Sample collection and Nucleus pulposus cell extraction
Informed consent is provided by the patients and their relatives before obtaining the intervertebral disc tissue in surgery. The experiment was authorized by the ethics committee of Second Military Medical University. Three normal intervertebral disc tissue sample were collected from lumbar trauma patients who underwent spinal fusion with no radiological sign of degeneration (Pfirrmann grade I, n=3, age 45 to 49 years, mean 47 years). And three degenerated disc tissue were collected from diagnosed lumbar herniation patients who underwent disc resection and fusion surgery to relieve symptom (Pfirrmann grade IV-V, n=3, age 46 to 50 years, mean 48 years). MRI T-2 weighted images were collected and the modified Pfirrmann grading system(3) was used to evaluate the degree of IDD.
For cell extraction, NP tissue specimens were washed twice with PBS, then minced and digested with 2U/mL protease in DMEM/F12 (Gibco, Grand island NY, USA) for 30 minutes at 37°C. NP cells were released from the tissues by treating with 0.25 mg/mL type II collagenase (Gibco, Cat. No. 17101-015) for 4 hours at 37°C.The resulting cell suspension was transferred into a 40μm cell strainer (BD Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) and centrifuged at 800 g for 5 minutes. The cell pellets were used subsequently in proteomic analysis or total RNA extraction.
RNA extraction and reverse transcription
RNA was extracted from human nucleus pulposus samples using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Concentration of total RNA was measured at 260 nm with a spectrophotometer (DU-800; Beckman Coulter, Brea, CA). First strand complementary DNA (cDNA) synthesis was performed with 500ng of total RNA in a 10μL final volume containing 2μL Primer Script RT Master Mix (Takara, RR036A, Japan) and 8μLof RNase-free dH2O and total RNA. The reverse transcription procedure was carried out according to manufacturer’s instructions.
Quantitative real-time polymerase chain reaction
Real-time PCR for KRT19, COL6A2, DPT, COL11A2, CLIP and CHI3L1 were performed by using SYBR premix Ex Taq™ (Takara Bio Inc., Shiga, Japan) with a Step One Plus real-time PCR system (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. GAPDH was used to normalize the gene expression of mRNAs. The relative amount of transcripts was calculated according to the comparative 2–∆∆CT method. All of the primers were synthesized by Invitrogen (Supplementary Data 6). Each experiment was repeated at least three times independently. Statistical significance was assessed by comparing mean values (±SD) using a Student's t test for independent groups and was assumed for *P<0.05.
Label-free proteomic profiling.
For protein extraction, tissues samples were transferred to a 1.5-mL screw capped tube and centrifuged at 10,000×g for 30 min at 4 °C. Next, 100 μL lysis buffer (7 M urea, 2 M thiourea) were added into each sample and then ultrasonic crushed to extract total proteins. Proteins were precipitated with trichloroacetic acid (TCA) for 30 min on ice and centrifuged at 40,000×g for 30 min. Protein concentration was determined using the Qubit fluorescent protein quantification kit (Invitrogen) according to the manufacturer’s instructions.
Protein were excised from the preparative tube and destained with 50 mM NH4HCO3 to the final concentration 0.5mg/mL. Following the addition of 100 mmol/L DTT （DL-Dithiothreitol）to its final concentration of 10 mmol/L, the protein fractions were mixed at 56℃ for 60 min, then diluted 10x with 250 mmol/L IAM （Iodoacetamide）and kept in dark for 60 min. Finally, the samples were digested with trypsin (substrate to enzyme mass to mass ratio at 50:1) at 37℃ for 12 h. Digested supernatant fractions were pressure-loaded onto a fused silica capillary column packed with 3-μm dionex C18 material (RP; Phenomenex). After desalting, a 5-mm, 300-μm C18 capture tip was placed in line with an Agilent 1100 quaternary HPLC （High Performance Liquid Chromatography）and analyzed using a 12-step separation. After the elution, they were electrosprayed directly into a micrOTOF-Q II mass spectrometer (BRUKER Scientific) with the application of a distal 180°C source temperature. The mass spectrometer was operated in the MS/MS (auto) mode. Survey MS scans were acquired in the TOF-Q II with the resolution set to a value of 20,000. Each survey scan (50~2,500) was followed by five data-dependent tandem mass (MS/MS) scans at 2HZ normalized scan speed.
Transcriptome data acquiring and bioinformatics analysis.
For IDD transcriptome sequencing data, we downloaded the processed data of GSE70362 from Gene Expression Omnibus (GEO) database (13). Samples were rearrange and combined according to Pfirrmann grade. Here we used Pfirrmann grade I and II samples as normal NP (IVD), and Pfirrmann grade III to V as degenerated NP (IDD) for further analysis. Unsupervised clustering using average linkage and median centering were sub sequentially performed and visualized with TreeView. Gene ontology (GO) analysis was carried out using annotations in Protein ANalysisTHrough Evolutionary Relationships (PANTHER) database v 6.1 (www.pantherdb.org). Pathway analysis were performed with the tools on the Kyoto Encyclopedia of Genes and Genome (KEGG) database (http://www.genome.jp/kegg/pathway.html), respectively. Other bioinformatics analyses were performed by Beijing BangFei Bioscience Co., Ltd. and Shanghai NovelBio Bio-Pharm Technology Co.,Ltd.
Co-expression network costruction.
By comparing the normal NP with degenerated NP data, we constructed a co-expression network for both groups according to the gene’s mRNA or protein expression trend during IDD (fold change). Both the mRNA and the protein data were used, and if a gene’s expression is reversed between mRNA and protein level, we confirm it as a noise. The relationship between the genes were calculated using KCore method, and the correlation of the two genes were calculated using the pearson correlation, and the candidate genes were selected according to the FDR and P value. The network is further constructed according to the KCore and the calculated Degree.
Immunohistochemistry was performed to localize KRT19, COL6A2, DPT, COL11A2, CLIP and CHI3L1 in NP samples of 3 Pfirrmann grade 1 (IVD group) and 3 NP samples of grade IV-V (IDD group). The procedure is according to the standard protocol, briefly, antigen retrieval was performed using trypsin for 30 min at 37°C, and the sections were blocked with 1% bovine serum albumin for 15 min at room temperature. Next, the sections were incubated at 4°C overnight with the rabbit polyclonal antibody against KRT19, COL6A2, DPT, COL11A2, CLIP and CHI3L1 (all using 1:200 dilution). Next, the secondary antibody peroxidase-conjugated goat anti-rabbit IgG (1:1000 dilution) (Proteintech) was applied to the sections and they were counterstained with hematoxylin, and imaged using a ZEISS microscope (ZEISS Axio Imager A2, Carl Zeiss microscopy GmbH, Jena, Germany).