WO2023093599A1 - 一种改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用 - Google Patents

一种改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用 Download PDF

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WO2023093599A1
WO2023093599A1 PCT/CN2022/132394 CN2022132394W WO2023093599A1 WO 2023093599 A1 WO2023093599 A1 WO 2023093599A1 CN 2022132394 W CN2022132394 W CN 2022132394W WO 2023093599 A1 WO2023093599 A1 WO 2023093599A1
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lysosome
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薛雪
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南开大学
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

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  • the invention relates to the field of cell repair, and relates to the application of modified lysosomes as drugs for treating protein misfolding or processing diseases.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • AD Alzheimer's Disease
  • senile plaques formed by abnormal deposition of ⁇ -amyloid outside the neurons and abnormal phosphorylation of Tau protein to form neurofibrillary tangles (Cheray M, Stratoulias V, Joseph B and Grabert K.
  • the Rules of Engagement Do Microglia Seal the Fate in the Inverse Relation of Glioma and Alzheimer's Disease? Front Cell Neurosci. 2019; 13:522.).
  • amyloid as a type I transmembrane protein, has a membrane receptor-like structure, and its molecules can undergo trans-dimerization to promote cell adhesion.
  • APP amyloid
  • ⁇ -amyloid protein the degradation product of APP, ⁇ -amyloid protein, can accelerate the aggregation of APP and trigger cell apoptosis.
  • Tau protein can promote tubulin polymerization to form microtubules, maintain microtubule stability, reduce the dissociation of tubulin molecules, and induce microtubule bundles.
  • the abnormal hyperphosphorylation of Tau protein will lose the role of maintaining microtubule stability, resulting in extensive destruction of microtubule structure, damage to normal axonal transport, loss of synapses, neuron function damage, and brain neurodegeneration. Accompanied by the abnormal folding and processing of amyloid precursor protein and Tau protein in the brain, neurosynaptic degeneration in AD patients, memory and other cognitive functions gradually decline.
  • Lysosomes are the major organelles in cells that perform degradative functions, and the degradative function of their internal hydrolytic enzymes is critical to many cellular processes, including nutrient digestion during starvation, elimination of damaged cellular components, termination of mitotic signaling, intracellular and Elimination of extracellular pathogens and cellular and tissue remodeling (Perera RM and Zoncu R. The Lysosome as a Regulatory Hub. Annu Rev Cell Dev Biol. 2016; 32:2 23-253.).
  • intracellular components such as misfolded proteins, aging and damaged organelles are delivered to the lysosome for degradation through autophagy; outside the cell, exogenous substances are delivered to the lysosome through endocytosis and phagocytosis degradation.
  • the v-ATPase on the surface of lysosomes uses the energy generated by the hydrolysis of ATP to pump hydrogen ions into the lumen, thereby generating the acidic pH of lysosomes and providing an acidic environment for hydrolyzing enzymes to hydrolyze lipids, polysaccharides, nucleic acids, and proteins.
  • lysosome-associated membrane proteins LAMP1 and LAMP2, lysosomal integral membrane protein 2 (LIMP2) and CD63, etc. are highly glycosylated on the luminal side and form glycocalyx to prevent hydrolysis in the cavity Enzymes self-digest membrane structures. These proteins are critical for lysosomal biogenesis, acidification, metabolite trafficking, and chaperone-mediated autophagy.
  • lysosomes play a key role in maintaining cell homeostasis
  • abnormalities in lysosomal proteins are closely related to the occurrence of various diseases, such as ATPase (ATPase) abnormalities promote the occurrence and development of Parkinson's syndrome; chloride voltage
  • the abnormality of gating channel 7 (CLC-7) promotes the occurrence and development of osteopetrosis; the abnormality of Cystinosin promotes the occurrence and development of cystinosis; the abnormality of lysosome-associated membrane protein 2 (LAMP2) promotes
  • the abnormality of mucolipin TRP cation channel (Mucolipin) promotes the occurrence and development of mucolipidosis IV;
  • the abnormality of cholesterol transporter 1 (NPC1) in NPC cells can promote the occurrence and development of Niemann's disease; Abnormalities in solute carrier family proteins lead to the development of sialiduria.
  • lysosomes have therapeutic effects on various neurodegenerative diseases including Alzheimer's disease, Parkinson's syndrome and Huntington's disease. See CN202010294396.0 A lysosome as an application in the field of preparing drugs for treating Alzheimer's disease and delaying mental decline in the elderly.
  • AD drugs currently entering the clinical stage to achieve satisfactory curative effects include drug off-target, delayed intervention time, and low drug action intensity in the brain.
  • Drug off-target and intervention time delay are caused by the characteristics of the antibody drug itself.
  • the lower drug effect in the brain is mainly due to the low concentration of the drug reaching the brain due to the existence of the blood-brain barrier. Therefore, the efficiency of drugs entering the brain is an important factor in determining the therapeutic effect of drugs on diseases.
  • the present invention is based on the further research and improvement of CN202010294396.0, a kind of lysosome as the application in the field of preparation of drugs for treating Alzheimer's disease and delaying senile mental decline.
  • the purpose is to increase their rate of entry into the brain and maximize the curative effect by modifying natural lysosomes and artificially synthesizing materials with lysosome functions.
  • the technical problem to be solved by the present invention is to select the natural lysosome species with the best effect, and further modify it to obtain higher brain targeting, enzyme activity, safety and stability, and prolong its lifespan.
  • the invention discloses the application of a modified lysosome as a medicine for treating protein misfolding or processing diseases.
  • the modified lysosome comes from brain microglial cells modified by external stimulation.
  • the external stimulus includes at least one of physical stimulus, biological stimulus, chemical stimulus, and environmental stimulus.
  • the physical stimulation includes thermal stimulation, force stimulation, light stimulation, and electrical stimulation
  • the biological stimulation includes stimulation with complement factors or chemokines, stimulation with polypeptides, and gene editing;
  • the chemical stimulation includes inflammation-inducing factor stimulation, autophagy activator stimulation, polymer modification surface modification, porous organic framework material encapsulation;
  • the environmental stimuli include hypoxic environmental stimuli, sugar-deficient environmental stimuli, and amino acid-deficient environmental stimuli.
