本文選題：Caspase-3 + 腦缺血��； 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的:本實(shí)驗(yàn)采用高血脂合并腦缺血大鼠模型,觀察腦缺血灶形態(tài)學(xué)(TTC染色和HE染色)、血脂四項(xiàng)(膽固醇、甘油三酯、高密度脂蛋白和低密度脂蛋白)含量和缺血皮質(zhì)區(qū)Caspase-3蛋白表達(dá)的變化,探討電針對(duì)高血脂合并腦缺血大鼠行為學(xué)、血脂四項(xiàng)、肝臟和腦組織形態(tài)學(xué)、缺血灶梗死體積和缺血皮質(zhì)區(qū)Caspase-3表達(dá)的影響,為闡明電針抗凋亡機(jī)制提供基礎(chǔ)研究。方法:分組:105只SD大鼠適應(yīng)性飼養(yǎng)1周后,隨機(jī)分為正常組、假手術(shù)組、模型組、電針Ⅰ組、電針Ⅱ組,每組21只,其中每組按隨機(jī)數(shù)字表抽取7只做TTC染色;剩余每組14只隨機(jī)分為1天組和7天組,各7只,做HE染色、血脂四項(xiàng)檢測(cè)和Caspase-3蛋白檢測(cè)。除正常組外,其余各組以高脂飼料喂養(yǎng)6周制備高血脂癥模型。6周后電針Ⅰ組開(kāi)始電針雙側(cè)"豐隆" "三陰交",1次/日,每次20 min,連續(xù)干預(yù)7天,模型組、電針Ⅱ組不干預(yù)。7天后模型組、電針Ⅰ組、電針Ⅱ組,采用FeCl3化學(xué)誘導(dǎo)血栓性閉塞大腦中動(dòng)脈模型,假手術(shù)組除不滴加FeCl3溶液外,其余手術(shù)步驟同模型組。造模后電針Ⅰ、Ⅱ組電針雙側(cè)"豐隆"、"三陰交"及針刺"水溝""百會(huì)",1次/日,每次20min,分別干預(yù)1、7天。造模后1、3、7天分別對(duì)各組大鼠進(jìn)行神經(jīng)行為學(xué)評(píng)分;造模后1天TTC染色,觀察腦梗死體積;造模后1、7天腹主動(dòng)脈取血,檢測(cè)血清中膽固醇(CHO)、甘油三酯(TG)、高密度脂蛋白(HDL-C)和低密度脂蛋白(LDL-C)含量,HE染色觀察大鼠肝臟和腦組織病理變化,免疫組化方法檢測(cè)缺血皮質(zhì)區(qū)Caspase-3蛋白的表達(dá)。結(jié)果:1.電針對(duì)各組大鼠神經(jīng)行為學(xué)的影響大鼠腦缺血造模后1、3、7天神經(jīng)功能評(píng)分:與同期正常組、假手術(shù)組比較,模型組、電針Ⅰ組和電針Ⅱ組神經(jīng)功能評(píng)分造模后1、3、7天均有明顯差異(P0.05);與同期模型組比較,電針Ⅰ、Ⅱ組造模后1、3、7天均有明顯差異(P0.05);與同期電針Ⅱ組比較,造模后1、3天,電針Ⅰ組評(píng)分有明顯差異(P0.05),造模后7天,電針Ⅰ、Ⅱ組無(wú)明顯差異(P0.05)。2.電針對(duì)各組大鼠血脂四項(xiàng)(膽固醇、甘油三酯、高密度脂蛋白和低密度脂蛋白)含量的影響血脂四項(xiàng)結(jié)果顯示,與正常組比較,假手術(shù)組、模型組造模后1、7天,CHO、TG、HDL-C和LDL-C含量均有明顯差異(P0.05);與同期模型組比較,電針Ⅰ組造模后1、7天,CHO、TG、HDL-C和LDL-C含量均有明顯差異(P0.05),電針ⅡI組造模后1天,TG含量有明顯差異(P0.05),CHO、HDL-C和LDL-C含量無(wú)明顯差異(P0.05),造模后7天,CHO、TG、HDL-C和LDL-C含量均有明顯差異(P0.05);與同期電針Ⅱ組比較,電針Ⅰ組造模后1天,CHO、HDL-C和LDL-C含量均有明顯差異(P0.05),TG含量無(wú)明顯差異(P0.05),造模后7天,CHO、TG、HDL-C和LDL-C含量均有明顯差異(P0.05);兩電針Ⅰ組比較,CHO、HDL-C和LDL-C含量有明顯差異(P0.05),TG含量無(wú)明顯差異(P0.05);兩電針Ⅱ組比較,CHO、TG和LDL-C含量均有明顯差異(P0.05),HDL-C含量無(wú)明顯差異(P0.05)。3.電針對(duì)各組大鼠肝組織形態(tài)學(xué)的影響肝組織HE染色結(jié)果顯示,造模后1、7天正常組和假手術(shù)組肝細(xì)胞未見(jiàn)變性壞死,細(xì)胞排列整齊,結(jié)構(gòu)清晰。模型組造模后1天,可見(jiàn)淋巴細(xì)胞聚集,肝板索狀結(jié)構(gòu)被破壞,肝血竇小而少,偶見(jiàn)點(diǎn)狀壞死灶;造模后7天,肝細(xì)胞腫脹明顯,肝竇結(jié)構(gòu)不清,可見(jiàn)脂肪變性,典型表現(xiàn)有廣泛的氣球樣變性,病理表現(xiàn)明顯比1天模型組嚴(yán)重。電針Ⅰ、Ⅱ組造模后1、7天,肝細(xì)胞病變較輕,異型性低,肝板、肝竇、肝小葉結(jié)構(gòu)稍不清,炎性細(xì)胞比同期模型組有明顯減少。4.電針各組大鼠腦梗死體積的影響TTC染色結(jié)果顯示,大鼠腦缺血造模后1天,缺血部位均呈典型的缺血性改變,表明該模型制備成功。與正常組、假手術(shù)組比較,模型組、電針Ⅰ、Ⅱ組梗死體占比均有明顯差異(P0.05);與模型組比較,電針Ⅰ組梗死體積占比有明顯差異(P0.05),電針Ⅱ組梗死體積占比無(wú)明顯差異(P0.05);與電針Ⅱ組比較,電針Ⅰ組梗死體積占有明顯差異(P0.05)。5.電針對(duì)各組大鼠腦組織形態(tài)學(xué)的影響腦組織皮質(zhì)區(qū)HE染色結(jié)果顯示:正常組和假手術(shù)組神經(jīng)細(xì)胞基本正常,排列整齊,核仁清晰。模型組造模后1天,可見(jiàn)膠質(zhì)細(xì)胞輕度增生,神經(jīng)元輕度腫脹,造模后7天可見(jiàn)間質(zhì)細(xì)胞水腫明顯,大量死亡神經(jīng)元,呈篩狀壞死,壞死組織周?chē)梢?jiàn)炎性細(xì)胞浸潤(rùn);海馬CA1區(qū)染色結(jié)果顯示:正常組和假手術(shù)組海馬CA1區(qū)結(jié)構(gòu)致密細(xì)胞核數(shù)量較多,細(xì)胞核形態(tài)基本正常。模型組造模后1天,整體未見(jiàn)軟化灶,海馬細(xì)胞萎縮較少,造模后7天海馬區(qū)神經(jīng)細(xì)胞數(shù)量減少,細(xì)胞核固縮,層次紊亂。電針Ⅰ、Ⅱ組神經(jīng)細(xì)胞受損情況較模型組明顯減輕,存活的神經(jīng)細(xì)胞數(shù)量增多,海馬結(jié)構(gòu)排列較整齊。6.電針各組大鼠缺血皮質(zhì)區(qū)Caspase-3蛋白表達(dá)的影響正常組和假手術(shù)組大鼠缺血皮質(zhì)區(qū)Caspase-3蛋白呈弱陽(yáng)性表達(dá)。