重復經顱磁刺激對局灶性腦缺血大鼠神經再生及功能恢復的相關研究
發(fā)布時間：2018-09-19 09:02
【摘要】：研究目的：利用重復經顱磁刺激對局灶性腦缺血再灌注成年大鼠進行干預,研究重復經顱磁刺激對腦缺血再灌注損傷大鼠運動功能及側腦室室管膜下區(qū)NSCs增殖的影響,并探索可能的機制。材料與方法：將45只成年雄性SD大鼠進行隨機分組,分成假手術組、模型組和rTMS組,每組15只。采用右側大腦中動脈栓塞(middle artery occlusion,MCAO)模型,梗阻80分鐘。rTMS組于造模成功后24h開始干預,每天一次,連續(xù)干預7天,期間假手術組和模型組不接受任何干預。每組大鼠在術后第1天和第7天給予神經功能評分(neurological severity scores,NSSs),部分大鼠(每組5只)在rTMS組最后一次治療結束后即刻開始進行腹腔內注射5-溴脫氧尿嘧啶核苷(5-Bromo-2-deoxy Uridine, BrdU)以標記增殖的細胞,每隔4小時注射一次,連續(xù)注射3次。另一部分大鼠(每組10只)在術后第7天取患側缺血腦組織。利用NSSs評分觀察rTMS對MCAO大鼠運動功能的影響,利用免疫組化熒光雙重標記技術檢測患側SVZ內源性NSCs增殖的情況,利用實時熒光定量反轉錄聚合酶鏈式反應(real-time fluorescent quantitative reverse transcription polymerase chain reaction,qRT-PCR)檢測microRNA-25(miR-25)相對表達量,利用免疫組織蛋白印跡(Western blotting)檢測miR-25增殖相關靶蛋白的變化。統(tǒng)計學數據分析采用單因素方差分析,組間兩兩比較采用Bonferroni檢驗。結果：1.模型組和rTMS組的大鼠術后1天時進行NSSs評分結果無顯著差異(p0.05)；術后7天時兩組大鼠NSSs評分于第1天相比較均得到明顯改善(p0.005),且rTMS組大鼠的神經功能改善程度優(yōu)于模型組大鼠(p=0.019)。2.利用BrdU+/Nestin+標記患側SVZ增殖的NSCs數,結果發(fā)現(xiàn)術后7天,模型組較假手術組NSCs增殖數增多且存在顯著的差異(p=0.044);rTMS組較模型組及假手術組NSCs增殖數目均有顯著增多且存在統(tǒng)計學意義(p0.005)。3. qRT-PCR的結果顯示術后7天時,模型組大鼠患側腦缺血組織內miR-25的表達明顯高于假手術組(p0.001); rTMS組患側腦缺血組織miR-25表達較其他兩組(p0.001)均顯著增高。4. Western blotting的結果發(fā)現(xiàn)術后7天時,miR-25靶蛋白p57和PTEN在假手術組以及模型組的表達無顯著性差異(p0.05)；而rTMS組大鼠患側腦缺血組織內p57的表達較其他兩組明顯減少(p0.01),PTEN的表達則較其他兩組顯著增加(p0.01)。結論：10Hz rTMS干預患側大腦皮層7天能促進局灶性腦缺血大鼠運動功能的恢復,并促進患側SVZ內源性NSCs的增殖,且這種促NSCs增殖的作用可能與上調miR-25的表達進而降低其靶蛋白p57的表達有關。研究目的：通過側腦室注射miR-25拮抗劑抑制miR-25的表達,探索參與調節(jié)NSCs增殖的miR-25/p57在rTMS促進局灶性腦缺血后患側SVZ內源性NSCs增殖中的作用,進一步證實rTMS對內源性NSCs的增殖效應與miR-25/p57密切相關。材料與方法：將成年雄性SD大鼠進行隨機分組,分成rTMS組、rTMS+拮抗劑組和rTMS+對照劑組,每組15只。造模方式為右側大腦中動脈栓塞(middle artery occlusion,MCAO),梗阻80分鐘。rTMS+拮抗劑組及rTMS+對照劑組分別予以不同劑量的miR-25拮抗劑和miR-25對照劑側腦室注射后進行右側MCAO造模。所有大鼠造模成功后24h開始給予rTMS干預,每天一次,連續(xù)干預7天。各組大鼠在術后7天取患側的腦組織,通過qRT-PCR檢測miR-106b-25家族的相對表達量,確定合適的拮抗劑劑量。在miR-25被特異性抑制后,部分大鼠(每組5只)在術后第7天取患側的腦組織進行Western blotting檢測相關靶蛋白的變化,另一部分大鼠(每組5只)在rTMS組最后一次治療后即刻開始進行腹腔注射BrdU以標記增殖的細胞,每隔4小時一次,連續(xù)3次。利用免疫組化熒光雙重標記技術檢測miR-25抑制后患側SVZ內源性NSCs的增殖情況。統(tǒng)計學數據分析采用單因素方差分析,組間兩兩比較采用Bonferroni檢驗。結果：1.miRNA拮抗劑可抑制相關miRNA表達,并表現(xiàn)出劑量依賴性。2. qRT-PCR結果顯示2.5nmol Ant-25可以顯著抑制miR-25的表達,且不影響miR-106b家族其他成員的表達。3.Western blotting結果顯示miR-25被抑制后,拮抗劑組的p57表達較rTMS組(p=0.018)和對照劑組(p=0.012)顯著提高,而p21的表達在各個組之間差異沒有統(tǒng)計學意義(p0.05)。4.利用BrdU+/Nestin+標記患側SVZ增殖的NSCs數,結果顯示miR-25被抑制后,rTMS組和對照劑組NSCs增殖數無顯著差異(p0.05)；拮抗劑組較rTMS組(p=0.006)及對照劑組(p=0.013)NSCs增殖數明顯減少且有統(tǒng)計學意義。結論：MiR-25特異性有效性的抑制后,靶蛋白p57表達升高,而大鼠腦缺血側SVZ內源性NSCs增殖降低,即10Hz rTMS可以通過干預miR-25來調節(jié)其靶蛋白p57的表達,進而促進NSCs的增殖。研究目的：利用rTMS對局灶性腦缺血大鼠進行干預,研究rTMS對腦缺血大鼠學習記憶功能的影響,并觀察患側海馬區(qū)神經再生和凋亡的情況。材料與方法：將成年雄性SD大鼠進行隨機分組,分成7天組和14天組,兩組再進行隨機分組,分成假手術組、模型組和rTMS三個亞組。采用右側MCAO模型,梗阻80分鐘。rTMS組于造模成功后24h開始干預,每天一次,直到相應治療時間結束,期間假手術組和模型組不接受任何干預。各組大鼠在術后第1天、第7天和第14天利用磁共振(magnetic resonance imaging,MRI)進行彌散加權成像(Diffusion-weighted images,DWI)檢查,并根據DWI獲取表觀彌散系數(apparent diffusion coefficient,ADC)值。7天組的大鼠(每組5只)在rTMS組最后一次治療結束后即刻開始進行腹腔內注射BrdU以標記增殖的細胞,每隔4小時注射一次,連續(xù)注射3次。另一部分大鼠(每組10只)在術后第7天取患側缺血腦組織。14天組的部分大鼠(每組5只)于術后第一天開始,每天腹腔注射一次BrdU來標記分化的細胞,連續(xù)注射一周。另一部分大鼠在術后第12天進行Morris水迷宮測試,并于第14天處死取患側海馬組織,分別進行Tunel染色(每組5只)和western blotting檢測(每組5只)。利用缺血側ADC值與健側ADC比值研究rTMS對MCAO大鼠腦梗體積的影響,采用Morris水迷宮實驗觀察rTMS對MCAO大鼠的學習記憶的影響,利用免疫組化熒光雙重標記技術檢測患側SGZ內源性NSCs增殖和分化的情況,利用Tunel染色觀察患側海馬神經元凋亡的情況,利用western blotting證實患側海馬凋亡相關蛋白B細胞淋巴瘤／白血病基因2(B cell lymphoma/leukemia gene 2,Bcl-2)和Bcl基因相關蛋白(Bcl2-associated protein X,Bax)的表達變化。統(tǒng)計學數據分析采用單因素方差分析,組間兩兩比較采用Bonferroni檢驗。結果：1.模型組和rTMS組的大鼠術后1天時梗死灶體積差異無統(tǒng)計學意義(p=0.878),但兩組大鼠都可見明顯梗死灶且有統(tǒng)計學意義(p0.001)。