CN113834937B - Diagnostic markers for distinguishing idiopathic inflammatory myopathy - Google Patents
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Abstract
The invention belongs to the technical field of medical biology, and particularly relates to a group of diagnostic markers for distinguishing idiopathic inflammatory myopathy. The group of diagnostic markers for differentiating the idiopathic inflammatory myopathy is as follows: receptor-interacting protein 3(RIP3), mixed kinase domain protein (MLKL).
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a group of diagnostic markers for distinguishing idiopathic inflammatory myopathy.
Background
Idiopathic Inflammatory Myopathies (IIMs) are a heterogeneous group of autoimmune diseases characterized by serum myopathy abnormalities, muscle weakness, muscle inflammation, etc. [1 ]. The molecular mechanism of IIMs myocyte death remains to be elucidated. There are studies reporting that apoptosis is not abnormal in muscle tissues of IIMs patients and may be associated with increased expression of anti-apoptotic proteins. In subsequent studies, researchers reported that endoplasmic reticulum stress, excessive activation of autophagy, is an important non-immunological mechanism of IIMs muscle injury [2,3 ]. Furthermore, swedish scholars demonstrated that CD28(null) T cells are capable of producing cytotoxic effects on autologous muscle cells in vitro and are achieved by secreting perforin [4] recently, french scholars demonstrated, through cell experiments and animal model studies, that anti-SRP antibodies and anti-HMGCR antibodies have direct pathological effects on IIMs muscle cells [5,6] these studies suggest that IIMs muscle injury may involve multiple factors, including immunological and non-immunological mechanisms. Further defines the muscle cell death mechanism of IIMs, and has important significance for discovering new therapeutic targets.
Muscle pathology analysis found that myocyte necrosis was the most common pathological change in muscle tissue in IIMs patients. At early stages, cell necrosis was considered a passive mode of cell death and was not regulated. With the discovery of programmed necrosis (necroptosis), cellular necrosis is no longer considered as an accidental cell death mode, but rather can be regulated [7] molecules involved in programmed necrosis mainly include receptor-interacting protein 1 (RIP 1), receptor-interacting protein 3(RIP3), mixed kinase domain protein (MLKL), which work together to form programmed necrotic bodies [7]. As a late event of programmed necrosis, RIP3 and MLKL proteins are phosphorylated and transported to the cell membrane, resulting in increased permeability of the cell membrane, disruption of the membrane structure and thus induction of cell necrosis. With apoptosis, damage-associated molecular patterns (DAMPs) including HMGB1, IL33, etc. are released outside the cell, further mediating inflammatory damage to tissues [7,8 ]. Muscle biopsy analysis confirmed that myocyte necrosis and inflammatory infiltration can be common in the muscle tissues of IIMs patients, but the molecular mechanisms remain to be elucidated.
The study is intended to study whether programmed necrosis is involved in the pathogenesis of IIMs by studying the expression levels of RIP3 and MLKL proteins in muscle tissues of IIMs patients, analyzing the association of the expression levels with the severity of diseases, and carrying out cell experiments. .
[ reference documents ]
1.Dalakas MC:Inflammatory muscle diseases.The New England journal of medicine 2015,372(18):1734-1747.
2.Nagaraju K,Casciola-Rosen L,Lundberg I,Rawat R,Cutting S,Thapliyal R,Chang J,Dwivedi S,Mitsak M,Chen YW et al:Activation of the endoplasmic reticulum stress response in autoimmune myositis:potential role in muscle fiber damage and dysfunction.Arthritis and rheumatism 2005,52(6):1824-1835.
3.Alger HM,Raben N,Pistilli E,Francia DL,Rawat R,Getnet D,Ghimbovschi S,Chen YW,Lundberg IE,Nagaraju K:The role of TRAIL in mediating autophagy in myositis skeletal muscle:a potential nonimmune mechanism of muscle damage.Arthritis and rheumatism 2011,63(11):3448-3457.
4.Pandya JM,Venalis P,Al-Khalili L,Shahadat Hossain M,Stache V,Lundberg IE,Malmstrom V,Fasth AE:CD4+and CD8+CD28(null)T Cells Are Cytotoxic to Autologous Muscle Cells in Patients With Polymyositis.Arthritis&rheumatology(Hoboken,NJ)2016,68(8):2016-2026.