與正常組比較,假手術(shù)組術(shù)后1、7天,Caspase-3陽(yáng)性細(xì)胞數(shù)量和平均光密度值均無(wú)明顯差異(P0.05);與同期假手術(shù)組比較,模型組造模后1、7天,Caspase-3陽(yáng)性細(xì)胞數(shù)量和平均光密度值均有明顯差異(P0.05);與同期模型組比較,電針Ⅰ組造模后1、7天,Caspase-3陽(yáng)性細(xì)胞數(shù)量和平均光密度值均有明顯差異(P0.05),電針Ⅱ組造模后1天Caspase-3陽(yáng)性細(xì)胞數(shù)量無(wú)明顯差異(P0.05),平均光密度值有明顯差異(P0.05),電針Ⅱ組造模后7天,Caspase-3陽(yáng)性細(xì)胞數(shù)量和平均光密度值有明顯差異(P0.05);與同期電針Ⅱ組比較,電針Ⅰ組造模后1、7天,Caspase-3陽(yáng)性細(xì)胞數(shù)量和平均光密度值均有明顯差異(P0.05);兩電針Ⅰ組比較,Caspase-3陽(yáng)性細(xì)胞數(shù)量和平均光密度值有明顯差異(P0.05);兩電針Ⅱ組比較,Caspase-3陽(yáng)性細(xì)胞數(shù)量和平均光密度值有明顯差異(P0.05)。結(jié)論:1.電針可以降低神經(jīng)功能評(píng)分,腦缺血早期干預(yù)對(duì)改善神經(jīng)功能癥狀作用明顯,即刻的針刺效應(yīng)對(duì)神經(jīng)功能改善也有一定作用。2.電針可以降低血清CHO、TG和LDL-C含量,升高HDL-C含量,隨著腦缺血時(shí)間延長(zhǎng)腦受損情況加重,電針對(duì)HDL-C作用逐漸不明顯。3.腦缺血可以加重高血脂癥誘導(dǎo)的肝損傷,電針對(duì)肝組織病理形態(tài)學(xué)變化有改善作用,可能通過(guò)減少炎性細(xì)胞降低肝損傷。4.電針對(duì)缺血區(qū)腦組織病理形態(tài)學(xué)變化有改善作用,可能通過(guò)減輕神經(jīng)元變性脫失現(xiàn)象,神經(jīng)細(xì)胞腫脹和炎性細(xì)胞浸潤(rùn),減輕腦損傷。5.早期電針干預(yù)可以減少大鼠缺血部位梗死體積,降低腦組織損傷。6.電針可以下調(diào)高血脂合并腦缺血大鼠缺血皮質(zhì)區(qū)Caspase-3蛋白的含量,且提前電針干預(yù)下調(diào)作用明顯,說(shuō)明其存在抗凋亡的作用,下調(diào)Caspase-3蛋白含量的表達(dá)可能是電針起效機(jī)制之一。
[Abstract]:Objective: in this experiment, we used hyperlipidemia and cerebral ischemia rat model to observe the morphology (TTC and HE staining) of cerebral ischemia, the content of four lipids (cholesterol, triglyceride, high-density lipoprotein and low density lipoprotein) and the change of Caspase-3 protein expression in ischemic cortex. Four terms of lipid, liver and brain histomorphology, infarct volume and Caspase-3 expression in ischemic cortex provide a basic study for the anti apoptosis mechanism of electroacupuncture. Methods: 105 SD rats were randomly divided into normal group, sham operation group, model group, electroacupuncture group I, group II, group II, each group of 21 rats, of which each group was in each group. A random number table was used to extract 7 TTC stains; 14 of the remaining groups were randomly divided into 1 days group and 7 day group, each 7, HE staining, four blood lipid testing and Caspase-3 protein detection. Except the normal group, the rest groups were fed with high fat feed for 6 weeks to prepare hyperlipidemia model.6 weeks after the electroacupuncture group began to electroacupuncture bilateral "Feng long" "Three Yin cross" ", 1 / day, each. 20 min, continuous intervention for 7 days, model group, electroacupuncture group II group did not interfere with.7 days model group, electroacupuncture group I, electroacupuncture group II, using FeCl3 chemical induced thrombotic occlusion of the middle cerebral artery model, sham operation group in addition to the FeCl3 solution, the rest of the procedure with the model group. Water ditch "hundred meetings", 1 times / day, each time 20min, intervention 1,7 days respectively. After the model 1,3,7 days, the rats were graded respectively on neurobehavioral score, and the volume of cerebral infarction was observed at 1 days after modeling, and the abdominal aorta was taken on 1,7 days