Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades
Abstract
:1. Introduction
2. Results
2.1. Genome-Wide Microarray Gene Expression Profiling
2.2. Determination of Glycosaminoglycan and Collagen Content
2.3. Stimulation Assay
3. Discussion
4. Materials and Methods
4.1. Sample Preparation for Genome-Wide Microarrays
4.2. Genome-Wide Microarray Gene Expression Profiling
4.3. Determination of Glycosaminoglycan (GAG) and Collagen Content
4.4. Stimulation Assay
4.5. RTD-PCR
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ACAN | Aggrecan |
AF | Anulus fibrosus |
BGLAP | Bone gamma-carboxcylglutamate protein (osteocalcin) |
BGN | Biglycan |
BMP | Bone morphogenetic protein |
CILP | Cartilage intermediate layer protein |
COL1A1 | Collagen type I, alpha 1 |
COL2A1 | Collagen type II, alpha 1 |
COL10A1 | Collagen type X, alpha 1 |
COMP | Cartilage oligomeric matrix protein |
CRTAP | Cartilage associated protein |
DCN | Decorin |
DMMB | Dimethyl methylene blue |
cDNA | Complementary deoxyribonucleic acid |
ECM | Extracellular matrix |
EDTA | Ethylenediaminetetraacetic acid |
FBS | Fetal bovine serum |
FC | Fold change |
GAG | Glycosaminoglycan |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
gDNA | Genomic deoxyribonucleic acid |
GDP | Gross domestic product |
HPRT1 | Hypoxanthine phosphoribosyltransferase 1 |
IBSP | Integrin-binding sialoprotein |
ID1 | Inhibitor of DNA Binding 1, HLH Protein |
IL1β | Interleukin 1β |
IVD | Intervertebral disc |
MMP | Matrix metalloproteinases |
NOG | Noggin |
NP | Nucleus pulposus |
POSTN | Periostin (osteoblast-specific factor) |
RNA | Ribonucleic acid |
RTD-PCR | Real-time detection polymerase chain reaction |
RUNX2 | RUNX family transcription factor 2 |
SEM | Standard error of the mean |
SERPINE1 | Serpin family E member 1 |
SMAD7 | SMAD Family Member 7 |
SOX9 | SRY (sex determining region Y)-box 9 |
SPP1 | Secreted phosphoprotein 1 (osteopontin) |
TGFβ1 | Transforming growth factor β1 |
TNFα | Tumor necrosis factor α |
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Affymetrix ID | Symbol | Accession No. | Mean Signal NA Mild | Mean Signal NA Severe | Native FC | Mean Signal ML Mild | Mean Signal ML Severe | Monolayer FC | Name |
---|---|---|---|---|---|---|---|---|---|
205679_x_at | ACAN | NM_001135 | 6901 | 2063 | 4.32 | 161 | 410 | −2.35 | aggrecan |
222608_s_at | ANLN | NM_018685 | 34 | 153 | −4.19 | 851 | 320 | 2.76 | anillin, actin binding protein |
227911_at | ARHGAP28 | NM_001010000 | 56 | 168 | −3.51 | 160 | 354 | −2.32 | Rho GTPase activating protein 28 |
219087_at | ASPN | NM_017680 | 20,240 | 8186 | 2.35 | 56 | 558 | −9.40 | asporin |
218899_s_at | BAALC | NM_001024372 | 653 | 154 | 5.12 | 238 | 87 | 2.66 | brain and acute leukemia, cytoplasmic |
207172_s_at | CDH11 | NM_001797 | 118 | 810 | −7.24 | 931 | 2050 | −2.05 | cadherin 11, type 2, OB-cadherin (osteoblast) |
204170_s_at | CKS2 | NM_001827 | 132 | 362 | −2.13 | 1146 | 508 | 2.14 | CDC28 protein kinase regulatory subunit 2 |
