Application method for revealing RIPK3 as osteoarthritis pathological index based on transcriptomics analysis

Abstract

The invention discloses an application method for revealing RIPK3 as a pathological index of osteoarthritis based on transcriptomics analysis, and belongs to the technical field of molecular biology and genetic engineering.The method comprises the following steps that S1, a Trizol reagent is used for conducting total RNA extraction on an articular cartilage specimen, and the RIN number gt is selected; 7.0, constructing a sequencing library by using the high-quality RNA sample; s2, carrying out quality inspection on the constructed library, and carrying out mRNA sequencing by using an Illumina NovaseqTM6000 sequencer after all the libraries are qualified in quality inspection; s3, carrying out bioinformatics analysis on data generated by high-throughput sequencing; s4, different parts of the cartilage tissue of the OA patient are labeled with RIPK3 through immunofluorescence, it is proved that expression of the RIPK3 in cartilage cells has strong correlation with the pathology of the OA focus, and the feasibility of taking the RIPK3 as a clinical pathology index is proved.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and genetic engineering, and specifically relates to an application method based on transcriptomics analysis to reveal RIPK3 as an osteoarthritis pathological indicator. Background Art

[0002] Osteoarthritis (OA) is the most common chronic degenerative joint disease in the world. Due to the many factors affecting OA and its unclear pathogenesis, the current treatment is mainly to relieve symptoms, and there is no effective means to prevent its progression. With the advent of an aging society, the incidence of OA is gradually increasing. Therefore, it is urgent to explore its pathogenesis and find effective treatments.

[0003] OA is mainly characterized by cartilage degeneration, and increasing evidence shows that pathological changes in subchondral bone are closely related to the progression of OA. During joint movement, subchondral bone can support the surface articular cartilage and absorb mechanical stress. Cartilage and subchondral bone maintain the structural and functional integrity of the joint through dynamic interaction. Articular cartilage is an avascular tissue with poor self-healing ability, and its injury and repair face arduous clinical challenges. Chondrocytes, as the only cell type in cartilage, are a key factor affecting the progression of OA. Abnormal apoptosis and inflammatory response of chondrocytes are closely related to matrix degradation and cartilage destruction in OA.

[0004] Necroptosis is a form of programmed cell death mediated by serine / threonine kinases receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). When necroptosis occurs, damage-associated signaling molecules (DAMPs) are released, causing the inflammation and other reactions of the tissues surrounding the damaged cells to increase, thereby causing organ damage. Therefore, necroptosis is closely related to the occurrence and development of a variety of inflammatory and damaging diseases. Studies have shown that necroptosis is an important way of chondrocyte death. The expression of RIPK3 and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) in highly degenerated cartilage tissue of human knee OA is increased. Inhibiting chondrocyte necrosis can significantly reduce the overall degree of cartilage damage. Therefore, inhibiting chondrocyte necrosis may be a new attempt to treat OA in the future. Using transcriptomes to screen possible genes related to pathological changes and immune infiltration in OA lesions is a traditional Chinese medicine method to study the pathological mechanism of osteoarthritis. However, the lack of high-quality transcriptome data has seriously hindered the pathological research of osteoarthritis.

[0005] Based on this, we proposed an application method based on transcriptomic analysis to reveal RIPK3 as an osteoarthritis pathological indicator, hoping to address the shortcomings of existing technologies. Summary of the invention

[0006] The purpose of the present invention is to provide an application method based on transcriptomics analysis to reveal RIPK3 as an osteoarthritis pathological indicator in response to existing problems.

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

[0008] A method for revealing RIPK3 as an osteoarthritis pathological indicator based on transcriptomic analysis, comprising the following steps:

[0009] S1. Total RNA was extracted from articular cartilage specimens using Trizol reagent, and high-quality RNA samples with RIN number > 7.0 were selected to construct sequencing libraries;

[0010] S2. Perform quality inspection on the constructed library. After passing the quality inspection, the average insert size of the final library is 300±50bp;

[0011] After all quality inspections were passed, Illumina Novaseq TM 6000 sequencer for mRNA sequencing;

[0012] S3, perform bioinformatics analysis on the data generated by high-throughput sequencing;

[0013] S4. Immunofluorescence labeling of RIPK3 in different parts of the cartilage tissue of OA patients confirmed that the expression of RIPK3 in chondrocytes was strongly correlated with the pathology of OA lesions, confirming the feasibility of using RIPK3 as a clinical pathological indicator.