after modeling, and serum cholesterol (CHO), triglyceride (TG), high-density lipoprotein (HDL-C) and low density lipoprotein (LDL-) were detected on 1,7 days after modeling. C) content, HE staining was used to observe the pathological changes of liver and brain tissue in rats. Immunohistochemical method was used to detect the expression of Caspase-3 protein in ischemic cortex. Results: 1. the effect of Electroacupuncture on neurobehavioral effects of rats in each group was measured by the 1,3,7 day nerve function score of the rats after the model of cerebral ischemia: compared with the normal group, sham operation group, model group, electroacupuncture group I and electroacupuncture. In group II, there were significant differences in 1,3,7 days after modeling (P0.05). Compared with the same model group, there was a significant difference between group I and group II after making model (P0.05). Compared with group II of electroacupuncture at the same time, the score of electroacupuncture group I was significantly different (P0.05), 7 days after modeling (P0.05), and there was no significant difference (P0.05).2. electroacupuncture at 7 days after modeling. The four results of blood lipid four items (cholesterol, triglyceride, high density lipoprotein and low density lipoprotein) showed that compared with the normal group, the contents of CHO, TG, HDL-C and LDL-C in the sham operation group were significantly different (P0.05) in the model group 1,7 days after model building, and compared with the model group, 1,7 days, CHO, TG after the model group I was built. The contents of HDL-C and LDL-C were significantly different (P0.05). There were significant differences in TG content (P0.05) at 1 days after electroacupuncture group II I. There was no significant difference in CHO, HDL-C and LDL-C content (P0.05). There were significant differences in the content of TG, TG, CHO, HDL-C and LDL-C after 7 days of modeling. There was no significant difference (P0.05), and there was no significant difference in TG content (P0.05). The content of CHO, TG, HDL-C and LDL-C were significantly different (P0.05) at 7 days after the model. The content of CHO, HDL-C and LDL-C was significantly different (P0.05), and there was no significant difference in the content of CHO, HDL-C and LDL-C in the two electroacupuncture group. The effect of.3. electroacupuncture on the liver histomorphology of rats in each group (P0.05) showed that the liver tissue HE staining results showed that there was no degeneration and necrosis in the normal group and the sham operation group after the model building, the cells arranged neatly and the structure was clear. 