5.Arouche-Delaperche L,Allenbach Y,Amelin D,Preusse C,Mouly V,Mauhin W,Tchoupou GD,Drouot L,Boyer O,Stenzel W et al:Pathogenic role of anti-signal recognition protein and anti-3-Hydroxy-3-methylglutaryl-CoA reductase antibodies in necrotizing myopathies:Myofiber atrophy and impairment of muscle regeneration in necrotizing autoimmune myopathies.Annals of neurology 2017,81(4):538-548.
6.Bergua C,Chiavelli H,Allenbach Y,Arouche-Delaperche L,Arnoult C,Bourdenet G,Jean L,Zoubairi R,Guerout N,Mahler M et al:In vivo pathogenicity of IgG from patients with anti-SRP or anti-HMGCR autoantibodies in immune-mediated necrotising myopathy.Annals of the rheumatic diseases 2019,78(1):131-139.
7.Zhou W,Yuan J:Necroptosis in health and diseases.Seminars in cell&developmental biology 2014,35:14-23.
8.Silke J,Rickard JA,Gerlic M:The diverse role of RIP kinases in necroptosis and inflammation.Nature immunology 2015,16(7):689-697.
9.Lundberg IE,
A,Bottai M,Werth VP,Pilkington C,Visser M,Alfredsson L,Amato AA,Barohn RJ,Liang MH et al:2017 European League Against Rheumatism/American College of Rheumatology classification criteria for adult and juvenile idiopathic inflammatory myopathies and their major subgroups.Annals of the rheumatic diseases 2017,76(12):1955-1964.
10.Hoogendijk JE,Amato AA,Lecky BR,Choy EH,Lundberg IE,Rose MR,Vencovsky J,de Visser M,Hughes RA:119th ENMC international workshop:trial design in adult idiopathic inflammatory myopathies,with the exception of inclusion body myositis,10-12 October 2003,Naarden,The Netherlands.Neuromuscular disorders:NMD 2004,14(5):337-345.
11.Varsani H,Charman SC,Li CK,Marie SK,Amato AA,Banwell B,Bove KE,Corse AM,Emslie-Smith AM,Jacques TS et al:Validation of a score tool for measurement of histological severity in juvenile dermatomyositis and association with clinical severity of disease.Annals of the rheumatic diseases 2015,74(1):204-210.
12.Wedderburn LR,Varsani H,Li CK,Newton KR,Amato AA,Banwell B,Bove KE,Corse AM,Emslie-Smith A,Harding B et al:International consensus on a proposed score system for muscle biopsy evaluation in patients with juvenile dermatomyositis:a tool for potential use in clinical trials.Arthritis and rheumatism 2007,57(7):1192-1201.
13.Zhang L,Xia Q,Li W,Peng Q,Yang H,Lu X,Wang G:The RIG-I pathway is involved in peripheral T cell lymphopenia in patients with dermatomyositis.Arthritis research&therapy 2019,21(1):131.
Disclosure of Invention
The invention firstly relates to the application of a group of molecular marker detection reagents in the preparation of a diagnostic reagent or a kit for identifying idiopathic inflammatory myopathy,
the group of molecular markers is as follows: receptor interacting protein 3(RIP3), mixed kinase domain protein (MLKL);
preferably, the detection reagent is: monoclonal or polyclonal antibodies.
The invention also relates to a diagnostic kit for identifying idiopathic inflammatory myopathy,
the kit comprises:
a detection reagent for detecting the following proteins: receptor interacting protein 3(RIP3), mixed kinase domain protein (MLKL).
Preferably, the detection reagent is: monoclonal or polyclonal antibodies.
Preferably, the detection kit is as follows: ELISA kit, chemiluminescence kit, and flow cytometry kit.
The invention also relates to the application of a detection reagent of a group of molecular markers in preparing a diagnostic reagent or a kit for distinguishing idiopathic inflammatory myopathy subtypes,
the group of molecular markers is as follows: receptor interacting protein 3(RIP3), mixed kinase domain protein (MLKL);
the differentiation of the idiopathic inflammatory myopathy subtypes is as follows: the idiopathic inflammatory myopathies were classified as:
subtype (1): dermatomyositis (DM) and/or immune-mediated necrotic myopathy (IMNM);
subtype (2): (amyopathic DM, ADM);
the differential subtype refers to:
when the group of molecular markers are highly expressed, the idiopathic inflammatory myopathy is divided into a subtype (1); when the molecular markers are low expressed, the idiopathic inflammatory myopathy is divided into subtypes (2).