第7天(p=0.246)和14天(p=0.132)時模型組和rTMS組之間梗死灶體積無統(tǒng)計學差異,但rTMS組大鼠腦梗死灶體積的改善較模型組的大鼠趨于更優(yōu)。此外,雖然模型組和rTMS組大鼠在術后7天及14天時,梗死灶體積均較第1天明顯減小(p0.001),但模型組(p=0.716)和rTMS組(p=0.264)第14天較第7天差異均沒有統(tǒng)計學意義。2. Morris水迷宮結果顯示,模型組大鼠與假手術組相比逃避潛伏期明顯延長(p=0.042),60s內穿越目標區(qū)域的次數也明顯減少(p=0.001); rTMS組較模型組大鼠逃避潛伏期顯著縮短(p=0.006),60s內穿越目標區(qū)域的次數也顯著增多(p=0.045)。3.利用BrdU+/Nestin+標記患側SGZ區(qū)增殖的NSCs數,結果顯示術后7天,模型組較假手術組增殖的NSCs數增多且有統(tǒng)計學意義(p=0.042);rTMS組較假手術組及模型組增殖的NSCs數均明顯增多且有顯著差異(p0.001)。4.利用BrdU+/NeuN+標記患側SGZ區(qū)分化的NSCs數,結果顯示術后14天,rTMS組較假手術組(p=0.006)及模型組(p=0.021)NSCs分化數目均顯著增多且有統(tǒng)計學意義。而模型組較假手術組NSCs分化數未見統(tǒng)計學差異(p0.05)。5. Tunel染色結果顯示術后14天,模型組大鼠患側海馬神經元凋亡數目較假手術組顯著增多且有統(tǒng)計學意義(p0.001),而rTMS組大鼠患側海馬神經元凋亡數目較模型組明顯減少且有統(tǒng)計學差異(p0.001)。6. Western blotting結果顯示,與假手術組相比,模型組患側海馬Bcl-2表達下降(p0.01), Bax表達升高(p0.01)。而rTMS組的Bcl-2表達較模型組明顯升高(p0.005), Bax的表達則較模型組顯著降低(p0.005)。結論：局灶性腦缺血大鼠學習記憶能力下降,但lOHz rTMS能改善局灶性腦缺血大鼠學習記憶的恢復,并能有效促進局灶性腦缺血大鼠患側SGZ內源性NSCs增殖和分化和通過調節(jié)凋亡相關蛋白Bcl-2和Bax抑制局灶性腦缺血大鼠患側海馬神經元的凋亡。
[Abstract]:AIM: To study the effects of repetitive transcranial magnetic stimulation on motor function and proliferation of NSCs in the subependymal zone of the lateral ventricle in rats with focal cerebral ischemia-reperfusion injury, and to explore the possible mechanism. Materials and Methods: Forty-five adult male SD rats were randomly divided into two groups. Fifteen rats in each group were divided into sham operation group, model group and rTMS group. The right middle cerebral artery occlusion (MCAO) model was used and the obstruction lasted for 80 minutes. Neurological severity scores (NSSs) were given on the 7th day. Some rats (5 rats in each group) were injected intraperitoneally with 5-bromo-2-deoxy Uridine (BrdU) to label proliferating cells, once every four hours, three times continuously after the last treatment in rTMS group. On the 7th day after operation, the ischemic brain tissue was taken from the affected side of the rats (10 rats in each group). The effects of rTMS on the motor function of MCAO rats were observed by NSS score. The proliferation of endogenous NSCs in the affected side of SVZ was detected by immunohistochemical fluorescence double labeling technique. Real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) was used. Verse transcription polymerase chain reaction (qRT-PCR) was used to detect the relative expression of microRNA-25 (microRNA-25) and Western blotting was used to detect the changes of proliferation-related target proteins of microRNA-25. There was no significant difference in NSS scores between the rTMS group and the control group on the 1st day after operation (p0.05). The NSS scores of the two groups were significantly improved on the 7th day after operation (p0.005), and the neurological function of the rTMS group was better than that of the model group (p = 0.019). 2. The number of NSCs proliferated by BrdU + / Nestin + labeling SVZ on the affected side was found. On the 7th day after operation, the proliferation of NSCs in the model group was significantly higher than that in the sham operation group (p = 0.044), and the proliferation of NSCs in the rTMS group was significantly higher than that in the model group and sham operation group (p 0.005). 3. The results of qRT-PCR showed that the expression of microwave-25 in the affected cerebral ischemic tissue of the model group was significantly higher than that in the sham operation group on the 7th day after operation. The expression of microRNA-25 in the affected cerebral ischemic tissues of the rTMS group was significantly higher than that of the other two groups (p0.001). 