  • the complement factor uses C5a
  • the chemokine uses CXC.
  • the gene editing is implemented by overexpressing the main functional proteins of lysosomes, and the overexpressed proteins include ATPase, cathepsin B, cathepsin D, cathepsin K, cathepsin L, acid amidase, acid Lipase, ⁇ -galactosidase, ⁇ -L-iduronidase, ⁇ -N-acetylgalactosaminidase, hyaluronidase, chloride voltage-gated channel CLC-7, cystine protease Cystinosin, lysosome-associated membrane protein LAMP1 and/or LAMP2, multifunctional transport intrinsic membrane protein LIMO-2, mucin TRP cation channel Mucolipin, NPC intracellular cholesterol transporter NPC1, solute carrier family 11 and/or 17.
  • the overexpressed proteins include ATPase, cathepsin B, cathepsin D, cathepsin K, cathepsin L, acid amidase, acid Lip
  • LPS is used as the inflammation-inducing factor.
  • the autophagy activator stimulation is the use of an activator of rapamycin targeting protein mTOR and/or an activator of transcription factor EB; wherein the activator includes carbohydrate compounds, glycoside compounds, ketone compounds , Antibiotic compounds.
  • the carbohydrate compound includes sucrose, trehalose; the glycoside compound includes digoxin; the ketone compound includes Torin1, and the antibiotic compound includes rapamycin.
  • the organelles used do not contain genetic material and will not have adverse effects on the receptors;
  • the modification method is simple, the cost is low, and there is no toxic or side effect.
  • Fig. 1 Test diagram of targeted aggregation of lysosomes from different cell sources in the brain
  • Figure 2 The targeted aggregation test diagram of brain microglial lysosomes in different organs
  • FIG. 3 Targeted aggregation test diagram of brain microglial lysosomes in different sample brains
  • Fig. 4 brain microglial cell lysosome cytotoxicity test table
  • Figure 6 Degradation test table of brain microglia lysosomes to toxic proteins in brain hippocampus
  • Fig. 7 Degradation test table of brain microglia lysosomes to cortical toxic proteins
  • Fig. 8 Degradation of toxic proteins in brain hippocampus by large concentration of brain microglia lysosomes.
  • Figure 13 LPS-modified brain microglial lysosomes in the brain targeting aggregation test diagram
  • Fig. 14 Test diagram of targeted aggregation test of C5a-modified brain microglia lysosomes in the brain.
  • mice 3 months old used as experimental animals were purchased from Weitong Lihua Co., Ltd. (Beijing, China).
  • mice were raised in the Experimental Animal Center of Nankai University; C57BL/6J-APP/PS1 Alzheimer's mice (12 months old) and C57BL/6J wild-type mice (6 months, 12 months old) were used as experimental animals. age) were purchased from Huafukang Co., Ltd. (Beijing, China).
  • the mice were bred in the Experimental Animal Center of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences.
  • Step 1 centrifugal washing. Digest with 0.25% trypsin to collect 2-5 ⁇ 10 7 cells, wash by centrifugation with PBS;
  • Step 2 cell wall breaking. Discard the supernatant after centrifugation, put the cell pellet on ice, add 400 ⁇ L of cell bursting buffer, and let it stand on ice for 10 minutes; use a homogenizer to homogenize for 30-40 times, and collect the homogenate product into a new centrifuge tube ;
  • Step 3 centrifuging the modified lysosome.
  • Centrifuge step by step in the following order, take the supernatant after each centrifugation, and transfer to a new centrifuge tube for the next step: centrifuge at 1000 ⁇ g for 5 minutes——centrifuge at 3000 ⁇ g for 10 minutes—centrifuge at 5000 ⁇ g for 10 minutes ——Centrifuge at 20,000 ⁇ g for 30 minutes; take the precipitate after the last centrifugation, suspend it with washing buffer, centrifuge again at 20,000 ⁇ g for 30 minutes, weigh the obtained precipitate and suspend it with the storage solution, which is the modified lysosome. Store in refrigerator at 4°C.
  • a lysosome labeling probe Li.otracker, Red
  • BV2 cells were heated at 39-42°C for 30 minutes; then cultured at 37°C for 24 hours, and then lysosomes were extracted.
  • the modified lysosome extraction method is the same as in Example 1.
  • mice were C57BL/6J-APP/PS1 Alzheimer's mice; the disease-free mice were C57BL/6J wild-type mice.
  • the comparative examples 1-5 are carried out as follows: randomly select the mice with specified requirements, and inject 30 mg/kg lysosome-labeled probe-labeled lysosomes from different sources into their tail veins respectively (the blank control example injects an equal amount of PBS), and perfuse with normal saline after 2h, and dissect out the mouse brain. Fluorescent imaging was performed using a small animal imaging system, and absorbance was measured by an enzyme-linked immunosorbent assay.
  • the comparative examples 6-8 were carried out as follows: randomly select mice with specified requirements, and inject 30 mg/kg lysosome-labeled probe-labeled lysosomes into their tail veins (inject the same amount of PBS in the blank control example) for 2 hours After perfusion with normal saline, the corresponding parts of the mice were dissected. Fluorescent imaging was performed using a small animal imaging system, and absorbance was measured by an enzyme-linked immunosorbent assay.
  • Brain microglia-derived lysosomes are enriched in targeted manner in the brains of Alzheimer's mice (Comparative Example 6);
  • Lysosomes derived from brain microglia for the targeting of mouse brain in different states
  • Comparative examples 6, 9, 10, and 11 were carried out as follows: randomly select mice with specified requirements, inject 30 mg/kg lysosome-labeled probe-labeled lysosomes into their tail veins (inject an equivalent amount of PBS), and perfuse with normal saline after 2h, and dissect out the mouse brain. Fluorescent imaging was performed using a small animal imaging system, and absorbance was measured by an enzyme-linked immunosorbent assay.