Preferably, the detection reagent is: monoclonal or polyclonal antibodies.
The invention also relates to a diagnostic kit for differentiating idiopathic inflammatory myopathy subtypes,
the differentiation of the idiopathic inflammatory myopathy subtypes is as follows: the idiopathic inflammatory myopathy was classified as:
subtype (1): dermatomyositis (DM) and/or immune-mediated necrotic myopathy (IMNM);
subtype (2): (amyopathic DM, ADM);
the differential subtype refers to:
when the group of molecular markers are highly expressed, the idiopathic inflammatory myopathy is divided into a subtype (1); when the group of molecular markers is low in expression, the idiopathic inflammatory myopathy is divided into a subtype (2);
the kit comprises:
a detection reagent for detecting the following proteins: receptor interacting protein 3(RIP3), mixed kinase domain protein (MLKL).
Preferably, the detection reagent is: monoclonal or polyclonal antibodies.
Preferably, the detection kit is as follows: ELISA kit, chemiluminescence kit, and flow cytometry kit.
Drawings
Figure 1, IIMs patient muscle tissue RIP3 and MLKL protein expression assay:
1A: carrying out western blot detection on RIP3 and MLKL protein expression of IIMs muscle tissues;
1B: carrying out western blot detection on expression of phosphorylation RIP3 and MLKL protein of IIMs muscle tissues;
1C: performing immunohistochemical analysis on the expression of RIP3 and MLKL protein and phosphorylation protein thereof;
1D: carrying out immunohistochemical analysis on RIP3 and MLKL protein by continuous sections;
1E: and (5) carrying out quantitative analysis on the Western blot result.
Figure 2, RIP3, MLKL protein expression level correlated with patient muscle lesion extent:
2A, 2B, 2C: typical necrosis scores were 0, 1, 2 HE staining of muscle tissue, respectively;
2D: the expression level of RIP3 and MLKL protein is obviously higher in the case that the necrosis score is 2 groups than in the case that the necrosis score is 0 and 1 groups;
2E: RIP3 and MLKL protein expression levels of IIMs patients are obviously positively correlated with CK and LDH of serum; significant negative correlation with the patient muscle force score MMT8 (r-0.43, P < 0.05; r-0.63, P < 0.01);
2F: expression of RIP3 and MLKL proteins in muscle tissues changes before and after treatment.
Figure 3, detection of expression of HMGB1 and IL33 in IIM muscle tissue:
3A: immunohistochemical detection of HMGB1 and IL33 expression in IIM patients and healthy control muscle tissue;
3B: carrying out immunohistochemical analysis on HMGB1 and MLKL protein by continuous sections;
3C: expression of HMGB1 protein in muscle tissue varies before and after treatment.
Figure 4, in vitro experimental study TNF α induces apoptosis overactivation of myoblast C2C 12:
4A: after 24 hours of stimulation, morphologically observing the number and the state of cells in each group;
4B: the cell activity determination experiment result shows that the percentage of the live cells of the TNF alpha + z-VAD group is obviously reduced, and the TNF alpha group, the TNF alpha + z-VAD + Nec-1s group and the control group have no difference;
4C: flow cytometry analysis proves that after TNF alpha + z-VAD (vascular endothelial growth factor) co-stimulation, the proportion of C2C12 positively stained by PI is obviously increased, and the TNF alpha group, the TNF alpha + z-VAD + Nec-1s group and a control group have no difference;
4D and 4E: the Western blot result shows that MLKL and phosphorylated MLKL protein of C2C12 cells in the TNF alpha + z-VAD co-stimulation group are remarkably up-regulated, and the TNF alpha group, the TNF alpha + z-VAD + Nec-1s group and the control group have no difference.
Figure 5, C2C12 cells with knockdown of MLKL gene resist TNF α -induced apoptosis:
5A: C2C12 cell MLKL gene is knocked down by a CRISPR/Cas9 gene editing technology, and the expression of the polyclonal cell MLKL protein is remarkably reduced through western blot analysis;
5B: the results of TNF alpha induced programmed necrosis experiments on MLKL knocked-down C2C12 cells and control C2C12 cells show that the percentage of live MLKL knocked-down C2C12 cells is remarkably higher than that of control C2C12 cells after TNF alpha + z-VAD co-stimulation.