[0014] Furthermore, step S1 further includes:

[0015] The quantity and purity of total RNA were checked using Bioanalyzer 2100 and RNA 6000Nano Lab Chip Kit;

[0016] mRNA was purified from 5ug of total RNA using Dyna beads Oligo, and two rounds of purification were performed;

[0017] After purification, the mRNA was fragmented into short fragments using divalent cations at elevated temperatures, reacting at 94°C for 7 min;

[0018] SuperScript TM II reverse transcriptase reverse transcribes the cleaved RNA fragment to produce cDNA, which is then used to synthesize a U-labeled second-strand cDNA strand using E. coli DNA polymerase I, RNase H, and dUTP solution;

[0019] The prepared double-stranded cDNA was repaired at the end, A-tailed, and connected to the sequencing adapter, and size-screened using AMPure XP beads;

[0020] The U-labeled second-strand DNA was treated with the heat-labile UDG enzyme, and the ligation product was amplified by PCR under the following conditions: pre-denaturation at 95°C for 3 min; denaturation at 98°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C for 30 sec, 8 cycles, and extension at 72°C for 5 min.

[0021] Furthermore, the bioinformatics analysis in step S3 includes:

[0022] (1) Data preprocessing and quality control:

[0023] The raw data generated by high-throughput sequencing were further filtered to remove reads containing adapters, remove reads containing polyA and polyG, remove reads containing more than 5% unknown nucleotides, and remove low-quality reads containing more than 20% low-quality bases;

[0024] FastQC was then used to verify sequence quality, including Q20, Q30, and GC content of the cleaned data;

[0025] Afterwards, a total of G bp of clean paired-end reads were generated to comprehensively evaluate the expression of cartilage samples through genomic alignment rate;

[0026] (2) Reference genome comparison:

[0027] Use HISAT2 to align the reads of all samples with the reference genome, first remove a portion of the reads based on the quality information attached to each read, and then map the reads to the reference genome;

[0028] (3) Quantification of gene abundance:

[0029] The comprehensive transcriptome was obtained using gffcompare software;

[0030] After the final transcriptome was generated, StringTie and ballgown were used to estimate the expression levels of all transcripts, and the expression abundance of mRNA was calculated by calculating the FPKM value;

[0031] (4) Analysis of differentially expressed genes:

[0032] Gene differential expression analysis was performed between two different groups by DESeq 2 software and between two samples by edgeR. Genes with FDR parameter < 0.05 and |FC| ≥ 2 were considered differentially expressed genes. GO function and KEGG pathway enrichment analysis were then performed on the differentially expressed genes.

[0033] (5) Principal component analysis:

[0034] Use R's princomp function to perform principal component analysis;

[0035] (6) PPI interaction analysis:

[0036] The screened differentially expressed genes were used to construct a PPI protein network interaction diagram through the STRING database and GeneMANIA.

[0037] Furthermore, step S4 also includes paraffin section preparation, paraffin section hematoxylin-eosin staining and immunofluorescence staining.

[0038] Furthermore, the paraffin section preparation includes:

[0039] (1) Place the samples of lesion tissue, junction tissue, and normal tissue of the case specimens in 15 ml centrifuge tubes, add 10 ml of 4% paraformaldehyde, and fix at room temperature for 24 h;

[0040] (2) Discard the fixative solution, transfer the fixed tissue to PBS solution, place it on a shaker at room temperature and wash for 10 min, repeat three times;

[0041] (3) gradient dehydration and clearing with ethanol, the specific steps are: 70% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 100% ethanol I for 1 hour, 100% ethanol II for 1 hour, xylene I for 0.5 hour, xylene II for 0.5 hour;

[0042] (4) Soak the transparent sample in benzene wax at 60°C for 0.5 h, paraffin I at 60°C for 1 h, and paraffin II at 60°C for 1 h. After soaking, use an embedding machine to embed the tissue in a conventional way. Place the dehydrated tissue block in an embedding box, pour in melted paraffin to completely cover the tissue and cover the groove of the embedding box, and cool on ice to form a wax block.

[0043] (5) Set the slicer program to a slice thickness of 5 nm;

[0044] Keep observing during the sectioning process. After cutting to the desired position, gently lift the wax slice with a brush and slowly place it in a 42°C water bath to fully spread the slice. Use a clean glass slide to pick up the slice, use absorbent paper to absorb moisture from the surface of the slide, and bake the slice in a 37°C oven.

[0045] Further, the paraffin section hematoxylin-eosin staining comprises:

[0046] (1) sequentially placing the sections in environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - and washing with distilled water;

[0047] (2) The sections were immersed in hematoxylin staining solution for 5 min and then washed thoroughly with distilled water;

[0048] (3) The sections were immersed in eosin staining solution for 5 min, washed with tap water for 10 min, differentiated with hydrochloric acid ethanol differentiation solution for 30 s, and rinsed with tap water for 10 min;

[0049] (4) 70% ethanol for 10 seconds, 80% ethanol for 10 seconds, 90% ethanol for 10 seconds, anhydrous ethanol for 10 seconds, xylene I for 5 minutes, and xylene II for 5 minutes for transparent sealing. Then, use neutral resin to seal the slides and observe and take pictures under a microscope.

