【摘要】：目的 本實驗通過建立大鼠大腦中動脈腦缺血再灌注模型，并運用Cathepsin D抑制劑pepstatin A對其進行干預，檢測Cathepsin D和pMLK3的蛋白表達變化，探討Cathepsin D與MLK3在大鼠腦缺血再灌注損傷過程中神經細胞凋亡的關系，為研究溶酶體參與腦缺血再灌注損傷神經細胞凋亡提供新的探索，為缺血性腦血管病提供新的治療靶點。 方法 67只清潔級健康雄性Sprague-Dawley大鼠，體重280±20g，隨機分為3組：假手術組（sham，n=7），模型組（M，n=30），干預組（I，n=30）：于再灌注前及再灌注后每12h腹腔注射溶酶體Cathepsin D抑制劑pepstatin A2ml/100g（而假手術組及模型組于相應時間腹腔注射生理鹽水2ml/100g）。其中模型組和干預組又分為2小時組（M-2h，n=6；I-2h，n=6），6小時組（M-6h，n=6；I-6h，n=6），24小時組（M-24h，n=6；I-24h，n=6），48小時組（M-48h，n=12；I-48h，n=12）。應用改良的Long線栓法制作大鼠大腦中動脈腦缺血再灌注模型，缺血2小時后恢復再灌注，使用Longa’s的5級評分法評價神經功能；使用TTC染色法、TUNEL凋亡檢測法及免疫組織化學方法，分別檢測腦梗死體積、細胞凋亡數量、CathepsinD與pMLK3的表達變化。 結果 1.模型成功率76.14%；模型組48小時相對腦梗死體積為37.36±1.44%，干預組48小時相對腦梗死體積為27.49±2.30%。 2.假手術組腦組織未見梗死灶，，模型組和干預組缺血側皮質及皮質下可見白色梗死灶，但干預組48小時相對梗死體積較模型組減少（p=0.000），且干預組神經功能缺損評分較模型組也得到明顯改善（p0.05）。 3.大鼠腦缺血側皮質區(qū)，假手術組凋亡細胞很少，模型組2小時可以觀察到凋亡細胞，6小時、24小時、48小時呈上升趨勢（p0.001）；干預組2小時凋亡細胞較模型組2小時無顯著改變（p=0.127），干預組6小時、24小時、48小時觀察到的凋亡細胞較模型組明顯減少（p0.05）。 4.大鼠腦缺血側皮質區(qū)，假手術組可見少量Cathepsin D的表達，模型組Cathepsin D的表達在再灌注2小時、6小時、24小時呈上升趨勢，24小時達高峰，48h組較24h組有所下降，但仍居高水平，均比假手術組高（p=0.000）；干預組各時間點Cathepsin D的表達較模型組明顯減少（p=0.000）。 5.大鼠腦缺血側皮質區(qū)，假手術組pMLK3少量表達，模型組pMLK3的表達在再灌注2小時即有大量表達，于6小時達高峰，之后逐漸下降，均高于假手術組（p=0.000）；干預組各時間點pMLK3的表達均較模型組明顯減少（p0.05）。 結論 1.大鼠腦缺血再灌注損傷過程中溶酶體Cathepsin D可能通過上調MLK3的磷酸化而介導神經細胞凋亡。 2.Pepstatin A可能通過抑制Cathepsin D而下調MLK3的磷酸化活性表達，從而減少細胞凋亡，減少腦梗死體積，起到神經保護作用。
[Abstract]:Objective to investigate the changes of Cathepsin D and pMLK3 protein expression in middle cerebral artery (MCAA) ischemia-reperfusion model of rats by using pepstatin A, a Cathepsin D inhibitor, to establish a rat model of cerebral ischemia-reperfusion. To investigate the relationship between Cathepsin D and MLK3 in the process of cerebral ischemia-reperfusion injury in rats, and to explore the role of lysosome in neuronal apoptosis during cerebral ischemia-reperfusion injury, and to provide a new therapeutic target for ischemic cerebrovascular disease. Methods Sixty-seven clean grade healthy male Sprague-Dawley rats, weighing 280 鹵20 g, were randomly divided into three groups: sham operation group (sham,n=7), model group (Mannan 30), intervention group (I), and control group (I). Nongma 30: lysosomal Cathepsin D inhibitor pepstatin A2ml/100g was injected intraperitoneally every 12 hours before and after reperfusion (while normal saline 2ml/100g was injected intraperitoneally in sham operation group and model group at the corresponding time). Among them, the model group and the intervention group were divided into two hours group (M-2hnntir 6L), 6-hour group (M-6hnntir 6hnc1hnc6), 24-hour group (M-24hntir 6I-24hncnc6), and 48-hour group (M-48hntrol 12), the model group and the intervention group were divided into three groups: the model group and the intervention group, respectively. I-48h ~ (12). The rat model of middle cerebral artery ischemia reperfusion was established by modified Long thread occlusion method, and the reperfusion was resumed 2 hours after ischemia. The nerve function was evaluated by Longa's 5 grade scoring method. TTC staining, TUNEL apoptosis detection and immunohistochemistry were used to detect the volume of cerebral infarction, the number of apoptosis and the expression of CathepsinD and pMLK3. Result 1. The relative volume of cerebral infarction was 37.36 鹵1.44 in the model group and 27.49 鹵2.30 in the intervention group at 48 hours. 2. No infarct was found in the cerebral tissue of sham-operation group, and white infarct was found in ischemic cortex and subcortical of model group and intervention group, but the relative infarct volume of 48 hours in intervention group was lower than that in model group (p0. 000). The neurological deficit score in the intervention group was significantly improved than that in the model group (p0.05). 3. There were few apoptotic cells in the cerebral ischemic cortex of rats in the sham-operation group. The apoptotic cells were observed in the model group at 2 hours, and increased at 6 hours, 24 hours and 48 hours (p0.001). There was no significant change in apoptotic cells in the intervention group at 2 hours compared with that in the model group (p0. 127). The apoptotic cells in the intervention group at 6 hours, 24 hours and 48 hours were significantly lower than those in the model group (p0.05). 4. A small amount of Cathepsin D expression was observed in the sham-operated group. The expression of Cathepsin D in the model group increased at 2 hours, 6 hours and 24 hours after reperfusion, and reached its peak at 24 hours. The expression of Cathepsin D in the 48 h group was lower than that in the 24 h group, but it was still at a high level. All of them were higher than those of sham operation group (p0. 000). The expression of Cathepsin D in the intervention group was significantly lower than that in the model group (P < 0. 000). 5. There was a little expression of pMLK3 in the cerebral ischemic cortex and sham-operation group. The expression of pMLK3 in the model group reached its peak at 6 hours after reperfusion and was higher than that in the sham-operation group (p0. 000). The expression of pMLK3 in the model group was higher than that in the sham operation group (P < 0. 000). The expression of pMLK3 in the intervention group was significantly lower than that in the model group at each time point (p0. 05). Conclusion 1. Lysosomal Cathepsin D may induce neuronal apoptosis by up-regulating the phosphorylation of MLK3 during cerebral ischemia-reperfusion injury in rats. 2.Pepstatin A may down-regulate the expression of phosphorylation of MLK3 by inhibiting Cathepsin D, thus reducing apoptosis and cerebral infarct volume, thus playing a neuroprotective role.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R743.3

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