Method for enhancing the ability of alpha-synuclein aggregates to damage cell membranes

Abstract

The present invention discloses a method for enhancing the ability of α-synuclein (α-Syn) aggregates to destroy cell membranes. 140 μL 0.01M PBS is used to dissolve 1 mg of recombinant human α-Syn, and then 70 μL PBS, healthy personnel, and PD patient plasma are added respectively and incubated at 37 ° C for 4 days under constant temperature oscillation. Acidic phospholipids 1-palmitoyl-2-oleoyl-sn-glycerol-3-phospho-L-serine (POPS) are used as raw materials. Small unilamellar liposomes (SUVs) are prepared by thin film dispersion-ultrasound method, and different concentrations of proteins are incubated with SUVs and SH-SY5Y cells respectively. The ability of α-Syn aggregates formed under different conditions to destroy cell membranes is observed by calcein leakage experiments. The results show that the α-Syn aggregates formed by incubation of PD patient plasma have stronger ability to destroy cell membranes.

Description

[Technical field]

[0001] The invention belongs to the technical field of alpha-synuclein aggregates and relates to a method for enhancing the cell membrane-destroying ability of alpha-synuclein aggregates. [Background technology]

[0002] Parkinson's disease (PD) is an α-synuclein disease with movement disorders as a common clinical manifestation. Its pathological characteristics are the progressive loss of a large number of dopaminergic neurons in the substantia nigra-striatum of the midbrain, and the presence of inclusion bodies (Lewy bodies, LBs) composed of abnormal aggregations of α-synuclein (α-synuclein, α-Syn) in the cell bodies of the remaining neurons. The clinical manifestations of PD are mainly motor symptoms such as bradykines and resting tremor, and non-motor symptoms such as mental, cognitive and sensory disorders.

[0003] α-Syn is a soluble protein composed of 140 amino acids, mostly distributed in the presynaptic terminals in the form of monomers. Under physiological conditions, α-Syn mainly exists in disordered and α-helical states; under pathological conditions, α-Syn is prone to aggregation, first forming soluble oligomers, and then assembling into ordered, β-fold-rich insoluble amyloid fibrils, which further aggregate to form intracellular inclusions, becoming the hallmark pathological structure of synucleinopathy. The results show that the stability of the hallmark pathological structures of different synucleinopathy varies. For example, compared with the hallmark pathological structure of PD, Lewy bodies (LBs), the hallmark pathological structure of MSA, glial cytoplasmic inclusions (GCIs), have stronger resistance to proteinase K (PK), and α-Syn derived from GCIs can cause more severe neuropathological changes in mice, suggesting that the α-Syn of the two may have different protein conformations. The conformational state of α-Syn is affected by pH, temperature, ionic strength, viscosity, etc. It can self-assemble and aggregate in vivo and in vitro. The formed aggregates can spread in a prion-like manner in the form of "seeds".

[0004] There have been studies on α-synuclein, such as Chinese patent application No. CN201610801424.7 on protofibril-binding antibodies and their use in the treatment and diagnosis of Parkinson's disease, Lewy body dementia and other α-synuclein diseases. The antibodies and fragments thereof have high affinity for human α-synuclein protofibrils and low binding to α-synuclein monomers. The composition comprises such antibodies or fragments, and such antibodies or fragments are used to detect α-synuclein protofibrils; prevent or delay the onset of neurodegenerative diseases with α-synuclein pathology or treat the disease; prepare pharmaceutical compositions for treating neurodegenerative diseases with α-synuclein pathology; diagnose or monitor the progression of neurodegenerative diseases with α-synuclein pathology, and reduce or inhibit α-synuclein aggregation.

[0005] α-Syn aggregates are the main neurotoxic substances in the pathogenesis of PD. Their neurotoxicity is closely related to their damage to cell membrane permeability and their intercellular transfer. They further damage the membranes of organelles such as lysosomes, Golgi bodies and mitochondria, leading to organelle dysfunction and ultimately neuronal damage. Existing research results suggest that α-Syn aggregates change the permeability of lipid membranes, destroy calcium homeostasis, and induce neuronal degeneration. α-Syn aggregates interfere with the normal structure of cell membranes by inserting into the lipid bilayer through their β-fragment-rich regions, forming cell membrane pore-like structures, changing the permeability of cell membranes, causing abnormal calcium influx, and leading to neuronal cell damage. Some research results show that the formation of α-Syn toxic aggregates is related to specific cell microenvironments. The plasma metabolic environment of PD patients can promote the formation of α-Syn aggregates, but the destructive effect of the aggregates formed by incubation on cell membranes is still unknown. [Summary of the invention]

[0006] In view of the problem that the formation of α-Syn toxic aggregates is related to a specific cell microenvironment in existing studies, and the metabolic environment of PD patient plasma can promote the formation of α-Syn aggregates, but the destructive effect of the aggregates formed by incubation on the cell membrane is still unknown, the present invention provides a method for enhancing the ability of α-synuclein aggregates to destroy cell membranes, and provides a basis for subsequent comparison of the differences in the ability of α-Syn aggregates incubated with phosphate buffered saline (PBS), healthy people's plasma (HC), and PD patient plasma to destroy cell membranes at the artificial lipid membrane and cell membrane levels.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for enhancing the ability of α-synuclein aggregates to damage cell membranes comprises the following steps:

[0009] 1) Preparation of α-Syn aggregates: 1 mg of recombinant human α-Syn was dissolved in 140 μL 0.01 M PBS, and then 70 μL of phosphate buffered saline (PBS), plasma from healthy subjects (HC group plasma), and plasma from patients with Parkinson's disease (PD patient plasma) were added to make the final concentration of α-Syn 2 μg / μL, and a preservative was added to prevent protein bacterial growth. The mixture was incubated in a constant temperature oscillating mixer at 37°C and 1800 g for 4 days, and stored in aliquots at -80°C.

