【摘要】：目的：吡非尼酮是近年來(lái)發(fā)現(xiàn)的抗纖維化新藥，其抗纖維化作用效果明確，因而其在纖維化機(jī)制導(dǎo)致的慢性腦積水的治療上有很大的應(yīng)用前景，其安全性尚無(wú)論證，本研究通過(guò)測(cè)定喂食吡非尼酮后大鼠血清及腦脊液中神經(jīng)元特異性烯醇化酶的含量，以觀察其對(duì)神經(jīng)的損傷作用。 方法： 1大鼠分組及處理：挑選清潔級(jí)SD健康成年雄性大鼠50只，體重220±10g，隨機(jī)分為10組，每組5只，分為空白對(duì)照組、50mg/kg組、100mg/kg組、300mg/kg組、500mg/kg組、700mg/kg組、900mg/kg組、1100mg/kg組、1300mg/kg組、1500mg/kg組，各組大鼠分別在灌胃前1天、灌胃后1小時(shí)、6小時(shí)、12小時(shí)、24小時(shí)經(jīng)眼眶采血，在灌胃后24小時(shí)采集腦脊液標(biāo)本，采集腦脊液標(biāo)本后均灌注取腦。 2大鼠灌胃：各組大鼠灌胃前均禁食12小時(shí)，用大鼠灌胃器抽取配制好的PFD混懸液，給予大鼠灌胃處理。 3大鼠行為學(xué)檢測(cè)：各組大鼠于灌胃前一天、當(dāng)天、后一天，分別用平衡木行走試驗(yàn)、轉(zhuǎn)W試驗(yàn)對(duì)大鼠行為能力進(jìn)行評(píng)分。 4血清標(biāo)本取材：各大鼠于預(yù)定時(shí)間經(jīng)大鼠眼眶后靜脈叢取血標(biāo)本，用ELISA法檢測(cè)血清中NSE的含量。 5腦脊液標(biāo)本取材：各大鼠于預(yù)定時(shí)間用枕大池穿刺法取腦脊液標(biāo)本，用ELISA法檢測(cè)腦脊液中NSE的含量。 6腦標(biāo)本取材：各大鼠于預(yù)定時(shí)間點(diǎn)灌注后斷頭取腦，腦標(biāo)本石蠟包埋后留存待檢。 7染色處理：用鍍銀染色法觀察大鼠腦皮層組織病理學(xué)改變。 8數(shù)據(jù)分析：應(yīng)用IBM SPSS Statistics version21統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果： 1一般狀態(tài)變化：空白組大鼠實(shí)驗(yàn)期間，一般狀態(tài)良好，精神好，進(jìn)食好，被毛有光澤，活動(dòng)靈活，未見(jiàn)明顯異常狀態(tài)，體重?zé)o明顯變化。實(shí)驗(yàn)組大鼠中，低劑量組大鼠一般狀態(tài)與空白組大鼠情況相近，隨劑量越大，大鼠反應(yīng)越重，一般狀態(tài)越差，精神萎靡、飲食攝水減少，活動(dòng)減少，隨著時(shí)間延長(zhǎng)，狀態(tài)漸恢復(fù)，但仍不及實(shí)驗(yàn)前表現(xiàn)。各組實(shí)驗(yàn)大鼠均未出現(xiàn)死亡情況。 2各組大鼠體重比較：各組體重灌胃后較實(shí)驗(yàn)前均有所下降，但各組前后體重差異均無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。其體重變化考慮為實(shí)驗(yàn)刺激應(yīng)激所致。 3各組大鼠平衡木試驗(yàn)比較：各組大鼠行非參數(shù)性檢驗(yàn)，灌胃前一天各組大鼠間比較均無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。在每一組的大鼠灌胃均有統(tǒng)計(jì)學(xué)顯著差異（P＜0.05）。灌胃后一天各組大鼠間比較均有統(tǒng)計(jì)學(xué)差異(P0.05)。各組大鼠組內(nèi)不同時(shí)相間進(jìn)行比較，空白組、50mg/kg組、100mg/kg組大鼠不同時(shí)間點(diǎn)之間平衡木試驗(yàn)記分差異有統(tǒng)計(jì)學(xué)意義（P0.05），300mg/kg組、500mg/kg組、700mg/kg組、1100mg/kg組、1300mg/kg組、1500mg/kg組各組大鼠在不同時(shí)間點(diǎn)之間其平衡木實(shí)驗(yàn)記分差別均有統(tǒng)計(jì)學(xué)意義（P0.05）。 4各組大鼠轉(zhuǎn)圈試驗(yàn)比較：各組大鼠記錄結(jié)果用非參數(shù)檢驗(yàn)進(jìn)行分析，灌胃前一天各組大鼠之間差異沒(méi)有統(tǒng)計(jì)學(xué)意義（P0.05）。灌胃當(dāng)天和灌胃后一天，各組大鼠之間的比較，差別均有統(tǒng)計(jì)學(xué)意義（P0.05）�？瞻捉M、50mg/kg組、100mg/kg組、300mg/kg組、500mg/kg組大鼠分別比較不同時(shí)相間差異，其差異均無(wú)統(tǒng)計(jì)學(xué)意義（P0.05），700mg/kg組、900mg/kg組、1100mg/kg組、1300mg/kg組、1500組大鼠分別比較不同時(shí)相間差異，其差異均有統(tǒng)計(jì)學(xué)意義（P0.05）。 5各組大鼠腦組織重量及密度比較：各組大鼠腦組織重量（g）組間兩兩進(jìn)行比較，大劑量組1100mg/kg組、1300mg/kg組、1500mg/kg組比其小劑量組50mg/kg組、100mg/kg組、300mg/kg組及空白組腦重量均小，且差異有統(tǒng)計(jì)學(xué)意義（P0.05）。各組大鼠腦組織體積（ml）組間兩兩進(jìn)行比較，大劑量組1100mg/kg組、1500mg/kg組比小劑量組300mg/kg組及空白組腦體積小，差異有統(tǒng)計(jì)學(xué)意義（P0.05），其它組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。各組大鼠腦組織密度（g/ml）組間兩兩進(jìn)行比較，差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。 6各組大鼠血清NSE比較：各組大鼠灌胃前一天血清中NSE含量組間兩兩進(jìn)行比較差異沒(méi)有統(tǒng)計(jì)學(xué)意義（P0.05）。各組大鼠灌胃后1小時(shí)血清中NSE含量，空白組、50mg/kg組、100mg/kg組、300mg/kg組之間差異沒(méi)有統(tǒng)計(jì)學(xué)意義（P0.05），500mg/kg組與700mg/kg組之間差異沒(méi)有統(tǒng)計(jì)學(xué)意義（P0.05），900mg/kg組與1100mg/kg組之間差異沒(méi)有統(tǒng)計(jì)學(xué)意義（P0.05），1500mg/kg組與其它組之間兩兩進(jìn)行比較差異有統(tǒng)計(jì)學(xué)意義（P0.05）。各組大鼠灌胃后6小時(shí)血清中NSE含量（ng/ml），空白組、50mg/kg組、100mg/kg組、300mg/kg組之間差異沒(méi)有統(tǒng)計(jì)學(xué)意義（P0.05），其余組兩兩組間進(jìn)行對(duì)比差別均有統(tǒng)計(jì)學(xué)意義（P0.05）。各組大鼠灌胃后12小時(shí)血清中NSE含量，空白組、50mg/kg組、100mg/kg組、300mg/kg組之間差異沒(méi)有統(tǒng)計(jì)學(xué)意義（P0.05），900mg/kg組與1100mg/kg組之間差異沒(méi)有統(tǒng)計(jì)學(xué)意義（P0.05），其余各組與之兩兩進(jìn)行比較差異有統(tǒng)計(jì)學(xué)意義（P0.05）。