Application of targeted granzyme B in immunotherapy medicine for neuronal degeneration of Parkinson's disease

Abstract

The invention discloses an application of targeted granzyme B in a drug for immunotherapy of neuronal degeneration of Parkinson's disease, relates to an application of the granzyme B in protection of dopaminergic neurons after silencing of the granzyme B in inflammatory helper Th17 cells, and belongs to the technical field of biological medicines. According to the application, a GZMB-shRNA plasmid is adopted to transfect Th17 cells, so that the GZMB expression of the Th17 cells is reduced, the transfected Th17 cells and the non-transfected Th17 cells are added into dopaminergic neurons cultured in vitro, and the apoptosis and necrosis conditions of the neurons are observed; the result shows that GZMB silencing in the Th17 cells can obviously inhibit the damage effect of the Th17 cells on dopaminergic neurons.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of targeted granzyme B in immunotherapy drugs for Parkinson's disease neuron degeneration. Background Art

[0002] Parkinson's disease (PD) is a neurodegenerative disease that occurs frequently worldwide. Although the exact etiology of PD remains unclear, studies of the body's immune response are believed to contribute to the progression of the disease. The link between human immune dysfunction and PD is supported by data showing that PD is associated with infiltration of peripheral immune cells into the central nervous system, chronic neuroinflammation, and immune-related gene mutations. The recent identification of autoreactive T cells targeting α-synuclein (α-Syn), the aggregation of which is a hallmark of PD-related neuropathology, suggests that regulating immune dysfunction can limit PD-related neurodegeneration, pointing to the role of immune dysfunction in PD. The field has been looking for further evidence to further confirm the involvement of the neuroimmune axis in PD, demonstrating that immune cells can directly promote the death of autologous neurons.

[0003] It has been reported that IL-17-producing T cells are drivers of PD-associated neurodegeneration. In the evaluation of T cell effector responses in PD patients, CD4 + The levels of IL-17 produced by T cells (commonly known as Th17 cells) were much higher. To determine whether IL-17 production could underlie T cell-mediated neuronal cell death, co-cultures of midbrain neurons and T cells were treated with IL-17 or IL-17 receptor (IL-17R) neutralizing antibodies, and it was found that the disappearance of IL-17 signals completely rescued T cell-mediated neuronal cell death in PD co-cultures, indicating that Th17 cells play a crucial role in neuronal cell death. In addition, in the PD model with high α-Syn expression, the lack of CD4 + Mice that developed T cells were also found to be protected against neurodegeneration.

[0004] These studies have opened up new areas of understanding of how T cell responses lead to neurodegeneration in PD. The molecular mechanisms that mediate the neurodegenerative properties of Th17 cells need to be further explored, which may provide new targets for immunotherapy of PD, a neurodegenerative disease. Summary of the invention

[0005] Technical issues solved:

[0006] In response to the deficiencies in the prior art, the present application provides an application of targeted granzyme B in immunotherapy for Parkinson's disease neuronal degeneration, wherein GZMB-shRNA plasmid is used to transfect Th17 cells to downregulate the expression of GZMB, and transfected and untransfected Th17 cells are added to dopaminergic neurons cultured in vitro to observe the apoptosis and necrosis of neurons. The results indicate that silencing GZMB in Th17 cells can significantly inhibit the damaging effect of Th17 cells on dopamine neurons (P<0.05).

[0007] Technical solution:

[0008] To achieve the above objectives, this application is implemented through the following technical solutions:

[0009] Application of targeted granzyme B in immunotherapy for neuronal degeneration in Parkinson's disease.

[0010] Furthermore, the Parkinson's disease neuron degeneration immunotherapy drug is in an injectable or non-injectable dosage form.

[0011] Furthermore, the Parkinson's disease neuron degeneration immunotherapy drug is an injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

[0012] Furthermore, the Parkinson's disease neuron degeneration immunotherapy drug includes targeted granzyme B and pharmaceutically acceptable excipients.