[0010] 2) Western blot was used to detect and analyze the differences in the formation of α-Syn aggregates in each group: 400 ng of phosphate buffered saline (PBS) group, healthy plasma (HC plasma) group, and Parkinson's disease patient plasma (PD patient plasma) group were separated and transferred to PVDF membranes by polyacrylamide gel electrophoresis. After blocking with 5% skim milk powder at room temperature for 90 minutes, the membranes were incubated with anti-α-Syn monoclonal antibodies for 2 hours, and further incubated with goat anti-mouse fluorescent secondary antibodies at room temperature for 1 hour. The membranes were scanned and imaged under the ODYSSEY dual-color infrared fluorescence imaging system. The protein bands were quantitatively analyzed by Image J software to compare the aggregation differences of α-Syn aggregates formed under different conditions.

[0011] 3) Preparation of small unilamellar vesicles (SUVs) encapsulating 100 mM calcein: 400 μL of 10 mg / mL acidic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) was dissolved in 10 mL of chloroform, and the mixture was ultrasonicated in a water bath for 30 minutes, and then placed in a rotary evaporator and evaporated under reduced pressure at 30°C for 30 minutes to obtain a lipid film; 5 mL of 25 mM PBS solution containing 100 mM calcein was added, and the mixture was rotated at 30°C for hydration for 1 hour; ultrasonicated in an ice bath at a power of 300 W, with an ultrasonication period of 5 seconds on and 5 seconds off, and then for 10 minutes, 3 times, with a rest period of 10 minutes each time; the SUVs were dialyzed in a 2000 Da dialysis bag to remove free calcein;

[0012] 4) Analyze the particle size and morphology using Malvern nanometer particle size potential analyzer and transmission electron microscope: Take 1 mL of the prepared SUVs and analyze the particle size and distribution of the SUVs;

[0013] 5) Imaging of SUVs under transmission electron microscopy using negative staining:

[0014] Hydrophilization treatment: add 10 μL of 1 g / L BSA solution onto the support surface of the copper grid wrapped with carbon film for 1 minute, and absorb the excess liquid;

[0015] Sample adsorption: add 5 μL SUVs sample to the support surface of the copper grid, keep it for 3 minutes, and repeat the addition of sample 5 times;

[0016] Washing: add 5 μL 25 mM PBS onto the grid, leave for 10-15 seconds, repeat twice;

[0017] Negative staining: add 5 μL of 3% uranyl acetate onto the grid, quickly aspirate, repeat 3 times, keep for 1 minute for the last time, dry under infrared light, and image under transmission electron microscope;

[0018] 6) Determination of calcein release ratio after SUVs dialysis: At an excitation wavelength of 485 nm and an emission wavelength of 535 nm, the 100% dye leakage fluorescence intensity F of SUVs treated with 1% Triton X-100 and untreated SUVs after dialysis for different time periods was measured. Triton and the original fluorescence intensity F Con According to the formula, % calcein release = (F Triton / F Con )×100 to calculate the calcein release ratio after dialysis;

[0019] 7) Determination of calcein release ratio of SUVs with different concentrations: The lipid concentration was set to 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM. At an excitation wavelength of 485 nm and an emission wavelength of 535 nm, the 100% dye leakage fluorescence intensity F of SUVs treated with 1% Triton X-100 and untreated SUVs was measured after different dialysis times. Triton and the original fluorescence intensity F Con According to the formula, % calcein release = (F Triton / F Con )×100 to calculate the calcein release ratio after dialysis;

[0020] 8) Determination of calcein release ratio of artificial SUVs treated with protein: α-Syn protein incubated with PBS, plasma of healthy people, and plasma of patients with Parkinson's disease was added to 5 μM SUVs, respectively, to make the final protein concentrations of 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM, incubated at room temperature for 2 hours, and the fluorescence intensity value was detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm; according to the formula, % calcein release = (F-F0 / F Triton -F0)×100 to calculate the release ratio, where F Triton is the fluorescence intensity value obtained by destroying SUVs with 1% Triton X-100 to obtain 100% dye leakage, F is the fluorescence intensity value measured after treatment with α-Syn aggregates in each group, and F0 is the background fluorescence value of SUVs without protein addition;

[0021] 9) Cell culture: 10,000 SH-SY5Y cells / well were seeded in a poly-lysine-coated 96-well cell culture plate and cultured in a DMEM medium containing 10% FBS and 1% PS at 37° C. in a cell culture incubator containing 5% CO 2 ;

[0022] 10) Calcein permeation test in cell membrane: cells were incubated with 100 μL 2 μM calcein-AM working solution at 37°C in dark for 30 minutes, different concentrations of α-Syn aggregates were added and incubated in dark for 1 hour, cells were rinsed with PBS once, and fluorescence intensity values ​​were recorded at an excitation wavelength of 485 nm and an emission wavelength of 535 nm; according to the formula, % intracellular calcein = (F-F0 / F Con -F0)×100 to calculate the calcein release ratio, where F is the fluorescence value measured in each group, F0 is the background fluorescence value, and F Con Fluorescence values ​​measured for the Con group, and cell imaging was performed using an inverted fluorescence microscope;

[0023] 11) Determination of cell viability by MTS method: After treating cells with 8 μM α-Syn aggregates in each group for 48 hours, 20 μL of MTS solution was added to each well, incubated at 37°C for 1 hour, and the absorbance was measured at a wavelength of 490 nm;

[0024] 12) Statistical methods: SPSS 17.0 software was used for data statistics and analysis. Image J and GraphPad Prism 9.0 software were used for graphics. Normally distributed quantitative data were expressed as mean ± standard deviation (x ± s). One-way analysis of variance was used for comparison among the groups. P < 0.05 was considered statistically significant.

