构建细粒棘球蚴荧光小鼠模型的方法学研究

发布时间：2018-04-21 03:14

  本文选题：细粒棘球蚴 + 荧光成像　； 参考：《石河子大学》2016年硕士论文

【摘要】：目的:1.尝试细粒棘球蚴体外荧光染色,通过红绿荧光信号强度分析不同药物对原头蚴活性的影响。2.构建细粒棘球蚴小鼠活体内荧光发光模型,并对小鼠肝脏中的细粒棘球蚴感染灶进行定位及量化分析。探讨构建小鼠活体内生物发光模型的可能性。方法:1.无菌分离提取原头蚴,将原头蚴分为对照组、二甲双胍处理组(将10 m M二甲双胍加入原头蚴培养基中)、联合用药处理组(将10 m M二甲双胍以及15μM阿苯达唑(相当于4.2μg/m L)联合加入原头蚴培养基中)。培养并动态监测原头蚴活性。2.采用JC-1荧光染料对不同处理组原头蚴进行荧光染色,并置于共聚焦荧光显微镜下采集成像并记录分析各组原头蚴荧光强度。3.将不同处理组的原头蚴置于24孔板中,连续培养并采用小动物发光成像仪对三组原头蚴荧光强度进行动态监测,直至荧光信号消失。4.将不同处理组的原头蚴染色后种植于CF-1雄性小鼠肝脏被膜下,将不同注射组的小鼠置于小动物发光成像仪中进行动态成像分析。选取对照组小鼠进行荧光梯度实验,分析荧光信号衰减趋势。结果:1.与对照组相比,Met药物处理组原头蚴活性出现一定程度的下降,ABZSO和Met药物联合使用组原头蚴活性下降最为显著。2.对照组原头蚴的红色荧光强度明显强于绿色荧光;Met药物处理组原头蚴红色荧光强度与绿色荧光没有显著差异;ABZSO和Met药物联合使用组原头蚴绿色荧光强度明显强于红色荧光。3.三组中原头蚴的红色荧光出现了明显的下降趋势,联合用药组的下降趋势最为明显,其次是二甲双胍药物处理组,对照组的下降趋势较为平缓。而绿色荧光在联合用药组与二甲双胍处理组中出现了明显的增强趋势,且联合用药组强于二甲双胍组,随后相应出现下降的趋势。对照组的绿色荧光从检测开始就处于较低水平,且有一定的下降趋势,实验组与对照组相比具有显著的统计学差异(P0.05)。4.成功种植进小鼠肝脏后,对照组的红色荧光区域明显大于绿色荧光,且红色荧光强度强于绿色荧光,红/绿荧光的强度比为4.1。二甲双胍组的红色荧光信号与绿色荧光相比,无论是荧光区域的大小还是荧光强度都没有明显的差异,且红/绿比值为1.2。联合用药组的红色荧光区域要小于绿色荧光,且红色荧光强度也弱于绿色荧光,红/绿比值为0.5。对照组小鼠肝区的红色荧光在观察初期强度较高,随后逐渐的减弱。绿色荧光在种植后出现一定范围内的上升趋势,随后逐渐消褪。红、绿荧光在36h检测后,信号完全消失。结论:1.我们成功构建了小鼠活体内细粒棘球蚴荧光模型,并可通过红/绿荧光强度比来检测原头蚴活性。
[Abstract]:Objective: 1. to try to stain the Echinococcus granulosus in vitro, and to analyze the effect of different drugs on the activity of echinococcosis through the intensity of red green fluorescence signal..2. construction of the fluorescent luminescence model in the living body of Echinococcus granulosus mice, and the localization and quantitative analysis of the infection foci of Echinococcus granulosus in the liver of mice. Method: 1. aseptic separation and extraction of the original cercariae were divided into the control group, the metformin treatment group (10 m M metformin was added to the metarcariae medium) and the combined treatment group (10 m M metformin and 15 mu M albendazole (equivalent to 4.2 mu m L) were joined in the culture medium of the original cercariae). The original cercariae active.2. was stained with JC-1 fluorescent dyes for different treatment groups. The fluorescence intensity of the original cercariae was collected and recorded under confocal fluorescence microscope, and the fluorescence intensity of the primary cercariae in each group was recorded and recorded by.3.. The original cercariae of different treatment groups were placed in the 24 foramen plate, and the fluorescence intensity of the three groups of cercariae was continuously cultured and the fluorescent intensity of small animals was used for the fluorescence intensity of the cercariae. Dynamic monitoring was carried out until the fluorescent signal disappeared.4. and the original cercariae of different treatment groups were stained under the membrane of the CF-1 male mice liver. The mice in the different injection groups were placed in the small animal luminescence imaging instrument for dynamic imaging. The control group was selected to carry out the fluorescence ladder test, and the attenuation trend of the fluorescence signal was analyzed. The results were as follows: 1 Compared with the control group, the activity of the primary cercariae in the Met treatment group decreased to a certain extent. The activity of the ABZSO and Met drugs in the combined use group was the most significant decrease in the red fluorescence intensity of the original cercariae in the.2. control group, and the red fluorescence intensity of the original cercariae in the Met drug treatment group was not significantly different from the green fluorescence; ABZSO The green fluorescence intensity of the primary cercariae in the combination group with the Met drug was significantly stronger than that of the red fluorescent.3. three group. The decline trend of the combined drug group was the most obvious, followed by the metformin drug treatment group and the control group. In the metformin treatment group, the obvious enhancement trend was found, and the combination group was stronger than the metformin group, and then the decrease trend correspondingly appeared. The green fluorescence of the control group was at a lower level from the beginning of detection, and there was a certain decline trend. Compared with the control group, the experimental group had a significant statistical difference (P0.05).4. successfully planted. The red fluorescence intensity of the control group was significantly greater than that of green fluorescence in the control group, and the red fluorescence intensity was stronger than the green fluorescence. The red / green fluorescence intensity ratio of the 4.1. metformin group was compared with the green fluorescence. The red / green ratio was 1.2, and the red / green ratio was 1.2. The red fluorescence region of the combined drug group was less than the green fluorescence, and the red fluorescence intensity was also weaker than the green fluorescence. The red / green ratio was high in the early observation of the liver region of the 0.5. control group, and then gradually weakened. After 36h detection, the signal completely disappeared. Conclusion: 1. we successfully constructed the fluorescence model of Echinococcus granulosus in vivo, and can detect the activity of the echinococcosis by red / green fluorescence intensity ratio.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R532.32;R-332
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本文编号：1780681