利用线虫模型研究紫娟普洱茶抗阿尔兹海默病的作用机制
发布时间：2018-09-04 18:41
【摘要】：阿尔兹海默病是一种神经系统退行性疾病,其主要的病理特征是脑中老年斑的沉积和神经纤维的缠结,但具体的发病机制至今尚不明确。随着人口老龄化进程的加快,患有阿尔兹海默病的人数也在逐年增多,因此急需开发出一种药物或治疗方法来减少阿尔兹海默病的发病几率,延缓阿尔兹海默病的发生和发展。目前治疗阿尔兹海默病的药物主要是胆碱酯酶抑制剂,这类药物只能缓解病人的症状,不能达到治疗根本的目的,因此科研工作者正致力于开发能够治疗阿尔兹海默病的新药物。已有大量的研究表明,具有抗氧化效果的天然产物可能具有治疗阿尔兹海默病的作用,天然产物作为源于自然受益自身的药物,具有良好的应用前景,因此本论文对天然产物进行了抗阿尔兹海默病的药物筛选。虽然阿尔兹海默病的发病机制尚不明确,但有几种假说受到大众的认可,尤其是Aβ级联假说,因此我们所选用的药物筛选模型为秀丽隐杆线虫(Caenorhabditis elegans,C.elegans)的Aβ模型CL4176转基因线虫株,针对Aβ假说筛选抗阿尔兹海默病的药物。经过一系列的筛选我们发现,多种天然产物都具有较好的效果,我们选择紫娟普洱茶粉作为主要的研究对象。实验设计了三个给药浓度,分别为0.1mg/ml、0.2mg/ml和0.4mg/ml,对于CL4176转基因线虫,三个浓度都具有延缓线虫瘫痪的作用,并且呈现浓度依赖,因此选择0.4mg/ml的紫娟普洱茶粉进行机制的研究,对EGCG和原花青素的效果进行验证发现0.4mg/ml的紫娟普洱茶粉中发挥主要作用的不是上述两种成分。首先对紫娟普洱茶粉发挥作用的水平进行检测,发现0.4mg/ml的紫娟普洱茶粉不影响CL4176线虫体内Aβm RNA的水平,但是能够抑制Aβ的沉积,说明药物是在转录后水平发挥作用的。其次对CL4176线虫进行给药预处理,发现给药组仍然具有延缓线虫瘫痪的作用,因此对线虫体内同抗逆性相关的一些转录因子进行qPCR检测,我们发现sir2.1有明显的上调。再次利用TJ356线虫追踪了0.4mg/ml的紫娟普洱茶粉对DAF-16入核的影响,结果发现给药后能够促进DAF-16的核转位,利用SOD-3::GFP共表达的线虫来进一步确认DAF-16的转录活性对线虫抵抗氧化应激的必要性,根据Aβ会引起线虫体内氧化应激增强,及给药与否CL4176线虫体内ROS的含量及SOD活性的变化,我们推测这可能是给药以后提高了线虫抵抗氧化应激的能力。最后对药物的毒性进行检测,发现紫娟普洱茶在安全的剂量下能够发挥延缓线虫瘫痪的作用。综上所述,本论文通过秀丽隐杆线虫筛选了抗阿尔兹海默病的药物,紫娟普洱茶粉能够抑制Aβ毒性,显著延缓线虫的瘫痪,并且这种抑制作用可能是与提高线虫抵抗Aβ引起的氧化应激的能力和抑制Aβ的沉积有关。
[Abstract]:Alzheimer's disease is a kind of neurodegenerative disease. The main pathological features of Alzheimer's disease are the deposition of senile plaques and the tangles of nerve fibers in the brain. However, the mechanism of Alzheimer's disease is still unclear. With the acceleration of population aging, the number of people suffering from Alzheimer's disease is also increasing year by year. Therefore, it is urgent to develop a drug or treatment to reduce the incidence of Alzheimer's disease and delay the occurrence and development of Alzheimer's disease. The current treatment of Alzheimer's disease is mainly cholinesterase inhibitors, such drugs can only alleviate the symptoms of patients, can not achieve the basic purpose of treatment. So researchers are working on new drugs that can treat Alzheimer's disease. A large number of studies have shown that natural products with antioxidant effects may have the effect of treating Alzheimer's disease, and natural products, as drugs derived from natural benefits themselves, have a good prospect of application. Therefore, natural products were screened for anti-Alzheimer's disease in this paper. Although the pathogenesis of Alzheimer's disease is still unclear, several hypotheses, especially the A 尾 cascade hypothesis, have been accepted by the public. Therefore, the drug screening model we selected is A 尾 CL4176 transgenic nematode strain of Caenorhabditis elegans,C.elegans. Screening of drugs against Alzheimer's disease based on A 尾 hypothesis. After a series of screening, we found that a variety of natural products have a better effect, we choose Zijuan Puer tea powder as the main research object. Three dosages were designed, 0.1 mg / ml 0.2 mg / ml and 0.4 mg / ml respectively. For CL4176 transgenic nematodes, the three concentrations could delay the paralysis of nematodes and present a concentration-dependent manner. Therefore, the mechanism of 0.4mg/ml powder was studied. The effects of EGCG and proanthocyanidins were verified. It was found that the main components of 0.4mg/ml were not the two components mentioned above. At first, the effect level of Puer tea powder from 0.4mg/ml was detected, and it was found that 0.4mg/ml did not affect the level of A 尾 m RNA in CL4176 nematode, but could inhibit the deposition of A 尾, indicating that the drug acted on the posttranscriptional level. Secondly, the pretreatment of CL4176 nematodes showed that the treatment group still had the effect of delaying the paralysis of nematodes. Therefore, some transcription factors related to stress resistance in nematodes were detected by qPCR. We found that sir2.1 was upregulated obviously. TJ356 nematode was used to track the effect of 0.4mg/ml 's Pu'er tea powder on the nucleation of DAF-16. The results showed that the nucleation of DAF-16 was promoted after administration. SOD-3::GFP co-expressed nematodes were used to further confirm the necessity of DAF-16 transcriptional activity for nematode resistance to oxidative stress. According to the effect of A 尾 on the increase of oxidative stress in nematode, the content of ROS and the change of SOD activity in CL4176 nematodes were determined. We speculate that this may increase the ability of nematodes to resist oxidative stress after administration. Finally, the toxicity of Pu'er tea was detected, and it was found that Zijuan Pu'er tea could delay the paralysis of nematodes in a safe dose. To sum up, the drug against Alzheimer's disease was screened by Rhizoma elegans. The tea powder of Zjuanjuan Pu'er can inhibit A 尾 toxicity and delay the paralysis of nematodes. The inhibition may be related to the ability of nematode to resist the oxidative stress induced by A 尾 and to inhibit the deposition of A 尾.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5;R-332
