SSTF对MPTP帕金森小鼠治疗作用的实验研究

发布时间：2018-06-25 12:45

本文选题：帕金森 + MPTP　；参考：《泰山医学院》2014年硕士论文

【摘要】：目的：通过腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）建立帕金森小鼠模型，观察对比各组小鼠的行为学改变及其中脑神经元凋亡的程度，探讨黄芩茎叶总黄酮（SSTF）对MPTP造成的黑质多巴胺能神经元损伤的保护作用及可能机制，并对SSTF的治疗效果进行评价，以期为SSTF治疗帕金森病的临床应用奠定一定的理论基础。方法：一：实验分组及MPTP帕金森小鼠模型的建立。实验动物为8-10周龄健康雄性C57BL/6J小鼠32只，随机分为4组。（1）A组：空白对照组，每天不予任何特殊处置。（2）B组：MPTP模型组，从实验第4天起，每天经腹腔注射25mg/Kg的MPTP，2h后给予等量生理盐水腹腔注射1次，，连续5天。（3）C组：SSTF预处理组，从实验第1天起，经腹腔注射给予SSTF1次/天，连续3天，从第4天起，给予小鼠腹腔注射SSTF2h后腹腔注射MPTP1次，连续5天。（4）D组：SSTF治疗组，从实验第4天起，经腹腔注射给予小鼠MPTP2h后腹腔注射SSTF1次/天，连续5天。二：小鼠行为学检测。每日观察小鼠行为学改变并按标准对实验小鼠震颤麻痹、悬挂实验、爬杆实验进行记录评分。三：实验标本的制作。将麻醉后的小鼠行心脏灌注。清洗和固定后断头取脑，切取小鼠中脑组织制作石蜡切块后保存备用。四：组织切片经过烤片、脱蜡、水化及抗原修复后，按说明书步骤行TH、Bcl-2、caspase-3和GFAP的免疫组化染色，倒置显微镜下观察各个分组的细胞形态并进行免疫阳性细胞计数。结果：一：B组（MPTP模型组）小鼠注射MPTP3-5min后均出现身体震颤、运动迟缓、弓背、步宽增加、步态不稳、竖尾、竖毛等行为学改变。持续30分钟左右症状开始减轻，24小时后基本恢复正常。于B组对比，C组（SSTF预处理组）和D组（SSTF治疗组）小鼠症状均有不同程度减轻，持续时间相对缩短。二：（1）震颤评分，悬挂能力和爬杆能力的测试结果均显示，C组与D组能明显改善小鼠运动协调能力，与B组（MPTP模型组）的差值均具有统计学意义（P0.05）。与A组（空白对照组）相比仍有统计学差异（P0.05）。C组与D组对比均无统计学差异（P0.05）。随着给药次数的增加，每天的差值无统计学意义（P0.05）。二：（1）TH免疫化学染色结果显示，A组可见大量TH阳性神经元，轴突明显，形态正常。B组TH阳性神经元数量明显减少，C、D两组TH阳性神经元数量较B组增加，较A组仍有差别，差值均具有统计学意义（P0.05）。C、D两组对比无统计学差异（P0.05）,B组细胞形态改变明显，胞体肿胀并轮廓不清，轴突减少或消失。（2）Bcl-2染色结果显示，A组阳性细胞数最多，棕黄色染色较深。B组阳性细胞数量明显减少， C、D两组阳性细胞数较B组明显增多，与正常组相比仍有差别，差值均具有统计学差异（P0.05）, C、D两组对比无统计学差异（P0.05）。（3）caspase-3免疫化学染色结果显示，B组的阳性细胞数比A组显著增多，差值具有统计学意义（P0.05）。C组、D组阳性细胞数较B组明显减少，差值具有统计学意义（P0.05），与A组相比阳性细胞数增加，有统计学差异（P0.05）。C、D两组对比差值无统计学意义（P0.05）。（4）GFAP免疫化学染色结果显示，B、C、D组阳性细胞数均有不同程度增加，与A组对比，有统计学差异（P0.05）。B组GFAP阳性细胞数最多，与C、D两组的差值具有统计学意义（P0.05）。C、D两组对比差值无统计学意义（P0.05）。B、C、D组细胞的形态均发生改变，染色与A组对比有不同程度的加深。结论：（1）SSTF可明显改善MPTP帕金森小鼠的症状。（2）SSTF可明显抑制MPTP帕金森小鼠中脑黑质神经元的凋亡。（3）相比后治疗小鼠，预防性使用SSTF的MPTP帕金森小鼠的症状无明显的改善。（4）相比后治疗组，预防性使用SSTF并不能更好的抑制帕金森小鼠中脑黑质神经元的凋亡。（5）SSTF在治疗帕金森病方面具有广阔的研究前景。
[Abstract]:Objective:
The Parkinson mice model was established by intraperitoneal injection of 1- methyl -4- phenyl -1,2,3,6- four hydropyridine (MPTP). The behavior changes and the degree of apoptosis of the mesencephalon neurons were observed and compared. The protective effect and possible mechanism of total flavonoids (SSTF) on dopaminergic neuron damage caused by MPTP in the Scutellaria baicalensis Scutellaria were investigated and the treatment of SSTF was also discussed. The therapeutic effect was evaluated in order to lay a theoretical foundation for the clinical application of SSTF in the treatment of Parkinson's disease.
Method:
First: experimental group and the establishment of MPTP Parkinson Parkinson mouse model. Experimental animals were 32 healthy male C57BL/6J mice of 8-10 weeks old. They were randomly divided into 4 groups. (1) group A: blank control group, without any special treatment every day. (2) group B: MPTP model group, MPTP from 25mg/Kg by intraperitoneal injection every day from fourth days of experiment, 2h was given to equal saline abdomen after 2H. 1 times, 1 consecutive days. (3) group C: SSTF preconditioning group, from the first days from the experiment, the abdominal injection was given SSTF1 times / day for 3 days. From fourth days, the abdominal injection of SSTF2h was given to the mice for 5 days for 5 days. (4) D group, SSTF treatment group, from fourth days, intraperitoneal injection of MPTP2h to mice MPTP2h to give SSTF1 times after MPTP2h to mice MPTP2h / intraperitoneal injection / peritoneal injection of mice MPTP2h after MPTP2h / after MPTP2h / Day, 5 days. Two: behavior test in mice. Observe the behavior changes of mice daily and record the tremor paralysis, suspension experiment and climbing pole experiment. Three: the experimental specimens were made. The mice after the anesthesia were perfused. After cleaning and fixing the head, the brain tissue was cut and cut into the paraffin cut block in the middle brain tissue of mice. Four: after the tissue section was roasted, dewaxing, hydrating and the antigen was repaired, the immuno histochemical staining of TH, Bcl-2, caspase-3 and GFAP were performed according to the instructions, and the cell morphology of each group was observed under the inverted microscope and the immunoreactive cells were counted.
