本文選題：帕金森 + MPTP　； 參考：《泰山醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的： 通過腹腔注射1-甲基-4-苯基-1,2,3,6-四氫吡啶（MPTP）建立帕金森小鼠模型，觀察對比各組小鼠的行為學(xué)改變及其中腦神經(jīng)元凋亡的程度，探討黃芩莖葉總黃酮（SSTF）對MPTP造成的黑質(zhì)多巴胺能神經(jīng)元損傷的保護(hù)作用及可能機(jī)制，并對SSTF的治療效果進(jìn)行評價，以期為SSTF治療帕金森病的臨床應(yīng)用奠定一定的理論基礎(chǔ)。 方法： 一：實驗分組及MPTP帕金森小鼠模型的建立。實驗動物為8-10周齡健康雄性C57BL/6J小鼠32只，隨機(jī)分為4組。（1）A組：空白對照組，每天不予任何特殊處置。（2）B組：MPTP模型組，從實驗第4天起，每天經(jīng)腹腔注射25mg/Kg的MPTP，2h后給予等量生理鹽水腹腔注射1次，，連續(xù)5天。（3）C組：SSTF預(yù)處理組，從實驗第1天起，經(jīng)腹腔注射給予SSTF1次/天，連續(xù)3天，從第4天起，給予小鼠腹腔注射SSTF2h后腹腔注射MPTP1次，連續(xù)5天。（4）D組：SSTF治療組，從實驗第4天起，經(jīng)腹腔注射給予小鼠MPTP2h后腹腔注射SSTF1次/天，連續(xù)5天。二：小鼠行為學(xué)檢測。每日觀察小鼠行為學(xué)改變并按標(biāo)準(zhǔn)對實驗小鼠震顫麻痹、懸掛實驗、爬桿實驗進(jìn)行記錄評分。三：實驗標(biāo)本的制作。將麻醉后的小鼠行心臟灌注。清洗和固定后斷頭取腦，切取小鼠中腦組織制作石蠟切塊后保存?zhèn)溆�。四：組織切片經(jīng)過烤片、脫蠟、水化及抗原修復(fù)后，按說明書步驟行TH、Bcl-2、caspase-3和GFAP的免疫組化染色，倒置顯微鏡下觀察各個分組的細(xì)胞形態(tài)并進(jìn)行免疫陽性細(xì)胞計數(shù)。 結(jié)果： 一：B組（MPTP模型組）小鼠注射MPTP3-5min后均出現(xiàn)身體震顫、運動遲緩、弓背、步寬增加、步態(tài)不穩(wěn)、豎尾、豎毛等行為學(xué)改變。持續(xù)30分鐘左右癥狀開始減輕，24小時后基本恢復(fù)正常。于B組對比，C組（SSTF預(yù)處理組）和D組（SSTF治療組）小鼠癥狀均有不同程度減輕，持續(xù)時間相對縮短。二：（1）震顫評分，懸掛能力和爬桿能力的測試結(jié)果均顯示，C組與D組能明顯改善小鼠運動協(xié)調(diào)能力，與B組（MPTP模型組）的差值均具有統(tǒng)計學(xué)意義（P0.05）。與A組（空白對照組）相比仍有統(tǒng)計學(xué)差異（P0.05）。C組與D組對比均無統(tǒng)計學(xué)差異（P0.05）。隨著給藥次數(shù)的增加，每天的差值無統(tǒng)計學(xué)意義（P0.05）。二：（1）TH免疫化學(xué)染色結(jié)果顯示，A組可見大量TH陽性神經(jīng)元，軸突明顯，形態(tài)正常。B組TH陽性神經(jīng)元數(shù)量明顯減少，C、D兩組TH陽性神經(jīng)元數(shù)量較B組增加，較A組仍有差別，差值均具有統(tǒng)計學(xué)意義（P0.05）。C、D兩組對比無統(tǒng)計學(xué)差異（P0.05）,B組細(xì)胞形態(tài)改變明顯，胞體腫脹并輪廓不清，軸突減少或消失。（2）Bcl-2染色結(jié)果顯示，A組陽性細(xì)胞數(shù)最多，棕黃色染色較深。B組陽性細(xì)胞數(shù)量明顯減少， C、D兩組陽性細(xì)胞數(shù)較B組明顯增多，與正常組相比仍有差別，差值均具有統(tǒng)計學(xué)差異（P0.05）, C、D兩組對比無統(tǒng)計學(xué)差異（P0.05）。（3）caspase-3免疫化學(xué)染色結(jié)果顯示，B組的陽性細(xì)胞數(shù)比A組顯著增多，差值具有統(tǒng)計學(xué)意義（P0.05）。C組、D組陽性細(xì)胞數(shù)較B組明顯減少，差值具有統(tǒng)計學(xué)意義（P0.05），與A組相比陽性細(xì)胞數(shù)增加，有統(tǒng)計學(xué)差異（P0.05）。C、D兩組對比差值無統(tǒng)計學(xué)意義（P0.05）。（4）GFAP免疫化學(xué)染色結(jié)果顯示，B、C、D組陽性細(xì)胞數(shù)均有不同程度增加，與A組對比，有統(tǒng)計學(xué)差異（P0.05）。B組GFAP陽性細(xì)胞數(shù)最多，與C、D兩組的差值具有統(tǒng)計學(xué)意義（P0.05）。C、D兩組對比差值無統(tǒng)計學(xué)意義（P0.05）。B、C、D組細(xì)胞的形態(tài)均發(fā)生改變，染色與A組對比有不同程度的加深。 結(jié)論： （1）SSTF可明顯改善MPTP帕金森小鼠的癥狀。（2）SSTF可明顯抑制MPTP帕金森小鼠中腦黑質(zhì)神經(jīng)元的凋亡。（3）相比后治療小鼠，預(yù)防性使用SSTF的MPTP帕金森小鼠的癥狀無明顯的改善。（4）相比后治療組，預(yù)防性使用SSTF并不能更好的抑制帕金森小鼠中腦黑質(zhì)神經(jīng)元的凋亡。（5）SSTF在治療帕金森病方面具有廣闊的研究前景。
[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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R742.5

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