【摘要】： 研究目的: 研究重組人粒細(xì)胞集落刺激因子(recombinant human granulocyte colonystimulating factor,rhG-CSF)對(duì)快速老化模型小鼠P10(senescence acceleratedmouse/prone 10,SAMP10)認(rèn)知障礙的改善、腦組織炎癥反應(yīng)的抑制及其抗凋亡作用,為G-CSF治療阿爾茨海默病(Alzheimer disease,AD)的進(jìn)一步研究提供依據(jù)。 研究方法: 1.隨機(jī)將48只SAMP10分為兩組,每組24只:rhG-CSF治療組:rhG-CSF100μg/(kg·d),皮下注射;對(duì)照組:等體積生理鹽水,皮下注射。給藥時(shí)間均為7d。給藥結(jié)束后第7天,14天,28天,56天時(shí)間點(diǎn)進(jìn)行各種指標(biāo)檢測(cè)。 2.認(rèn)知功能測(cè)驗(yàn):各組小鼠于給藥前5天及處死前5天進(jìn)行Morris水迷宮訓(xùn)練,實(shí)驗(yàn)歷時(shí)5天,觀察并記錄其逃避潛伏期和目的象限游泳距離百分比。 3.Morris水迷宮訓(xùn)練結(jié)束后心臟灌注處死小鼠,取腦,制作石蠟切片,進(jìn)行免疫熒光染色,檢測(cè)小鼠額葉腦組織腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和半胱氨酸蛋白酶-3(caspase-3)的表達(dá),采用Image-pro Plus圖像分析軟件,分析TNF-α、iNOS和caspase-3陽(yáng)性細(xì)胞百分率、總面積及灰度值。 4.采用SPSS13.0 for Windows軟件包進(jìn)行統(tǒng)計(jì)學(xué)處理,各組數(shù)據(jù)以(?)±s表示,差異顯著性檢驗(yàn)用方差分析和兩樣本t檢驗(yàn),P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果: 1.認(rèn)知功能測(cè)驗(yàn) 1.1定位航行實(shí)驗(yàn)結(jié)果對(duì)照組于第14天,28天,56天,逃避潛伏期較給藥前明顯延長(zhǎng)(P＜0.05);rhG-CSF治療組于不同時(shí)間點(diǎn)與給藥前相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05),第14天逃避潛伏期較對(duì)照組縮短(P＜0.05)。 1.2空間探索實(shí)驗(yàn)結(jié)果對(duì)照組小鼠目的象限游泳距離百分比于給藥后第28天,56天較給藥前減少(P＜0.05);rhG-CSF治療組目的象限游泳距離百分比于各個(gè)時(shí)間點(diǎn)之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05),在第14天,28天較對(duì)照組增加(P＜0.05)。 2.免疫熒光染色法檢測(cè)額葉TNF-α表達(dá)陽(yáng)性反應(yīng)物主要分布于大腦額葉皮質(zhì),為紅色熒光標(biāo)記,位于胞漿,細(xì)胞核不著色。細(xì)胞核用DAPI復(fù)染,為藍(lán)色熒光標(biāo)記。對(duì)照組治療組于各時(shí)間點(diǎn)均可見額葉皮質(zhì)TNF-α免疫陽(yáng)性細(xì)胞表達(dá),rhG-CSF治療組于第14天、28天、56天TNF-α陽(yáng)性細(xì)胞率,陽(yáng)性細(xì)胞總面積及灰度值均明顯小于對(duì)照組(P＜0.05)。 3.免疫熒光染色法檢測(cè)額葉iNOS的表達(dá)陽(yáng)性反應(yīng)物主要分布于大腦額葉皮質(zhì),為紅色熒光標(biāo)記,位于胞漿,細(xì)胞核不著色。細(xì)胞核用DAPI復(fù)染,為藍(lán)色熒光標(biāo)記。對(duì)照組治療組于各時(shí)間點(diǎn)均可見額葉皮質(zhì)iNOS免疫陽(yáng)性細(xì)胞表達(dá),iNOS陽(yáng)性細(xì)胞率于治療第14天、28天、56天明顯少于對(duì)照組(P＜0.05),陽(yáng)性細(xì)胞總面積于各時(shí)間點(diǎn)均小于對(duì)照組(P＜0.05)而灰度值于第14天與對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。 4.免疫熒光染色法檢測(cè)額葉caspase-3表達(dá)陽(yáng)性反應(yīng)物主要分布于大腦額葉皮質(zhì),為紅色熒光標(biāo)記,位于胞漿亦可見于胞膜胞核。細(xì)胞核用DAPI復(fù)染,為藍(lán)色熒光標(biāo)記。對(duì)照組治療組于各時(shí)間點(diǎn)均可見額葉caspase-3免疫陽(yáng)性細(xì)胞表達(dá),陽(yáng)性細(xì)胞率于治療第14天、28天明顯少于對(duì)照組(P＜0.05),陽(yáng)性細(xì)胞總面積于28天小于對(duì)照組(P＜0.05),灰度值于第28天與對(duì)照組有差異(P＜0.05)。 結(jié)論: 1.rhG-CSF能夠改善SAMP10的認(rèn)知障礙,延緩其學(xué)習(xí)記憶功能的衰退。 2.rhG-CSF能減少SAMP10額葉皮質(zhì)TNF-α和iNOS陽(yáng)性細(xì)胞表達(dá),抑制小鼠腦組織炎癥反應(yīng)。 3.rhG-CSF可以減少SAMP10額葉皮質(zhì)caspase-3陽(yáng)性細(xì)胞的表達(dá),具有抑制神經(jīng)元凋亡的作用。
[Abstract]:The purpose of the study: The effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the cognitive disorder of mouse P10 (SMP10) in the rapid aging model, the inhibition of inflammatory response of brain tissue and the anti-apoptotic effect of rhG-CSF were studied, and the G-CSF was used to treat Alzheimer's disease (Alzheimer's disease). se, AD) On the basis of. Methods:1.48 SAMP10 were randomly divided into two groups: rhG-CSF (rhG-CSF100. mu.g/ (kg 路 d), subcutaneous injection, control group, etc. Brine water, subcutaneous injection. The administration time was 7 d. Day 7,14,28, and 56 days after the end of the administration 2. Cognitive function test: The Morris water maze training was performed at 5 days before and 5 days before the administration of each group of mice. The experiment lasted for 5 days to observe and record the escape latent period. Objective To determine the percentage of swimming distance in the target quadrant.3. After the end of the Morris water maze training, the mice were sacrificed, the brain was taken, the paraffin section was made, and the immunofluorescent staining was performed to detect the tumor necrosis factor-1 (TNF-1) and the inducible nitric oxide synthase (iNOS) in the frontal lobe of the mouse. Expression of c-oxide synthase (iNOS) and caspase-3 (caspase-3), and image-pro Plus image analysis software was used to analyze the effects of TNF-1, iNOS and caspase-3. Percentage of sex cells, total area, and gray values.4. SPSS13.0 for Windows The software package was statistically processed, and each group of data was expressed in (?)