4. Western blotting results showed that there was no significant difference in the expression of microRNA-25 target protein p57 and PTEN between the sham-operated group and the model group at 7 days after operation (p0.05); however, the expression of p57 in the affected cerebral ischemic tissues of the rTMS group was higher than that of the other groups (p0.001). Conclusion: 10Hz rTMS can promote the recovery of motor function and the proliferation of SVZ endogenous NSCs in the affected cerebral cortex in rats with focal cerebral ischemia for 7 days, and this effect may be related to the up-regulation of the expression of microRNA-25 and the down-regulation of its target. Objective: To investigate the role of microRNAs-25/p57 in promoting the proliferation of endogenous NSCs in SVZ after focal cerebral ischemia by rTMS through inhibiting the expression of microRNAs-25 by intracerebroventricular injection of microRNAs-25 antagonist, and further confirm that the proliferation effect of rTMS on endogenous NSCs is closely related to microRNAs-25/p57. Methods: Adult male SD rats were randomly divided into rTMS group, rTMS + antagonist group and rTMS + control group with 15 rats in each group. All rats were treated with rTMS once a day for 7 days. The brain tissues of each group were taken from the affected side 7 days after operation. The relative expression of the microRNA106 B-25 family was detected by qRT-PCR to determine the appropriate antagonist dose. After the microRNA25 was specifically inhibited, part of the brain tissues were partially inhibited. Western blotting was used to detect the changes of target proteins in the brain tissues of rats (5 rats in each group) on the 7th day after operation. BrdU was injected intraperitoneally to label proliferative cells immediately after the last treatment in the rTMS group, once every 4 hours for three consecutive times. Results: 1. MiRNA antagonists inhibited the expression of related microRNAs and showed a dose-dependent effect. 2. qRT-PCR results showed that 2.5 nmol Ant-25 significantly inhibited the expression of microRNAs-25. Western blotting results showed that the expression of p57 in the antagonist group was significantly higher than that in the rTMS group (p = 0.018) and the control group (p = 0.012), but the expression of p21 was not significantly different among the groups (p 0.05). 4. NSCs proliferated in the affected side of SVZ were labeled with BrdU + / Nestin +. The results showed that there was no significant difference in NSCs proliferation between rTMS group and control group (p0.05) after inhibition of microwave irradiation. The number of NSCs proliferation in antagonist group was significantly lower than that in rTMS group (p = 0.006) and control group (p = 0.013). Conclusion: The expression of target protein p57 was increased after inhibition of specificity and validity of MiR-25, but the endogenous SVZ in cerebral ischemic side of rats was significantly lower than that in rTMS group (p = 0.006) and control group (p = 0.01 Objective: To study the effects of rTMS on learning and memory function in rats with focal cerebral ischemia, and to observe the regeneration and apoptosis of hippocampal neurons. METHODS: Adult male SD rats were randomly divided into 7-day group and 14-day group. The rats were divided into sham-operation