[0050] Furthermore, the immunofluorescence staining comprises:

[0051] (1) sequentially placing the sections in environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - and washing with distilled water;

[0052] (2) The tissue sections were placed in a repair box filled with EDTA antigen repair buffer in a microwave oven for antigen repair. The pH of the buffer was 8.0.

[0053] Medium heat for 8 minutes, stop for 8 minutes and switch to medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented and the slides should not be dried;

[0054] After cooling naturally, the slides were placed in PBS with a pH of 7.4 and washed three times on a decolorizing shaker, each time for 5 min;

[0055] (3) After the slices are slightly dried, use a tissue pen to draw a circle around the tissue;

[0056] (4) Add BSA to the circle and incubate for 30 min;

[0057] (5) Gently shake off the blocking solution, add rabbit RIPK3 primary antibody diluted with PBS to the slices, and incubate the slices flat in a humidified chamber at 4°C overnight;

[0058] (6) The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time.

[0059] After the slices are slightly dried, add the secondary antibody diluted with the corresponding species of the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 minutes;

[0060] (7) After the slices are slightly dried, DAPI staining solution is added to the circle and incubated at room temperature in the dark for 10 min;

[0061] (8) After the slices are slightly dried, add the autofluorescence quencher in the circle for 5 minutes and rinse with running water for 10 minutes;

[0062] (9) The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time. The sections were slightly dried and sealed with an anti-fluorescence quenching sealing medium.

[0063] (10) Place the slices under a scanner to collect images or take photos under a fluorescence microscope.

[0064] Compared with the prior art, the present invention has the following advantages:

[0065] The present invention collected 12 clinical specimens of cartilage tissue from OA patients, and screened 140 significantly differentially expressed genes between normal tissue and junctional tissue of cartilage pathological specimens by high-throughput transcriptome sequencing (Log2|FC|>1, P<0.05). GO and KEGG analysis showed that the differentially expressed genes were mainly enriched in immunoglobulin complex, immune response, material metabolism, etc., and the possible genes related to the pathological changes and immune infiltration of OA lesions were screened out: ITLN1, CCL19, SIRPG, IL12, XCL1, RIPK3, among which RIPK3 was enriched and annotated to multiple signaling pathways, including necroptosis, TNF signaling pathway, NOD-like receptor signaling pathway, and cytoplasmic DNA sensing pathway. Among them, RIPK3 protein is a key regulatory factor in cell necroptosis, and forms necrosomes under the stimulation of inflammatory factors such as TNF, thereby promoting the progression of various inflammatory diseases. Death signals induce the activation of RIPK3 kinase, which then phosphorylates the specific execution protein MLKL of cell necrosis. Phosphorylated MLKL (p-MLKL) oligomerizes and translocates to the cell membrane structure, leading to the destruction of the cell membrane and organelle membrane, resulting in cell death and leakage of intracellular substances. Apoptosis is a gene-controlled autonomous orderly death that can remove differentiated cartilage, while necroptosis is a pathological form of death that causes cell membrane rupture, releases inflammatory factors, triggers a strong inflammatory response, and has a harmful effect on surrounding tissues. Chondrocytes secrete the extracellular matrix that constitutes the main component of cartilage. Therefore, in the process of OA, abnormal activation of chondrocyte apoptosis is an important factor causing cartilage degeneration.

[0066] The present invention determined multiple pathways through which RIPK3 may play a role in the occurrence and development of OA based on transcriptome sequencing technology, and further confirmed that the expression of RIPK3 in chondrocytes was strongly correlated with the pathology of OA lesions by immunofluorescence labeling of RIPK3 in different parts of the cartilage tissue of OA patients. The present invention confirmed the feasibility of using RIPK3 as a clinical pathological indicator. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 is the distribution diagram of differential gene expression;

[0068] Figure 2 This is the result diagram of differential gene function enrichment analysis;

[0069] Figure 3 This is the prediction result diagram of PPI interaction of differentially expressed genes;

[0070] Figure 4 These are the results of HE staining and immunofluorescence staining of OA pathological cartilage tissue specimens. DETAILED DESCRIPTION

[0071] In order to further explain the present invention, it is described below in conjunction with the following specific embodiments.