[0013] Furthermore, the auxiliary materials include one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

[0014] Principle explanation: The application of granzyme B (GZMB) silencing in inflammatory helper T (Th) 17 cells in protecting dopaminergic neurons. The release of GZMB by Th17 cells with silencing of the GZMB gene is reduced, which weakens the production of α-synuclein (α-Syn) and apoptosis and necrosis of dopaminergic neurons, and enhances the expression of tyrosine hydroxylase TH and cell activity of neurons.

[0015] Beneficial effects:

[0016] The present application provides the use of targeted granzyme B in immunotherapy for Parkinson's disease neuron degeneration, which has the following beneficial effects compared with the prior art:

[0017] 1. It has been reported in our previous studies that Th17 cells can release the inflammatory factor IL-17A to act on microglia to aggravate neuroinflammation, thereby aggravating dopamine neuron degeneration. This application further verifies that Th17 cells can directly induce dopamine neuron degeneration by releasing the cytotoxic molecule granzyme B (GZMB);

[0018] 2. There has been no significant progress in the effect of immunotherapy targeting IL-17A in the process of Parkinson's disease neurodegeneration. This application can provide experimental basis for the combined targeted cytotoxic molecule granzyme B (GZMB) treatment of dopamine neuron degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a graph showing that Th17 cells in the present invention cause dopaminergic neuron death by releasing GZMB; wherein A is a graph showing changes in the content of GZMB in the culture supernatant of neurons with different treatments; B is a graph showing the activity detection of neurons in different treatment groups; C is a graph showing the level of α-Syn expressed by neurons; D is a graph showing the level of α-Syn secreted by neurons; E is a graph showing changes in the proportion of TH immunoreactive positive cells in neurons, wherein E1 is a graph showing staining of TH immunoreactive positive cells, and E2 is a graph showing the counting of TH positive neurons; FIG. F is a graph showing changes in the proportion of apoptotic cells stained with TUNEL in neurons, wherein F1 is a graph showing staining of TUNEL positive cells, and F2 is a graph showing the counting of TUNEL positive neurons; Figure 1 is a quantitative graph showing the proportion of apoptosis-related proteases caspase-3 and caspase-9 in neurons; Figure G is a graph showing the changes in the expression of apoptosis-related proteases caspase-3 and caspase-9 in neurons, where G1 is an immunoblot of caspase-3 and caspase-9, and G2 is a statistical graph showing the optical density values ​​of activated fragments of caspase-3 and caspase-9; Figure H is an evaluation graph showing neuronal toxicity damage under different treatments; Figure I is a graph showing the changes in the proportion of necrotic cells positively stained with PI in neurons, where I1 is a graph showing the staining of SYTO-13 and PI-positive neurons, and I2 is a statistical graph showing the proportion of PI-positive neurons; **p<0.01; ***p<0.001. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but it should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods and reagents without specifying the specific conditions in the examples are all in accordance with the conventional conditions in the art.

[0021] Example 1

[0022] Application of targeted granzyme B in immunotherapy of neuronal degeneration in Parkinson's disease, in vitro cultured mouse MN9D dopaminergic neurons. GZMB-shRNA, a plasmid silencing the GZMB gene, was transfected into isolated and purified Th17 cells to downregulate the expression of GZMB. Th17 cells were co-cultured with MN9D dopaminergic neurons at a ratio of 2:1, and the supernatant and neurons were collected after 24 hours to observe the activity and death of neurons.