  • Brain microglia-derived lysosomes are enriched in the brains of Alzheimer's mice (Comparative Example 6);
  • Brain microglia-derived lysosomes are enriched in the brains of aged disease-free mice (Comparative Example 10);
  • Brain microglia-derived lysosomes will not be enriched in the brains of young disease-free mice (Comparative Example 11);
  • Brain microglia-derived lysosomes can be targeted to enter the lesion in diseased mice; lysosomes may play a role in delaying memory loss in the elderly.
  • the primary neurons of neonatal mice were extracted according to conventional methods, and the primary neurons were seeded in a 96-well plate at a density of 5 ⁇ 10 6 /well, 100 ⁇ L per well, and incubated in a CO 2 incubator at 37°C.
  • Half of the solution was changed every 2-3 days, and different concentrations of lysosomes (100, 200, 300, 400, 500 ⁇ g/mL) were added to the 96-well plate on the 7th day.
  • the absorbance was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent detector, and the cell viability was calculated. As shown in Figure 4, graph pad software was used for statistical analysis, and t-test was used for comparison between groups, and there was no significant difference.
  • N2a cells were seeded in 6-well plates and incubated in a CO2 incubator at 37 °C. When the cells cover 80-90% of the well plate, at 0, 4, 6, 8, 10, and 12 hours, replace the old medium in the corresponding 6-well plate with the medium containing 50 ⁇ g/mL lysosome During cultivation, the incubation was continued in a CO 2 incubator at 37°C. Take the culture medium of each well into a centrifuge tube, and use a pH tester to measure the pH value of the culture medium at different times (0, 2, 4, 6, 8, 12 h) of lysosome action. (See Table 3 and accompanying drawing 5, wherein the abscissa of accompanying drawing 5 represents the length of time that lysosomes are added, and the values from left to right correspond to the time points of adding lysosomes in Table 4 respectively)
  • Graph pad software was used for statistical analysis, and t-test was used for comparison between groups, and there was no significant difference.
  • Lysosomes do not affect the pH of the extracellular environment.
  • APP antibody Biolegend, #SIG-39320
  • p62 antibody Biolegend Biotechnology, PM045
  • LC3 antibody Cell Signal Technology, #2775
  • Actin antibody Biolegend, BE0021-100
  • Tetramer Antibody i.e. APP antibody, Biolegend, #SIG-39320
  • Tail vein injection of brain microglia-derived lysosomes can promote the degradation of toxic proteins expressed in the brain of Alzheimer's disease mice.
  • High-dose test group extract the primary neurons of APP/PS1 neonatal mice according to conventional methods, inoculate the primary neurons in a 6-well plate at a density of 5 ⁇ 10 6 /well, and incubate in a CO2 incubator at 37°C. Change half of the solution every 2-3 days, give 500 ⁇ g/ml lysosome at one time after 7 days, and extract protein after 7 days.
  • Normal primary neuron group the primary neurons of normal wild-type neonatal mice were extracted according to conventional methods, and the primary neurons were seeded in 6-well plates at a density of 5 ⁇ 10 6 /well, and incubated in a CO2 incubator at 37°C. Change half solution every 2-3 days, extract protein after 14 days.
  • Blank group similar to the high-dose test group, the difference is that lysosomes are not used for administration.
  • 293T cells were seeded in 24-well plates and incubated in a CO2 incubator at 37°C. When the cells cover 80-90% of the well plate, transfect the cells with CathepsinB (CatB)-GFP plasmid and CathepsinD (CatD)-GFP plasmid, mix the CathepsinB-GFP plasmid and CathepsinD-GFP plasmid with Lipofectamine 2000 respectively, and let the After 20 minutes, they were added to the cells, and after 6 hours, the cell culture medium was replaced with fresh medium. After 24 hours, the overexpression of lysosomal cathepsin B and cathepsin D in the cells was observed, and the results were shown in Figure 9. .
  • Randomly select 9 yielderly disease-free, no drug administration
  • inject the same volume of PBS as in Example 3 into the tail vein of the mouse according to the weight of the mouse, and other conditions are the same as in Comparative Example 6 (elderly diseased, drug administration).
  • mice Put the mice into the test box without any objects in turn to adapt to the experimental environment, for 2 consecutive days, twice a day.
  • 2Familiarization stage put two identical objects (A and B) at a distance of 10 cm from the side wall, put the mice in with their backs facing the objects, and take them out after 10 minutes of free exploration.
  • the frequency of object detection 3 consecutive days, 2 times a day.
  • 3Testing stage test after 24 hours, replace two of the same objects B with another object C, that is, the familiar object (A) and the novel object (C), and record the mouse’s 10-min exploration time and sniffing times of the two objects .
  • the positions of A and C were randomly permuted to avoid positional bias.
  • Exploration activity was defined as the behavior when the mouse's nose pointed directly at the object or directly touched the object within a range of ⁇ 5 cm. When the mouse turned its head or sat on the object, it was not considered to be exploring the object.
  • Graph pad software was used for statistical analysis, and one-way was used for comparison between groups. P ⁇ 0.05 means the difference is statistically significant.
  • Tail vein injection of lysosomes can improve the ability of Alzheimer's mice to recognize things and improve hippocampus-related learning and memory functions.
  • mice 2 hours before the nocturnal rhythm of the mice, put some 2cm ⁇ 2cm square paper towels (a total of three kitchen papers) in the mouse cage. After 24 hours, the nesting situation of the mice was counted and recorded by taking pictures.
  • the scoring criteria are as follows:
  • the paper towel spreads evenly and forms loose clusters: 2 points;
  • Paper towels spread evenly in 1/3 of the cage and form clusters is 4 points;
  • Graph pad software was used for statistical analysis, and one-way was used for comparison between groups.
  • the nesting score of the administration group was higher, proving that tail vein injection of lysosomes would improve the social behavior of Alzheimer's mice.