[0025] In the present invention:

[0026] The preservative in step 1) is selected from 0.02% v / v Proclin 300 preservative.

[0027] The anti-α-Syn monoclonal antibody in step 2) is diluted at a ratio of 1:10000; the goat anti-mouse fluorescent secondary antibody is diluted at a ratio of 1:10000.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1. The method of enhancing the ability of α-synuclein aggregates to destroy cell membranes described in the present invention, the results of the destruction of SUVs showed that the calcein induced by each group of α-Syn aggregates increased with the increase of protein concentration. When the protein concentration was 8 μM, the PD group released more calcein compared with the PBS and HC groups, and the difference was statistically significant (P<0.05), indicating that the α-Syn aggregates incubated with PD patient plasma had a stronger ability to destroy artificial liposome membranes (SUVs) than the α-Syn aggregates incubated with PBS and HC plasma.

[0030] 2. The method for enhancing the ability of α-synuclein aggregates to destroy cell membranes according to the present invention shows that the results of cell membrane destruction show that with the increase of protein concentration, after the α-Syn aggregates in each group destroyed the SH-SY5Y cell membrane, the intracellular calcein fluorescence value of each group gradually decreased. When the protein concentration was 8 μM, the intracellular calcein fluorescence value of the PD group was significantly lower than that of the PBS group and the HC group, and the difference was statistically significant (P<0.05), indicating that the α-Syn aggregates incubated with PD patient plasma had a stronger ability to destroy cell membranes than the α-Syn oligomers incubated with PBS and HC plasma.

[0031] 3. The method of enhancing the cell membrane destruction ability of α-synuclein aggregates described in the present invention, the results of cell viability experiments showed that the cell viability of each group treated with α-Syn aggregates was significantly decreased, and the cell viability of the PD group decreased less than that of the PBS group and the HC group, and the difference was statistically significant (P<0.05), indicating that after destroying the cell membrane, α-Syn aggregates further affected the survival of cells, and α-Syn aggregates incubated with PD patient plasma induced more cell death and had a greater cytotoxic effect.

Brief Description of the Drawings

[0032] Figure 1 is a graph comparing the aggregation of α-Syn aggregates incubated under different conditions in the experimental examples of the present invention;

[0033] Figure 2 is a diagram of the structural characteristics of SUVs in the experimental examples of the present invention;

[0034] Figure 3 is a graph showing the dialysis effect of SUVs in the experimental examples of the present invention;

[0035] Figure 4 is a graph comparing the destructive effects of various groups of α-Syn aggregates on SUVs in the experimental examples of the present invention;

[0036] Figure 5 is a diagram comparing the cell membrane-damaging effects of different α-Syn aggregates in the experimental examples of the present invention;

[0037] Figure 6 It is a graph showing the results of cell viability assayed by the MTS method in the experimental examples of the present invention. [Specific implementation method]

[0038] The specific implementation modes of the present invention are further described below in conjunction with examples.

[0039] Example:

[0040] A method for enhancing the ability of α-synuclein aggregates to damage cell membranes comprises the following steps:

[0041] 1) Preparation of α-Syn aggregates: 1 mg of recombinant human α-Syn was dissolved in 140 μL 0.01 M PBS, and then 70 μL of phosphate buffered saline (PBS), healthy plasma (HC group plasma), and Parkinson's disease patient plasma (PD patient plasma) were added to make the final concentration of α-Syn 2 μg / μL, and 0.02% v / v Proclin 300 preservative was added to prevent protein bacterial growth, and incubated in a constant temperature shaking mixer at 37°C and 1800g for 4 days, and stored in aliquots at -80°C;

[0042] 2) Western blot was used to detect and analyze the differences in the formation of α-Syn aggregates in each group: 400 ng of phosphate buffered saline (PBS) group, healthy plasma (HC plasma) group, and Parkinson's disease patient plasma (PD patient plasma) group were separated and transferred to PVDF membranes by polyacrylamide gel electrophoresis. After blocking with 5% skim milk powder at room temperature for 90 minutes, the membranes were incubated with anti-α-Syn monoclonal antibodies (1:10000) for 2 hours, and further incubated with goat anti-mouse fluorescent secondary antibodies (1:10000) at room temperature for 1 hour. The membranes were scanned and imaged under the ODYSSEY dual-color infrared fluorescence imaging system. The protein bands were quantitatively analyzed in grayscale using Image J software to compare the aggregation differences of α-Syn aggregates formed under different conditions.