各組大鼠灌胃后24小時(shí)血清中NSE含量，各組間兩兩進(jìn)行比較差異有統(tǒng)計(jì)學(xué)意義（P0.05）�？瞻捉M、50mg/kg組、100mg/kg組、300mg/kg組大鼠各時(shí)間點(diǎn)血清中NSE含量差別沒(méi)有統(tǒng)計(jì)學(xué)差異（P0.05）。500mg/kg組、700mg/kg組、1100mg/kg組大鼠各時(shí)間點(diǎn)血清中NSE含量相比較，6小時(shí)、12小時(shí)最高，1小時(shí)、24小時(shí)次之，灌胃前最低，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。900mg/kg組大鼠各時(shí)間點(diǎn)血清中NSE含量相比較，12小時(shí)最高，6小時(shí)次之，1小時(shí)、24小時(shí)再次之，灌胃前最低，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。1300mg/kg組大鼠各時(shí)間點(diǎn)血清中NSE含量相比較，各時(shí)間點(diǎn)差異有統(tǒng)計(jì)學(xué)意義（P0.05）。1500mg/kg組大鼠各時(shí)間點(diǎn)血清中NSE含量相比較，各時(shí)間點(diǎn)兩兩不相同，，差異有統(tǒng)計(jì)學(xué)意義（P0.05），由小到大依次為灌胃前、1小時(shí)、24小時(shí)、6小時(shí)、12小時(shí)。 7各組大鼠腦脊液NSE比較：各組大鼠灌胃后24小時(shí)腦脊液中NSE含量，各組間兩兩進(jìn)行比較差異有統(tǒng)計(jì)學(xué)意義（P0.05）。將其與24小時(shí)血清中NSE含量進(jìn)行比較，其含量要大，且差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 8各組大鼠大腦鍍銀染色比較：空白對(duì)照組大鼠腦組織神經(jīng)細(xì)胞數(shù)量及形態(tài)結(jié)構(gòu)分布正常。給予灌胃PFD的大鼠，小劑量組（50mg/kg、100mg/kg、300mg/kg）其腦組織神經(jīng)細(xì)胞數(shù)量及形態(tài)結(jié)構(gòu)分布與正常大鼠相近，大劑量組（500mg/kg）大鼠腦組織神經(jīng)可見(jiàn)壞死，隨劑量增大，壞死程度加重，正常神經(jīng)元結(jié)構(gòu)減少。 結(jié)論： 1NSE可作為大鼠腦損傷的重要檢測(cè)指標(biāo)，且其含量與損傷程度有相關(guān)性； 2NSE在大鼠腦損傷后有其分布規(guī)律，腦脊液中含量要高于血清中含量； 3PFD對(duì)大鼠腦神經(jīng)有一定損傷作用； 4PFD對(duì)大鼠腦神經(jīng)損傷有其劑量規(guī)律，50mg/kg、100mg/kg、300mg/kg對(duì)大鼠腦神經(jīng)的作用不明顯，500mg/kg以及更大劑量對(duì)大鼠腦神經(jīng)的損傷作用呈正相關(guān)性；
[Abstract]:Objective: To study the effect of unifenone in the treatment of chronic hydrocephalus which has been found in recent years, and its anti-fibrosis effect is clear, so it has a great application prospect in the treatment of chronic hydrocephalus caused by fibrosis mechanism. In this study, the content of neuron-specific enolase in the serum and cerebrospinal fluid of rats fed with non-nitrone was determined to observe the damage to the nerve. square Method:1 rat group and treatment:50 healthy adult male rats with clean-grade SD were selected, weighing 220 to 10 g, and randomly divided into 10 groups, each group was divided into a blank control group, a 50 mg/ kg group, a 100 mg/ kg group, a 300 mg/ kg group, a 500 mg/ kg group, a 700 mg/ kg group, a 900 mg/ kg group, a 1100 mg/ kg group, a 1300 mg/ kg group, and a 1500 mg/ kg group. The rats were divided into four groups:1 day,1 hour,6 hours,12 hours and 24 hours after the intragastric administration respectively. The samples of the cerebrospinal fluid were collected 24 hours after the intragastric administration, and the cerebrospinal fluid samples were collected and the samples were collected. injection of the brain. Rats were given intragastric administration: the rats were fasted for 12 hours before the intragastric administration, and the prepared PFD suspension was extracted with the rats in the rat, and the rats were given a large amount Rats were treated by intragastric administration.3 The behavior of rats: the day before and after the administration of the rats, the day of the day, the day after the administration of the rats, the balance wood walking test was used for each group, and the rats were treated with the W test. The ability was scored.4 serum samples: the rats were collected from the rat's orbit after the scheduled time, and the blood samples were collected