Specimens Collection
The research program was approved by the Institutional Review Committee of Tianjin Union Medical Center, and all procedures are based on the Helsinki Declaration. When patients underwent posterior lumbar surgery, the full thickness of the LF was removed from L4/5 segments. (p > 0.05, Table 3). There was no significant difference in baseline data between groups (p > 0.05, Table 1). After conservative treatment for at least three months, no obvious symptoms improved in all patients. All patients had no ossification of LF, secondary adhesive arachnoiditis, polyneuritis, no history of lumbar surgery, history of intraspinal invasive treatment such as epidural, etc. Considering the influence of diabetes and hypertension on the hypertrophy of the LF, none of the selected patients had a history of diabetes, hypertension, and underlying diseases that could have a potential impact on LF. We divided LF tissue into three groups: simple lumbar disc herniation (LDH), single-segment spinal stenosis (SLSS), double-segment spinal stenosis (DLSS). The dorsal and dural LF were detected for each group. Western blot and qPCR were used to detect S100 and P16 and their expression products.
MRI Measurement
The measurement method of LF was done as in our previous study [8]. We consider the ligamentum flavum> 3 mm as hypertrophy of ligamentum flavum in imaging measurement. The thickness of the LF was measured from the mid-point of the LF to the ventral side of the inner rim. The lumbar spinal canal oblique diameter is measured from the midpoint of the dorsal side of the ligamentum flavum to the midpoint of the posterior margin of the vertebral body. Relative thickness of LF (RT)(%) was calculated as the percentage of the thickness of LF to the oblique diameter of lumbar spinal canal. The average of three independent measurements of three surgeons were taken to determine the relative thickness of a single sample.
Western Blot
The dorsal and ventral LF of LDH, SLSS and DLSS were ground in liquid nitrogen. Extraction of total protein using RIPA lysate containing PMSF (Solarbio, USA). The CBA protein quantitative kit (Biosciences, USA) was diluted with 5 × buffer at 1:5 and denatured at 95 ℃ for 10 min To prepare SDS-PAGE gel (biorbyt, UK), 20 μL of total protein was taken, and the protein band was separated by 120V electrophoresis for 1 h, then 100mA transferred it to PVDF membrane (Millipore, USA). After 5% skimmed milk powder was sealed for 3 h, the PVDF membrane and the first antibody were incubated overnight at 4 ℃. After washing the membrane 3 times with tris-buffered saline with Tween 20 (TBST, Solarbio, USA), the secondary antibodies labeled by Horseradish peroxidase (HRP, Solarbio, USA) were incubated at 37 ℃ for 1 h. The ChemiDocMP chemiluminescence imaging system (Bio-rad, USA) was exposed to observe the bands on the PVDF film.
Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted using Trizol reagent (Takara, Japan). SuperScriptIIIRT kit (Gibco, USA) is used for reverse transcription. Sybr qPCR mix (TOYOBO, Japan) was used for quantitative PCR analysis. PCR amplification conditions: 95 ℃, 2 min, 94 ℃, 20 s, 60 ℃, 20 s, 72 ℃, 30 s, a total of 40 cycles. Quantitative analysis was performed on ABI7900PCR instrument. Using Actin as the control, the primer sequences of quantitative mRNA, are provided in Table 2. The gene expression level was calculated using the 2-ΔΔCt method relative to the actin gene. 2-ΔΔCt> 2 or <1/2 is considered statistically significant.
Statistical Analysis
The average values were taken from three biological repeats in each experiment. We determined the relationships between the thickness and the expression of P16 and S100 using Pearson’s correlation coefficient test. A one-way ANOVA was used to analyze the differences among the three groups. Use t-test for comparison between two groups. And all statistical analyses were performed by Statistical Program for Social Sciences (SPSS) software (version 21.0, IBM, New York, USA). All the analyses were statistically significant at p < 0.05.