丁苯酞注射液对VCI大鼠海马神经元Calbindin-D28k表达的影响

发布时间：2018-06-04 23:14

本文选题：VCI + 丁苯酞注射液　；参考：《湖南师范大学》2013年硕士论文

【摘要】：目的：本研究在Wistar大鼠认知功能障碍(VCI)模型的基础上,结合病理检测、认知功能检测和免疫组化蛋白定量、荧光定量PCR-mRNA检测、流式细胞仪Ca2+定量,以及用丁苯酞注射液干预后的Calbindin-D28k (CaBP)表达的对比。初步探讨CaBP在凋亡神经细胞中的作用及丁苯酞注射液的保护作用。方法：所有动物造模前先用Morris水迷宫定位航行实验进行筛选,淘汰学习记忆能力差的大鼠,获得250g-300g健康雄性Wistar大鼠36只。将36只合格大鼠称重,排序,采用随机数字表法分为3组：正常组(normal group)(n=4)、VCI模型组(model group)(n=16)、药物组(drug inerwention group)(n=16)。利用双侧颈总动脉永久结扎术(2-VO)建立大鼠VCI模型。按照造模后处死时间将3组随机分为一周组(1W)、2周组(2W)、4周组(4W)、8周组(8W)四个亚组,正常组每组1只,其余分组每个亚组4只动物。用Morris水迷宫定位航行及空间探索实验对8W大鼠的记忆及学习能力进行测定；采用HE染色观察病理学变化；应用免疫组织化学检测CaBP在神经细胞内表达水平的动态变化；采用荧光定量PCR检测细胞内CaBP-mRNA表达的变化。采用流式细胞仪测定海马细胞内Ca2+浓度。结果： 1、免疫组化：正常组各时间点可见CaBP免疫阳性细胞正常表达,模型组1W时表达高于正常组,1W以后免疫阳性细胞表达逐渐下降,且均低于正常组。药物组较正常组高表达,1W-8W逐渐下降,至8W时降至正常或稍低于正常组(P0.05)。其中1W、2W、4W组与正常组差异有统计学意义(P0.05)。同时间点药物组与模型组比较,药物组CaBP免疫阳性细胞数目较多,表达差异均有显著意义(P0.05)。 2、荧光定量PCR:CaBP-mRNA的荧光定量PCR检测结果示1-8W总体表达逐渐下降,药物组1W、2W高表达,4W组稍高表达,8W组降至正常或稍低于正常组(P0.05),与免疫组化结果吻合。模型组总体表达亦呈下降趋势,1W时高表达,1W后明显低于正常组。药物组1、2、4W组与正常组表达有差异(P0.05),8W组与正常组同一水平(P0.05)。模型组1W、4W、8W组较正常组表达有差异(P0.05),2W组与正常组差异无统计学意义。模型组与药物组各时间点表达均有差异,表达差异均有统计学差异意义(P0.05)。 3、细胞内Ca2+：模型组各时间点均高表达,较正常组、与药物组有明显的差异(P0.05)。药物组1W、2W组稍高表达,较正常组无明显差异(P0.05),4W、8W组高表达,较正常组表达差异有统计学意义(P0.05)。结论： 1、大鼠慢性脑缺血损伤后CaBP参与了大鼠脑缺血神经元凋亡的病理生理过程,可降低细胞内Ca2+浓度,避免钙超载,起到神经保护作用； 2、丁苯酞注射液可促进海马细胞内CaBP的表达,改善血管性认知功能障碍大鼠的学习和记忆能力。
[Abstract]:Objective: In this study, based on the Wistar rat model of cognitive dysfunction, combined with pathological examination, cognitive function test and immunohistochemical protein quantification, fluorescence quantitative PCR-mRNA detection, flow cytometry Ca2 quantitative analysis. The expression of Calbindin-D28k was compared with that of butyphthalide injection. To explore the role of CaBP in apoptosis of nerve cells and the protective effect of butyphthalide injection. Methods: The Morris water maze navigation experiment was used to screen all the animals before modeling, and 36 healthy male Wistar rats with 250g-300g were obtained by eliminating the rats with poor learning and memory ability. Thirty-six eligible rats were weighed and sequenced. They were divided into 3 groups by random number table method: normal group (normal group), normal group (n = 4), model group (n = 16) and drug group (inerwention) group (n = 16). Rat VCI model was established by permanent ligation of bilateral common carotid artery (2-VOO). According to the time of death, the three groups were randomly divided into four subgroups, one in each group and four animals in each subgroup. The memory and learning ability of 8W rats were measured by Morris water maze navigation and space exploration experiment, the pathological changes were observed by HE staining, the dynamic changes of CaBP expression in nerve cells were detected by immunohistochemistry. Fluorescence quantitative PCR was used to detect the expression of CaBP-mRNA. The concentration of Ca2 in hippocampal cells was measured by flow cytometry. Results: 1Immunohistochemistry: normal expression of CaBP immunoreactive cells was observed at each time point in the normal group, and the expression of CaBP immunoreactive cells in the model group was lower than that in the normal group after 1 week, and was lower than that in the normal group. The high expression of 1W-8W in the drug group was gradually decreased than that in the normal group, and decreased to normal or slightly lower than that of the normal group at 8W. Among them, there was a significant difference between the 1W 2 W 4 W group and the normal group (P 0.05). At the same time, the number of CaBP immunoreactive cells in the drug group was more than that in the model group, and the difference was significant (P 0.05). 2. The results of fluorescence quantitative PCR:CaBP-mRNA showed that the total expression of 1-8W decreased gradually, and that of the 4W group decreased to normal or slightly lower than that of the normal group, which coincided with the immunohistochemical results. The overall expression of the model group also showed a downward trend after 1 W high expression was significantly lower than that of the normal group. There was significant difference in the expression of P0. 05 and P0. 05 between the two groups (P 0. 05) and the normal group (P 0. 05) at the same level as that in the normal control group (P 0. 05 and P 0. 05). There was no significant difference between the model group and the normal group. There were significant differences in expression between model group and drug group at each time point, and there was significant difference in expression between model group and drug group (P 0.05). (3) intracellular Ca2: the expression of Ca2 in the model group was higher than that in the normal group, and there was a significant difference between the model group and the drug group (P 0.05). There was no significant difference between the drug group and the control group in the expression of 1W ~ 2W group, but there was no significant difference between the two groups (P 0.05 ~ 4W ~ 8W group), and there was a significant difference between the two groups in the expression of P _ (0.05) P _ (0.05) P _ (0.05). Conclusion: 1. CaBP was involved in the pathophysiological process of apoptosis of rat cerebral ischemic neurons after chronic cerebral ischemia injury, which could reduce the concentration of intracellular Ca2, avoid calcium overload and play a neuroprotective role. 2, butyphthalide injection can promote the expression of CaBP in hippocampal cells and improve the learning and memory ability of rats with vascular cognitive impairment.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.13

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