節(jié)苷脂鈉（monosialotetrahexosylganglioside,GM-1）對(duì)缺血性腦損傷大鼠的保護(hù)作用及機(jī)制，并通過(guò)H2O2介導(dǎo)的體外人臍靜脈內(nèi)皮細(xì)胞（human umbilical vein endothelial cells，HUVEC）損傷模型，從體外實(shí)驗(yàn)角度探討GM-1對(duì)血管內(nèi)皮細(xì)胞的保護(hù)作用，初步明確磷脂酰肌醇3激酶（phosphatidylinositol3kinase,PI3K）/糖原合成酶激酶-3（glycogen synthasekinase3,GSK-3）信號(hào)通路在腦缺血性損傷中的作用。 方法 本研究從體內(nèi)實(shí)驗(yàn)和體外細(xì)胞培養(yǎng)2個(gè)方面進(jìn)行探討，主要方法如下： 1.動(dòng)物實(shí)驗(yàn) 將大鼠隨機(jī)分為假手術(shù)組（Sham組）、腦缺血組（Model組）及GM-1治療組3組，每組10只，除Sham組外，其余2組大鼠均采用改良線栓法阻塞大鼠右側(cè)大腦中動(dòng)脈復(fù)制局灶性腦缺血模型，GM-1治療組于造模成功當(dāng)天腹腔注射GM-110mg/kg體重，每天兩次，連續(xù)給藥7d。Sham組及腦缺血組腹腔注射生理鹽水1ml/kg體重，每天兩次，連續(xù)給藥7d。3組大鼠自手術(shù)清醒后每日采用改良NSS評(píng)分標(biāo)準(zhǔn)（16分制）進(jìn)行神經(jīng)功能評(píng)分；術(shù)后第8天處死大鼠，取腦組織行常規(guī)HE染色及免疫組化檢測(cè)各組大鼠腦組織CD31、PI3K及GSK-3表達(dá)，結(jié)果用Image J圖像分析軟件進(jìn)行半定量分析。 2.體外實(shí)驗(yàn) 將常規(guī)培養(yǎng)的HUVEC分為對(duì)照組（CTRL組），H2O2處理組及GM-1低、中、高劑量（1、5、10mg/l）處理組，分別用CCK-8試劑盒檢測(cè)細(xì)胞增殖能力，用流式細(xì)胞術(shù)進(jìn)行細(xì)胞周期分析，Bradford法測(cè)定樣本總蛋白濃度，以免疫熒光法、Western blot法檢測(cè)各組培養(yǎng)細(xì)胞PI3K、Akt及GSK-3蛋白表達(dá)情況；并分別檢測(cè)各組HUVEC細(xì)胞漿與細(xì)胞核中NF-κb的表達(dá)。 進(jìn)一步將HUVEC分為對(duì)照組（CTRL組），H2O2處理組，CHIR-99021處理組及LY294002處理組，用Western blot法和Real-time PCR法檢測(cè)各組培養(yǎng)細(xì)胞細(xì)胞漿與細(xì)胞核中PI3K、Akt及GSK-3蛋白及mRNA含量。 結(jié)果 1. GM-1對(duì)MCAO致大鼠腦缺血損傷的保護(hù)作用 1.1各組大鼠一般狀況及神經(jīng)功能評(píng)分比較 動(dòng)物實(shí)驗(yàn)結(jié)果顯示，假手術(shù)組大鼠精神狀態(tài)良好，活動(dòng)正常，皮毛光澤，術(shù)后無(wú)神經(jīng)功能障礙表現(xiàn)，未見任何反射異常；腦缺血組大鼠精神狀態(tài)較差，活動(dòng)減少，出現(xiàn)不同程度行為學(xué)障礙，隨著飼養(yǎng)時(shí)間的延長(zhǎng)，上述癥狀有不同程度的恢復(fù)；GM-1組大鼠于造模初期，上述癥狀與腦缺血組相似，隨給藥時(shí)間延長(zhǎng)，，腦缺血癥狀改善明顯，自給藥第4天起，與腦缺血組比較，差異有統(tǒng)計(jì)學(xué)意義（P0.01）。 1.2各組大鼠腦組織HE染色結(jié)果 Sham組大鼠腦組織神經(jīng)元及膠質(zhì)細(xì)胞數(shù)量、形態(tài)分布正常，細(xì)胞膜完整，胞漿均勻，細(xì)胞核位于中央，染色質(zhì)分布較均勻；腦缺血組大鼠腦組織在光鏡下的主要改變是不同程度的神經(jīng)元變性、壞死和膠質(zhì)細(xì)胞反應(yīng)。鏡下可見缺血灶中心區(qū)以神經(jīng)元變性、壞死為主，缺血灶周圍膠質(zhì)細(xì)胞增生活躍，并逐漸向缺血灶中心移動(dòng)，缺血區(qū)膠原成分增加。GM-1治療組大鼠腦組織神經(jīng)變性及壞死情況較腦缺血組有不同程度的改善。 1.3各組大鼠腦組織CD31免疫組化結(jié)果 CD31陽(yáng)性細(xì)胞呈棕色或棕黃色。在Sham組大鼠，CD31陽(yáng)性細(xì)胞呈單個(gè)散在分布或排列成細(xì)條索狀排列。與Sham組比較，腦缺血組大鼠缺血區(qū)周圍CD31表達(dá)明顯增加，GM-1組陽(yáng)性信號(hào)增加，與腦缺血組比較，差異具有顯著性（P0.01）。 