[0072] Example 1

[0073] A method for revealing RIPK3 as an osteoarthritis pathological indicator based on transcriptomic analysis, comprising the following steps:

[0074] (1) Clinical sample collection

[0075] Specimens from 12 patients with knee osteoarthritis who were admitted to the Fifth People's Hospital of Ningxia Hui Autonomous Region (No. 1-No. 12) were collected and divided into three locations according to the specimen conditions: lesion tissue (LT), junctional tissue (JT), and normal tissue (NT). 1 cm of cartilage tissue was collected from each location. 3 , placed in a 1.5 ml EP tube, quickly frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator;

[0076] (2) Library construction

[0077] 1) Total RNA was extracted from articular cartilage specimens using Trizol reagent to detect RNA integrity. The amount and purity of total RNA were checked using Bioanalyzer 2100 and RNA 6000Nano Lab Chip Kit. Sequencing libraries were constructed using high-quality RNA samples with RIN numbers > 7.0. Since RNA in tissues at the lesion site was severely degraded and could not be used for subsequent sequencing experiments, the junction tissues and normal tissues of 6 case specimens numbered NO.2.4.6.7.10.12 were selected for high-throughput sequencing;

[0078] 2) Purify mRNA from 5ug total RNA using Dyna beads Oligo(dT) and perform two rounds of purification;

[0079] 3) After purification, mRNA was fragmented into short fragments using divalent cations at elevated temperature at 94°C for 7 min;

[0080] 4)SuperScript TM II reverse transcriptase reverse transcribes the cleaved RNA fragment to produce cDNA, which is then used to synthesize a U-labeled second-strand cDNA strand using E. coli DNA polymerase I, RNase H, and dUTP solution;

[0081] 5) The prepared double-stranded cDNA is repaired at the end, A-tailed, and connected to the sequencing adapter, and size-screened using AMPureXP beads;

[0082] 6) Treat the U-labeled second-strand DNA with the thermolabile UDG enzyme, and amplify the ligation product by PCR under the following conditions: pre-denaturation at 95°C for 3 min; denaturation at 98°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C for 30 sec, 8 cycles, and extension at 72°C for 5 min;

[0083] 7) The constructed library was quality checked and the average insert size of the final library was 300±50bp after passing the quality check;

[0084] After all quality inspections were passed, Illumina Novaseq TM 6000 sequencer for mRNA sequencing;

[0085] (3) Bioinformatics analysis

[0086] 1) Data preprocessing and quality control:

[0087] The raw data (Raw reads) generated by high-throughput sequencing were further filtered to remove reads containing adapters, remove reads containing polyA and polyG, remove reads containing more than 5% unknown nucleotides (N), and remove low-quality reads containing more than 20% low-quality (Q value ≤ 20) bases;

[0088] FastQC ( http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / , 0.11.9) was then used to verify sequence quality, including Q20, Q30, and GC content of the cleaned data;

[0089] Afterwards, a total of G bp of clean paired-end reads were generated to comprehensively evaluate the expression of cartilage samples through genomic alignment rate;

[0090] 2) Reference genome alignment:

[0091] HISAT2 (https: / / daehwankimlab.github) was used to align the reads of all samples with the reference genome. First, a portion of the reads were removed based on the quality information attached to each read, and then the reads were mapped to the reference genome.

[0092] 3) Quantification of gene abundance:

[0093] The comprehensive transcriptome was obtained using gffcompare software ( http: / / ccb.jhu.edu / software / stringtie / gffcompare.shtml , version: gffcompare-0.9.8);

[0094] After the final transcriptome was generated, StringTie and ballgown ( http: / / www.bioconductor.org / packages / release / bioc / html / ballgown.html ) were used to estimate the expression levels of all transcripts, and the expression abundance of mRNA was calculated by calculating the FPKM value;

[0095] 4) Differentially expressed genes (DEG) analysis:

[0096] Gene differential expression analysis was performed between two different groups (and between two samples by edgeR) using DESeq 2 software, and genes with FDR parameter < 0.05 and |FC| ≥ 2 were considered differentially expressed genes. GO function and KEGG pathway enrichment analysis were then performed on the differentially expressed genes.