  • Exogenous lysosomes have a therapeutic effect on the misfolding or processing of therapeutic proteins represented by Alzheimer's disease;
  • the therapeutic effect has no toxic and side effects
  • the inventors further improved the lysosome to enhance the efficiency and targeting of the drug into the brain. And tested the following:
  • Randomly get Application Example 1 inject LPS into its lateral ventricle, and inject 30 mg/kg lysosome-labeled probe-labeled lysosomes ( Add 0.1 ⁇ g/mL LPS to the culture medium at a ratio of 1:1000 12 hours before cell extraction of lysosomes, and the rest of the steps are the same as normal lysosome extraction steps), and 2 hours later, use normal saline perfusion to cut out the mouse brain.
  • the brain microglia-derived lysosomes modified by LPS have better enrichment and targeting in the brain than directly extracted brain microglia-derived lysosomes.
  • Randomly take Application Example 2 inject LPS into its lateral ventricle, and inject 30 mg/kg lysosome-labeled probe-labeled lysosomes ( 0.5h before cell extraction of lysosomes, the concentration of C5a after mixing was 10nmol/L, and the rest of the steps were the same as the normal lysosome extraction steps), and 2h later, the mouse brain was perfused with normal saline.
  • the brain microglia-derived lysosomes modified by C5a have better enrichment and targeting in the brain than directly extracted brain microglia-derived lysosomes.

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Abstract

提供了改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，该改性溶酶体的靶向性更强，利用的细胞器不含有遗传物质，不会对受体产生不良影响；改性方法简单，成本低，无毒副作用。

Description

一种改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用 技术领域
本发明细胞修复领域，涉及改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用。
背景技术
神经系统的复杂性使很多药物无法进入大脑，而神经细胞的自我修复能力有限，这造成许多神经系统疾病的不可逆转和进行性，最终导致结果的严重性，造成巨大的经济损失和社会负担(Fernandes LF,Bruch GE,Massensini AR and Frézard F.Recent Advances in the Therapeutic and Diagnostic Use of Liposomes and Carbon Nanomaterials in Ischemic Stroke.Front Neurosci.2018；12:453.)。阿尔兹海默症(Alzheimer’s disease,AD)、帕金森综合征(Parkinson’s disease,PD)、亨廷顿氏症(Huntington’s disease,HD)都是高发病率的中枢神经系统疾病，这些疾病的病因都是蛋白异常折叠或加工，导致神经元死亡，病情发展后严重影响病人的生活和寿命。
AD是痴呆症最常见的一种形式，它的主要症状有两个：β-淀粉样蛋白在神经元细胞外异常沉积形成老年斑和Tau蛋白异常磷酸化形成神经元纤维缠结(Cheray M,Stratoulias V,Joseph B and Grabert K.The Rules of Engagement:Do Microglia Seal the Fate in the Inverse Relation of Glioma and Alzheimer's Disease？Front Cell Neurosci.2019；13:522.)。
其致病原因在于：淀粉样蛋白(APP)作为I型跨膜蛋白，具有膜受体样结构，分子间可以进行反式二聚化，促进细胞黏附。但APP的降解产物β-淀粉样蛋白可以加速APP的聚集，引发细胞凋亡。Tau蛋白作为微管相关蛋白可促进微管蛋白聚合形成微管，维持微管稳定性，降低微管蛋白分子的解离，并诱导微管成束。而Tau蛋白异常过度磷酸化，会丧失维持微管稳定的作用，导致微管结构广泛破坏，正常轴突转运受损，引起突触丢失神经元功能损伤，发生脑神经退行性病变。伴随着脑内淀粉样前体蛋白和Tau蛋白的异常折叠与加工，AD患者神经突触变性，记忆力和其他认知功能逐渐下降。