Detailed Description
Materials and methods
Grouping patient information
The study was reviewed by the ethical committee of the well-friendly hospital (ethical approval No. 2019-25-K19.) all patients' clinical data were used anonymously and all patients signed informed consent. The study included 26 diagnosed IIM patients treated in the rheumatoid and immunogical department of the Zhongri friendly Hospital in 2017 + 2019, and the diagnosis criteria of the patients adopted the EULAR/ACR criteria in 2017 [9 ]. According to the classification criteria, 5 patients were diagnosed with Dermatomyositis (DM), and 4 patients were diagnosed with myopathic dermatomyositis (ADM). In addition, 17 patients were diagnosed with immune-mediated necrotizing myopathy (IMNM) [10 ]. All patients were examined for muscle biopsies and clearly diagnosed pathologically. In this study, 4 muscle tissues of trauma patients without muscle disease were collected as healthy controls.
Muscle histopathology scoring
The pathological scoring of the patient's muscle biopsies was performed according to the published scoring tools for child dermatomyositis muscle biopsies [11,12], with a muscle necrosis score defined as three grades, 0 (no necrotic muscle cells in the biopsy), 1 (proportion of necrotic muscle cells 1% -5%), 2 (proportion of necrotic muscle cells ≥ 5%).
Western blot analysis
Total protein extracted from patient muscle tissue or in vitro cultured cells was subjected to SDS-PAGE gel electrophoresis of 12% or 15%, after transferring to PVDF membrane, blocked with 5% nonfat dry milk for 2 hours, and then PVDF membrane was incubated with antibodies against RIP3(ProSci, Poway, CA, USA), anti-MLKL (Abcam, Cambridge, UK) and anti-GAPDH (Abcam, Cambridge, UK) at 4 ℃ overnight.
Horseradish peroxidase (HRP) -labeled goat-anti-mouse (Abcam, Cambridge, UK) and goat-anti-rabbit (Abcam, Cambridge, UK) secondary antibodies were incubated at room temperature for 1h and imaged. ImageJ (National Institute of Health, USA) was used to analyze immunoblot band intensity.
Immunohistochemical analysis
Preparing frozen sections of IIM and HCs skeletal muscle, and incubating overnight at 4 ℃ with primary antibody; washing the membrane with PBS-T for 3 times the next day, and incubating for 1h at room temperature with goat anti-mouse/rabbit anti-antibody; after washing the membrane with PBS-T for 3 times, 3,3' -diaminobenzidine is developed and read by a microscope.
Primary antibodies used in this study included:
polyclonal anti-RIP 3 antibody (ProSci, Poway, CA, USA),
an anti-MLKL monoclonal antibody (Abcam),
anti-RIP 3(phospho S227) monoclonal antibody (Abcam),
an anti-MLKL (phospho S358) monoclonal antibody (Abcam),
anti-HMGB 1 polyclonal antibody (Proteintech),
anti-IL 33 monoclonal antibody (Abcam).
Cell culture and stimulation
C2C12 cells were cultured using a 100mm petri dish (Corning, New York, USA). The C2C12 medium was DMEM (Gibco, Grand Island, USA), 15% fetal bovine serum (FBS; Gibco, Grand Island, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, USA).
Cell stimulating agents used in this study include:
recombinant TNF α protein (Biolegend, San Diego, CA, USA) was used at a concentration of 100 ng/ml;
z-VAD-FMK (Promega, Madison, Wis., USA) used at a concentration of 40. mu.M;
nec-1s (Biovision, San Francisco, Calif., USA) was used at a concentration of 50. mu.M.
Cell death assay
By using
The level of cell death was measured using a luminometric cell viability assay kit (Promega, Madison, WI, USA). C2C12 were seeded into opaque 96-well plates, 1 × 104 cells per well. 24 hours after the addition of the corresponding cell stimulating agent, the agent was added to each well
Mu.l of reagent, and then shaking on a horizontal shaker for 10 minutes. Luminescence was measured for each well in a 96-well plate using a multifunctional microplate reader (Tecan, Mannedorf, Switzerland). Cell activity was calculated by the ratio of test wells to control wells.