group, model group and rTMS subgroup. Diffusion-weighted imaging (DWI) was performed on the first day, the seventh day and the fourteenth day after operation, and the apparent diffusion coefficient (ADC) was obtained on the basis of DWI. After the last treatment, BrdU was injected intraperitoneally to label the proliferative cells, once every four hours, three times in succession. On the seventh day after operation, the ischemic brain tissue was taken from the affected side in another group of rats (10 rats in each group). Morris water maze test was performed on the 12th day after operation, and the hippocampus was sacrificed on the 14th day after operation. Tunel staining (5 in each group) and Western blotting (5 in each group) were used to detect the volume of cerebral infarction in MCAO rats. Morris water maze test was used to observe the effect of rTMS on learning and memory in MCAO rats. The proliferation and differentiation of endogenous NSCs in SGZ were detected by immunohistochemical double labeling technique. The apoptosis of neurons in the affected hippocampus was observed by Tunel staining. The apoptosis-related protein B cells in the affected hippocampus were confirmed by Western blotting. The expression of B cell lymphoma/leukemia gene 2 (Bcl-2) and Bcl 2-associated protein X (Bax) was analyzed by one-way ANOVA. Bonferroni test was used to compare the two groups. Results: 1. The infarct volume of the model group and rTMS group was different at 1 day after operation. There was no significant difference in infarct volume between the model group and the rTMS group on the 7th day (p = 0.246) and the 14th day (p = 0.132), but the improvement of infarct volume in the rTMS group was better than that in the model group. The infarct volume of rats in MS group decreased significantly on the 7th and 14th day after operation (p0.001), but there was no significant difference between model group (p = 0.716) and rTMS group (p = 0.264) on the 14th day and the 7th day. The escape latency of rTMS group was significantly shorter than that of model group (p = 0.006), and the number of NSCs proliferating within 60 seconds was also significantly increased (p = 0.045). 3. The number of NSCs proliferating in SGZ of affected side was marked by BrdU + / Nestin +, and the results showed that the number of NSCs proliferating in model group was significantly higher than that in sham operation group 7 days after operation. The number of NSCs differentiated by BrdU+/NeuN+ labeling of SGZ on the affected side was significantly higher in rTMS group than that in sham operation group and model group (p = 0.042), and the number of NSCs differentiated by BrdU+/NeuN+ labeling was significantly higher in rTMS group than that in sham operation group (p = 0.006) and model group (p = 0.021). There was no significant difference in the number of NSCs differentiated between the model group and the sham group (p0.05). 5. Tunel staining showed that the number of neuronal apoptosis in the hippocampus of the model group was significantly higher than that of the sham group at 14 days after operation (p0.001). The results of Western blotting showed that the expression of Bcl-2 decreased (p0.01) and the expression of Bax increased (p0.01) in the affected hippocampus of the model group compared with the sham-operated group. The expression of Bcl-2 in the rTMS group was significantly higher than that in the model group (p0.005), but the expression of Bax was significantly lower than that in the model group (p0.005). However, lOHz rTMS can improve the recovery of learning and memory in rats with focal cerebral ischemia, and can effectively promote the proliferation and differentiation of endogenous NSCs in SGZ and inhibit the apoptosis of hippocampal neurons in rats with focal cerebral ischemia by regulating apoptosis-related proteins Bcl-2 and Bax.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R743.3