by ELISA. The content of NSE in serum was measured in 5 CSF specimens. The content of NSE in the cerebrospinal fluid (CSF) was measured. and the paraffin embedded in the brain specimen is stored for the to-be-tested.7 dyeing treatment: dyeing with silver-plated Methods To observe the pathological changes of the cerebral cortex of the rat.8 Data analysis: The application of the IBM SPSS Statistics ver Sio Statistical analysis of n21 statistical software. Results:1 General status change: during the experimental period of the blank group rats, the normal condition was good, the spirit was good, the food was good, and the hair was lustrous. In the experimental group, the normal state of the rats in the low-dose group was similar to that of the blank group. The larger the dosage, the more the rats reacted, the worse the general state, the listlessness, the decrease of the diet and the decrease of the activity. The time is extended, the state is gradually restored, but it still does not And before and after the experiment, there was no death in each group of experimental rats. The weight of each group was compared with that of each group. However, there was no significant difference in body weight before and after each group ( (P0.05). The weight change of each group was considered to be due to the experimental stimulation of stress. There was no statistical difference between the rats in the day before and after the stomach (P0.05). There was a statistically significant difference in each group of rats (P <0.05). There was no statistical difference between each group of rats in the day after the stomach (P0.05). There was no significant difference in the scores of the balance wood between the groups in the group, the blank group, the 50 mg/ kg group and the 100 mg/ kg group (P0.05), the 300 mg/ kg group, the 500 mg/ kg group and the 700 mg/ kg group. Rats in the kg group,1100 mg/ kg group,1300 mg/ kg group and 1500 mg/ kg group at different time points There was a significant difference in the experimental scores of the balance wood in each group (P0.05). There was no significant difference in the difference between the rats in the previous day (P0.05). The day of intragastric administration and the day after the gastric administration There was no significant difference between the rats in the blank group, the 50 mg/ kg group, the 100 mg/ kg group, the 300 mg/ kg group and the 500 mg/ kg group, respectively. The difference was not significant (P0.05), the 700 mg/ kg group,900 mg/ kg group,1100 mg/ kg group,1300 mg/ kg group and 1500 rats were respectively The difference of brain tissue weight and density of each group was statistically significant (P0.05). The weight and density of brain tissue in each group were compared with that of the group. The weight and density of brain tissue in each group were compared with that of the group of the rats in the group of 100 mg/ kg,1300 mg/ kg and 1500 mg/ kg in the group of 50mg/ kg, 100mg/ kg and 300mg/ kg. The weight of the brain in the kg group and the blank group was small, and the difference was significant (P0.05). (P0.05). There was no significant difference between the other groups (P0.05). The density of brain tissue in each group (P0.05). There was no significant difference between the two