[0097] 5) Principal Component Analysis:

[0098] Principal component analysis (PCA) was performed using the princomp function of R (http: / / www.r-project.org / );

[0099] 6) GO enrichment analysis:

[0100] All DEGs were mapped to GO entries in the Gene Ontology database (http: / / www.geneontology.org / ), and the number of genes in each entry was calculated. The GO entries that were significantly enriched in DEGs compared with the genomic background were determined by hypergeometric test. GO terms that met this condition and p < 0.05 were defined as significantly enriched GO entries in DEGs; this analysis can identify the main biological functions performed by DEGs;

[0101] 7) KEGG enrichment analysis:

[0102] Genes often interact with each other and play a role in certain biological functions. Pathway-based analysis helps to further understand the metabolic pathways or signal transduction pathways that are significantly enriched in DEGs compared with the whole genome, and the metabolic pathways or signal transduction pathways that are significantly enriched in DEGs compared with the whole genome background;

[0103] 8) PPI interaction analysis:

[0104] Protein-Protein Interaction Networks (PPI) are composed of proteins that interact with each other and participate in various aspects of life processes such as biological signal transmission, gene expression regulation, energy and material metabolism, and cell cycle regulation.

[0105] The screened DEGs were used to construct the PPI protein network interaction diagram through the STRING database (https: / / cn.string-db.org) and GeneMANIA (https: / / genemania.org);

[0106] (4) Preparation of paraffin sections

[0107] 1) Fixed:

[0108] A total of 18 samples of lesion tissue, junctional tissue, and normal tissue from 10 cases were placed in 15 ml centrifuge tubes, added with 10 ml of 4% paraformaldehyde, and fixed at room temperature for 24 h;

[0109] 2) Decalcification and cleaning:

[0110] Discard the fixative solution, transfer the fixed tissue to PBS solution, place it on a shaker at room temperature and wash for 10 minutes, repeat three times;

[0111] Add 10 ml of 25% EDTA decalcification solution to each tube and shake at 37°C for one to three weeks. When the needle can penetrate the bone tissue, proceed to the next step.

[0112] PBS solution was used for washing. 10 ml of washing solution was added to each tube. The centrifuge tube was placed on a shaker for 10 min and washed. This was repeated three times.

[0113] 3) Tissue dehydration and transparency:

[0114] Gradient dehydration with ethanol was started, and the specific steps were: 70% ethanol (1 h), 80% ethanol (1 h), 90% ethanol (1 h), 100% ethanol I (1 h), 100% ethanol II (1 h), xylene I (0.5 h), xylene II (0.5 h);

[0115] 4) Infiltration and embedding:

[0116] Soak the transparent samples in benzene wax (60℃, 0.5h), paraffin I (60℃, 1h), and paraffin II (60℃, 1h). After soaking, use an embedding machine to embed the tissue routinely. Put the dehydrated tissue block into an embedding box, pour melted paraffin to completely cover the tissue and cover the groove of the embedding box, and cool it on ice to form a wax block. The tissue wax block can be stored in a -20℃ refrigerator for a long time.

[0117] 5) Slicing and gluing:

[0118] Set the slicer program and the slice thickness is 5nm;

[0119] Keep observing during the sectioning process. When the desired position is reached, gently lift the wax slice with a brush and slowly place it in a 42°C water bath to fully spread the slice. Use a clean glass slide to pick up the slice, use absorbent paper to absorb the moisture on the surface of the glass slide, and bake the slice in a 37°C oven.

[0120] (5) Hematoxylin-eosin staining of paraffin sections

[0121] 1) Dewaxing to water:

[0122] Sequentially put the slices into environmentally friendly dewaxing solution for 10 min-environmentally friendly dewaxing solution for 10 min-environmentally friendly dewaxing solution for 10 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-anhydrous ethanol III for 5 min-distilled water for washing;

[0123] 2) Hematoxylin staining:

[0124] The sections were immersed in hematoxylin staining solution for 5 min and then washed thoroughly with distilled water;

[0125] 3) Eosin counterstaining: The sections were immersed in eosin staining solution for 5 minutes, washed with tap water for 10 minutes, differentiated with hydrochloric acid ethanol differentiation solution for 30 seconds, and rinsed with tap water for 10 minutes;

[0126] 4) Transparent sealing: 70% ethanol (10s), 80% ethanol (10s), 90% ethanol (10s), anhydrous ethanol (10s), xylene I (5min), xylene II (5min), then use neutral resin to seal the slide and observe and take pictures under a microscope;

[0127] (6) Immunofluorescence staining

[0128] 1) Dewaxing to water:

[0129] Sequentially put the slices into environmentally friendly dewaxing solution for 10 min-environmentally friendly dewaxing solution for 10 min-environmentally friendly dewaxing solution for 10 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-anhydrous ethanol III for 5 min-distilled water for washing;

[0130] 2) Antigen retrieval:

[0131] The tissue sections were placed in a repair box filled with EDTA antigen repair buffer (pH 8.0) in a microwave oven for antigen repair;

[0132] Medium heat for 8 minutes, stop for 8 minutes and switch to medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented and the slides should not be dried;

[0133] After cooling naturally, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker, each time for 5 min;

[0134] 3) Draw a circle:

[0135] After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away);

[0136] 4) Serum blocking:

[0137] BSA was added dropwise into the circle and incubated for 30 min;

[0138] 5) Add the first antibody:

[0139] Gently shake off the blocking solution, add rabbit RIPK3 primary antibody diluted with PBS on the slices, and incubate the slices flat in a humidified box at 4°C overnight (add a small amount of water in the humidified box to prevent the antibody from evaporating);

[0140] 6) Add secondary antibody:

[0141] The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker, each time for 5 min;

[0142] After the slices are slightly dried, add the secondary antibody diluted with the corresponding species of the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 minutes;

[0143] 7) DAPI counterstaining of cell nuclei:

[0144] After the slices were slightly dried, DAPI stain was added to the circle and incubated at room temperature in the dark for 10 min;

[0145] 8) Autofluorescence quenching:

[0146] After the slices were slightly dried, an autofluorescence quencher was added to the circle for 5 min and then rinsed with running water for 10 min;

[0147] 9) Sealing:

[0148] The slides were placed in PBS (pH 7.4) and washed on a decolorizing shaker for 3 times, 5 min each time. The sections were slightly dried and sealed with anti-fluorescence quenching sealing medium.

[0149] 10) Microscopic examination and photography:

[0150] The slices were placed under a scanner to collect images or photographed under a fluorescence microscope. The DAPI ultraviolet excitation wavelength is 330-380nm, the emission wavelength is 420nm, and it emits blue light; the CY3 excitation wavelength is 510-560, the emission wavelength is 590nm, and it emits red light. The cell nuclei stained with DAPI are blue under ultraviolet excitation, and positive expression is red light labeled with the corresponding fluorescent marker.

[0151] Example 2

[0152] Results Characterization

[0153] 1. Differential gene analysis

[0154] The sequencing results showed that in the articular cartilage tissue of OA cases, there were 140 genes differentially expressed between the junction tissue and the normal tissue (Log2|FC|>1, P<0.05), of which 84 were up-regulated and 56 were down-regulated, such as Figure 1 As shown in A, Figure 1 B is the volcano plot of differential gene expression. Figure 1 C Heat map shows the expression intensity of differentially expressed genes in the two groups of samples, indicating that the cartilage tissues in the normal area and the junction area have significantly different gene expression patterns.

[0155] Table 1 shows some of the significantly up-regulated and down-regulated differentially expressed genes.

[0156] Table 1 Some significantly differentially expressed genes

[0157]

[0158]

[0159] Figure 1 Middle: Distribution of differential gene expression. A. Bar graph of the number of differentially expressed genes, red for up-regulated and blue for down-regulated; B. Volcano graph of differentially expressed genes (Log2|FC|>1, P<0.05); C. Cluster heat map of differentially expressed genes. JT: Junction tissue, NT: Normal tissue.

[0160] 2. Correlation analysis of differentially expressed genes

[0161] In order to analyze the relationship between the biological processes, molecular functions, cellular components, signaling pathways and diseases of the differentially expressed genes, GO and KEGG functional enrichment analysis was performed on the differentially expressed genes.

[0162] like Figure 2 As shown in A, the results of GO analysis showed that compared with normal tissue, the differentially enriched items of genes in OA lesion border tissue were mainly in leukotriene metabolism and positive regulation of granulocyte migration in terms of biological processes; in terms of cellular components, they were mainly enriched in immunoglobulin complexes; and in terms of molecular functions, they were mainly enriched in chemokine receptor binding.

[0163] like Figure 2 As shown in B, the results of KEGG analysis showed that the differentially expressed genes were mainly annotated to signaling pathways such as necroptosis, endocytosis, cytokine-receptor interaction, metabolic pathways, and chemokine signaling.

[0164] Figure 2 Middle: A. GO analysis bubble chart of differentially expressed genes; B. KEGG analysis item bar chart of differentially expressed genes.

[0165] 3. Screening of differentially expressed genes

[0166] Among the significantly differentially expressed genes, most of the down-regulated genes are regulatory non-coding RNAs or unannotated RNAs, and the up-regulated genes are mainly related to cytokine activity, chemokine receptor binding, and granulocyte migration. Based on the GeneMANIA and STRING protein interaction databases, the non-directional scores of each node in the protein-protein interaction network of the up-regulated differentially expressed genes are calculated, and the protein interaction network is visualized and analyzed according to the obtained degree values, such as Figure 3 A. Further screening of possible genes related to pathological changes and immune infiltration of OA lesions: ITLN1, CCL19, SIRPG, IL12, XCL1, RIPK3, and constructing protein interaction network prediction diagrams for the above differentially expressed genes, as shown in Figure 3 As shown in B, there is an interaction between them, and they may jointly participate in the occurrence, progression and immune regulation of OA.