[Abstract]:AIM: To study the effects of repetitive transcranial magnetic stimulation on motor function and proliferation of NSCs in the subependymal zone of the lateral ventricle in rats with focal cerebral ischemia-reperfusion injury, and to explore the possible mechanism. Materials and Methods: Forty-five adult male SD rats were randomly divided into two groups. Fifteen rats in each group were divided into sham operation group, model group and rTMS group. The right middle cerebral artery occlusion (MCAO) model was used and the obstruction lasted for 80 minutes. Neurological severity scores (NSSs) were given on the 7th day. Some rats (5 rats in each group) were injected intraperitoneally with 5-bromo-2-deoxy Uridine (BrdU) to label proliferating cells, once every four hours, three times continuously after the last treatment in rTMS group. On the 7th day after operation, the ischemic brain tissue was taken from the affected side of the rats (10 rats in each group). The effects of rTMS on the motor function of MCAO rats were observed by NSS score. The proliferation of endogenous NSCs in the affected side of SVZ was detected by immunohistochemical fluorescence double labeling technique. Real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) was used. Verse transcription polymerase chain reaction (qRT-PCR) was used to detect the relative expression of microRNA-25 (microRNA-25) and Western blotting was used to detect the changes of proliferation-related target proteins of microRNA-25. There was no significant difference in NSS scores between the rTMS group and the control group on the 1st day after operation (p0.05). The NSS scores of the two groups were significantly improved on the 7th day after operation (p0.005), and the neurological function of the rTMS group was better than that of the model group (p = 0.019). 2. The number of NSCs proliferated by BrdU + / Nestin + labeling SVZ on the affected side was found. On the 7th day after operation, the proliferation of NSCs in the model group was significantly higher than that in the sham operation group (p = 0.044), and the proliferation of NSCs in the rTMS group was significantly higher than that in the model group and sham operation group (p 0.005). 3. The results of qRT-PCR showed that the expression of microwave-25 in the affected cerebral ischemic tissue of the model group was significantly higher than that in the sham operation group on the 7th day after operation. The expression of microRNA-25 in the affected cerebral ischemic tissues of the rTMS group was significantly higher than that of the other two groups (p0.001). 