2. GM-1對(duì)PI3K/GSK-3信號(hào)轉(zhuǎn)導(dǎo)通路的影響 2.1各組大鼠腦組織PI3K免疫組化結(jié)果 在Sham組大鼠，PI3K主要在正常神經(jīng)元細(xì)胞漿中表達(dá)，膠質(zhì)細(xì)胞及內(nèi)皮細(xì)胞中亦有少量表達(dá)；在腦缺血組，腦缺血7d后，缺血灶中PI3K表達(dá)量減少，陽(yáng)性信號(hào)主要集中在血管內(nèi)皮細(xì)胞及增生的膠質(zhì)細(xì)胞細(xì)胞漿，與Sham組比較，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；GM-1組大鼠缺血灶中膠質(zhì)細(xì)胞增生明顯，PI3K仍主要表達(dá)于增生的膠質(zhì)細(xì)胞及內(nèi)皮細(xì)胞胞漿中，與腦缺血組比較，差異具有顯著性（P0.01）。 2.2各組大鼠腦組織GSK-3免疫組化結(jié)果 Sham組大鼠腦組織中GSK-3表達(dá)量為6.42±0.58%，GSK-3陽(yáng)性信號(hào)主要分布神經(jīng)元、膠質(zhì)細(xì)胞及內(nèi)皮細(xì)胞細(xì)胞漿中；在腦缺血組，腦缺血7d后，缺血灶中GSK-3表達(dá)量明顯增加，且主要集中在血管內(nèi)皮細(xì)胞及增生的膠質(zhì)細(xì)胞細(xì)胞漿中，殘存的神經(jīng)元細(xì)胞中亦有表達(dá)，與Sham組比較，GSK-3表達(dá)量增加，差異有顯著性（P0.05）；GM-1組大鼠缺血灶GSK-3仍主要表達(dá)于增生的膠質(zhì)細(xì)胞及內(nèi)皮細(xì)胞胞漿中，與腦缺血組比較，其表達(dá)量明顯降低，差異具有顯著性（P0.01）。 3. H2O2與血管內(nèi)皮細(xì)胞氧化損傷 3.1CCK-8檢測(cè)結(jié)果 以對(duì)照組細(xì)胞為100%，H2O2處理組細(xì)胞增殖率為75.97%，明顯低于對(duì)照組（P0.001）；GM-1中、高劑量細(xì)胞增殖能力明顯高于H2O2處理組，差異顯著（P0.001，0.05），而與H2O2處理組比較，GM-1低劑量組細(xì)胞增殖能力差異不顯著。 3.2流式細(xì)胞術(shù)結(jié)果 對(duì)照組50.87%細(xì)胞處于G2/M期和S期，與H2O2處理組（13.52%）比較，組間差異顯著（P0.0001）。GM-1低、中、高劑量組處于G2/M期和S期的細(xì)胞比例分別為33.95%，30.70%和28.68%，與H2O2處理組比較，差異顯著（P0.001，0.01）。 4. PI3K/GSK-3信號(hào)轉(zhuǎn)導(dǎo)途徑與內(nèi)皮細(xì)胞氧化損傷 4.1PI3K及GSK-3Western blot檢測(cè)結(jié)果 與對(duì)照組比較，H2O2處理組HUVEC PI3K表達(dá)量明顯降低（P0.01），與H2O2模型組比較，GM-1中、高劑量組PI3K表達(dá)明顯增加（P0.05）；與對(duì)照組比較，H2O2處理組細(xì)胞GSK-3/β表達(dá)明顯升高（P0.01）；在GM-1各處理組，隨著GM-1應(yīng)用濃度降低，GSK-3/β蛋白表達(dá)量逐漸升高，與H2O2損傷組比較，GM-1高劑量組GSK-3/β蛋白表達(dá)量明顯下降，差異有顯著性（P0.05）。 與H2O2處理組比較，CHIR-99021組及CHIR-99021+H2O2組PI3K和p-Akt蛋白表達(dá)量明顯增加（P0.01），GSK-3表達(dá)量則明顯降低（P0.01）；LY294002+H2O2組PI3K和p-GSK-3表達(dá)量顯著降低（P0.01）；LY294002組和LY294002+H2O2組p-Akt表達(dá)量明顯增加（P0.01）。 4.2Real-time PCR結(jié)果 與對(duì)照組比較，H2O2處理組PI3K、Akt mRNA表達(dá)明顯降低，差異有顯著性（P0.01），而GSK-3mRNA表達(dá)量明顯升高（P0.01）。與H2O2組比較，CHIR-99021組及CHIR-99021+H2O2組PI3K、Akt mRNA表達(dá)量明顯增加（P0.01），GSK-3表達(dá)量則明顯降低（P0.01）；LY294002+H2O2組PI3K和GSK-3表達(dá)量顯著降低（P0.01）；LY294002組和LY294002+H2O2組Akt mRNA表達(dá)量明顯增加（P0.01）。 5. NF-κb與內(nèi)皮細(xì)胞損傷 5.1NF-κb Western blot檢測(cè)結(jié)果 各組HUVEC細(xì)胞胞漿中NF-κb表達(dá)量差異無(wú)顯著性，而各組細(xì)胞NF-κb在細(xì)胞核中表達(dá)量差異有顯著性，H2O2處理組NF-κb核/漿比值為12.07±2.96，與對(duì)照組比較，差異有顯著性（P0.05）。與H2O2處理組比較，GM-1中、高劑量NF-κb核/漿比明顯升高（P0.01）。 5.2免疫熒光雙重染色結(jié)果 NF-κb在體外培養(yǎng)的HUVEC中均有表達(dá)，但各組細(xì)胞NF-κb表達(dá)部位有差異，H2O2處理組NF-κb主要在HUVEC胞漿表達(dá)，而在胞核有較低表達(dá)。