[0167] Figure 3 A. Upregulated differentially expressed genes; B. Upregulated differentially expressed genes after screening: ITLN1, CCL19, SIRPG, IL12, XCL1, and RIPK3.

[0168] 4. Immunofluorescence Validation

[0169] Immunofluorescence was used to verify the expression of ITLN1, CCL19, SIRPG, IL12, XCL1, and RIPK3 in different parts of OA specimens. The expression of RIPK3 in lesion tissues was higher than that in border tissues, and further higher than that in normal tissues. Figure 4 As shown. RIPK3 is significantly differentially expressed in different OA cartilage tissues. The present invention confirms the feasibility of using RIPK3 as an indicator of OA pathological specimens. RIPK3 is also enriched and annotated into multiple signaling pathways, including necroptosis, cytoplasmic DNA sensing pathway, TNF signaling pathway, NOD-like receptor signaling pathway, and other pathways that may be related to the occurrence and development of OA.

[0170] In summary, the present invention screened out the RIPK3 differentially expressed genes through high-throughput transcriptional sequencing, and further verified the differences of RIPK3 in lesion tissues, junctional tissues, and normal tissues in OA case specimens through immunofluorescence, verified the feasibility of RIPK3 as a pathological indicator, and indicated the degree of lesions in pathological specimens of OA patients by the expression level of RIPK3.

[0171] Figure 4 Middle: A. Normal tissue; B. Border tissue; C. Lesion tissue; Immunofluorescence blue fluorescence: DIPA, red fluorescence: RIPK3.

[0172] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An application method based on transcriptomic analysis to reveal RIPK3 as an osteoarthritis pathological indicator, characterized in that: The steps include: S1. Total RNA was extracted from articular cartilage specimens using Trizol reagent, and high-quality RNA samples with RIN number > 7.0 were selected to construct sequencing libraries; S2. Perform quality inspection on the constructed library. After passing the quality inspection, the average insert size of the final library is 300±50bp; After all quality inspections were passed, Illumina Novaseq TM 6000 sequencer for mRNA sequencing; S3, perform bioinformatics analysis on the data generated by high-throughput sequencing; S4. Immunofluorescence labeling of RIPK3 in different parts of the cartilage tissue of OA patients confirmed that the expression of RIPK3 in chondrocytes was strongly correlated with the pathology of OA lesions, confirming the feasibility of using RIPK3 as a clinical pathological indicator.

2. According to claim 1, a method for revealing RIPK3 as an osteoarthritis pathological indicator based on transcriptomic analysis, characterized in that: Step S1 also includes: The quantity and purity of total RNA were checked using Bioanalyzer 2100 and RNA 6000Nano Lab Chip Kit; mRNA was purified from 5ug of total RNA using Dyna beads Oligo, and two rounds of purification were performed; After purification, the mRNA was fragmented into short fragments using divalent cations at elevated temperatures, reacting at 94°C for 7 min; SuperScript TM II reverse transcriptase reverse transcribes the cleaved RNA fragments to produce cDNA, which is then used to synthesize a U-labeled second-strand cDNA chain using E. coli DNA polymerase I, RNase H, and dUTP solution; The prepared double-stranded cDNA was repaired at the end, A-tailed, and connected to the sequencing adapter, and size-screened using AMPure XP beads; The U-labeled second-strand DNA was treated with the heat-labile UDG enzyme, and the ligation product was amplified by PCR under the following conditions: pre-denaturation at 95°C for 3 min; denaturation at 98°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C for 30 sec, 8 cycles, and extension at 72°C for 5 min.

3. According to claim 1, a method for using RIPK3 as an osteoarthritis pathological indicator based on transcriptomic analysis, characterized in that: The bioinformatics analysis in step S3 includes: (1) Data preprocessing and quality control: The raw data generated by high-throughput sequencing were further filtered to remove reads containing adapters, remove reads containing polyA and polyG, remove reads containing more than 5% unknown nucleotides, and remove low-quality reads containing more than 20% low-quality bases; FastQC was then used to verify sequence quality, including Q20, Q30, and GC content of the cleaned data; Afterwards, a total of G bp of clean paired-end reads were generated to comprehensively evaluate the expression of cartilage samples through genomic alignment rate; (2) Reference genome comparison: Use HISAT2 to align the reads of all samples with the reference genome, first remove a portion of the reads based on the quality information attached to each read, and then map the reads to the reference genome; (3) Quantification of gene abundance: The comprehensive transcriptome was obtained using gffcompare software; After the final transcriptome was generated, StringTie and ballgown were used to estimate the expression levels of all transcripts, and the expression abundance of mRNA was calculated by calculating the FPKM value; (4) Analysis of differentially expressed genes: Gene differential expression analysis was performed between two different groups by DESeq 2 software and between two samples by edgeR. Genes with FDR parameter < 0.05 and |FC| ≥ 2 were considered differentially expressed genes. GO function and KEGG pathway enrichment analysis were then performed on the differentially expressed genes. (5) Principal component analysis: Use R's princomp function to perform principal component analysis; (6) PPI interaction analysis: The screened differentially expressed genes were used to construct a PPI protein network interaction diagram through the STRING database and GeneMANIA.