[0023] The specific process is as follows:

[0024] 1. Isolation and purification of Th17 cells in vitro

[0025] Male C57B / 6 mice aged 6 to 8 weeks (provided by the Experimental Animal Center of Nantong University) were quickly killed by cervical dislocation, and the abdominal skin was disinfected with ethanol. The spleen of the mice was removed by aseptic operation and ground to prepare a single-cell suspension. The filtered single-cell suspension was washed and added with red blood cell lysis buffer, mixed and allowed to stand, and complete culture medium was added to stop digestion. According to the instructions of the kit, the selected cells were added to the culture medium and resuspended, and magnetic beads were added to mix thoroughly, and then cooled on ice and incubated for 30 minutes. After adding base medium and centrifugation, the cells were resuspended and placed in 1 ml EP tubes for thorough mixing. After placing on a magnetic stand for 5 minutes, the supernatant was carefully aspirated and placed in a new centrifuge tube. The sorted CD4 + T cells were inoculated into culture plates, and CD28 and CD3 monoclonal antibodies (1 μg / ml) and cytokines IL-23, IL-1β, TNF-α, and IL-6 that induce Th17 cell polarization were added, and then cultured in a CO2 incubator for 5 days.

[0026] 2. Nuclear transfection of GZMB-shRNA to silence the GZMB gene on Th17 cells

[0027] First, the in vitro activated Th17 cells were collected by centrifugation and counted, and the prepared transfection solution was added to the cells in equal proportions and divided into four groups (preheated for 30 minutes in advance). Except for the control group, the remaining three groups were added with GzmB-shRNA1, GzmB-shRNA2, and GzmB-shRNA33 micrograms, respectively. After gently blowing and mixing, they were added to the disposable electrotransfer cup with a small dropper. Turn on the electrotransfer instrument, put it in the electrotransfer cup, press X-001 to start the electrotransfer instrument, and after the electrotransfer is completed, add the transfected solution to 500 microliters of preheated complete medium for culture. After 48 hours of culture, the cells were moved to an inverted microscope for observation. The uniform fluorescence showed that the transfection effect was good. The relative expression of the GZMB gene after Th17 cell transfection was then verified by quantitative PCR.

[0028] 3. MN9D dopaminergic neuronal cell culture

[0029] Take out the MN9D cells purchased from Gioni and quickly put them in a constant temperature water bath to shake and dissolve. Pay attention to aseptic operation before taking out the cells. Add the cell resuscitation solution to the cell culture medium and mix well, then centrifuge, remove the cell supernatant, mix the remaining cell pellet with fresh complete preparation, and evenly inoculate it in a disposable cell culture bottle. When the cells grow to a suitable density, digest them with trypsin, plate the cells with a 6-well plate or 24-well plate coated with lysine, observe the cell viability, and conduct subsequent experiments after the cell density is uniform. Add transfected and untransfected Th17 cells to the MN9D cell culture of appropriate density (cell ratio 2:1), and collect the cell supernatant and MN9D cells after 24 hours for corresponding detection.

[0030] 4.CCK8 detection of cell activity

[0031] After MN9D cells were treated with different methods for 24 h, 10 μl of CCK8 reagent was added to each well of cells, and the 96-well plate was placed in a 37°C, 5% CO2 cell culture incubator for further 2 h. The 96-well plate was placed in an ELISA reader to detect the absorbance value at a wavelength of 450 nm.

[0032] 5.ELISA detection of GZMB and α-Syn levels in cell supernatant

[0033] Take out the ELISA kit, take out the ELISA plate sealing bag, restore to room temperature and take out the ELISA plate, add the PBS buffer prepared in the kit to the well of the ELISA plate, shake off the ELISA plate completely after 15 seconds, and gently pat the PBS buffer remaining in the well of the ELISA plate on a napkin, repeat 4 times. Take out the standard powder and add it to the sample diluent. After fully dissolving, dilute the prepared standard in multiples and add it to the corresponding wells. Add an equal amount of sample diluent to the blank wells, and add an equal amount of sample to the other wells. Next, prepare the Biotin-Conjugate solution, add 100μl Biotin-Conjugate solution to each well, take out the special adhesive tape for sealing the plate after adding, seal the reaction, mix with an oscillator, and incubate for 2h. After taking the plate, wash the plate again 5 times as before. Add 100μl Streptavidin-HRP solution to each well, shake the plate after sealing, and incubate for 1h. After incubation, wash the plate 5 times, and finally add 100μl TMB solution to each well of the ELISA plate, place it at room temperature, and incubate it for 10min away from light. Finally, add 100 μl of Stop Solution to each well to terminate the reaction. Immediately after the reaction, measure the OD450 value within 3 minutes. Prepare a standard curve as required, and calculate the sample concentration based on the standard curve given in the kit instructions.