[Abstract]:Alzheimer's disease is a kind of neurodegenerative disease. The main pathological features of Alzheimer's disease are the deposition of senile plaques and the tangles of nerve fibers in the brain. However, the mechanism of Alzheimer's disease is still unclear. With the acceleration of population aging, the number of people suffering from Alzheimer's disease is also increasing year by year. Therefore, it is urgent to develop a drug or treatment to reduce the incidence of Alzheimer's disease and delay the occurrence and development of Alzheimer's disease. The current treatment of Alzheimer's disease is mainly cholinesterase inhibitors, such drugs can only alleviate the symptoms of patients, can not achieve the basic purpose of treatment. So researchers are working on new drugs that can treat Alzheimer's disease. A large number of studies have shown that natural products with antioxidant effects may have the effect of treating Alzheimer's disease, and natural products, as drugs derived from natural benefits themselves, have a good prospect of application. Therefore, natural products were screened for anti-Alzheimer's disease in this paper. Although the pathogenesis of Alzheimer's disease is still unclear, several hypotheses, especially the A 尾 cascade hypothesis, have been accepted by the public. Therefore, the drug screening model we selected is A 尾 CL4176 transgenic nematode strain of Caenorhabditis elegans,C.elegans. Screening of drugs against Alzheimer's disease based on A 尾 hypothesis. After a series of screening, we found that a variety of natural products have a better effect, we choose Zijuan Puer tea powder as the main research object. Three dosages were designed, 0.1 mg / ml 0.2 mg / ml and 0.4 mg / ml respectively. For CL4176 transgenic nematodes, the three concentrations could delay the paralysis of nematodes and present a concentration-dependent manner. Therefore, the mechanism of 0.4mg/ml powder was studied. The effects of EGCG and proanthocyanidins were verified. It was found that the main components of 0.4mg/ml were not the two components mentioned above. At first, the effect level of Puer tea powder from 0.4mg/ml was detected, and it was found that 0.4mg/ml did not affect the level of A 尾 m RNA in CL4176 nematode, but could inhibit the deposition of A 尾, indicating that the drug acted on the posttranscriptional level. Secondly, the pretreatment of CL4176 nematodes showed that the treatment group still had the effect of delaying the paralysis of nematodes. Therefore, some transcription factors related to stress resistance in nematodes were detected by qPCR. We found that sir2.1 was upregulated obviously. TJ356 nematode was used to track the effect of 0.4mg/ml 's Pu'er tea powder on the nucleation of DAF-16. The results showed that the nucleation of DAF-16 was promoted after administration. SOD-3::GFP co-expressed nematodes were used to further confirm the necessity of DAF-16 transcriptional activity for nematode resistance to oxidative stress. According to the effect of A 尾 on the increase of oxidative stress in nematode, the content of ROS and the change of SOD activity in CL4176 nematodes were determined. We speculate that this may increase the ability of nematodes to resist oxidative stress after administration. Finally, the toxicity of Pu'er tea was detected, and it was found that Zijuan Pu'er tea could delay the paralysis of nematodes in a safe dose. To sum up, the drug against Alzheimer's disease was screened by Rhizoma elegans. The tea powder of Zjuanjuan Pu'er can inhibit A 尾 toxicity and delay the paralysis of nematodes. The inhibition may be related to the ability of nematode to resist the oxidative stress induced by A 尾 and to inhibit the deposition of A 尾.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5;R-332
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