阅读说明：本技术 一种溶酶体作为制备治疗阿尔兹海默症及延缓老年智力衰退药物领域的应用 (Application of lysosome in preparation of medicine for treating Alzheimer's disease and delaying senile mental decline ) 是由 薛雪 李天琪 齐一琳 于 2020-04-14 设计创作，主要内容包括：本发明属细胞修复领域,具体涉及一种溶酶体作为制备治疗阿尔兹海默症及延缓老年智力衰退药物领域的应用。其有益效果在于：提供了一种新的治疗治疗蛋白错误折叠或加工类疾病的途径；原料获取容易,成本低；具有靶向性,且不存在细胞毒性；利用的细胞器不含有遗传物质,不会对受体产生不良影响。(The invention belongs to the field of cell repair, and particularly relates to application of lysosomes in the field of preparation of medicaments for treating Alzheimer's disease and delaying senile mental deterioration. The beneficial effects are that: provides a new approach for treating protein misfolding or processing diseases; the raw materials are easy to obtain, and the cost is low; targeting property is realized, and cytotoxicity does not exist; the utilized organelles do not contain genetic materials and do not have adverse effects on receptors.)

1. An application of lysosome in the field of preparing a medicament for treating Alzheimer's disease and delaying senile mental deterioration.

2. Use of the lysosome according to claim 1 for the preparation of a medicament for treating alzheimer's disease and delaying the decline of the aged intelligence, characterized in that the lysosome is extracted from BV2 cells.

3. Use of the lysosome according to claim 1 for the preparation of a medicament for treating alzheimer's disease and delaying the decline of the aged intelligence, characterized in that the concentration of the lysosome is not higher than 500 μ g/mL.

4. The use of the lysosome of claim 1 for the manufacture of a medicament for treating alzheimer's disease and delaying the decline of aged intelligence, wherein the concentration of lysosome is 5-55 μ g/mL.

5. Use of the lysosome according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of Alzheimer's disease and for delaying the decline of the mental capacity of the elderly, characterized by its use for the cognitive repair and prevention of Alzheimer's disease in the elderly.

Technical Field

The invention belongs to the field of cell repair, and relates to application of lysosomes in the field of preparing medicaments for treating Alzheimer's disease and delaying senile mental deterioration.

Background

The complexity of the nervous system prevents many drugs from entering the brain, while the nerve cells have limited self-repair capacity, which results in irreversible and progressive nature of many nervous system diseases, ultimately leading to severity of the outcome, causing huge economic losses and social burden (FernandesLF, Bruch GE, Massensini AR and Frezard F. Recentrative Advances in the Therapeutic and Diagnostic of lipometers and CarbonNanomatics in Ischemic stress. front neurosci.2018; 12: 453.). Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease (HD) are high incidence central nervous system diseases, the causes of these diseases are abnormal folding or processing of protein, which leads to neuronal death, and the disease after development seriously affects life and life span of patients.

AD is one of the most common forms of dementia, with two main symptoms: amyloid beta deposits abnormally outside neurons to form senile plaques and Tau protein phosphorylates abnormally to form neurofibrillary tangles (Cheray M, Stratoulis V, Joseph B and Grabert K. the Rules of Engagement: Do microgliaal the face in the Inverse relationship of Glioma and Alzheimer's Disease.

The pathogenic reasons are as follows: amyloid protein (APP) is used as a type I transmembrane protein, has a membrane receptor-like structure, and can perform trans-dimerization among molecules to promote cell adhesion. However, the degradation product of APP, namely beta-amyloid, can accelerate the aggregation of APP and trigger apoptosis. The Tau protein serving as the microtubule-associated protein can promote the polymerization of tubulin to form microtubules, maintain the stability of the microtubules, reduce the dissociation of tubulin molecules and induce the microtubules to form bundles. And Tau protein is abnormally over-phosphorylated, the effect of maintaining microtubule stability is lost, so that the microtubule structure is widely damaged, normal axon transport is damaged, synapses are lost, the function of neurons is damaged, and cerebral neurodegenerative diseases occur. With abnormal folding and processing of amyloid precursor protein and Tau protein in the brain, AD patients experience gradual decline in neurosynaptic degeneration, memory and other cognitive functions.

PD is a chronic progressive neurodegenerative disease characterized by the widespread spread of the intracellular protein alpha-synuclein and early prominent death of dopaminergic neurons in the compact part of the substantia nigra (Radhishnan DM and Goyal V.Parkinson' S disease: A review. neuron India.2018; 66: S26-S35.).

The pathogenic reasons are as follows: the normal alpha-synuclein is in a disordered monomer state and plays a role in adjusting the component structure of membrane lipid and the transport process of vesicles. Under the influence of various factors, alpha-synuclein is abnormally expressed and aggregated, participates in the formation of lewy bodies, generates oxidative stress and can cause the death of dopaminergic neurons (Kalia LV, diagnostic biologizers for Parkinson's disease: focus on alpha-synuclein in diabetic nerve fluid. Parkinson Relat disease.2019; 59: 21-25.).