1 days after the model group, the lymphocyte aggregation, the liver plate cable structure was destroyed, the hepatic sinusoids were small and small. On the 7 day after the model, the liver cell swelling was obvious, the hepatic sinusoid structure was not clear, the fatty degeneration was seen, the typical manifestation had the extensive balloon denaturation, the pathological manifestation was obviously more serious than the 1 day model group. The electroacupuncture group I, group II, after 1,7 days, the liver cell lesion was light, the heteromorphosis was low, the liver plate, hepatic sinus, hepatic lobule structure was slightly unclear, inflammatory cells were compared with the same The effect of the model group on the cerebral infarction volume of the.4. electroacupuncture group was significantly reduced by TTC staining. The results showed that 1 days after the cerebral ischemia, the ischemic parts were all typical ischemic changes, which showed that the model was successfully prepared. Compared with the normal group, the sham operation group, the model group, the electroacupuncture I, and the group II were significantly different (P0.05). Compared with group I, there was a significant difference in the infarct volume ratio in the electroacupuncture group (P0.05), and there was no significant difference in the infarct volume in the electroacupuncture group (P0.05). Compared with the electroacupuncture group, the infarct volume in the group I was significantly different (P0.05). The effect of.5. electroacupuncture on the brain histomorphology of the rats in each group showed that the result of HE staining in the cortex area of the cerebral cortex showed that the normal group and the artificial hand were the result of HE staining. The nerve cells in the operation group were basically normal, arranged neatly, and the nucleolus were clear. On the 1 day after the model group, the glial cells were slightly proliferated and the neurons were swollen slightly. The interstitial cell edema was visible on the 7 day after the model building. A large number of dead neurons were necrotic and inflammatory cells infiltrated around the necrotic tissue; the hippocampal CA1 staining results showed that the normal group was the normal group. The number of dense nuclei in the hippocampal CA1 area of the sham operation group was more than that of the model group, and the nucleus morphology was basically normal. The model group had no softening focus on the whole of the model group 1 days after the model building. The hippocampal cells atrophied less, the number of neurons in the hippocampus decreased in the 7 day after model building, the nucleus retraction and the level disorder. The damage of nerve cells in group I, group II was significantly reduced than that of the model group. The number of surviving nerve cells increased, and the expression of Caspase-3 protein in the ischemic cortex of the rats with.6. electroacupuncture group was more orderly. The expression of Caspase-3 protein in the ischemic cortex of the normal group and the sham operation group was weakly positive. Compared with the normal group, the number of Caspase-3 positive cells and the mean light density value of the Caspase-3 positive cells in the sham operation group were 1,7 days