[0034] 6. Cell Immunofluorescence Staining

[0035] MN9D cells from each treatment group were taken, rinsed with PBS for 5 min × 3 times, and then fixed with 4% formaldehyde fixative for 30 min. After the cells were fixed, they were washed 3 times as before, and the slides were incubated with blocking permeabilization solution for 30 min. Then TH primary antibody (1:500) was added, and after incubation in a 4°C refrigerator for 24 h, they were rinsed with PBS 3 times, 5 min / time, and then the prepared FITC-labeled secondary antibody was added. After incubation at room temperature for 4-6 h, the cells were rinsed 3 times with PBS, and the cell nuclei were counterstained with Hoechst. Finally, the slides were sealed with glycerol phosphate and observed under a fluorescence microscope to calculate the proportion of cells with TH positive reaction.

[0036] 7. TUNEL staining to observe apoptosis of MN9D cells

[0037] Take MN9D cells from each treatment group, rinse with PBS for 5min×3 times, and then fix with 4% formaldehyde fixative for 30min. After the cells are fixed, wash them again 3 times as before, block the slides with blocking solution for 30min, add freshly prepared TUNEL working solution and incubate at 37 degrees in the dark for 1h. After the incubation, take out the slides in the dark, rinse with PBS 3 times, observe the green fluorescence under a fluorescence microscope, and count the number of TUNEL-positive cells. Take 3 fields of view for each slide, and calculate the average number of TUNEL-positive cells in the field of view of each slide.

[0038] 8.Western blot

[0039] 1) Sample preparation

[0040] Collect MN9D cells, add a certain proportion of tissue or cell lysate, shake well, centrifuge the lysate at 12000rpm, 4℃, 15min, boil for 10min, aspirate the supernatant into a new EP tube, and store at -70℃ for later use.

[0041] 2) Protein content determination

[0042] Take 5 μl of the protein standard and the sample to be tested and dilute them with normal saline (the dilution ratio is 1:10), and detect the protein content with a nucleic acid protein analyzer.

[0043] 3) SDS-polyacrylamide gel electrophoresis

[0044] Use vertical flat plate, discontinuous, denaturing polyacrylamide gel electrophoresis system (SDS-PAGE). Prepare 10% separation gel (3.2ml double distilled water, 2.64ml 30% reserve gel, 2ml 1.5mM Tris-HCL, 0.08ml 10% SDS, 0.08ml 10% AP, 4μl TEMED), 4% concentration gel (1.4ml ultrapure water, 0.33ml 30% reserve gel, 2.5ml 1.0mM Tris-HCL, 0.02ml 10% SDS, 0.02ml 10% AP, 2μl TEMED), add electrophoresis buffer to the upper and lower electrophoresis tanks. Take the supernatant of the above different groups of tissue lysates and mix with appropriate amount of buffer, boil for 3-5min. Take one lane and add pre-stained standard molecular weight protein.

[0045] 4) Western blot detection

[0046] After electrophoresis, constant current wet electrotransfer was performed, and the standard protein lane of the transferred PVDF membrane was cut out. The rest was washed with 1×TBS membrane wash buffer for 5 min × 3 times, and blocked with a blocking solution containing 5% skim milk powder at 4°C overnight. Washed with TBS / T membrane wash buffer for 5 min × 3 times, immersed in primary antibodies α-Syn (1:1000), caspase-3 (1:500), caspase-9 (1:1000) and β-actin (1:5000), and coated at 4°C overnight. Fluorescently labeled goat anti-mouse IgG (1:5000) and fluorescently labeled goat anti-rabbit IgG (1:5000) were incubated at room temperature for 1 hour. Washed with TBS / T membrane wash buffer for 5 min × 3 times, and scanned and analyzed with the Odyssey dual infrared laser scanning system.