211343_s_at | COL13A1 | NM_001130103 | 205 | 1454 | −5.61 | 288 | 89 | 2.94 | collagen, type XIII, alpha 1 |
225647_s_at | CTSC | NM_001114173 | 101 | 471 | −4.78 | 1430 | 547 | 2.35 | cathepsin C |
203666_at | CXCL12 | NM_000609 | 461 | 2751 | −5.53 | 317 | 1576 | −4.00 | chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) |
201289_at | CYR61 | NM_001554 | 10,725 | 5011 | 2.37 | 3354 | 7457 | −2.19 | cysteine-rich, angiogenic inducer, 61 |
202481_at | DHRS3 | NM_004753 | 697 | 266 | 2.46 | 395 | 1167 | −2.52 | dehydrogenase/reductase (SDR family) member 3 |
226281_at | DNER | NM_139072 | 1458 | 497 | 5.00 | 828 | 355 | 3.10 | delta/notch-like EGF repeat containing |
232204_at | EBF1 | NM_024007 | 300 | 686 | −2.06 | 276 | 573 | −2.13 | early B-cell factor 1 |
219134_at | ELTD1 | NM_022159 | 348 | 960 | −3.38 | 110 | 46 | 2.41 | EGF, latrophilin and seven transmembrane domain containing 1 |
40665_at | FMO3 | NM_001002294 | 38 | 173 | −3.68 | 61 | 135 | −3.59 | flavin containing monooxygenase 3 |
202709_at | FMOD | NM_002023 | 24,906 | 7901 | 3.15 | 273 | 592 | −2.54 | fibromodulin |
204984_at | GPC4 | NM_001448 | 79 | 433 | −4.67 | 126 | 277 | −2.08 | glypican 4 |
209170_s_at | GPM6B | NM_001001994 | 201 | 573 | −5.92 | 121 | 336 | −3.25 | glycoprotein M6B |
214091_s_at | GPX3 | NM_002084 | 11,915 | 1856 | 5.79 | 1232 | 2315 | −3.27 | glutathione peroxidase 3 (plasma) |
227314_at | ITGA2 | NM_002203 | 133 | 450 | −2.46 | 267 | 114 | 2.18 | integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) |
219949_at | LRRC2 | NM_024512 | 279 | 116 | 2.64 | 29 | 66 | −2.42 | leucine rich repeat containing 2 |
226210_s_at | MEG3 | NR_002766 | 560 | 161 | 4.13 | 121 | 51 | 2.19 | maternally expressed 3 (non-protein coding) |
203434_s_at | MME | NM_000902 | 32 | 109 | −3.17 | 126 | 444 | −3.25 | membrane metallo-endopeptidase |
227394_at | NCAM1 | NM_000615 | 68 | 1034 | −5.12 | 84 | 27 | 3.17 | neural cell adhesion molecule 1 |
218039_at | NUSAP1 | NM_001129897 | 82 | 214 | −2.46 | 572 | 178 | 2.83 | nucleolar and spindle associated protein 1 |
213125_at | OLFML2B | NM_015441 | 386 | 2864 | −6.96 | 530 | 1360 | −2.35 | olfactomedin-like 2B |
204040_at | RNF144A | NM_014746 | 211 | 525 | −2.64 | 120 | 246 | −2.00 | ring finger protein 144A |
209773_s_at | RRM2 | NM_001034 | 31 | 159 | −4.32 | 891 | 203 | 3.48 | ribonucleotide reductase M2 |
204051_s_at | SFRP4 | NM_003014 | 496 | 2665 | −5.92 | 1749 | 5272 | −3.43 | secreted frizzled-related protein 4 |
229151_at | SLC14A1 | NM_001128588 | 1420 | 253 | 4.59 | 854 | 128 | 10.00 | solute carrier family 14 (urea transporter), member 1 (Kidd blood group) |
225987_at | STEAP4 | NM_024636 | 447 | 1962 | −3.22 | 1030 | 1855 | −2.19 | STEAP family member 4 |
213247_at | SVEP1 | NM_153366 | 107 | 511 | −4.09 | 316 | 1184 | −3.46 | sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 |
209228_x_at | TUSC3 | NM_006765 | 136 | 299 | −2.54 | 1121 | 614 | 2.00 | tumor suppressor candidate 3 |