% s, and the variance significance test was performed with an analysis of variance and two different t-tests Inspection, P <0.05 is the difference Results:1. The results were as follows:1. The results of 1.1.1. The experimental results of the cognitive function test (1.1) were significantly prolonged (P <0.05) on the 14th day, the 28th day and the 56 th day, and the rhG-CSF treatment group was different from those of the control group. The difference was not statistically significant (P> 0.0 5) The escape latency of the 14th day was shorter than that of the control group (P <0.05). The percentage of the swimming distance in the control group of the control group in the experimental group was less than that of the control group (P <0.05) on the 28th day after the administration (P <0.05), and the target quadrant of the rhG-CSF treatment group was swimming. The difference between the distance percentage and the time point was not statistically significant (P> 0.05) In day 14 and 28, the control group increased (P <0.05).2. Immunofluorescence staining method for detecting the expression of TNF-antigen in the frontal lobe. The substance is mainly distributed in the frontal cortex of the brain and is a red fluorescent label. In the control group, the expression of TNF-antigen-positive cells in the frontal cortex was observed at all time points, and the rhG-CSF treatment group was in the 14th day, the 28th day, the 56-day TNF-positive-positive cells. The positive cell rate, the total area of positive cells and the gray level were significantly lower than that in the control group (P <0.05).3. The expression of iNOS in the frontal lobe was detected by immunofluorescence staining. The reactive reactant is mainly distributed in the frontal cortex of the brain and is red In the control group, the expression of iNOS, iNOS and iNOS in the frontal cortex were observed in the control group at all time points. The positive cell rate was less than that of the control group (P <0.05) at the 14th day, the 28th day and the 56 th day. The total area of the positive cells was less than that of the control group (P <0.05). The difference of the gray value on the 14th day and the control group was not statistically significant (P> 0.05).4. The caspase-3 in the frontal lobe was detected by the immunofluorescence staining method. The positive reactant is mainly distributed in the frontal cortex of the brain. In the control group, the expression of caspase-3-positive cells in the frontal lobe was observed in the treatment group of the control group. The positive rate of the positive cells in the treatment group was 14 days and 28 days. In less than control group (P <0.05), the total area of positive cells was 28 Day is less than In the control group (P <0.05), the gray value of the control group was different from the control group (P <0.05). Conclusion:1. rhG-CSF can improve the cognitive function of SAMP10 and delay the function of learning and memory. HG-CSF can reduce the expression of TNF-1 and iNOS positive cells in the frontal cortex of the SAMP10, and inhibit the inflammatory reaction of the brain tissue of the mouse.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R-332

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