4. Western blotting results showed that there was no significant difference in the expression of microRNA-25 target protein p57 and PTEN between the sham-operated group and the model group at 7 days after operation (p0.05); however, the expression of p57 in the affected cerebral ischemic tissues of the rTMS group was higher than that of the other groups (p0.001). Conclusion: 10Hz rTMS can promote the recovery of motor function and the proliferation of SVZ endogenous NSCs in the affected cerebral cortex in rats with focal cerebral ischemia for 7 days, and this effect may be related to the up-regulation of the expression of microRNA-25 and the down-regulation of its target. Objective: To investigate the role of microRNAs-25/p57 in promoting the proliferation of endogenous NSCs in SVZ after focal cerebral ischemia by rTMS through inhibiting the expression of microRNAs-25 by intracerebroventricular injection of microRNAs-25 antagonist, and further confirm that the proliferation effect of rTMS on endogenous NSCs is closely related to microRNAs-25/p57. Methods: Adult male SD rats were randomly divided into rTMS group, rTMS + antagonist group and rTMS + control group with 15 rats in each group. All rats were treated with rTMS once a day for 7 days. The brain tissues of each group were taken from the affected side 7 days after operation. The relative expression of the microRNA106 B-25 family was detected by qRT-PCR to determine the appropriate antagonist dose. After the microRNA25 was specifically inhibited, part of the brain tissues were partially inhibited. Western blotting was used to detect the changes of target proteins in the brain tissues of rats (5 rats in each group) on the 7th day after operation. BrdU was injected intraperitoneally to label proliferative cells immediately after the last treatment in the rTMS group, once every 4 hours for three consecutive times. Results: 1. MiRNA antagonists inhibited the expression of related microRNAs and showed a dose-dependent effect. 2. qRT-PCR results showed that 2.5 nmol Ant-25 significantly inhibited the expression of microRNAs-25. Western blotting results showed that the expression of p57 in the antagonist group was significantly higher than that in the rTMS group (p = 0.018) and the control group (p = 0.012), but the expression of p21 was not significantly different among the groups (p 0.05). 4. NSCs proliferated in the affected side of SVZ were labeled with BrdU + / Nestin +. The results showed that there was no significant difference in NSCs proliferation between rTMS group and control group (p0.05) after inhibition of microwave irradiation. The number of NSCs proliferation in antagonist group was significantly lower than that in rTMS group (p = 0.006) and control group (p = 0.013). Conclusion: The expression of target protein p57 was increased after inhibition of specificity and validity of MiR-25, but the endogenous SVZ in cerebral ischemic side of rats was significantly lower than that in rTMS group (p = 0.006) and control group (p = 0.01 Objective: To study the effects of rTMS on learning and memory function in rats with focal cerebral ischemia, and to observe the regeneration and apoptosis of hippocampal neurons. METHODS: Adult male SD rats were randomly divided into 7-day group and 14-day group. The rats were divided into sham-operation group, model group and rTMS subgroup. Diffusion-weighted imaging (DWI) was performed on the first day, the seventh day and the fourteenth day after operation, and the apparent diffusion coefficient (ADC) was obtained on the basis of DWI. After the last treatment, BrdU was injected intraperitoneally to label the proliferative cells, once every four hours, three times in succession. On the seventh day after operation, the ischemic brain tissue was taken from the affected side in another group of rats (10 rats in