RhG-CSF干预快速老化模型小鼠P10的实验研究
发布时间：2019-05-28 17:52
【摘要】： 研究目的: 研究重组人粒细胞集落刺激因子(recombinant human granulocyte colonystimulating factor,rhG-CSF)对快速老化模型小鼠P10(senescence acceleratedmouse/prone 10,SAMP10)认知障碍的改善、脑组织炎症反应的抑制及其抗凋亡作用,为G-CSF治疗阿尔茨海默病(Alzheimer disease,AD)的进一步研究提供依据。 研究方法: 1.随机将48只SAMP10分为两组,每组24只:rhG-CSF治疗组:rhG-CSF100μg/(kg·d),皮下注射;对照组:等体积生理盐水,皮下注射。给药时间均为7d。给药结束后第7天,14天,28天,56天时间点进行各种指标检测。 2.认知功能测验:各组小鼠于给药前5天及处死前5天进行Morris水迷宫训练,实验历时5天,观察并记录其逃避潜伏期和目的象限游泳距离百分比。 3.Morris水迷宫训练结束后心脏灌注处死小鼠,取脑,制作石蜡切片,进行免疫荧光染色,检测小鼠额叶脑组织肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和半胱氨酸蛋白酶-3(caspase-3)的表达,采用Image-pro Plus图像分析软件,分析TNF-α、iNOS和caspase-3阳性细胞百分率、总面积及灰度值。 4.采用SPSS13.0 for Windows软件包进行统计学处理,各组数据以(?)±s表示,差异显著性检验用方差分析和两样本t检验,P＜0.05为差异有统计学意义。 结果: 1.认知功能测验 1.1定位航行实验结果对照组于第14天,28天,56天,逃避潜伏期较给药前明显延长(P＜0.05);rhG-CSF治疗组于不同时间点与给药前相比,差异无统计学意义(P＞0.05),第14天逃避潜伏期较对照组缩短(P＜0.05)。 1.2空间探索实验结果对照组小鼠目的象限游泳距离百分比于给药后第28天,56天较给药前减少(P＜0.05);rhG-CSF治疗组目的象限游泳距离百分比于各个时间点之间差异无统计学意义(P＞0.05),在第14天,28天较对照组增加(P＜0.05)。 2.免疫荧光染色法检测额叶TNF-α表达阳性反应物主要分布于大脑额叶皮质,为红色荧光标记,位于胞浆,细胞核不着色。细胞核用DAPI复染,为蓝色荧光标记。对照组治疗组于各时间点均可见额叶皮质TNF-α免疫阳性细胞表达,rhG-CSF治疗组于第14天、28天、56天TNF-α阳性细胞率,阳性细胞总面积及灰度值均明显小于对照组(P＜0.05)。 3.免疫荧光染色法检测额叶iNOS的表达阳性反应物主要分布于大脑额叶皮质,为红色荧光标记,位于胞浆,细胞核不着色。细胞核用DAPI复染,为蓝色荧光标记。对照组治疗组于各时间点均可见额叶皮质iNOS免疫阳性细胞表达,iNOS阳性细胞率于治疗第14天、28天、56天明显少于对照组(P＜0.05),阳性细胞总面积于各时间点均小于对照组(P＜0.05)而灰度值于第14天与对照组差异无统计学意义(P＞0.05)。 4.免疫荧光染色法检测额叶caspase-3表达阳性反应物主要分布于大脑额叶皮质,为红色荧光标记,位于胞浆亦可见于胞膜胞核。细胞核用DAPI复染,为蓝色荧光标记。对照组治疗组于各时间点均可见额叶caspase-3免疫阳性细胞表达,阳性细胞率于治疗第14天、28天明显少于对照组(P＜0.05),阳性细胞总面积于28天小于对照组(P＜0.05),灰度值于第28天与对照组有差异(P＜0.05)。 结论: 1.rhG-CSF能够改善SAMP10的认知障碍,延缓其学习记忆功能的衰退。 2.rhG-CSF能减少SAMP10额叶皮质TNF-α和iNOS阳性细胞表达,抑制小鼠脑组织炎症反应。 3.rhG-CSF可以减少SAMP10额叶皮质caspase-3阳性细胞的表达,具有抑制神经元凋亡的作用。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R-332
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R-332
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