圖像分析結(jié)果顯示，與H2O2處理組比較，GM-1高劑量組NF-κb在細(xì)胞核中表達(dá)明顯增加，差異具有顯著性（P0.05）。 結(jié)論 1．GM-1可有效保護(hù)大腦缺血性損傷，血管內(nèi)皮細(xì)胞可能為其實(shí)現(xiàn)此作用的一個(gè)重要治療靶點(diǎn)。 2．GM-1刺激血管內(nèi)皮細(xì)胞增生的作用可能與其激活PI3K/GSK-3信號(hào)轉(zhuǎn)導(dǎo)通路及NF-κb的核轉(zhuǎn)位有關(guān)。 創(chuàng)新點(diǎn) 1．本研究從內(nèi)皮細(xì)胞損傷修復(fù)角度探討GM-1對(duì)腦缺血性損傷的保護(hù)作用，國(guó)內(nèi)外未見報(bào)道。 2．GM-1保護(hù)腦損傷的機(jī)制相關(guān)研究較多，但本研究首次探討其對(duì)PI3K/GSK-3信號(hào)通路及NF-κb的影響。
[Abstract]:objective
Middle cerebral artery occlusion (middle cerebral artery oclusion, MCAO) rat model of focal cerebral ischemia was established to investigate single sialic acid ganglioside sodium (monosialotetrahexosylganglioside, GM-1) on the protective effect and mechanism of ischemic brain injury in rats, and mediated by H2O2 in human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVEC) damage model, to investigate the protective effect of GM-1 on vascular endothelial cells in vitro, initially identified phosphatidylinositol 3 kinase (phosphatidylinositol3kinase, PI3K) / glycogen synthase kinase -3 (glycogen synthasekinase3 GSK-3) signaling pathway in brain ischemic injury.
Method
This study is discussed from 2 aspects: in vivo experiment and in vitro cell culture. The main methods are as follows:
1. animal experiments
The rats were randomly divided into sham operation group (group Sham), cerebral ischemia group (Model group) and GM-1 treatment group, 3 groups, 10 rats in each group, except Sham group, the other 2 rats were treated with modified artery occlusion model of focal cerebral ischemia was copied on the right side of rat brain, GM-1 in the treatment group rats by intraperitoneal injection of GM-110mg/kg on weight, two times a day, continuous administration of 7d.Sham group and cerebral ischemia group by intraperitoneal injection of saline 1ml/kg weight, two times a day, continuous administration of 7d.3 group rats awake after modified NSS daily operation standard for evaluation (16 points) neurological score big; rats were sacrificed eighth days after operation, take the brain tissue by HE staining and immunohistochemistry in brain tissue of rats with CD31, the expression of PI3K and GSK-3, the results of semi quantitative analysis using Image J image analysis software.