after operation. There was no significant difference (P0.05). Compared with the sham operation group in the same period, the number of Caspase-3 positive cells and the mean light density of the model group were significantly different at 1,7 days after model building (P0.05). Compared with the model group, the number of Caspase-3 positive cells and the number of light density were significantly different at 1,7 days after the model group (P0.05), and the model of electroacupuncture group II was made by the model group (P0.05). There was no significant difference in the number of Caspase-3 positive cells in the last 1 days (P0.05), and the mean light density was significantly different (P0.05). The number of Caspase-3 positive cells and the mean light density were significantly different (P0.05) at the 7 day after the electroacupuncture group II group. Compared with the Electroacupuncture group II group, the number of Caspase-3 positive cells and the mean optical density value of the Caspase-3 positive cells after the electroacupuncture group I made the model. There were significant differences (P0.05). Two electroacupuncture group I compared the number of Caspase-3 positive cells and average optical density (P0.05); two electroacupuncture group II compared, the number of Caspase-3 positive cells and the mean light density value is significantly different (P0.05). Conclusion: 1. electroacupuncture can reduce the neurological function score, cerebral ischemia early intervention to improve nerve work The effect of symptomatic effect is obvious. The immediate acupuncture effect also has certain effect on the improvement of nerve function..2. electroacupuncture can reduce the content of serum CHO, TG and LDL-C, increase the content of HDL-C. With the prolongation of cerebral ischemia time, the condition of brain damage is aggravated. The effect of Electroacupuncture on HDL-C is gradually not obvious in.3. cerebral ischemia, which can aggravate the liver injury induced by hyperlipidemia, the Electroacupuncture of the liver. The changes of histopathology can be improved by reducing the injury of inflammatory cells and reducing the liver injury..4. electroacupuncture can improve the pathological changes of the brain tissue in the ischemic area. It may reduce the degeneration and loss of neurons, the swelling of the nerve cells and the infiltration of inflammatory cells, and reduce the brain injury in the early stage of.5. by Electroacupuncture intervention to reduce the deficiency of rats. .6. electroacupuncture can reduce the content of Caspase-3 protein in the ischemic cortex of rats with hyperlipidemia and cerebral ischemia, and the effect of early electro acupuncture intervention is obvious, indicating the effect of anti apoptosis, and the expression of Caspase-3 protein can be one of the mechanism of electroacupuncture.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R245

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