[0043] 3) Preparation of small unilamellar vesicles (SUVs) encapsulating 100 mM calcein: 400 μL of 10 mg / mL acidic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) was dissolved in 10 mL of chloroform, and the mixture was ultrasonicated in a water bath for 30 minutes, and then placed in a rotary evaporator and evaporated under reduced pressure at 30°C for 30 minutes to obtain a lipid film; 5 mL of 25 mM PBS solution containing 100 mM calcein was added, and the mixture was rotated at 30°C for hydration for 1 hour; ultrasonicated in an ice bath at a power of 300 W, with an ultrasonication period of 5 seconds on and 5 seconds off, and then for 10 minutes, 3 times, with a rest period of 10 minutes each time; the SUVs were dialyzed in a 2000 Da dialysis bag to remove free calcein;

[0044] 4) Analyze the particle size and morphology using Malvern nanometer particle size potential analyzer and transmission electron microscope: Take 1 mL of the prepared SUVs and analyze the particle size and distribution of the SUVs;

[0045] 5) Imaging of SUVs under transmission electron microscopy using negative staining:

[0046] Hydrophilization treatment: add 10 μL of 1 g / L BSA solution onto the support surface of the copper grid wrapped with carbon film for 1 minute, and absorb the excess liquid;

[0047] Sample adsorption: add 5 μL SUVs sample to the support surface of the copper grid, keep it for 3 minutes, and repeat the addition of sample 5 times;

[0048] Washing: add 5 μL 25 mM PBS onto the grid, leave for 10-15 seconds, repeat twice;

[0049] Negative staining: add 5 μL of 3% uranyl acetate onto the grid, quickly aspirate, repeat 3 times, keep for 1 minute for the last time, dry under infrared light, and image under transmission electron microscope;

[0050] 6) Determination of calcein release ratio after SUVs dialysis: At an excitation wavelength of 485 nm and an emission wavelength of 535 nm, the 100% dye leakage fluorescence intensity F of SUVs treated with 1% Triton X-100 and untreated SUVs after dialysis for different time periods was measured. Triton and the original fluorescence intensity F Con According to the formula, % calcein release = (F Triton / F Con )×100 to calculate the calcein release ratio after dialysis;

[0051] 7) Determination of calcein release ratio of SUVs with different concentrations: The lipid concentration was set to 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM. At an excitation wavelength of 485 nm and an emission wavelength of 535 nm, the 100% dye leakage fluorescence intensity F of SUVs treated with 1% Triton X-100 and untreated SUVs was measured after different dialysis times. Triton and the original fluorescence intensity F Con According to the formula, % calcein release = (F Triton / F Con )×100 to calculate the calcein release ratio after dialysis;

[0052] 8) Determination of calcein release ratio of artificial SUVs treated with protein: α-Syn protein incubated with PBS, plasma of healthy people, and plasma of patients with Parkinson's disease was added to 5 μM SUVs, respectively, to make the final protein concentrations of 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM, incubated at room temperature for 2 hours, and the fluorescence intensity value was detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm; according to the formula, % calcein release = (F-F0 / F Triton -F0)×100 to calculate the release ratio, where F Triton is the fluorescence intensity value obtained by destroying SUVs with 1% Triton X-100 to obtain 100% dye leakage, F is the fluorescence intensity value measured after treatment with α-Syn aggregates in each group, and F0 is the background fluorescence value of SUVs without protein addition;

[0053] 9) Cell culture: 10,000 SH-SY5Y cells / well were seeded in a poly-lysine-coated 96-well cell culture plate and cultured in a DMEM medium containing 10% FBS and 1% PS at 37° C. in a cell culture incubator containing 5% CO 2 ;

[0054] 10) Calcein permeation test in cell membrane: cells were incubated with 100 μL 2 μM calcein-AM working solution at 37°C in dark for 30 minutes, different concentrations of α-Syn aggregates were added and incubated in dark for 1 hour, cells were rinsed with PBS once, and fluorescence intensity values ​​were recorded at an excitation wavelength of 485 nm and an emission wavelength of 535 nm; according to the formula, % intracellular calcein = (F-F0 / F Con -F0)×100 to calculate the calcein release ratio, where F is the fluorescence value measured in each group, F0 is the background fluorescence value, and F Con Fluorescence values ​​measured for the Con group, and cell imaging was performed using an inverted fluorescence microscope;

[0055] 11) Determination of cell viability by MTS method: After treating cells with 8 μM α-Syn aggregates in each group for 48 hours, 20 μL of MTS solution was added to each well, incubated at 37°C for 1 hour, and the absorbance was measured at a wavelength of 490 nm;

[0056] 12) Statistical methods: SPSS 17.0 software was used for data statistics and analysis. Image J and GraphPad Prism 9.0 software were used for graphics. Normally distributed quantitative data were expressed as mean ± standard deviation (x ± s). One-way analysis of variance was used for comparison among the groups. P < 0.05 was considered statistically significant.

[0057] Experimental example:

[0058] Materials and Methods

[0059] 1. Experimental Materials and Reagents

[0060] 1. Sources of cases and blood samples PD group: Peripheral blood samples of 10 patients with Parkinson's disease diagnosed in the Department of Neurology, Affiliated Hospital of Guilin Medical College from June 2020 to December 2020 were collected, including 5 males and 5 females. Inclusion criteria for PD patients: Meet the clinical diagnostic criteria for Parkinson's disease published by the International Parkinson and Movement Disorders Society in 2015. The normal plasma group selected healthy people with matched age and gender and without serious underlying diseases such as hypertension and diabetes. Use heparin-containing anticoagulant tubes to collect 10mL of peripheral venous blood from the above HC group and PD group, centrifuge at 1000rpm, collect supernatant plasma and blood cells in EP tubes, and store them at -80℃ for later use.