现有技术中，包括抗炎药、他汀类药物、激素疗法和螯合剂等在内的治疗方法均未能取得成功；而降低神经变性速率或停止疾病进程的疾病缓解疗法仍然难以捉摸(Waite LM.Treatment for Alzheimer’s disease:has anything changed？Aust Prescr.2015；38(2):60–63.)。
溶酶体是细胞中发挥降解作用的主要细胞器，其内部水解酶的降解功能对许多细胞过程至关重要，包括饥饿期间的营养消化、受损细胞成分的消除、有丝分裂信号的终止、细胞内和细胞外病原体的消除以及细胞和组织重塑(Perera RM and Zoncu R.The Lysosome as a Regulatory Hub.Annu Rev Cell Dev Biol.2016；32:2 23-253.)。在细胞内部，细胞内成分如错误折叠的蛋白、衰老及损伤的细胞器通过自噬传递到溶酶体被降解；在细胞外部，外源性物质通过内吞作用和吞噬作用被传递到溶酶体降解。
溶酶体表面的v-ATPase利用ATP水解产生的能量将氢离子泵入管腔，从而产生溶酶体的酸性pH，为水解酶提供水解脂质、多糖、核酸和蛋白质的酸性环境。其上的部分膜蛋白(溶酶体相关膜蛋白LAMP1和LAMP2、溶酶体整合膜蛋白2(LIMP2)及CD63等)在其腔侧被高度糖基化并形成糖萼，防止腔内的水解酶自消化膜结构。这些蛋白质对于溶酶体的生物发生、酸化、代谢物运输以及分子伴侣介导的自噬至关重要。
由于溶酶体在维持细胞稳态中发挥关键作用，因此溶酶体蛋白的异常与多种疾病的发生紧密相关，如ATP酶(ATPase)的异常促进帕金森综合征的发生发展；氯化物电压门控通道7(CLC-7)的异常促进骨硬化病的发生发展；胱氨酸蛋白酶(Cystinosin)的异常促进胱氨酸病的发生发展；溶酶体相关膜蛋白2(LAMP2)的异常促进糖原储存病的发生发展；粘蛋白TRP阳离子通道(Mucolipin)的异常促进粘脂沉积症IV的发生发展；NPC细胞内胆固醇转运蛋白1(NPC1)的异常会促进尼曼氏病的发生发展；溶质载体家族蛋白的异常会导致唾液酸尿症的发生发展。
本发明的发明人在此前的研究工作中，发现了溶酶体对于包括阿尔茨海默症、帕金森综合征和亨廷顿病在内的多种神经退行性疾病有治疗作用。参见CN202010294396.0一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。
目前进入临床阶段的AD药物未能达到令人满意的疗效的主要原因包括药物脱靶、干预时间延迟及脑内药物作用强度较低等。药物脱靶和干预时间延迟是抗体药物自身特性所导致，然而，脑内药物作用强度较低主要是由于血脑屏障的存在使药物到达脑内浓度过低。因此，药物的入脑效率是决定药物对疾病治疗效果的重要因素。
本发明是基于CN202010294396.0一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的进一步研究和改进。
目的是通过改造天然溶酶体及人工合成具有溶酶体功能的材料，提升它们的入脑率，使疗效最大化。
发明内容
本发明所要解决的技术问题是选择出效果最好的天然溶酶体种类，并对其进行进一步改性，以得到更高的脑靶向性、酶活性、安全性及稳定性，延长其在动物体内的血液循环时间及在体外环境中的保存时间，并赋予其载药性能。
本发明公开了一种改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，所述改性溶酶体来自经外界刺激改性的脑部小胶质细胞。
进一步的，所述外界刺激包括物理方式刺激、生物方式刺激、化学方式刺激、环境方式刺激中至少一种。
进一步的，所述物理方式刺激包括热刺激、力刺激、光刺激、电刺激；
所述生物方式刺激包括利用补体因子或趋化因子刺激、利用多肽刺激、基因编辑；
所述化学方式刺激包括炎症诱导因子刺激、自噬激活剂刺激、聚合物修饰表面修饰、多空有机框架材料封装；
所述环境刺激包括乏氧环境刺激、乏糖环境刺激、乏氨基酸环境刺激。
优选的，所述补体因子采用C5a，所述趋化因子采用CXC。
优选的，所述基因编辑，采用过表达溶酶体主要功能蛋白的方式实施，过表达的蛋白包括ATP酶、组织蛋白酶B、组织蛋白酶D、组织蛋白酶K、组织蛋白酶L、酸性酰胺酶、酸性脂肪酶、α-半乳糖苷酶、α-L-艾杜糖醛酸酶、α-N-乙酰半乳糖胺酶、透明质酸酶、氯化物电压门控通道CLC-7、胱氨酸蛋白酶Cystinosin、溶酶体相关膜蛋白LAMP1和/或LAMP2、多功能转运内在膜蛋白LIMO-2、粘蛋白TRP阳离子通道Mucolipin、NPC细胞内胆固醇转运 蛋白NPC1、溶质载体家族11和/或17。
优选的，所述炎症诱导因子采用LPS。
优选的，所述自噬激活剂刺激，是采用雷帕霉素靶向蛋白mTOR的激活剂和/或转录因子EB的激活剂；其中，激活剂包括糖类化合物、苷类化合物、酮类化合物、抗生素类化合物。
更优选的，所述自噬激活剂刺激的所述激活剂中，糖类化合物包括蔗糖、海藻糖；苷类化合物包括地高辛；酮类化合物包括Torinl、抗生素类化合物包括雷帕霉素。
本发明的有益效果在于：
1、提供了一种新的治疗蛋白错误折叠或加工类疾病的途径；
2、靶向性更强；
3、利用的细胞器不含有遗传物质，不会对受体产生不良影响；
4、改性方法简单，成本低，无毒副作用。
附图说明
图1不同细胞来源的溶酶体在脑部靶向性聚集测试图；
图2脑部小胶质细胞溶酶体在不同器官中的靶向性聚集测试图；
图3脑部小胶质细胞溶酶体在不同样本脑部中的靶向聚集测试图；
图4脑部小胶质细胞溶酶体细胞毒性测定表；
图5脑部小胶质细胞溶酶体对细胞外环境的影响表；
图6脑部小胶质细胞溶酶体对脑部海马的毒性蛋白的降解测试表；
图7脑部小胶质细胞溶酶体对皮层的毒性蛋白的降解测试表；
图8大浓度脑部小胶质细胞溶酶体对脑部海马的毒性蛋白的降解作用。
图9溶酶体水解酶过表达的细胞荧光图
图10溶酶体改善阿尔兹海默症小鼠对事物的识别能力数据表；
图11溶酶体改善阿尔兹海默症小鼠对事物的记忆能力数据表；
图12溶酶体改善阿尔兹海默症小鼠筑巢能力数据表；
图13 LPS改性的脑部小胶质细胞溶酶体在脑部靶向性聚集测试图；
图14 C5a改性的脑部小胶质细胞溶酶体在脑部靶向性聚集测试图.