[0047] 9. LDH method to detect cell death

[0048] The supernatant of MN9D cell culture in each group was collected and diluted with PBS buffer (1:10). 10 μl of sample or standard was added to the well plate. The oxidized coenzyme I solution and substrate buffer were mixed at a ratio of 1:5. 60 μl of the mixture was added to each microwell respectively. After mixing thoroughly, the mixture was placed in a 37°C water bath for 15 min. 50 μl of 2,4-dinitrophenylhydrazine was added. After mixing thoroughly, the mixture was placed in a 37°C water bath for 15 min again. 150 μl of the termination reaction reagent was added. The mixture was mixed thoroughly. After standing at room temperature for 3 min, the absorbance of the sample well was measured at a wavelength of 490 nm. The enzyme activity unit was calculated. Cellular LDH release (%) = enzyme activity unit / (enzyme activity unit measured by cell lysate + enzyme activity unit measured by cell culture medium) × 100%.

[0049] 10.PI / SYTO-13 staining

[0050] Take MN9D cells from each treatment group, rinse with PBS for 5 min × 3 times, add freshly prepared PI dye solution and incubate at 37°C in the dark for 20 min. After incubation, rinse again with PBS for 5 min × 3 times, add SYTO-13 dye solution (1:1000) diluted with PBS and incubate again at 37°C in the dark for 15 min. Wash the slides with PBS, seal the slides, and observe under a fluorescence microscope.

[0051] 11. Statistical Analysis

[0052] All results were expressed as mean ± standard deviation (M ± SD). SPSS16.0 statistical software package was used for data analysis and processing, and one-way analysis of variance (ANOVA) was used to test the significance of differences between groups, and Student-Newman-Keul's test was used to test the significance of differences between groups. P < 0.05 was considered significant.

[0053] The results are as follows Figure 1 As shown in the figure, the release of GZMB by Th17 cells with GZMB gene silenced was reduced, the production of α-synuclein (α-Syn) and apoptosis and necrosis of dopaminergic neurons were weakened, and the expression of tyrosine hydroxylase (TH) and cell activity of neurons were enhanced. The results of one-way ANOVA showed that the differences between the groups were statistically significant (P < 0.05). **p < 0.05; ***p < 0.01.

[0054] The examples selected in the above materials are for ease of understanding, not to limit the process. People in the same technical field can easily modify the process or transfer it to other cases without creative changes. If these changes also belong to the same claims or similar technologies of the present invention, then the original intention of the invention also includes these changes.

Claims

1. Application of targeted granzyme B in immunotherapy for neuronal degeneration in Parkinson's disease.

2. The use of targeted granzyme B in immunotherapy for Parkinson's disease neuron degeneration according to claim 1, characterized in that: The Parkinson's disease neuron degeneration immunotherapy drug is in an injectable dosage form or a non-injectable dosage form.

3. The use of targeted granzyme B in immunotherapy for Parkinson's disease neuron degeneration according to claim 1, characterized in that: The Parkinson's disease neuron degeneration immunotherapy drug is an injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

4. The use of targeted granzyme B in immunotherapy for Parkinson's disease neuron degeneration according to claim 1, characterized in that: The Parkinson's disease neuron degeneration immunotherapy drug comprises targeted granzyme B and pharmaceutically acceptable excipients.

5. The use of targeted granzyme B in immunotherapy for Parkinson's disease neuron degeneration according to claim 4, characterized in that: The auxiliary materials include one or more of diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

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