参知健脑方对拟血管性痴呆大鼠的治疗作用及其对胆碱能系统的影响

【作者】 林海；

【导师】 孙塑伦；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2005， 博士

【摘要】 血管性痴呆(VD)在我国由于脑血管病的多发而成为高发性疾病,是老年期痴呆的主要类型之一。现代医学在血管性痴呆病理及病理生理研究方面已取得了很大进展,并且从多角度、多层次对其进行了研究,而随着近年来中医药基础和临床研究的深入,中医药在本病的防治上日益显示出优势。 论文的综述部分从中医角度对 VD 病因病机、辨证分型及治疗等方面进行探讨,结合导师的临床经验,提出“正虚毒侵”为 VD 的病因病机;“扶正解毒”为其治则治法;参知健脑方的提出既符合痴呆现代药理学的研究,又是针对“正虚毒侵”的病因病机提出的“扶正解毒“治则治法的体现。从现代医学的角度,对 VD 发病的危险因素、病理变化、诊断治疗及胆碱能调控系统等方面进行研究,推论出胆碱能系统对 VD 的发生发展起重要作用;可以通过有效地消除危险因素,控制和改善心脑血管障碍,达到治疗和预防 VD 的目的。 实验研究分为以下四部分: 实验一、行为学实验 ①通过反复夹闭、再通双侧颈总动脉同时腹腔注射硝普钠制造大鼠拟血管性痴呆模型,通过 Morris 水迷宫检验大鼠的学习、记忆功能,并反证模型的成功建立。②使用参知健脑方大、中、小剂量对拟血管性痴呆大鼠进行治疗,通过 Morris 水迷宫来探讨参知健脑方对拟血管性痴呆模型大鼠的作用机理,并确定最佳剂量。 实验二、病理学实验 ①通过制作假手术组、模型组、参知健脑方大、中、小剂量组和阳性药组大鼠脑组织的病理切片并使用 HE 染色来观察拟血管性痴呆造模后大鼠海马 CA1 区整体形态和细胞的变化,使用阳性药和参知健脑方大、中、小剂量治疗后大鼠海马 CA1 区的整体形态和细胞是否有所好转。②计数 HE 染色病理切片中 100μm2内各组大鼠脑组织切片海马 CA1区的细胞数,并进行比较。③通过制作假手术组、模型组、参知健脑方大、中、小剂量组和阳性药组大鼠脑组织的病理切片并使用尼氏体甲苯胺蓝染色来观察拟血管性痴呆造模后大鼠海马 CA1 区整体形态及细胞的变化,使用阳性药和参知健脑方大、中、小剂量治疗后大鼠海马 CA1 区整体形态及细胞是否有所好转。④计数尼氏体甲苯胺蓝染色病理切片中 100μm2内各组大鼠脑组织切片海马 CA1 区的细胞数,并进行比较。 实验三、胆碱能系统部分 ①通过病理学方法制作假手术组、模型组、参知健脑方中剂量组大鼠脑组织的病理切片,并使用免疫组化的方法观察胆碱乙酰转移酶(ChAT)在各组大鼠海马 CA1 区的表达情况。②通过病理学方法制作假手术组、模型组、参知健脑方中剂量组大鼠脑组织的病理切片并使用免疫组化的方法观察乙酰胆碱酯酶(AchE)在各组大鼠海马 CA1 区的表达情况。③通过高效液相电化学工作站检测假手术组、模型组、中剂量参知健脑方治疗组大鼠海马内乙酰胆碱含量的变化。④通过胆碱能系统变化进一步探讨血管性痴呆发生的病理机制及参知健脑方对其的治疗作用。进而进一步论治“正虚”的致病机理及“扶正”的治则治法。 2 参知健脑方对拟血管性痴呆大鼠的治疗作用及其对胆碱能系统的影响实验四、Bax 凋亡相关蛋白的测定通过病理学方法制作假手术组、模型组、参知健脑方中剂量组大鼠脑组织的病理切片并使用免疫组化的方法观察凋亡相关蛋白 Bax 在各组大鼠海马 CA1 区的表达情况并且探测其光密度情况和阳性细胞数。进而进一步论治“毒侵”的致病机理及“解毒”的治则治法。结果:拟血管性痴呆模型组大鼠与假手术组大鼠相比,模型组大鼠较学习、记忆能力有显著的降低,海马 CA1 区中段 100μm2内的的细胞数明显减少,海马 CA1 区的 ChAT和 AchE 表达明显的减少,Bax 表达明显增多,海马内 Ach 含量明显减少。阳性药和小剂量组大鼠与模型组比较没有十分明显的改观,大剂量组和中剂量组大鼠有明显恢复,尤其中剂量组大鼠恢复的最佳。结论:拟血管性痴呆模型大鼠的海马和胆碱能调控系统均受到了严重损害,这正符合我们认为血管性痴呆“正虚”的病因病机;同时,模型大鼠海马 CA1 区中段 Bax 的表达明显增加,而 Bax 能够促进凋亡,从另一角度说明血管性痴呆“毒侵”的病因病机。采用参知健脑方可以明显改善大鼠的学习、记忆行为,减轻海马 CA1 区神经细胞所受的损伤,上调模型大鼠海马 CA1 区 ChAT 和 AchE 的表达,下调模型大鼠海马 CA1 区 Bax的蛋白表达,增加拟血管性痴呆大鼠海马内 Ach 含量。说明参知健脑方通过改善拟血管性痴呆模型大鼠的胆碱能调控系统以及降低海马 CA1 区凋亡相关蛋白 Bax 的表达,达到“扶正解毒”的治疗目的。关键词:血管性痴呆、正虚、毒侵、扶正、解毒、参知健脑方更多还原