Result:
1: B group (MPTP model group) mice were injected with MPTP3-5min after injection of MPTP3-5min, and the symptoms of the C group (SSTF preconditioning group) and the group of D (SSTF treatment group) were different. In the same degree, the duration was relatively shortened. Two: (1) the test results of the tremor score, the suspension ability and the ability of climbing pole showed that the C group and the D group could obviously improve the coordination ability of the mice, and the difference between the group of B (MPTP model group) was statistically significant (P0.05). Compared with the group A (blank control group), there were still statistical differences (P0.05).C and D groups There was no statistical difference (P0.05). Two: (1) TH immunochemistry staining showed that a large number of TH positive neurons were found in group A, the axon was obvious, the number of TH positive neurons in the normal.B group decreased obviously, and the number of TH positive neurons in C and D two groups increased in the C and D group than that in the A group. The difference was statistically significant (P0.05).C, and there was no statistical difference between the two groups of D (P0.05), the cell morphology of the B group was obviously changed, the body swelling and the outline were not clear, the axon decreased or disappeared. (2) the results of Bcl-2 staining showed that the number of positive cells in the A group was the most, the number of positive cells in the dark.B group decreased obviously, C and D two positive groups were positive. Compared with the B group, the number of cells was significantly higher than that in the normal group (P0.05), the difference between the two groups was statistically different (P0.05), and there was no statistical difference between the two groups (P0.05). (3) the results of Caspase-3 immunochemistry showed that the number of positive cells in the B group was significantly higher than that in the A group, and the difference was statistically significant (P0.05).C group, and the number of positive cells in D group was more obvious than that of the B group. The difference was statistically significant (P0.05). Compared with the A group, the number of positive cells increased (P0.05).C, and the difference between the D two groups was not statistically significant (P0.05). (4) GFAP immunochemistry staining results showed that the number of positive cells in B, C and D increased in varying degrees, and the number of positive cells was statistically different from that of A group. At most, the difference between the two groups with C and D was statistically significant (P0.05).C, and the contrast difference between the two groups of D was not statistically significant (P0.05).B, C, and D group cells were all changed, and the contrast between the staining and A groups was deepened to a different degree.
Conclusion:
(1) SSTF can obviously improve the symptoms of MPTP Parkinson mice. (2) SSTF can obviously inhibit the apoptosis of the neurons in the midbrain of MPTP mice. (3) compared with the post treatment mice, the symptoms of the SSTF MPTP Parkinson mice are not significantly improved. (4) compared to the post treatment group, the preventive use of SSTF can not better inhibit Parkinson mice. Apoptosis of substantia nigra neurons in the midbrain. (5) SSTF has broad research prospects in the treatment of Parkinson's disease.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

【参考文献】