4. According to claim 1, a method for revealing RIPK3 as an osteoarthritis pathological indicator based on transcriptomic analysis, characterized in that: Step S4 also includes paraffin section preparation, paraffin section hematoxylin-eosin staining and immunofluorescence staining.

5. The method according to claim 4, wherein the method is characterized in that: The paraffin section preparation comprises: (1) Place the samples of lesion tissue, junction tissue, and normal tissue of the case specimens in 15 ml centrifuge tubes, add 10 ml of 4% paraformaldehyde, and fix at room temperature for 24 h; (2) Discard the fixative solution, transfer the fixed tissue to PBS solution, place it on a shaker at room temperature and wash for 10 min, repeat three times; (3) gradient dehydration and clearing with ethanol, the specific steps are: 70% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 100% ethanol I for 1 hour, 100% ethanol II for 1 hour, xylene I for 0.5 hour, xylene II for 0.5 hour; (4) Soak the transparent sample in benzene wax at 60°C for 0.5 h, paraffin I at 60°C for 1 h, and paraffin II at 60°C for 1 h. After soaking, use an embedding machine to embed the tissue in a conventional way. Place the dehydrated tissue block in an embedding box, pour in melted paraffin to completely cover the tissue and cover the groove of the embedding box, and cool on ice to form a wax block. (5) Set the slicer program to a slice thickness of 5 nm; Keep observing during the sectioning process. After cutting to the desired position, gently pick up the wax slice with a brush and slowly place it in a 42°C water bath to fully spread the slice. Use a clean glass slide to pick up the slice, use absorbent paper to absorb moisture from the surface of the slide, and bake the slice in a 37°C oven.

6. According to claim 4, a method for using RIPK3 as an osteoarthritis pathological indicator based on transcriptomic analysis, characterized in that: The paraffin section hematoxylin-eosin staining comprises: (1) sequentially placing the sections in environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - and washing with distilled water; (2) The sections were immersed in hematoxylin staining solution for 5 min and then washed thoroughly with distilled water; (3) The sections were immersed in eosin staining solution for 5 min, washed with tap water for 10 min, differentiated with hydrochloric acid ethanol differentiation solution for 30 s, and rinsed with tap water for 10 min; (4) 70% ethanol for 10 seconds, 80% ethanol for 10 seconds, 90% ethanol for 10 seconds, anhydrous ethanol for 10 seconds, xylene I for 5 minutes, and xylene II for 5 minutes for transparent sealing. Then, use neutral resin to seal the slides and observe and take pictures under a microscope.

7. According to claim 4, a method for revealing RIPK3 as an osteoarthritis pathological indicator based on transcriptomic analysis, characterized in that: The immunofluorescence staining comprises: (1) sequentially placing the sections in environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - and washing with distilled water; (2) The tissue sections were placed in a repair box filled with EDTA antigen repair buffer in a microwave oven for antigen repair. The pH of the buffer was 8.

0. Medium heat for 8 minutes, stop for 8 minutes and switch to medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented and the slides should not be dried; After cooling naturally, the slides were placed in PBS with a pH of 7.4 and washed three times on a decolorizing shaker, each time for 5 min; (3) After the slices are slightly dried, use a tissue pen to draw a circle around the tissue; (4) Add BSA to the circle and incubate for 30 min; (5) Gently shake off the blocking solution, add rabbit RIPK3 primary antibody diluted with PBS to the slices, and incubate the slices flat in a humidified chamber at 4°C overnight; (6) The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time. After the slices are slightly dried, add the secondary antibody diluted with the corresponding species of the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 minutes; (7) After the slices are slightly dried, DAPI staining solution is added to the circle and incubated at room temperature in the dark for 10 min; (8) After the slices are slightly dried, add the autofluorescence quencher in the circle for 5 minutes and rinse with running water for 10 minutes; (9) The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time. The sections were slightly dried and sealed with an anti-fluorescence quenching sealing medium. (10) Place the slices under a scanner to collect images or take photos under a fluorescence microscope.

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