具体实施方式
下面结合实施例对本发明的具体实施方式作进一步描述，以下实施例仅用于更加清楚地说明本发明的技术实施例，而不能以此来限制本发明的保护范 围。
作为实验动物的C57BL/6J小鼠(3月龄)购自维通利华有限公司(北京，中国)。实验期间小鼠在南开大学实验动物中心饲养；作为实验动物的C57BL/6J-APP/PS1阿尔茨海默症小鼠(12月龄)和C57BL/6J野生型小鼠(6月龄、12月龄)购自华阜康有限公司(北京，中国)。实验期间小鼠在中国医学科学院放射医学研究所实验动物中心饲养。
需要说明的，本发明所述实施例均使用下述改性溶酶体提取方法，根据市售溶酶体提取试剂盒说明书进行。具体步骤如下：
步骤1、离心洗涤。0.25％胰蛋白酶消化收集2-5×10 7个细胞，用PBS离心洗涤；
步骤2、细胞破壁。离心后弃上清，将细胞沉淀置于冰上，加入400μL细胞胀破缓冲液，在冰上静置10min；利用匀浆器匀浆30-40下，收集匀浆产物至一新离心管中；
步骤3、离心分离改性溶酶体。按下述顺序梯次离心，每次离心后均取上清液，并移至新离心管中进行下一步：1000×g下离心5min——3000×g下离心10min——5000×g下离心10min——20000×g下离心30min；最后一次离心后取沉淀，并用洗涤缓冲液悬起，再次20000×g下离心30min，得到沉淀称重后利用储存液悬起，即为改性溶酶体。置于冰箱中4℃保存。
改性溶酶体的标记：
为了方便进一步测试改性溶酶体的作用机理与效果，通过如下步骤对改性溶酶体进行标记：向步骤3得到的产物中加入溶酶体标记探针(Lysotracker，Red)，以体积份数计，标记探针：溶酶体悬液＝1:20000，2h后取5μL溶酶体悬液，即为标记后的改性溶酶体。将其滴于载玻片上，盖上盖玻片，封片后在共聚焦显微镜下鉴定。
实施例1
改性溶酶体的预处理
39-42℃条件下加热BV2细胞30min；后置于37℃环境下培养24h，后提取溶酶体。
实施例2-19
改性溶酶体的预处理：
参见表1。
提取改性溶酶体：
改性溶酶体提取方法与实施例1相同。
表1溶酶体的预处理参数
Figure PCTCN2022132394-appb-000001
Figure PCTCN2022132394-appb-000002
应用例1
随机选取C57BL/6J-APP/PS1阿尔茨海默症小鼠(12月龄)，并向其尾静脉注射30mg/kg实施例6制备得到的标记改性溶酶体。
应用例2
随机选取C57BL/6J-APP/PS1阿尔茨海默症小鼠(12月龄)，并向其尾静脉注射30mg/kg实施例7制备得到的标记改性溶酶体。
应用例3
随机选取C57BL/6J-APP/PS1阿尔茨海默症小鼠(12月龄)，并向其尾静脉注射30mg/kg实施例14制备得到的标记改性溶酶体。
应用例4
随机选取C57BL/6J-APP/PS1野生小鼠(12月龄)，并向其尾静脉注射30mg/kg实施例6制备得到的标记改性溶酶体。
应用例5
随机选取C57BL/6J-APP/PS1野生小鼠(12月龄)，并向其尾静脉注射30mg/kg实施例7制备得到的标记改性溶酶体。
应用例6
随机选取C57BL/6J-APP/PS1野生小鼠(12月龄)，并向其尾静脉注射30mg/kg实施例14制备得到的标记改性溶酶体。
为了充分说明本发明的有益效果，注射标记改性溶酶体后2h使用生理盐水灌流，并取出鼠脑进行检测，并设置对比例。应用例与对比例的实验参数如下：
特设置如下对比例：
表2测试参数汇总表
Figure PCTCN2022132394-appb-000003
Figure PCTCN2022132394-appb-000004
注：1、患病情况中“是”表示患有阿尔茨海默症小鼠；“否”表示普通野生小鼠；
2、患病小鼠选用C57BL/6J-APP/PS1阿尔茨海默症小鼠；无病小鼠选用C57BL/6J野生型小鼠。
测试结果及分析如下：
一、不同部位细胞来源的溶酶体对于脑部的靶向性
将对比例1-5进行如下操作：随机选取规定要求的小鼠，分别向其尾静脉注射30mg/kg溶酶体标记探针标记后的不同来源的溶酶体(空白对比例注入等量的PBS)，2h后使用生理盐水灌流，剖出小鼠脑部。使用小动物成像系统进行荧光成像，酶联免疫检测仪测定吸光度。
如附图1所示，由左向右依次分别为对比例1-5。
观察发现：对比例2脑部小胶质细胞来源的溶酶体对于脑部的靶向性最强。
二、脑部小胶质细胞来源的溶酶体对于不同部位的靶向性
将对比例6-8进行如下操作：随机选取规定要求的小鼠，向其尾静脉注射30mg/kg溶酶体标记探针标记后的溶酶体(空白对比例注入等量的PBS)，2h 后使用生理盐水灌流，剖出小鼠相应部位。使用小动物成像系统进行荧光成像，酶联免疫检测仪测定吸光度。
如附图2所示，由上到下依次为脑、心、肺、肝、肾，其中肝的右侧较小的为脾，由左向右依次分别为对比例7,6,8。
观察发现：
脑部小胶质细胞来源溶酶体会在阿尔兹海默症小鼠脑部(对比例6)靶向性富集；
三、脑部小胶质细胞来源的溶酶体对于不同状态的鼠脑部靶向性
将对比例6、9、10、11进行如下操作：随机选取规定要求的小鼠，向其尾静脉注射30mg/kg溶酶体标记探针标记后的溶酶体(空白对比例注入等量的PBS)，2h后使用生理盐水灌流，剖出小鼠脑部。使用小动物成像系统进行荧光成像，酶联免疫检测仪测定吸光度。
如附图3所示，由上到下依次为对比例9、对比例10、对比例6、对比例11。实验数据如表3：
表3溶酶体对于不同状态的鼠脑部靶向性实验数据表
Figure PCTCN2022132394-appb-000005
观察发现：
脑部小胶质细胞来源溶酶体会在阿尔兹海默症小鼠脑部(对比例6)富集；
脑部小胶质细胞来源溶酶体会在老年无病小鼠脑部(对比例10)富集；
脑部小胶质细胞来源溶酶体不会在青年无病小鼠脑部(对比例11)富集；
结合老年小鼠和患病小鼠都会在脑部产生异常蛋白和糖缀合物，可得到如下结论：
脑部小胶质细胞来源溶酶体可以靶向进入患病小鼠病灶部位；溶酶体可能 会对延缓老年记忆力衰退产生作用。
四、脑部小胶质细胞来源溶酶体对原代神经元细胞毒性测试