Flow cytometry analysis
After the in vitro cultured C2C12 cells were treated with the stimulating agent, the cells were collected, washed with PBS, stained with PI stain (BD Pharmingen, San Diego, California, USA) for 20 minutes, centrifuged, washed with PBS, resuspended in 500 μ L PBS solution, and subjected to flow cytometry (FACS JAZZ, BD, San Diego, California, USA). Data were analyzed by the FASC software.
C2C12 cell MLKL gene knockout
MLKL gene knock-down is carried out on C2C12 cells by using a CRISPR/Cas9 gene editing system, and the specific method is disclosed in an earlier published article [13] of the subject group; the sgRNA sequence finally adopted is 5'-GTCTCTGGAGAGGCTGTAGC-3'; the annealed sgrnas were cloned into PX458 plasmid, and the recombinant plasmid was transfected into C2C12 cells using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA). Fluorescence positive cells were sorted by flow cytometry (BD Aria III FACS system). And carrying out western blot analysis on the sorted polyclonal cells to detect the expression of the MLKL protein, and using the MLKL knock-down cells as MLKL knock-down cells for follow-up research.
Statistical analysis
Those with continuous variables that fit a normal distribution are described by means of the mean ± standard deviation, and those with non-normal distributions are described by the median and the interquartile range (IQR). Categorical variables are expressed as percentages and absolute frequencies. Comparison between the measurement data groups applied either the independent sample t-test or the Mann-Whitney U-test. Spearman correlation analysis statistics for inter-data correlation. Data management and statistical analysis were performed using SPSS 23.0(SPSS institute, USA) and GraphPad Prism 5.0(GraphPad Software, USA).
Example 1 in IIM patients in muscle tissue, apoptosis necrosis factor overactivation
RIP3 and MLKL are key molecules involved in programmed necrosis. To investigate the extent of activation of programmed necrosis in the muscle tissue of IIM patients, we examined the expression levels of RIP3 and MLKL protein in IIM muscle tissue of 24 IIM patients. The clinical and laboratory examination data for these 24 patients are presented in table 1.
Western blot analysis results showed that expression levels of RIP3 and MLKL proteins in muscle tissue of Dermatomyositis (DM) and immune-mediated necrotic myopathy (IMNM) patient groups were significantly higher than that of myopathic dermatomyositis (ADM) patient groups and healthy control groups (fig. 1A). The expression levels of phosphorylated RIP3 and MLKL proteins were also higher in the DM and IMNM groups than in the ADM group and healthy control group (fig. 1B). The results show that it is possible to display,
healthy control, dermatomyositis without myopathy (ADM), Dermatomyositis (DM), immune-mediated necrotic myopathy (INMN),
the relative expression amounts of RIP3 protein are respectively 0.143 + -0.165, 0.053 + -0.05, 0.268 + -0.184 and 0.214 + -0.178;
the relative expression amounts of the MLKL protein are 0.179 +/-0.156, 0.145 +/-0.034, 0.949 +/-0.105 and 0.57 +/-0.389 respectively.
It can be seen that the expression levels of muscle tissue RIP3 and MLKL protein in Dermatomyositis (DM), immune-mediated necrotic myopathy (INMN) patients were significantly higher than those in dermatomyositis without myopathy (ADM) patients and healthy controls (. about.p < 0.05).
Immunohistochemistry results showed that RIP3, MLKL protein, and phosphorylated RIP3, MLKL protein were significantly up-regulated in muscle tissue of DM and IMNM patients, whereas no positive staining was seen in ADM patients and healthy control muscle tissue (fig. 1C).
Further immunohistochemical analysis of RIP3 and MLKL protein using serial sections revealed that RIP3 and MLKL protein were co-expressed in necrotic muscle cells (fig. 1D).
Quantitative analysis of western blot results, we found that RIP3, MLKL protein were significantly positively correlated in relative expression levels in muscle tissues of IIMs patients (r ═ 0.75, P <0.001) (fig. 1E). These results demonstrate that RIP3, the MLKL protein, is significantly highly expressed in muscle tissue of IIMs patients with myocyte necrosis, suggesting over-activation of programmed necrosis.