[0061] 2. Cells and main reagents: Human neuroblastoma SH-SY5Y cells (Chinese Academy of Sciences, China); recombinant human α-Syn protein and mouse anti-α-Syn monoclonal antibody (3D5) were kindly donated by Professor Yu Shun of Xuanwu Hospital, Capital Medical University; goat anti-mouse fluorescent secondary antibody (LI-COR, USA); Dulbecco's Modified Eagle Medium (DMEM) culture medium (Gibco, USA); fetal bovine serum (FBS) (Gibco, USA); 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) (Merck, USA); Calcein-AM (Shanghai Bio-Technology Co., Ltd., China); Calcein (Shanghai Yuanye Biotechnology Co., Ltd., China); chloroform (Xilong Scientific Co., Ltd., China); polyvinylidene fluoride (PVDF) (Millipore, USA); penicillin-streptomycin (PS) (Gibco, USA); bovine serum albumin (BSA) (Amresco, USA).

[0062] 3. Main instruments: electrophoresis apparatus, electrotransfer apparatus (Bio-Rad, USA); ODYSSEY two-color infrared fluorescence imaging system (LI-COR, USA); constant temperature oscillating mixer (Thermo Mixer F1.5) (Thermo Scientific, USA); microplate reader (Molecular Devices, USA); rotary evaporator (Zhengzhou Great Wall Science and Technology and Trade Co., Ltd., China); transmission electron microscope (Hitachi High-Tech, Japan); nanoparticle size potentiometer (Malvern, UK); inverted fluorescence microscope (Nikon, Japan); ultrasonic cell crusher (Shanghai Jingxin Industrial Development Co., Ltd., China).

[0063] 2. Experimental Methods

[0064] 1. Preparation of α-Syn aggregates: Dissolve 1 mg of recombinant human α-Syn in 140 μL 0.01 M PBS, then add 70 μL PBS, HC group plasma, and PD patient plasma respectively to make the final concentration of α-Syn 2 μg / μL, and add 0.02% v / v Proclin 300 preservative to prevent protein bacterial growth, incubate in a constant temperature oscillating mixer at 37°C and 1800g for 4 days, and store in aliquots at -80°C;

[0065] 2. Western blot was used to detect and analyze the differences in the formation of α-Syn aggregates in each group: 400 ng of α-Syn aggregates in the PBS group, HC group, and PD group were separated and transferred to a PVDF membrane by polyacrylamide gel electrophoresis, and then blocked with 5% skim milk powder at room temperature for 90 minutes, incubated with anti-α-Syn monoclonal antibody (1:10000) for 2 hours, and further incubated with goat anti-mouse fluorescent secondary antibody (1:10000) at room temperature for 1 hour. The membrane was scanned and imaged under the ODYSSEY two-color infrared fluorescence imaging system, and the protein bands were quantitatively analyzed by Image J software to compare the aggregation differences of α-Syn aggregates formed under different conditions.

[0066] 3. Preparation of small unilamellar vesicles (SUVs) encapsulating 100 mM calcein: 400 μL 10 mg / mL POPS was dissolved in 10 mL chloroform, and the mixture was ultrasonicated in a water bath for 30 minutes. The mixture was placed in a rotary evaporator and evaporated under reduced pressure at 30°C for 30 minutes to obtain a lipid film; 5 mL 25 mM PBS solution containing 100 mM calcein was added, and the mixture was rotated at 30°C for hydration for 1 hour; ultrasonicated in an ice bath with an ultrasonic power of 300 W, ultrasonicated for 5 seconds on and 5 seconds off, and ultrasonicated for 10 minutes, 3 times, with a rest period of 10 minutes each time, and the SUVs were dialyzed with a 2000 Da dialysis bag to remove free calcein. The particle size and morphology were analyzed using a Malvern nanoparticle size potentiometer and a transmission electron microscope;

[0067] 4. Malvern Nano Particle Size Potentiometer to measure the particle size of SUVs: Take 1 mL of the prepared SUVs and analyze the particle size and distribution of SUVs;

[0068] 5. Transmission electron microscopy: Negative staining was used to image SUVs under transmission electron microscopy. Hydrophilic treatment: Add 10 μL of 1g / L BSA solution on the support surface of the copper grid wrapped with carbon film for 1 minute, and remove the excess liquid; Sample adsorption: Add 5 μL of SUVs sample on the support surface of the copper grid, retain for 3 minutes, and repeat the addition of samples 5 times; Washing: Add 5 μL of 25mM PBS on the grid, retain for 10-15 seconds, and repeat 2 times; Negative staining: Add 5 μL of 3% uranyl acetate on the grid, quickly absorb, repeat 3 times, retain for 1 minute for the last time, dry under infrared light, and image under transmission electron microscope.

[0069] 6. Determination of calcein release ratio after SUVs dialysis: At an excitation wavelength of 485 nm and an emission wavelength of 535 nm, the 100% dye leakage fluorescence intensity F of SUVs treated with 1% Triton X-100 and untreated SUVs after dialysis for different time periods was measured. Triton and the original fluorescence intensity F Con According to the formula % calcein release = (F Triton / F Con )×100 to calculate the calcein release ratio after dialysis.