2. in vitro experiment
The cultured HUVEC were divided into control group (CTRL group), H2O2 treatment group and GM-1 low, high dose treatment group (1,5,10mg/l), respectively with CCK-8 assay for cell proliferation, cell cycle was analyzed by flow cytometry, the determination of total protein sample concentration Bradford, immunofluorescence Western blot, detected and cultured PI3K cells, the expression of Akt and GSK-3 protein were detected; and the expression of NF- kappa B were HUVEC cytoplasm and nucleus.
HUVEC was further divided into control group (group CTRL), H2O2 treatment group, CHIR-99021 treatment group and LY294002 treatment group. Western, blot and Real-time PCR methods were used to detect the contents of PI3K, Akt and protein and Western in cytoplasm and nucleus of cultured cells.
Result
Protective effect of 1. GM-1 on cerebral ischemia injury in rats induced by MCAO
1.1 comparison of general state and neurological function score of rats in each group
Animal experimental results showed that the rats in the sham operation group were in good spirits, normal activities, shiny coat, no postoperative nerve dysfunction, no abnormal reflex; cerebral ischemia rats, poor mental state, reduced activity, with varying degrees of behavioral disorder, with the feeding time, the symptoms have different degrees of recovery GM-1 group; rats in early stage, the symptoms and cerebral ischemia were similar, with the prolongation of injection, cerebral ischemia symptoms improved significantly, self medicine fourth days, compared with ischemia group, the difference was statistically significant (P0.01).
1.2 HE staining results of brain tissue of rats in each group
The number of neurons and glial cells in brain tissue of rats in group Sham, normal distribution, cell membrane integrity, uniform cytoplasm, the nucleus is located in the central, chromatin distribution is uniform; the main change of brain tissue in rats with cerebral ischemia group under light microscope is the degeneration of neurons in different degree, necrosis and glial cell reaction under the microscope. Visible focal ischemia in the central area of neuronal degeneration, necrosis, active focal ischemia surrounding glial cell proliferation, and gradually move to the center of focal ischemia, ischemia area of collagen increased in.GM-1 treatment group rat brain nerve degeneration and necrosis compared with ischemic group have different degrees of improvement.
1.3 immunohistochemical results of CD31 in rat brain tissue
CD31 positive cells showed brown or brown yellow. The rats in the Sham group, CD31 positive cells were scattered in a single strip or arranged in cords. Compared with group Sham, significantly increased the expression of CD31 around ischemic area of rats with cerebral ischemia group, GM-1 group, the positive signal increased, compared with ischemic group, the difference was significant (of P0.01).
Effect of 2. GM-1 on PI3K/GSK-3 signal transduction pathway
2.1 immunohistochemical results of PI3K in rat brain tissue
The rats in the Sham group, PI3K expressed mainly in the cytoplasm of normal neurons, glial cells and endothelial cells also expressed a little; in cerebral ischemia group, 7d after cerebral ischemia, reduce the amount of PI3K expression in focal ischemia, glial cell cytoplasm positive signals mainly in vascular endothelial cells and hyperplasia, compared with in Sham group, the difference was statistically significant (P0.05); glial cell hyperplasia of focal ischemia in rats of GM-1 group, PI3K is mainly expressed on the proliferation of glial cells and endothelial cells in the cytoplasm, compared with ischemia group, the difference was significant (P0.01).
2.2 immunohistochemical results of GSK-3 in rat brain tissue
The expression of GSK-3 was 6.42 + 0.58% in brain tissue in Sham rats, GSK-3 positive signals were mainly distributed in neurons, glial cells and endothelial cells in the cytoplasm; in the cerebral ischemia group, 7d after cerebral ischemia, increased the expression of GSK-3 in focal ischemia, glial cells and mainly in vascular endothelial cells and hyperplasia in the remaining neurons also expressed, compared with Sham group, the expression of GSK-3 increased significantly (P0.05); group GM-1 rat focal ischemia GSK-3 is still mainly expressed on the proliferation of glial cells and endothelial cells, and cerebral ischemia group, the expression decreased obviously the difference was significant (P0.01).
