Introduction
Materials and methods
Tissue samples
Tissue samples for Immunohistochemical analysis
Post-mortem tissue
Laboratory number | Source of tissue | Sex | Age (y) | Clinical diagnosis | Disc level | Histological grade |
---|---|---|---|---|---|---|
1 | Post-mortem | Male | 53 | No data | L4/5 | 1 |
2 | Post-mortem | Male | 53 | No data | L5/S1 | 1 |
3 | Surgical | Male | 44 | Relatively normal | L4/5 | 1 |
4 | Surgical | Male | 47 | Relatively normal | L4/5 | 2 |
5 | Post-mortem | Male | 75 | No data | L5/S1 | 3 |
6 | Surgical | Male | ? | Disc degeneration | L5/S1 | 3 |
7 | Surgical | Male | 48 | Disc degeneration | L4/5 | 3 |
8 | Surgical | Male | 64 | Disc degeneration | L5/S1 | 3 |
9 | Surgical | Male | 46 | Disc degeneration | L5/S1 | 4 |
10 | Surgical | Male | 21 | Disc degeneration | L5/S1 | 4 |
11 | Surgical | Female | 36 | Disc degeneration | L5/S1 | 4 |
12 | Surgical | Male | 39 | Disc degeneration | L4/5 | 5 |
13 | Surgical | Female | 32 | Disc degeneration | L5/S1 | 5 |
14 | Surgical | Female | 36 | Disc degeneration | L4/5 | 5 |
15 | Surgical | Male | 25 | Disc degeneration | L4/5 | 5 |
16 | Surgical | Female | 35 | Disc degeneration | L4/5 | 6 |
17 | Surgical | Male | 40 | Disc degeneration | L4/5 | 6 |
18 | Post-mortem | Female | 73 | No data | L5/S1 | 6 |
19 | Surgical | Male | 25 | Disc degeneration | L5/S1 | 6 |
20 | Surgical | Female | 55 | Disc degeneration | L5/S1 | 7 |
21 | Post-mortem | Female | ? | No data | L4/5 | 7 |
22 | Surgical | Female | 58 | Disc degeneration | L4/5 | 7 |
23 | Surgical | Male | 34 | Disc degeneration | L4/5 | 8 |
24 | Surgical | Female | 24 | Disc degeneration | L5/S1 | 8 |
25 | Surgical | Female | 33 | Disc degeneration | L5/S1 | 9 |
26 | Post-mortem | Female | 73 | No data | L4/5 | 9 |
27 | Surgical | Male | 68 | Disc degeneration | L5/S1 | 10 |
28 | Post-mortem | ? | 47 | No data | L5/S1 | 10 |
29 | Post-mortem | ? | 47 | No data | L5/S1 | 11 |
30 | Surgical | Male | 39 | Disc degeneration | L4/5 | 12 |
Surgical tissue
Treatment of tissue specimens
Tissue samples for in vitrocell studies
Production and localization of IL-1 family proteins
Image analysis
Statistical analysis
Investigation of the effect of IL-1 on human IVD cells in alginate culture
Issolation of Disc cells
Alginate bead culture
Assessment of re-differentiated state in alginate
Image analysis
Statistical analysis
Treatment of cells with IL-1, RNA extraction, and cDNA formation
Real-time PCR
Target | Forward primer | Probe | Reverse primer | Threshold |
---|---|---|---|---|
GAPDH | PDAR | PDAR | PDAR | 0.047 |
Collagen type I | 5' CAG CCG CTT CAC CTA CAG C 3' | 5' CCG GTG TGA CTC GTG CAG CCA TC 3' | 5' TTT TGT ATT CAA TCA CTG TCT TGC C 3' | 0.078 |