TABLE 1 IIM patient and health control clinical and laboratory examination data
ADM, sarcoidosis-free dermatomyositis; CK is creatine kinase; DM is dermatomyositis; IMNM immune-mediated necrotic myopathy, MMT 8: myometric assessment score
Example 2 RIP3, MLKL protein expression level correlated with patient muscle lesion level
By scoring the pathological changes of the musculature of IIMs patients, we divided IIMs patients into three groups: necrosis score 0 group, 1 group, 2 group. Fig. 2A, B, C show HE staining of muscle tissue with typical necrosis scores of 0, 1, 2, respectively.
By comparing the expression levels of RIP3, the MLKL protein in three groups of patients, we found that the higher the necrosis score, the higher the RIP3, MLKL protein level (fig. 2D). In addition, RIP3, MLKL protein expression levels of IIMs patients were significantly positively correlated with serum CK, LDH (fig. 2E); significant negative correlation with the patient muscle score MMT8 (r ═ 0.43, P < 0.05; r ═ 0.63, P <0.01) (fig. 2E).
We analyzed the expression changes of RIP3, MLKL protein before and after treatment in their muscle tissue for 2 IMNM patients with good treatment response (see table 2 for clinical and laboratory examination data), and found that RIP3, MLKL protein expression was significantly reduced in muscle tissue after 6 months of treatment and disease remission (fig. 2F).
As a control group, the expression level of RIP1 protein in the muscle tissue of 2 ADM patients and 6 IMNM patients was also detected by western blotting, and the results showed that the relative expression levels of RIP1 in the two groups of patients were 0.069 +/-0.01 and 0.138 +/-0.08 respectively; IMNM patients were suggested to express RIP1 at slightly higher levels than ADM patients, but not statistically significant. It is suggested that the changes in the expression levels of RIP3 and MLKL protein in patients are specific.
TABLE 2, 2 clinical and laboratory data before and after treatment of IMNM patients
IMNM immune-mediated necrotic myopathy
Example 3 increased expression of injury-associated molecular patterns in muscle tissue of IIMs patients with programmed necrotic overactivation
Necrotic cells resulting from activation of programmed necrosis release a variety of injury-associated molecular markers including HMGB1, IL33, etc. [8 ].
We found a significant increase in HMGB1 and IL33 protein expression in muscle tissue of IMNM and DM patients by immunohistochemical analysis, whereas no positive expression was seen in ADM and healthy control muscle tissue (fig. 3A). Immunohistochemical analysis of HMGB1 and MLKL proteins was performed by using serial sections, and the results showed that HMGB1 and MLKL proteins were co-expressed in necrotic muscle cells (fig. 3B), which suggested that the expression level of HMGB1 in muscle cells over-activated by programmed necrosis was significantly up-regulated. Furthermore, we also found that in post-treatment IMNM patients, there was a significant reduction in muscle tissue HMGB1 compared to pre-treatment (fig. 3C).
Example 4 TNF α Induction of programmed necrotic overactivation of myoblasts C2C12
The literature reports that TNF alpha can induce various kinds of apoptosis over-activation in vitro, so that the TNF alpha protein is utilized to stimulate myoblasts C2C12 cultured in vitro and study the apoptosis activation and cell death level.
The results show that a significant decrease in the number of C2C12 cells was observed with the addition of the pan-cystatin z-VAD simultaneously with TNF α stimulation (fig. 4A); the results of the cell viability assay showed that the percentage of viable cells in the TNF α + z-VAD co-stimulated group was significantly reduced, while the percentage of viable cells in the TNF α -stimulated group alone was not different from the control group (FIG. 4B).
We further treated the cells with the apoptosis inhibitor, necrostatin-1s (Nec-1s), and found that the percent of viable cells was significantly increased by the addition of Nec-1s while TNF α + z-VAD was co-stimulating C2C12 (FIGS. 4A, 4B).
Flow cytometric analysis also confirmed that the proportion of C2C12 staining positive for PI was significantly increased following TNF α + z-VAD co-stimulation, and the addition of Nec-1s was able to significantly reduce the proportion of PI-positive staining C2C12 induced by TNF α + z-VAD stimulation (fig. 4C).