首先按常规方法提取新生鼠原代神经元，将原代神经元按5×10 6/孔密度接种于96孔板中，每孔100μL，在37℃的CO 2培养箱孵育。
每2-3天换半液，第7天时向96孔板中加入不同浓度的溶酶体(100、200、300、400、500μg/mL)。
24小时后，吸弃培养基，向每孔加入1mg/mL的MTT溶液100μL，在37℃的CO 2培养箱避光孵育4小时。
吸弃MTT，向每孔加入150μL二甲基亚砜(DMSO)，在摇床上避光溶解10分钟。
使用酶联免疫检测仪在490nm波长下测定吸光度，计算细胞存活率。如附图4所示，应用graph pad软件进行统计学分析，组间比较采用t-test比较，无显著性差异。
结果显示：
脑部小胶质细胞来源溶酶体溶酶体不会影响细胞的存活率，对原代神经元细胞无毒性。
五、脑部小胶质细胞来源溶酶体对细胞外环境的影响测试
将N2a细胞接种于6孔板中，在37℃的CO 2培养箱孵育。待细胞铺满孔板80-90％时，在第0、4、6、8、10、12h，分别用含50μg/mL溶酶体的培养基替换相对应的6孔板中的旧培养基培养，期间于37℃的CO 2培养箱中继续孵育。取各孔培养基至离心管中，用pH测试仪测定溶酶体作用不同时间(0、2、4、6、8、12h)培养基的pH值。(见表3及附图5，其中附图5横坐标表示溶酶体加入的时间长度，从左到右数值分别依次对应表4的加入溶酶体的时间点)
表4溶酶体对环境影响实验数据表
Figure PCTCN2022132394-appb-000006
应用graph pad软件进行统计学分析，组间比较采用t-test，无显著性差异。
结果显示：
溶酶体不会影响细胞外环境的pH值。
六、脑部小胶质细胞来源溶酶体在患病脑部中降解毒性蛋白测试
取对比例7(老年有病空白组，WT组)、对比例10(老年无病给药组，APP/PS1组)、对比例12(老年有病给药组，APP/PS1LY组)，尾静脉注射0.5mg/kg溶酶体(空白组注射等量PBS)，每3天注射一次，连续注射一个月。给药结束后，麻醉实验小鼠，用0.9％生理盐水灌流小鼠，提取脑部海马和皮层蛋白质。
分别使用APP抗体(Biolegend，#SIG-39320)、p62抗体(博尔迈生物技术，PM045)、LC3抗体(细胞信号科技，#2775)、Actin抗体(柏奥易杰，BE0021-100)和Tetramer抗体(即APP抗体，Biolegend，#SIG-39320)，通过蛋白质印迹分析不同组别样品蛋白质的变化。(如附图6-7)。
结果显示：
尾静脉注射脑部小胶质细胞来源溶酶体可促进阿尔兹海默症小鼠脑内表达的毒性蛋白的降解。
七、脑部小胶质细胞来源溶酶体有效剂量测试
大剂量测试组：按常规方法提取APP/PS1新生鼠原代神经元，将原代神经元按5×10 6/孔密度接种于6孔板中，在37℃的CO2培养箱孵育。每2-3天换半液，7天后一次性给500μg/ml溶酶体，7天后提取蛋白质。
为了对比说明大浓度溶酶体对阿尔兹海默症小鼠原代神经元毒性蛋白的降解及自噬途径的作用，特设置如下对比组：
正常原代神经元组：按常规方法提取正常野生型新生鼠原代神经元，将原代神经元按5×10 6/孔密度接种于6孔板中，在37℃的CO2培养箱孵育。每2-3天换半液，14天后提取蛋白质。
空白组：与大剂量测试组相似，区别在不使用溶酶体进行给药。
分别使用APP抗体(Biolegend，#SIG-39320)、p62抗体(博尔迈生物技术，PM045)、Actin抗体(柏奥易杰，BE0021-100)，通过蛋白质印迹分析不同组别样品蛋白质的变化。(如附图8，从左到右分别为正常原代神经元组、空白组、大剂量测试组)
结果显示：
一次性外源给予大量溶酶体无法有效降解APP蛋白。
八、溶酶体水解酶过表达测试
将293T细胞接种于24孔板中，在37℃的CO2培养箱孵育。待细胞铺满孔板80-90％时，用CathepsinB(CatB)-GFP质粒及CathepsinD(CatD)-GFP质粒转染细胞，将CathepsinB-GFP质粒及CathepsinD-GFP质粒与Lipofectamine 2000分别混匀，静置20min后分别加入至细胞中，6h后将细胞培养基换为新鲜的培养基，24h后观察细胞内部的溶酶体组织蛋白酶B及组织蛋白酶D的过表达情况，得到结果如图9所示。
九、阿尔兹海默症鼠行为学-NOR测试
取对比例6(老年有病、给药)、对比例7(老年无病，不给药)、对比例9(老年无病，不给药)进行测试。
随机选取对比例6(老年有病、给药)小鼠，并根据小鼠体重向其尾静脉注射0.5mg/kg的溶酶体标记探针标记后的溶酶体，每3天注射一次，连续注射一个月。
随机选取对比例7(老年无病，不给药)，并根据小鼠体重向其尾静脉注射与取对比例6(老年有病、给药)等体积的PBS，其他条件与对比例6(老年有病、给药)相同。
随机选取9(老年无病，不给药)，并根据小鼠体重向其尾静脉注射与实施例3等体积的PBS，其他条件与对比例6(老年有病、给药)相同。
连续注射一个月后，进行如下测试：
新事物识别实验(NOR)
应用旷场测试箱，利用啮齿类动物喜欢接触和探索新奇事物的天性检测动物非空间记忆能力。实验分为三步：
①适应阶段：将小鼠依次放入没有任何物体的测试箱中，适应实验环境，连续2天，每天2次。
②熟悉阶段：在距侧壁10cm处放入两个相同物体(A和B)，将小鼠以背对物体方向放入，自由探索10min后将小鼠取出，记录小鼠物体探索时间及嗅探物体次数，连续3天，每天2次。
③测试阶段：24h后进行测试，将两个相同物体中B替换成另一物体C，即熟悉物体(A)和新奇物体(C)，记录小鼠10min对两物体的探索时间及嗅探次数。A和C的位置随机置换，以避免位置偏倚。
探索活动定义为：小鼠鼻子在≤5cm范围内直指向物体或直接接触物体时的行为，小鼠转头或坐在物体上时不被认为是对物体的探索。应用graph pad软件进行统计学分析，组间比较采用one-way。P<0.05为差异有统计学意义。
结果如附图10-11所示：
尾静脉注射溶酶体可改善阿尔兹海默症小鼠对事物的识别能力，改善海马相关学习记忆功能。
筑巢实验：
小鼠夜节律前2h，在鼠笼内放一些2cm×2cm的正方形纸巾(共三张厨房用纸)，24h后统计鼠的筑巢情况并拍照记录。