3. H2O2 and oxidative damage of vascular endothelial cells
3.1CCK-8 detection results
The cells in control group was 100%, H2O2 group cell proliferation rate was 75.97%, significantly lower than the control group (P0.001); high dose GM-1, cell proliferation was significantly higher than that in H2O2 group, significant difference (P0.001,0.05), while the treatment group compared with H2O2 group, low dose GM-1 cell proliferation ability was not significant difference.
Results of 3.2 flow cytometry
The 50.87% cells in control group were in G2/M phase and S stage. Compared with H2O2 treatment group (13.52%), there was significant difference between groups (P0.0001),.GM-1 was low, and the proportion of cells in G2/M phase and S phase was 33.95%, 30.70% and 28.68%, respectively, which was significantly different from H2O2 treatment group (P0.001,0.01).
4. PI3K/GSK-3 signal transduction pathway and oxidative damage of endothelial cells
4.1PI3K and GSK-3Western blot detection results
Compared with the control group, H2O2 treatment group HUVEC PI3K expression significantly decreased (P0.01), compared with the H2O2 model in the GM-1 group, high dose group significantly increased expression of PI3K (P0.05); compared with the control group, H2O2 treatment group GSK-3/ beta cell expression was significantly increased (P0.01); GM-1 in all treatment group, with GM-1 application of lower concentration, the expression of GSK-3/ beta protein was gradually increased, compared with the H2O2 model group, the expression of GM-1 in high dose group GSK-3/ beta protein significantly decreased, there was significant difference (P0.05).
Compared with H2O2 treatment, the expression of CHIR-99021 and CHIR-99021+H2O2 groups of PI3K and p-Akt protein were significantly increased (P0.01), the expression of GSK-3 was significantly decreased (P0.01); the expression of LY294002+H2O2 PI3K and p-GSK-3 group was significantly decreased (P0.01); the expression of LY294002 in group LY294002+H2O2 and group p-Akt were significantly increased (P0.01).
4.2Real-time PCR results
Compared with the control group, H2O2 treatment group PI3K, Akt mRNA expression was significantly reduced, there were significant differences (P0.01), and the expression of GSK-3mRNA was significantly increased (P0.01). Compared with H2O2 group, CHIR-99021 group and CHIR-99021+H2O2 PI3K group, Akt mRNA expression was significantly increased (P0.01), the expression of GSK-3 decreased significantly (P0.01 the expression of LY294002+H2O2 and PI3K); group GSK-3 was significantly decreased (P0.01); the expression of LY294002 group and LY294002+H2O2 group Akt mRNA were significantly increased (P0.01).
5. NF- kappa B and endothelial cell injury
Detection results of 5.1NF- kappa B Western blot
HUVEC was in the cytoplasm of NF- kappa B expression had no significant difference, while the cells were in the nucleus NF- kappa B expression had significantly difference, H2O2 group NF- kappa B nuclear / cytoplasm ratio was 12.07 + 2.96, compared with the control group, there was significant difference (P0.05) in GM-1 group. And with H2O2, high dose of NF- kappa B nuclear / cytoplasm ratio was significantly increased (P0.01).
5.2 double immunofluorescence double staining results
Showed the expression of NF- K B in HUVEC cultured in vitro, but each cell NF- kappa B expression sites between H2O2 treatment group NF- kappa B mainly in HUVEC cytoplasm, and in nuclei with low expression. Image analysis results showed that compared with H2O2, GM-1 NF- K B in high dose group the nucleus was significantly increased, the difference was significant (P0.05).
conclusion
1.GM-1 can effectively protect ischemic brain damage, and vascular endothelial cells may be an important therapeutic target for this effect.
The effect of 2.GM-1 on the proliferation of vascular endothelial cells may be related to the activation of the PI3K/GSK-3 signal transduction pathway and the nuclear translocation of NF- kappa B.
innovation point
1. this study explored the protective effect of GM-1 on ischemic brain damage from the angle of repair of endothelial cell injury, and no reports have been reported at home and abroad.
There are many studies on the mechanism of 2.GM-1 for the protection of brain injury. However, this study is the first to discuss the effect of PI3K/GSK-3 signaling pathway and NF- kappa B.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 余靖,鄧艷秋,楊瑩,張家玉,張業(yè)平,張少華,王建枝;GSK-3在阿爾茨海默病樣細(xì)胞骨架蛋白過(guò)度磷酸化中的作用(英文)[J];生物化學(xué)與生物物理進(jìn)展;2004年06期

2 寧娜;陳乃宏;;神經(jīng)節(jié)苷脂的生物學(xué)活性[J];生理科學(xué)進(jìn)展;2009年01期

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