230083_at | USP53 | NM_019050 | 1201 | 333 | 4.13 | 213 | 411 | −2.18 | ubiquitin specific peptidase 53 |
Gene | EMBL Accession No. | Sequence (5′-3′)/ Thermo Fisher Scientific Assay No. | PCR Product Size (bp) |
---|---|---|---|
GAPDH | NM_002046 | Cat. No. Hs99999905_m1 | 122 |
ACAN | NM_001135 | Cat. No. Hs00153936_m1 | 91 |
COMP | NM_000095 | Cat. No. Hs00164359_m1 | 101 |
RUNX2 | NM_001015051 | Cat. No. Hs00298328_s1 | 124 |
BGLAP | NM_199173 | Cat. No. Hs00609452_g1 | 74 |
SPP1 | NM_000582 | Cat. No. Hs00167093_m1 | 65 |
COL1A1 | NM_000088 | Cat. No. Hs01076780_g1 | 109 |
HPRT1 | NM_000194 | F: CTTTGCTGACCTGCTGGATT R: CTGCATTGTTTTGCCAGTGT | 211 |
ID1 | NM_002165 | F: GCTGCTCTACGACATGAACG R: CCAACTGAAGGTCCCTGATG | 131 |
SERPINE1 | NM_000602 | F: CTTTCAGACCAAGAGCCTCTC R: CCATGCGGGCTGAGACTAT | 118 |
SOX9 | NM_000346 | F: GCAAGCTCTGGAGACTTCTGA R: CTGCAGCGCCTTGAAGAT | 206 |
NOG | NM_005450 | F: GTGCAAGCCGTCCAAGTC R: GCTAGAGGGTGGTGGAACTG | 219 |
SMAD7 | NM_00119082 | F: GGCTTTCAGATTCCCAACTTCTT R: ATTTTGCTCCGCACCTTCT | 217 |
COL1A1 | NM_000088 | F: GGCAACGATGGTGCTAAGG R: GACCAGCATCACCTCTGTCA | 139 |
COL2A1 | NM_001844 | F: GATGGCTGCACGAAACATACC R: AAGAAGCAGACCGGCCCTAT | 155 |
COL10A1 | NM_000493 | F: CACCATAAAGAGTAAAGGTATAGCAGT R: GCACACCTGGTTTCCCTACA | 194 |
COMP | NM_000095 | F: CCAACTCAAGGCTGTGAAGTC R: GTCCTTCCAACCCACGTTTC | 130 |
ACAN | NM_001135 | F: GACACCCCATGCAATTTGAGAA R: CCGCACCAGGGAATTGATCT | 242 |
BGN | NM_001711 | F: CTCCCAGACCTCAAGCTCC R: GGGGTTGTTGAAGAGGCTGAT | 138 |
IL1β | NM_000576 | F: CCCTAAACAGATGAAGTGCTCC R: AGAAGGTGCTCAGGTCATTCTC | 197 |
MMP3 | NM_002422 | F: CTATCAGAGGAAATGAGGTACGAGC R: GCCTGGCTCCATGGAATTTC | 179 |
MMP13 | NM_002427 | F: CCCCAGGCATCACCATTCAA R: CAGGTAGCGCTCTGCAAACT | 150 |
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Stich, S.; Jagielski, M.; Fleischmann, A.; Meier, C.; Bussmann, P.; Kohl, B.; Schmidt, J.; Krüger, J.-P.; Endres, M.; Cabraja, M.; et al. Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades. Int. J. Mol. Sci. 2020, 21, 2165. https://doi.org/10.3390/ijms21062165
Stich S, Jagielski M, Fleischmann A, Meier C, Bussmann P, Kohl B, Schmidt J, Krüger J-P, Endres M, Cabraja M, et al. Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades. International Journal of Molecular Sciences. 2020; 21(6):2165. https://doi.org/10.3390/ijms21062165
Chicago/Turabian StyleStich, Stefan, Michal Jagielski, Anja Fleischmann, Carola Meier, Patricia Bussmann, Benjamin Kohl, Julia Schmidt, Jan-Philipp Krüger, Michaela Endres, Mario Cabraja, and et al. 2020. "Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades" International Journal of Molecular Sciences 21, no. 6: 2165. https://doi.org/10.3390/ijms21062165
APA StyleStich, S., Jagielski, M., Fleischmann, A., Meier, C., Bussmann, P., Kohl, B., Schmidt, J., Krüger, J. -P., Endres, M., Cabraja, M., Reimann, K., Laue, D., Ertel, W., & Sittinger, M. (2020). Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades. International Journal of Molecular Sciences, 21(6), 2165. https://doi.org/10.3390/ijms21062165