The subjects were cervical spine surgery patients admitted to Yijishan Hospital, Wannan Medical College (Wuhu, China) between February 2011 and August 2012. A total of 31 cases of cervical spondylosis (the cervical spondylosis group) were selected, with 19 males and 12 females, aged 38 to 72 years. The average age was 52 years. Seventeen cases of fracture and dislocation patients (the control group) were also selected, with 11 males and 6 females, aged 23 to 36 years. The average age was 30 years. Patients with tumors, tuberculosis, diabetes, infections and metabolic bone diseases were excluded. All the patients underwent magnetic resonance imaging (MRI) examination prior to surgery. The degeneration of pathological grade cartilage of the end-plate and intervertebral disc was classified according to Miller (7) and Thompson et al (8). Representative MRI results are shown in Fig. 1. The control group had 5 cases of Miller grade 0 and 12 cases of Miller grade 1 according to MRI examination of the cartilage end-plate prior to surgery. After surgery, 7 patients did not exhibit pathological degeneration. A total of 10 cases of pathological disc degeneration were Thompson grade 1. The cervical spondylosis group had 8 cases of Miller grade 2 and 23 cases of Miller grade 3 according to MRI examination of the cartilage end-plate prior to surgery. In addition, 6 cases were Thompson grade 3, 15 cases were Thompson grade 4 and 10 cases were Thompson grade 5 according to MRI examination of disc degeneration. This study was conducted in accordance with the Declaration of Helsinki and with approval from the Ethics Committee of Wannan Medical College. Written informed consent was obtained from all participants.

The cartilage end-plate tissue samples of the patients were excised from the disc and immediately sent to the central laboratory (Yijishan Hospital, Wannan Medical College, Wuhu, China). The samples were placed under a dissecting microscope (magnification, ×4) and processed by aseptic technique in a biological safety cabinet. The cartilage end-plate, nucleus pulposus and annulus of the samples were separated carefully. The chondrocytes were isolated (Type II collagenase and trypsin; HyClone, Logan, UT, USA) and cultured as in preliminary studies (9). The seeding density and culture conditions of the two groups of cells were the same. Changes in morphology and growth were observed regularly under an inverted microscope (Olympus, Tokyo, Japan).

The two groups of cells were mounted on a coverslip. On day 9 of cell culture, ~60% fusion was observed. The cells were then removed, seeded and stained. i) Toluidine blue staining: the coverslip was washed thrice with phosphate buffer and fixed with 4% paraformaldehyde for 30 min. The coverslip was washed with water for 15 min and stained with toluidine blue for 4 h. After the coverslip was washed with tap water, the cells were air-dried, neutral gum was added and the cells were observed under a microscope (Olympus, Japan). ii) H&E staining: the coverslip was washed thrice with phosphate buffer and fixed with 4% paraformaldehyde for 30 min. The coverslip was washed with water for 15 min and stained with H&E. After the coverslip was washed with tap water, the cells were air-dried, neutral gum was added and the cells were observed under a microscope.

The two groups of cells were passaged and cultured. When the logarithmic growth phase of the cells reached a density of 70–80%, the digested cells were mixed and blown into a single-cell suspension. Cells (10 μl) were collected and counted at 5×10⁴/ml cells per well, with an additional 2 ml for each well. The cells were cultured for 24 h after adhesion and washed twice for 5 min with phosphate-buffered saline (PBS). The cells were treated with MDC (0.05 mmol/l; Sigma, St. Louis, MO, USA) diluted with PBS at 37°C for 15 min and then washed with PBS. The cells were observed under a fluorescence microscope (Leica, Wetzlar, Germany) with an emission filter of 356 and 545 nm.