groups (P0.05). There was no significant difference between the two groups (P0.05). The levels of NSE in serum, blank group,50 mg/ kg group,100 mg/ kg group and 300 mg/ kg group were not statistically significant (P0.05). There was no statistical difference between the group of 500 mg/ kg and the group of 700 mg/ kg (P0.05), and there was no statistical difference between the group of 900 mg/ kg and the group of 1100 mg/ kg (P0.05), and 1500 mg. There was no significant difference (P> 0.05) between the/ kg group and the other groups (P <0.05). The serum NSE content (ng/ ml), the blank group, the 50 mg/ kg group, the 100 mg/ kg group and the 300 mg/ kg group were not statistically significant (P0. There was no significant difference between the two groups (P0.05). The levels of NSE, blank,50 mg/ kg,100 mg/ kg and 300 mg/ kg in the group were not significant (P0.05), and there was no statistical difference between the group of 900 mg/ kg and 1100 mg/ kg (P0.05). 05) There was a significant difference between the two groups (P0.05). The levels of NSE in the serum of the rats in the blank group, the 50 mg/ kg group, the 100 mg/ kg group and the 300 mg/ kg group were not statistically different (P0.05). The NSE content in the serum of the rats at the time points in the 500 mg/ kg group, the 700 mg/ kg group and the 1100 mg/ kg group was not statistically significant (P0.05). The levels of NSE in the serum of the rats in the 900 mg/ kg group were the highest and the difference was statistically significant (P0.05). The level of NSE in the serum of the rats at the 900 mg/ kg group was the highest, followed by 6 hours,1 hour and 24 hours, and the difference was statistically significant (P0. The levels of NSE in the serum of the rats at all time points in the 1300 mg/ kg group were statistically significant (P0.05). The levels of NSE in the serum of the 1500 mg/ kg group were not the same at each time point, and the difference was statistically significant (P0.05). 5) The levels of NSE in the cerebrospinal fluid of each group were as follows:1 hour,24 h,6 h and 12 h from small to large. The level of NSE in the spinal fluid and the difference between the two groups were statistically significant (P0.05). It was compared with the 24-hour serum. The content of NSE was higher and the difference was significant (P0.05). The results showed that the number of nerve cells and the morphological structure of the rats in the blank control group were normal, and the number of nerve cells and the morphological structure of the rats were similar to those of the normal rats and the large-dose group (500 mg/ kg). ) Rats The neurovisible necrosis of the brain tissue increases with the increase of the dose, the degree of necrosis is increased, and the normal neuronal structure is reduced. Conclusion: 1NSE can be used as an important test index for brain injury in rats, and its content and damage degree of correlation; NSE in rat brain The distribution of the brain in rats was higher than that in the serum, and the 3-D PFD had a certain damage to the rat's cranial nerve. The dose of the pfd to the rat's cranial nerve was 50 mg/ kg,100 mg/ kg and 300 mg/ kg.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R965

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