【作者】 周赛男；

【导师】 蔡光先；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2012， 博士

【摘要】 目的：在建立局灶性脑缺血大鼠模型基础上,通过动物实验探讨补阳还五汤对脑缺血后大鼠神经保护与促神经细胞增殖作用及对TLR2、TLR4、MyD88、NF-κBp65表达的影响,为补阳还五汤治疗脑缺血性中风临床用药提供实验依据。方法：采用大脑中动脉线栓法建立左侧局灶性脑缺血大鼠模型,将198只动物随机分为假手术组、模型组、补阳还五汤组三大组,每组66只,各大组内分三个时间点即于7d、14d和21d处死动物,每组动物每个时间点取5只动物腹腔注射Brdu,取10只神经功能评分,取5只做TTC计算其梗死面积,取6只HE染色以观察各组动物海马、皮质组织细胞形态,取6只进行WB、RT-PCR指标检测。采用免疫组织化学法观察BrdU标记干细胞/祖细胞增生情况,采用免疫组织化学法、WB、RT-PCR法检测补阳还五汤对大鼠脑缺血后TLR2、TLR4、NF-κBp65.MyD88的影响。结果：(1)与模型组比较补阳还五汤可减轻大鼠局灶性脑缺血后神经功能缺损(P<0.05),减小脑梗死面积(P<0.05),改善缺血区及周围组织细胞形态。(2)补阳还五汤组与相同时间相模型组相比BrdU阳性细胞数显著增多(P<0.05)。(3)模型组各时间点相比,随大鼠局灶性脑缺血后时间延长神经功能缺损、脑梗死面积有减小趋势,缺血区及周围组织细胞形态有所改善。(4)与相同时间相假手术组相比模型组BrdU阳性细胞数增多(P<0.05);大鼠缺血脑后TLR2、TLR4、MyD88、NF-κBp65表达水平升高(P<0.05)。(5)与相同时间相模型组相比较补阳还五汤可下调大鼠缺血脑组织TLR4、MyD88、NF-κBp65表达(P<0.05),上调TLR2表达(P<0.05)。结论：(1)补阳还五汤对大鼠脑缺血病理损伤具有神经保护作用,并促进缺后神经干细胞增殖,后者可能在其促进缺血后神经功能恢复中发挥作用。(2)大鼠脑缺血损伤后存在自我修复机制,可激活神经干细胞增殖,部分代偿神经功能缺损。(3)TLR2可能是大鼠脑缺血后的保护因素。补阳还五汤可增强大鼠脑缺血后TLR2表达,这可能是其实现脑保护与促神经再生机制之(4)TLR4/MyD88信号通路可能是大鼠脑缺血后损伤机制。补阳还五汤可抑制大鼠脑缺血后TLR4、MyD88、NF-κBp65表达,可能通过抑制TLR4／MyD88信号传导通路而减轻脑缺血后损伤及促神经再生。更多还原

【Abstract】 Objectives:To explore the effects of Buyang Huanwu Decoction on proliferation of nerve cell and expressions of TLR2、TLR4、NF-κB、 MyD88in rats with cerebral ischemia.Methods:Rat focal cerebral isehemia was induced by MCAO. Rats were randomly divided into following group including sham control, cerebral ischemia,cerebral ischemia with BYHW decoction.The group was divided into subgroups,animals in each subgroup were sacrificed in7,14or21day after operation. Neurological score of animals, the TTC calculating of their infarct area, to observe each group of animals the cell morphology in the hippocampus and cortex with HE staining, to observe BrdU labeling stem cells/progenitor cell proliferation using immunohistochemical staining and detecting the expression level of TLR2and TLR4.NF-κB and MyD88after cerebral ischemia using immunohistochemistry、the WB and RT-PCR were measured. Result:1) Compared with cerebral ischemia group BYHW decoction treatment can reduce the neurological deficit after focal cerebral ischemia, reduce the infarct size, improve the cellular morphology of the ischemic area and the surrounding tissue.2)Compared with cerebral ischemia group cerebral ischemia with BYHW decoction the BrdU positive cells increased significantly.3)Compared with the cerebral ischemia group at all time points each other neurological deficit and infarct area after focal cerebral ischemia had a trend of decreasing over time, morphology of ischemic area and the surrounding tissue are improved.4)Compared with the sham control, the number of BrdU positive cells was enhanced after ischemia in cerebral ischemia group.5)Compared with the sham group the expression of TLR2、TLR4、NF-κB and MvD88was increased after ischemia.6)Compared with the cerebral ischemia group, BYHW decoction treatment can lower the expression of TLR4, NF-κB and MyD88in ischemic brain tissue.7)Compared with the cerebral ischemia group BYHW decoction treatment can increase the expression of TLR2in ischemic brain tissue.Conclusion:1) Rats afer cerebral ischemia has self-repair mechanisms and can activate neural stem cell proliferation,that can compensate neurological deficit to a certain extent.2) BYHW decoction treatment has neuroprotective effects, and promotes proliferation of the neural stem cell after ischemia, which may play a role in its promotion of ischemic recovery of neurological function.3)TLR4/MyD88signaling pathway may be the mechanism of injury after cerebral ischemia.4)TLR2may be a protective factor after focal cerebral ischemia.5) BYHW decoction treatment can enhance the TLR2expression after cerebral ischemia, which may be one of mechanism of its neuroprotection and promoting nerve regeneration.6) BYHW decoction treatment can inhibit the expression of TLR4MyD88, NF-kappa B after cerebral ischemia,which may be a mechanism of its neuroprotective effects and promoting nerve regeneration through mitigating TLR4/of MyD88signaling pathway.更多还原