each group). Morris water maze test was performed on the 12th day after operation, and the hippocampus was sacrificed on the 14th day after operation. Tunel staining (5 in each group) and Western blotting (5 in each group) were used to detect the volume of cerebral infarction in MCAO rats. Morris water maze test was used to observe the effect of rTMS on learning and memory in MCAO rats. The proliferation and differentiation of endogenous NSCs in SGZ were detected by immunohistochemical double labeling technique. The apoptosis of neurons in the affected hippocampus was observed by Tunel staining. The apoptosis-related protein B cells in the affected hippocampus were confirmed by Western blotting. The expression of B cell lymphoma/leukemia gene 2 (Bcl-2) and Bcl 2-associated protein X (Bax) was analyzed by one-way ANOVA. Bonferroni test was used to compare the two groups. Results: 1. The infarct volume of the model group and rTMS group was different at 1 day after operation. There was no significant difference in infarct volume between the model group and the rTMS group on the 7th day (p = 0.246) and the 14th day (p = 0.132), but the improvement of infarct volume in the rTMS group was better than that in the model group. The infarct volume of rats in MS group decreased significantly on the 7th and 14th day after operation (p0.001), but there was no significant difference between model group (p = 0.716) and rTMS group (p = 0.264) on the 14th day and the 7th day. The escape latency of rTMS group was significantly shorter than that of model group (p = 0.006), and the number of NSCs proliferating within 60 seconds was also significantly increased (p = 0.045). 3. The number of NSCs proliferating in SGZ of affected side was marked by BrdU + / Nestin +, and the results showed that the number of NSCs proliferating in model group was significantly higher than that in sham operation group 7 days after operation. The number of NSCs differentiated by BrdU+/NeuN+ labeling of SGZ on the affected side was significantly higher in rTMS group than that in sham operation group and model group (p = 0.042), and the number of NSCs differentiated by BrdU+/NeuN+ labeling was significantly higher in rTMS group than that in sham operation group (p = 0.006) and model group (p = 0.021). There was no significant difference in the number of NSCs differentiated between the model group and the sham group (p0.05). 5. Tunel staining showed that the number of neuronal apoptosis in the hippocampus of the model group was significantly higher than that of the sham group at 14 days after operation (p0.001). The results of Western blotting showed that the expression of Bcl-2 decreased (p0.01) and the expression of Bax increased (p0.01) in the affected hippocampus of the model group compared with the sham-operated group. The expression of Bcl-2 in the rTMS group was significantly higher than that in the model group (p0.005), but the expression of Bax was significantly lower than that in the model group (p0.005). However, lOHz rTMS can improve the recovery of learning and memory in rats with focal cerebral ischemia, and can effectively promote the proliferation and differentiation of endogenous NSCs in SGZ and inhibit the apoptosis of hippocampal neurons in rats with focal cerebral ischemia by regulating apoptosis-related proteins Bcl-2 and Bax.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R743.3
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