【Abstract】 Vascular dementia(VD) has become a fredquently encountered disease and one of the main styles of the aged dementias because of the high incidence of cerebrovascular disease.The modern medicine has maken great development in the pathology and pathology-biology research on VD,many researchs in different angles and different levels have been made on VD.Recently, as more and more basic and clinic researchs on Traditional Chinese Medicine(TCM) treating VD have been made,more and more privileges have appeared. In summarise of this thesis I discussed the pathogeny、types and the treatments of VD in TCM’s angle of view.Combining the clinical experience of my tutor ,I put forward that “ positive Qi become weak and toxic Qi invade ”is the pathogeny of VD,“increase the positive Qi and decrease the toxic Qi”is the rule and method of the treatment to VD,“ShenZhiJianNaoFang”is the appearance of the rule and method of “increase the positive Qi and decrease the toxic Qi”,suit to the modern pharmacology research on VD.From the angle of modern medicine ,I researched the dangerous acts 、 pathology changes 、 diagnose and treatment of VD and the acethylcholine systeme’s control action ,make the conclusion that acethylcholine systeme make a great role to the happenness and development of VD,we can reach the aim of treat and prevent VD by decrease the dangerous acts、control and improve the vascular occlusion etc. The experiment part is departed into four parts: Part one :Behavior part.①Make the vascular dementia model by repeated close and open the rat’s common carotid arterys in both sides and intraperitoneal inject sodium nitroprusside(SNP), using Morris water-mazed palace to test the rat’s ability of study and memory and prove the succession of making the model.②Treat the model rats with large、medium、small dose of ShenZhiJianNaoFang and use Morris water-mazed palace to test the effection of ShenZhiJianNaoFang and make sure of the best dose. Part two:Pathology part. ①Divid the rats into controlled group、model group、positive drug group、ShenZhiJianNaoFang large、medium、small dose group. Make all the rats’pathology slices and dye in HE dying methods.Observe the model’s change in CA1 district in hippocampus and the treatment’s effection of the medicines.②Take count of the cells in CA1 district in hippocampus of all the HE dyed slices and compare it.③Make all the rats’pathology slices and dye in Nissle’s dying methods.Observe the model’s change in CA1 district in hippocampus and the treatment’s effection of the medicines.④Take count of the cells in CA1 district in hippocampus of all the Nissle’s dyed slices and compare Part three:Part of acethylcholine (ACh)system.①Make pathology slices of the rats in the controlled group、model group and ShenZhiJianNaoFang medium dose group.Observe the appearance of cholinacetyltranslase(ChAT) in CA1 district in hippocampus in immuo-histochemical method.②Make pathology slices of the rats in the controlled group 、 model group and ShenZhiJianNaoFang medium dose group.Observe the appearance of acethylcholine esterase(AChE) in CA1 district in hippocampus in immuo-histochemical method. ③ Observe the appearance of acethylcholine (ACh) in hippocampus of controlled group 、 model group 、ShenZhiJianNaoFang medium dose group’s rats by high performance liquid chromatograph-electrochemical detector.④Detect the pathology changes of VD and ShenZhiJianNaoFang’s treating effection through acethylcholine system’change. Discuss the principle of“positive Qi become weak”in VD’s occur and the principle of “increase the positive Qi”in VD’s treatment. Part four:Part of Bax, the protein associated with apoptosis. Make pathology slices of the rats in the controlled group、model group and ShenZhiJianNaoFang medium dose group.Observe the appearance of Bcl-2 associated X protein(Bax) in CA1 district in hippocampus in immuo-histochemical method.Discuss the principle of“toxic Qi invade”in VD’s occur and the principle of “decrease the toxic Qi”in VD’s treatment. Result:C更多还原