[0070] 7. Determination of calcein release ratio of SUVs with different concentrations: The lipid concentration was set to 5μM, 10μM, 20μM, 40μM, and 80μM. At an excitation wavelength of 485nm and an emission wavelength of 535nm, the 100% dye leakage fluorescence intensity F of SUVs treated with 1% Triton X-100 and untreated was measured after different dialysis times. Triton and the original fluorescence intensity F Con According to the formula % calcein release = (F Triton / F Con )×100 to calculate the calcein release ratio after dialysis;

[0071] 8. Determination of calcein release ratio of artificial SUVs treated with protein: α-Syn protein incubated with PBS, plasma of healthy people, and plasma of PD patients was added to 5 μM SUVs, respectively, to make the final concentration of protein 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM, incubated at room temperature for 2 hours, and the fluorescence intensity value was detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. According to the formula, % calcein release = (F-F0 / F Triton -F0)×100 to calculate the release ratio, where F Triton is the fluorescence intensity value obtained by destroying SUVs with 1% Triton X-100 to obtain 100% dye leakage, F is the fluorescence intensity value measured after treatment with α-Syn aggregates in each group, and F0 is the background fluorescence value of SUVs without protein addition;

[0072] 9. Cell culture: 10,000 SH-SY5Y cells / well were inoculated in a poly-lysine-coated 96-well cell culture plate and cultured in a cell culture incubator at 37°C and 5% CO2 using DMEM medium containing 10% FBS and 1% PS;

[0073] 10. Calcein permeation test in cell membrane: Incubate cells with 100 μL 2 μM calcein-AM working solution at 37°C in dark for 30 minutes, add different concentrations of α-Syn aggregates and continue incubating in dark for 1 hour, rinse cells once with PBS, record fluorescence intensity at excitation wavelength of 485 nm and emission wavelength of 535 nm; according to the formula, % intracellular calcein = (F-F0 / F Con -F0)×100 to calculate the calcein release ratio, where F is the fluorescence value measured in each group, F0 is the background fluorescence value, and F Con Fluorescence values ​​measured for the Con group, and cell imaging was performed using an inverted fluorescence microscope;

[0074] 11. Determination of cell viability by MTS method: After treating cells with 8 μM α-Syn aggregates in each group for 48 hours, add 20 μL MTS solution to each well, incubate at 37°C for 1 hour, and measure the absorbance at a wavelength of 490 nm.

[0075] 3. Statistical Methods

[0076] SPSS 17.0 software was used for data statistics and analysis. Image J and GraphPad Prism 9.0 software were used for graphics. Normally distributed quantitative data were expressed as mean ± standard deviation (x ± s). One-way analysis of variance was used for comparison among the groups. P < 0.05 was considered statistically significant.

[0077] result

[0078] 1. General Information

[0079] The average ages of the patients in the HC group and the PD group were (64.1±6.12) and (61.1±5.84) years, respectively, with no significant difference (t=1.130, P=0.300). The average disease duration of the PD group was (6.25±5.32) years.

[0080] 2. Formation and differences of α-Syn aggregates in PBS, HC, and PD groups

[0081] The relative molecular mass of α-Syn protein monomer is about 18000Da. The α-Syn aggregates formed by oscillating incubation in each group had significant differences in aggregation level and size. The aggregates in the PBS group were mainly concentrated at a relative molecular weight of about 36000Da (dimer); in addition to the distribution of dimers, the aggregates formed in the HC and PD groups formed higher molecular weight aggregates at relative molecular weights of 72000Da (tetramer), 90000Da (pentamer) and 108000Da (hexamer). Compared with the PBS and HC groups, the PD group formed more aggregates, and the difference was statistically significant (P<0.05).

[0082] Figure 1 Comparison of the aggregation of α-Syn aggregates incubated under different conditions (A: Western blotting shows the protein band results; B: quantitative comparison of the dimer protein (relative molecular weight 35000Da-40000Da) formed by the three groups of incubation; C: quantitative comparison of the multimer protein (relative molecular weight>40000Da) formed by the three groups of incubation). Compared with the PBS group, a P<0.05; compared with HC group, b P<0.05.

[0083] 3. Particle size distribution and electron microscopy results of SUVs

[0084] The particle size and distribution of SUVs were analyzed on a Malvern nanoparticle size potentiometer. The results showed that the average particle size of SUVs was 70.1 nm, the dispersion coefficient was 0.233, the particle size distribution of SUVs was narrow, and the peak was normally distributed, indicating that the particle size of the prepared SUVs was relatively concentrated and uniform ( Figure 2 A); The morphology of SUVs was observed under an electron microscope using the negative staining method. The results showed that the background of the image was light black, and the spherical structures of varying sizes, darker in the middle, and white around were SUVs ( Figure 2 B), the particle size is consistent with the results of the Malvern Nano-Particle Size Potentiometer particle size distribution range.

[0085] Figure 2 Structural characteristics of SUVs (A: The particle size of the sample was detected by nanoparticle size potentiometer, and the average particle size of SUVs was 70.1nm; B: Transmission electron microscopy result of SUVs (scale bar, 100nm)).

[0086] 4. SUVs Dialysis

[0087] When there is less free calcein outside, the fluorescence emitted by the internal calcein after being released into the external medium is stronger. In order to remove the free calcein, we first perform dialysis. The dialysis effect was evaluated by using 1% Triton X-100 to break the membrane to obtain the ratio of the fluorescence intensity value of 100% dye leakage to the fluorescence intensity value of untreated SUVs. The results showed that the fluorescence intensity ratio gradually increased with the extension of dialysis time, indicating that the free calcein content continued to decrease ( Figure 3 A); however, the results of the membrane rupture experiment with different concentrations of SUVs showed that at the same dialysis time, there was no significant difference in the fluorescence intensity ratio after membrane rupture with different concentrations of SUVs ( Figure 3 B).

[0088] Figure 3 Dialysis effect of SUVs (A: relationship between the fluorescence intensity ratio of SUVs after membrane rupture and dialysis time; B: comparison of the fluorescence intensity ratio of SUVs of different concentrations after membrane rupture after 168 hours of dialysis).