Western blot analysis of lysates of C2C12 cells in each stimulation group showed that TNF α + z-VAD was significantly upregulated in cooperation with MLKL and phosphorylated MLKL proteins in C2C12 cells in the stimulation group, whereas the expression levels of both were not different from those in the control group after Nec-1s treatment (FIGS. 4D and 4E). These results demonstrate that TNF α is able to induce in vitro an overactivation of myoblast C2C12 programmed necrosis, and thus an increase in myoblast necrosis.
Example 5 MLKL Gene knockdown of C2C12 cells were resistant to TNF α -induced apoptosis
Through a CRISPR/Cas9 gene editing technology, a C2C12 polyclonal cell with an MLKL gene knocked down is obtained, and Western blot analysis proves that the expression of MLKL protein of the polyclonal cell is remarkably reduced (FIG. 5A). The results of TNF α -induced apoptosis experiments on MLKL-knocked-down C2C12 cells and control C2C12 cells show that the percentage of live MLKL-knocked-down C2C12 cells was significantly higher than control C2C12 cells after TNF α + z-VAD co-stimulation (fig. 5B). This suggests that C2C12 is able to resist TNF α -induced apoptosis following MLKL knockdown.
Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.
Claims (4)
1. The application of a group of detection reagents of molecular markers in preparing a diagnostic reagent or a kit for identifying idiopathic inflammatory myopathy,
the group of molecular markers is as follows: receptor-interacting protein 3(RIP3), mixed kinase domain protein (MLKL).
2. The use of claim 1, wherein the detection reagent is: monoclonal or polyclonal antibodies.
3. The application of a group of detection reagents of molecular markers in preparing a diagnostic reagent or a kit for distinguishing idiopathic inflammatory myopathy subtypes,
the group of molecular markers is as follows: receptor interacting protein 3(RIP3), mixed kinase domain protein (MLKL);
the differentiation of the idiopathic inflammatory myopathy subtype is to differentiate the idiopathic inflammatory myopathy into:
subtype (1): dermatomyositis (DM) and/or immune-mediated necrotic myopathy (IMNM);
subtype (2): dermatomyositis inopathica (amyopathic DM, ADM);
the differential subtype refers to:
when the group of molecular markers is highly expressed, the idiopathic inflammatory myopathy is divided into a subtype (1);
when the group of molecular markers is low in expression, the idiopathic inflammatory myopathy is classified into a subtype (2).
4. The use of claim 3, wherein the detection reagent is: monoclonal or polyclonal antibodies.
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| WO2010022444A1 (en) * | 2008-08-25 | 2010-03-04 | The Walter And Eliza Hall Institute Of Medical Research | Methods and agents for modulating kinase signalling pathways through modulation of mixed-lineage kinase domain-like protein (mlkl) |
| CN102516384A (en) * | 2011-11-03 | 2012-06-27 | 北京生命科学研究所 | MLKL protein and application of MLKL protein as target point of cell necrosis inhibitor |
| JP6464436B2 (en) * | 2014-02-07 | 2019-02-06 | アジュ ユニバーシティー インダストリー−アカデミック コーオペレイション ファウンデーションＡｊｏｕ Ｕｎｉｖｅｒｓｉｔｙ Ｉｎｄｕｓｔｒｙ−Ａｃａｄｅｍｉｃ Ｃｏｏｐｅｒａｔｉｏｎ Ｆｏｕｎｄａｔｉｏｎ | An anti-cancer adjuvant composition comprising a receptor-coupled protein kinase-3 (RIP3) expression promoter as an active ingredient, and an anti-cancer adjuvant that promotes the sensitivity of the anti-cancer drug by promoting the expression of receptor-coupled protein kinase-3 (RIP3) Screening method and anticancer drug sensitivity monitoring method |
| KR101640068B1 (en) * | 2015-04-30 | 2016-07-18 | 아주대학교산학협력단 | pharmaceutical composition comprising RIP3-MLKL pathway blocker for preventing or treating of skin cell necrosis disease |
| CN107519165A (en) * | 2017-09-05 | 2017-12-29 | 中山大学中山眼科中心 | Applications of the Necrostatin 1 in nervus retrogression ocular disease drug is treated |
| CN107926860A (en) * | 2017-12-22 | 2018-04-20 | 武汉轻工大学 | A kind of method for building up of the lipopolysaccharide-induced procedural Necrosis Model of piglet liver cell |
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