HD is an autosomal dominant hereditary neurodegenerative disease characterized by abnormal amplification of the glutamine sequence (polyQ) in the N-terminal sequence of Huntington protein (HTT) (Saudou F and Humbert S. the Biology of Huntingtin. neuron. 2016; 89(5): 910-26.).

The pathogenic reasons are as follows: the normal HTT protein is an important factor for spindle orientation and neurogenesis, and a cytosine-adenine-guanine trinucleotide repetitive sequence in a specific region of a mutant HTT protein genome is pathologically amplified due to dynamic mutation to cause abnormal extension of a polyQ chain, so that dance-like movement accompanied with gait disorder, bradykinesia and stiffness, cognitive slowing, and reduced attention and psychological flexibility are caused. In addition to HD, trinucleotide repeat disease also has spinal bulbar atrophy, dentatorubral-rednucleus-protein globus easy atrophy, spinocerebellar ataxia.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a medicament and a treatment method which have low cytotoxicity, safety and effectiveness.

The invention discloses application of lysosomes in the field of preparing medicaments for treating Alzheimer's disease and delaying senile intelligence deterioration, and is used for treating protein misfolding or processing diseases.

Further, the therapeutic protein misfolding or processing-type disease includes at least one of alzheimer's disease, parkinson's syndrome, huntington's disease, mad cow disease, creutzfeldt-jakob syndrome, corticobasal degeneration, amyotrophic lateral sclerosis, spinocerebellar ataxia, cystic fibrosis, hereditary cataract, type ii diabetes, autosomal dominant hereditary tubulointerstitial nephropathy.

The invention also discloses a medicament for treating protein misfolding or processing diseases, including lysosomes.

Preferably, the lysosome is extracted from BV2 cells.

Preferably, the concentration of the lysosome is 5-55 μ g/mL.

Preferably, the concentration of the lysosome is not higher than 500. mu.g/mL.

The invention has the beneficial effects that:

1. provides a new approach for treating protein misfolding or processing diseases;

2. the raw materials are easy to obtain, and the cost is low;

3. targeting property is realized, and cytotoxicity does not exist;

4. the utilized organelles do not contain genetic materials and do not have adverse effects on receptors.

Drawings

FIG. 1 test pattern of lysosomes aggregation in different organs;

figure 2 targeted aggregation test pattern of lysosomes in different sample brains;

FIG. 3 Table of lysosomal cytotoxicity assays;

FIG. 4 is a table of the effect of lysosomes on the extracellular environment;

FIG. 5 shows the degradation test table of toxic proteins of lysosomes on brain hippocampus;

FIG. 6 is a table of the degradation test of toxic proteins from the cortex by lysosomes;

figure 7 data table of lysosomes improving the recognition ability of alzheimer's disease mice for things;

figure 8 data table of lysosomes improving memory ability of alzheimer's disease mice to things;

figure 9 data table of lysosomal improvement in nesting ability of alzheimer's disease mice;

figure 10 degradation of toxic proteins in the brain hippocampus by large concentrations of lysosomes.

Detailed Description

The following examples are given to illustrate the technical examples of the present invention more clearly and should not be construed as limiting the scope of the present invention.

Lysosome extraction

The lysosome extraction method was performed according to the instructions of the commercially available lysosome extraction kit. The method comprises the following specific steps:

1. centrifugal washing 0.25% trypsinization collection 2-5 × 10⁷Cells were washed centrifugally with PBS;

2. breaking cell wall. Centrifuging, removing supernatant, placing the cell precipitate on ice, adding 400 μ L cell bursting buffer solution, and standing on ice for 10 min; homogenizing for 30-40 times by using a homogenizer, and collecting the homogenate product into a new centrifuge tube;

3. lysosomes were separated by centrifugation. Centrifuging in steps according to the following sequence, taking supernatant after each centrifugation, and transferring the supernatant into a new centrifuge tube for the next step: centrifuging at 1000 Xg for 5 min-3000 Xg for 10 min-5000 Xg for 10 min-20000 Xg for 30 min; and (4) taking the precipitate after the last centrifugation, suspending the precipitate by using washing buffer, centrifuging the precipitate for 30min at 20000 Xg again, weighing the obtained precipitate, suspending the precipitate by using storage solution, and storing the suspension in a refrigerator at 4 ℃.

4. And marking and identifying. Adding a lysosome labeling probe (Lysotracker, Red) into the product obtained in the step 3, wherein the labeling probe comprises the following components in parts by volume: and (3) taking 5 mu L of lysosome suspension after 2h, namely the marked lysosome. The mixture is dripped on a glass slide, covered with a cover glass, and identified under a confocal microscope after being sealed.

B6C3F1 alzheimer mice (12 months old) and B6C3F1 wild-type mice (6 months old, 12 months old) as experimental animals were purchased from carfukang ltd (beijing, china). Mice were housed in the laboratory animal center of the institute of radiology and medicine, academy of Chinese medical sciences during the experiment.