[0089] 5. Destructive effects of α-Syn aggregates formed under different incubation conditions on SUVs

[0090] In order to verify the destructive effect of α-Syn aggregates on SUVs, we incubated different concentrations of α-Syn aggregates in each group with 5μM SUVs at room temperature for 2 hours. The results showed that the calcein released by each group of α-Syn aggregates increased with the increase of protein concentration. When the protein concentration was <4μM, the amount of calcein released induced by α-Syn aggregates in each group increased slowly, and there was no significant difference between the groups. At a protein concentration of 8μM, the amount of calcein released induced by the PD group was significantly increased compared with the PBS and HC groups, and the difference was statistically significant (P<0.05). At a protein concentration of 16μM, the calcein released induced by the PBS group, HC group and PD group all increased significantly ( Figure 4 A). Among them, when the protein concentration was 8 μM, the calcein released by the PD group was the most different from that of the PBS group and the HC group, and the difference was statistically significant (P<0.05) ( Figure 4 B).

[0091] Figure 4 Comparison of the destructive effect of α-Syn aggregates in each group on SUVs (A: Comparison of calcein leakage after incubation of different concentrations of α-Syn aggregates in each group with 5μM SUVs, with the maximum fluorescence intensity value of 100% obtained by adding 1% Triton X-100 to break the membrane as the compared value; B: Comparison of calcein leakage of SUVs when the concentration of α-Syn aggregates was 8μM), compared with the PBS group, a P<0.05; compared with HC group, b P<0.05.

[0092] 6. Damage to cell membranes by α-Syn aggregates formed under different incubation conditions

[0093] To explore the differences in cell membrane damage caused by α-Syn aggregates formed by incubation under different conditions, we treated SH-SY5Y cells pre-incubated with calcein-AM with each group of α-Syn aggregates. The results of fluorescence detection showed that the intracellular calcein fluorescence values ​​of each group decreased with the increase of protein concentration. When the protein concentration was <4μM, the intracellular calcein fluorescence values ​​of each group decreased slowly after treatment with α-Syn aggregates, and there was no significant difference between the groups. When the protein concentration was 8μM, the intracellular calcein fluorescence value of the PD group decreased significantly compared with the PBS group and HC group, and the difference was statistically significant (P<0.05). When the protein concentration was 16μM, the intracellular calcein fluorescence values ​​of the PBS group, HC group and PD group all decreased significantly ( Figure 5 A). Among them, when the protein was 8 μM, the intracellular calcein fluorescence value of the PD group was the most different from that of the PBS group and the HC group, and the difference was statistically significant (P<0.05) ( Figure 5 B).

[0094] Furthermore, the fluorescence intensity of intracellular calcein after treatment with 8 μM of each group protein was observed using an inverted fluorescence microscope. The results showed that compared with the control group (Con), the fluorescence intensity of the PBS, HC and PD groups was significantly weakened, among which the fluorescence intensity of the PD group was the weakest ( Figure 5 C). The statistical results showed that compared with the Con group, the relative fluorescence intensity values ​​of the PBS, HC and PD groups were significantly weakened, and the difference was statistically significant (P<0.05). Among them, compared with the PBS group and the HC group, the relative fluorescence intensity value of the PD group was lower, and the difference was statistically significant (P<0.05) ( Figure 5 D).

[0095] Figure 5 Comparison of the cell membrane damage effects of different α-Syn aggregates (A: Comparison of intracellular calcein fluorescence values ​​after incubation of SH-SY5Y cells with different concentrations of α-Syn aggregates, with the fluorescence intensity of the untreated group cells as the comparison value; B: Comparison of intracellular calcein fluorescence values ​​when the protein concentration is 8 μM, compared with the Con group, a P<0.05; compared with PBS group, b P<0.05; compared with HC group, c P<0.05; C: Calcein fluorescence intensity results after incubation of 8μM α-Syn aggregates in each group with SH-SY5Y cells (scale bar, 10μm); D: Quantitative comparison of the fluorescence intensity values ​​of the images), compared with the Con group, aP<0.05; compared with PBS group, b P<0.05; compared with HC group, c P<0.05.

[0096] 7. Determination of cell viability after treatment with α-Syn aggregates formed under different incubation conditions

[0097] To explore the effect of α-Syn aggregates in each group on cell survival after damaging the cell membrane, we measured the cell viability of SH-SY5Y cells after 48 hours of treatment with α-Syn aggregates. The results showed that compared with the Con group, the cell viability of each group treated with α-Syn aggregates was significantly decreased, among which the PD group decreased most significantly, and the difference was statistically significant (P<0.05). Compared with the PBS group and the HC group, the cell viability of the PD group decreased less, and the difference was statistically significant (P<0.05) ( Figure 6 ).

[0098] Figure 6 The cell viability results determined by MTS method were compared with those of the Con group. a P<0.05; compared with PBS group, b P<0.05; compared with HC group, c P<0.05.