Collagen type II | 5' GGC AAT AGC AGG TTC ACG TAC A 3' | 5' CCG GTA TGT TTC GTG CAG CCA TCC T 3' | 5' CGA TAA CAG TCT TGC CCC ACT T 3' | 0.100 |
Aggrecan | 5' TCG AGG ACA GCG AGG CC 3' | 5' ATG GAA CAC GAT GCC TTT CAC CAC GA 3' | 5' TCG AGG GTG TAG CGT GTA GAG A 3' | 0.050 |
SOX9 | 5' GAC TTC CGC GAC GTG GAC 3' | 5' CGA CGT CAT CTC CAA CAT CGA GAC 3' | 5' GTT GGG CGG CAG GTA CTG 3' | 0.0562 |
SOX6 | 5' CCG TGA GAT AAT GAC CAG TGT TAC TT 3' | 5' AAC CCC AGA GCG CCG CAA A 3' | 5' GTC CAC CAC ATC GGC AAG AC 3' | 0.052 |
IL-1α | PDAR | PDAR | PDAR | 0.107 |
IL-1β | PDAR | PDAR | PDAR | 0.122 |
IL-1Ra | 5' CCT GCA GGG CCA AGC A 3' | 5' AGC CTC GCT CTT GGC AGG TAC TCA GT 3' | 5' GCA CCC AAC ATA TAC AGC ATT CA 3' | 0.122 |
IL-1RI | 5' ATT TCT GGC TTC TAG TCT GGT GTT C 3' | 5' ACT TGA TTT CAG GTC AAT AAC GGT CCC C 3' | 5' AAC GTG CCA GTG TGG AGT GA 3' | 0.163 |
MMP-3 | 5' TGA AGA GTC TTC CAA TCC TAC TGT TG 3' | 5' CGT GGC AGT TTG CTC AGC CTA TCC AT 3' | 5' CTA GAT ATT TCT GAA CAA GGT TCA TGC A 3' | 0.108 |
MMP-9 | 5' CCC GGA GTG AGT TGA ACC A 3' | 5' CCA AGT GGG CTA CGT GAC CTA TGA CAT CC 3' | 5' CAG GAC GGG AGC CCT AGT C 3' | 0.041 |
MMP-13 | 5' GGA CAA GTA GTT CCA AAG GCT ACA A 3' | 5' CTC CAA GGA CCC TGG AGC ACT CAT GTT 3' | 5' CTT TTG CCG GTG TAG GTG TAG ATA G 3' | 0.108 |
ADAMTS-4 | 5' ACT GGT GGT GGC AGA TGA CA 3' | 5' ATG GCC GCA TTC CAC GGT G 3' | 5' TCA CTG TTA GCA GGT AGC GCT TT 3' | 0.052 |
ADAMTS-5 | 5' GGA CCT ACC ACG AAA GCA GAT C 3' | 5' CCC AGG ACA GAC CTA CGA TGC CAC C 3' | 5' GCC GGG ACA CAC GGA GTA 3' | 0.122 |
Statistical analysis
Results
Immunohistochemical localisation
Immunohistochemical staining and quantification of immunopositive cells
Assessment of redifferentiated state in alginate
Effect of IL-1 on human IVD cells
Target | General trend | Tissues affected | Significant changesa (P < 0.05) | |
---|---|---|---|---|
Origin of cells | Disease state | |||
MMP-3 | Increase (5- to 10-fold) | NP, AF | N, D | Non-degenerate NP and AF |
MMP-13 | Increase (5- to 10-fold) | NP | N, D | Non-degenerate NP |
ADAMTS-4 | Increase (8-fold) | NP | D | Degenerate NP |
ADAMTS-5 | No real trend | - | - | None |
SOX6 | Decrease (3- to 9-fold) | NP, AF | N | Non-degenerate NP |
SOX9 | Decrease (3-fold) | NP, AF | N | None |
Collagen I | Decrease (5- to 10-fold) | NP, AF | N, D | Degenerate NP |
Collagen II | Decrease (5- to 50-fold) | AF | N, D | None |
Aggrecan | Decrease (3- to 7-fold) | NP, AF | N, D | Non-degenerate NP and AF |
IL-1α | Increase (100-fold) | NP, AF | D | Degenerate NP |
IL-1β | Increase (100-fold) | NP, AF | D | Degenerate NP |
IL-1Ra | No real trend | - | - | None |
IL-1RI | Decrease (2- to 10-fold) | NP, AF | N | None |