Logarithmic phase cells were trypsinized and the precipitate was collected following centrifugation. The cells were drawn up using a pipette and mixed with a RPMI-1640 medium without antibiotics, producing a final count of 2×10⁵ cells per well. Immunofluorescence was obtained following cell adhesion for 24 h. The specific steps were as follows. Fixing: The cells were washed thrice with PBS for 5 min each time, then fixed with 4% paraformaldehyde for 15 min. Blocking: The cells were washed thrice with PBS for 5 min each time. Fetal bovine serum albumin (5%) was added to the blocking solution at 37°C for 2 h. The cells were washed thrice with PBS for 5 min each time, anti-LC3B antibody (5 μg/ml, Sigma) was added at 4°C and the cells were incubated overnight. The cells were then washed thrice with PBS for 5 min each time. The diluted fluorescent secondary antibody [Alexa Fluor^® 555 donkey anti-rabbit IgG (H+L); 10 μg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA] was added to the cells at 37°C for 1.5 h. The cells were washed thrice with PBS for 5 min each time. MDC (50 μM) was added to the cells as a dye at 4°C for 30 min and kept in the dark. A confocal laser scanner (Molecular Devices, Silicon Valley, CA, USA) was used to observe and capture photos of the cells the following day.

One-step TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from cells. The purity and concentration were measured using an ultraviolet spectrophotometer (Olympus, Japan). A total of 3 μg RNA was extracted to synthesize cDNA. Aggrecan, type II collagen and β-actin were amplified using 3 μl cDNA as a template. β-actin was used as the internal reference. The gene primer sequences and the amplified fragment sizes are shown in Table I. The reaction conditions were as follows: predenaturation at 94°C for 5 min, denaturation at 94°C for 1 min and annealing at 56.1°C for 30 sec. Type II collagen was maintained at 60°C for 30 sec. β-actin was maintained at 51.9°C for 30 sec, extended at 72°C for 40 sec and amplified for 35 cycles. The reaction was terminated at 72°C after 5 min. The products were stored at 4°C. The PCR products were separated through 1.5% agarose gel electrophoresis. The Tanon Gel Image system 1D (Tanon, Shanghai, China) was used for semi-quantitative analysis. The relative band intensity percentages of the target gene and its internal reference were considered as the relative expression levels of the target gene mRNA for statistical analysis.

Radioimmunoprecipitation assay buffer and phenylmethanesulfonyl fluoride were used to extract protein from the two groups of cells. The total protein was measured using the bicinchoninic acid method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the protein. Human anti-LC3B, and anti-β-actin were diluted (1:1,000) in TBS-Tween 20 containing 1% BSA. The membranes were incubated for 2 h at 37°C with the primary antibody, washed three times in TBS-Tween 20 and for 1 h with the secondary antibody goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP (1:5,000). Subsequent detection was performed using the ECL western blotting system (Amersham Biosciences, Chalfont St Giles, UK) according to the manufacturer’s instructions. Specificity of the antibody was assessed by omitting the first antibody in western blotting experiments.

Experimental data are expressed as mean ± SD. The two groups were compared using two independent sample Student’s t-tests. The results were determined using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant result.

Under an inverted microscope, it was observed that the majority of the control cells were polygonal. The cells from the patients with cervical spondylosis gradually stretched and the majority of these cells were shuttle type. The growth rate of the cells in the cervical spondylosis group was significantly slower than that of the cells in the control group. H&E staining demonstrated that the cytoplasms contained particulate matter in the control group; the nuclei were large and round, and two to three nucleoli per cell were observed. The spindle was the primary morphology of the cell nucleus in the cervical spondylosis group. The nucleolus was unclear and the gap between cells increased. The cytoplasm and cells surrounded by chondrocytes were stained and metachromatic in the two groups (Fig. 2). This result confirmed that the cells used in this experiment were end-plate chondrocytes.

Under a dark field fluorescence microscope, the autophagosome absorbed MDC and appeared as a green granular structure. The control and cervical spondylosis groups both contained fluorescent granular autophagosomes (Fig. 3).

Laser scanning confocal microscopy showed that the LC3 proteins of the autophagosome were primarily concentrated in the intracellular and perinuclear regions (Fig. 4).

The mRNA expression levels of the aggrecan gene (0.715±0.194) and the type II collagen gene (0.628±0.254) in the cervical spondylosis group were much lower than those in the control group (0.913±0.254 and 0.845±0.186, respectively). The difference between the two groups was statistically significant (both P<0.05; Fig. 5).

The expression of LC3-II in the cervical spondylosis group was downregulated compared with that in the control group. LC3-I was upregulated, whereas the ratio LC3-II/LC3-I was also decreased. The expression of β-actin did not differ between the two groups (Fig. 6).