[0099] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for enhancing the ability of α-synuclein aggregates to damage cell membranes, characterized in that: The steps include: 1) Preparation of α-Syn aggregates: Dissolve 1 mg of recombinant human α-Syn in 140 μL 0.01 M PBS, then add 70 μL of plasma from Parkinson's disease patients to make the final concentration of α-Syn 2 μg / μL, add preservatives to prevent protein bacterial growth, incubate in a constant temperature oscillating mixer at 37°C and 1800g for 4 days, and store in aliquots at -80°C; 2) Western blot analysis of the formation difference of α-Syn aggregates: 400 ng of α-Syn aggregates in the plasma group of Parkinson's disease patients were separated by polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk powder at room temperature for 90 minutes, anti-α-Syn monoclonal antibody was incubated for 2 hours, and goat anti-mouse fluorescent secondary antibody was further incubated at room temperature for 1 hour. The membrane was scanned and imaged under the ODYSSEY two-color infrared fluorescence imaging system. The protein bands were quantitatively analyzed by Image J software to compare the aggregation differences of α-Syn aggregates formed under different conditions. 3) Preparation of small unilamellar liposomes encapsulating 100 mM calcein: 400 μL of 10 mg / mL acidic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine was dissolved in 10 mL of chloroform, and the mixture was ultrasonicated in a water bath for 30 minutes. The mixture was placed in a rotary evaporator and evaporated under reduced pressure at 30°C for 30 minutes to obtain a lipid film; 5 mL of 25 mM PBS solution containing 100 mM calcein was added, and the mixture was rotated at 30°C for hydration for 1 hour; the mixture was ultrasonicated in an ice bath at a power of 300 W, with an ultrasonication period of 5 seconds on and 5 seconds off, and then for 10 minutes, 3 times, with a rest period of 10 minutes each time; the SUVs were dialyzed in a 2000 Da dialysis bag to remove free calcein; 4) Analyze the particle size and morphology using Malvern nanometer particle size potential analyzer and transmission electron microscope: Take 1 mL of the prepared SUVs and analyze the particle size and distribution of the SUVs; 5) Imaging of SUVs under transmission electron microscopy using negative staining: Hydrophilization treatment: add 10 μL of 1 g / L BSA solution onto the support surface of the copper grid wrapped with carbon film for 1 minute, and absorb the excess liquid; Sample adsorption: add 5 μL SUVs sample to the support surface of the copper grid, keep it for 3 minutes, and repeat the addition of sample 5 times; Washing: add 5 μL 25 mM PBS onto the grid, leave for 10-15 seconds, repeat twice; Negative staining: add 5 μL of 3% uranyl acetate onto the grid, quickly aspirate, repeat 3 times, keep for 1 minute for the last time, dry under infrared light, and image under transmission electron microscope; 6) Determination of calcein release ratio after SUVs dialysis: At an excitation wavelength of 485 nm and an emission wavelength of 535 nm, the 100% dye leakage fluorescence intensity F of SUVs treated with 1% Triton X-100 and untreated SUVs after dialysis for different time periods was measured. Triton and the original fluorescence intensity F Con According to the formula, % calcein release = (F Triton / F Con )×100 to calculate the calcein release ratio after dialysis; 7) Determination of calcein release ratio of SUVs with different concentrations: The lipid concentration was set to 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM. At an excitation wavelength of 485 nm and an emission wavelength of 535 nm, the 100% dye leakage fluorescence intensity F of SUVs treated with 1% Triton X-100 and untreated SUVs was measured after different dialysis times. Triton and the original fluorescence intensity F Con According to the formula, % calcein release = (F Triton / F Con )×100 to calculate the calcein release ratio after dialysis; 8) Determination of calcein release ratio of protein-treated artificial SUVs: α-Syn protein incubated with plasma from Parkinson's disease patients was added to 5 μM SUVs to make the final protein concentrations 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM, respectively. The mixture was incubated at room temperature for 2 hours, and the fluorescence intensity was detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. According to the formula, % calcein release = (F-F0 / F Triton -F0)×100 to calculate the release ratio, where F Triton is the fluorescence intensity value obtained by destroying SUVs with 1% Triton X-100 to obtain 100% dye leakage, F is the fluorescence intensity value measured after treatment with α-Syn aggregates, and F0 is the background fluorescence value of SUVs without protein addition; 9) Cell culture: 10,000 SH-SY5Y cells / well were seeded in a poly-lysine-coated 96-well cell culture plate and cultured in a DMEM medium containing 10% FBS and 1% PS at 37° C. in a cell culture incubator containing 5% CO 2 ; 10) Calcein permeation test in cell membrane: cells were incubated with 100 μL 2 μM calcein-AM working solution at 37°C in dark for 30 minutes, different concentrations of α-Syn aggregates were added and incubated in dark for 1 hour, cells were rinsed with PBS once, and fluorescence intensity values ​​were recorded at an excitation wavelength of 485 nm and an emission wavelength of 535 nm; according to the formula, % intracellular calcein = (F-F0 / F Con -F0)×100 to calculate the calcein release ratio, where F is the measured fluorescence value, F0 is the background fluorescence value, and F Con Fluorescence values ​​measured for the Con group, and cell imaging was performed using an inverted fluorescence microscope; 11) Determination of cell viability by MTS method: After treating cells with 8 μM α-Syn aggregates for 48 hours, add 20 μL MTS solution to each well, incubate at 37°C for 1 hour, and measure the absorbance at a wavelength of 490 nm; 12) Statistical methods: SPSS 17.0 software was used for data statistics and analysis, Image J and GraphPad Prism 9.0 software was used for drawing. Normally distributed quantitative data were expressed as mean ± standard deviation, x ± s. One-way analysis of variance was used for comparison among the groups. P < 0.05 was considered statistically significant.

2. The method for enhancing the cell membrane destructive ability of α-synuclein aggregates according to claim 1, characterized in that: The preservative in step 1) is selected from 0.02% v / v Proclin 300 preservative.

3. The method for enhancing the cell membrane destructive ability of α-synuclein aggregates according to claim 1, characterized in that: The anti-α-Syn monoclonal antibody in step 2) is diluted at a ratio of 1:10000; the goat anti-mouse fluorescent secondary antibody is diluted at a ratio of 1:10000.

Images