评分标准如下：
纸巾均匀铺展且未形成团簇为1分；
纸巾均匀铺展且形成松散团簇为2分；
纸巾均匀铺展在笼内1/2且形成团簇为3分；
纸巾均匀铺展在笼内1/3且形成团簇为4分；
纸巾均匀铺展在笼内1/4且形成团簇为5分。
应用graph pad软件进行统计学分析，组间比较采用one-way。
结果如附图12所示：
给药组筑巢评分较高，证明尾静脉注射溶酶体会改善阿尔兹海默症小鼠的社会性行为。
综合上述测试，可以得出如下结论：
1、外源溶酶体对于阿尔兹海默症为代表的治疗蛋白错误折叠或加工类疾病有治疗作用；
2、该治疗作用无毒副作用；
3、各种细胞来源的溶酶体中，脑部小胶质细胞来源效果最佳；
为了追求治疗效果的最优化，发明人进一步对溶酶体进行了改进，以增强药物的入脑效率和靶向性。并对进行了如下测试：
LPS预处理的脑部小胶质细胞来源的溶酶体靶向性测试
取应用例1(改性给药)、对比例6(未改性给药)、对比例7(空白对照)进行如下测试：
随机取应用例1，向其侧脑室注射LPS，在注射后6h向其尾静脉注射30mg/kg溶酶体标记探针标记后的LPS处理过的脑部小胶质细胞来源的溶酶体(细胞提取溶酶体前12h按1:1000的比例向培养基中加入0.1μg/mL的LPS，其余步骤同正常溶酶体提取步骤)，2h后使用生理盐水灌流，剖出小鼠脑部。
为了证明改造后的溶酶体针对病灶部位的靶向性更强，特设置如下对比例：
取对比例6，向其侧脑室注射LPS，在注射后6h向其尾静脉注射等量正常脑部小胶质细胞来源的溶酶体；其他条件与应用例1相同；
取对比例7，向其侧脑室注射LPS，在注射后6h向其尾静脉注射等体积PBS；其他条件与应用例1相同。
使用小动物成像系统进行荧光成像，得到结果如图13所示(从左到右依次分别为对比例7、对比例6、应用例1)：
LPS改性后的脑部小胶质细胞来源的溶酶体在脑部的富集靶向性优于直接提取的脑部小胶质细胞来源的溶酶体。
C5a预处理的脑部小胶质细胞来源的溶酶体靶向性测试
取应用例2(改性给药5nM)、应用例3(改性给药10nM)、对比例6(未改性给药)进行如下测试：
随机取应用例2，向其侧脑室注射LPS，在注射后6h向其尾静脉注射30mg/kg溶酶体标记探针标记后的C5a处理过的脑部小胶质细胞来源的溶酶体(细胞提取溶酶体前0.5h，混合后C5a浓度为10nmol/L，其余步骤同正常溶酶体提取步骤)，2h后使用生理盐水灌流，剖出小鼠脑部。
取应用例3，向其侧脑室注射LPS，在注射后6h向其尾静脉注射等量溶酶体标记探针标记后的C5a处理过的脑部小胶质细胞来源的溶酶体，混合后C5a浓度为10nmol/L，其他条件与应用例2相同；
取应用例6，向其侧脑室注射LPS，在注射后6h向其尾静脉注射等量正常脑部小胶质细胞来源的溶酶体；其他条件与应用例2相同。
使用小动物成像系统进行荧光成像，得到结果如图14所示(从左到右依次分别为对比例6、应用例2、应用例3)：
C5a改性后的脑部小胶质细胞来源的溶酶体在脑部的富集靶向性优于直接提取的脑部小胶质细胞来源的溶酶体。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (10)

  1. 一种改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述改性溶酶体来自经外界刺激改性的脑部小胶质细胞。
  2. 根据权利要求1所述的改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述外界刺激包括物理方式刺激、生物方式刺激、化学方式刺激、环境方式刺激中至少一种。
  3. 根据权利要求2所述的改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述物理方式刺激包括热刺激、力刺激、光刺激、电刺激。
  4. 根据权利要求2所述的改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述生物方式刺激包括利用补体因子或趋化因子刺激、利用多肽刺激、基因编辑。
  5. 根据权利要求2所述的改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述化学方式刺激包括炎症诱导因子刺激、自噬激活剂刺激、聚合物修饰表面、多孔有机框架材料封装。
  6. 根据权利要求2所述的改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述环境刺激包括乏氧环境刺激、乏糖环境刺激、乏氨基酸环境刺激。
  7. 根据权利要求4所述的改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述补体因子采用C5a，所述趋化因子采用CXC。
  8. 根据权利要求4所述的改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述基因编辑，采用过表达溶酶体主要功能蛋白的方式实施，过表达的蛋白包括ATP酶、组织蛋白酶B、组织蛋白酶D、组织蛋白酶K、组织蛋白酶L、酸性酰胺酶、酸性脂肪酶、α-半乳糖苷酶、α-L-艾杜糖醛酸酶、α-N-乙酰半乳糖胺酶、透明质酸酶、氯化物电压门控通道CLC-7、胱氨酸蛋白酶Cystinosin、溶酶体相关膜蛋白LAMP1和/或LAMP2、多功能转运内在膜蛋白LIMP-2、粘蛋白TRP阳离子通道Mucolipin、NPC细胞内胆固醇转运蛋白NPC1、溶质载体家族11和/或17。
  9. 根据权利要求5所述的改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述炎症诱导因子采用LPS。
  10. 根据权利要求5所述的改性溶酶体作为制备治疗蛋白错误折叠或加工类疾病药物的应用，其特征在于，所述自噬激活剂刺激，是采用雷帕霉素靶向蛋白mTOR的激活剂和/或转录因子EB的激活剂；其中，激活剂包括糖类化合物、苷类化合物、酮类化合物、抗生素类化合物。
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