Transfection of lentivirus-bone morphogenetic protein 2 and lentivirus-inhibitor of differentiation 1 into nucleus pulposus for delaying intervertebral disc degeneration in an<i>in vivo</i> rabbit model _Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LEI Tao ,  QUAN Zhengxue , ZHANG Yuan , LUO Xiaoji , YU Chang , ZHOU Qiang

Department of Orthopeadics, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, P.R.China;

Corresponding?author：

QUAN Zhengxue, Email: quanzx18@126.com

Keywords：

Intervertebral disc degeneration; gene therapy; bone morphogenetic protein 2; inhibitor of differentiation 1

DOI：

10.7507/1002-1892.201609035

Video：

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Objective To investigate if the course of intervertebral disc degeneration (IDD) is delayed by injecting lentivirus (Lv) vector carrying bone morphogenetic protein 2 (BMP-2) and inhibitor of differentiation 1 (Id1) genes directly into the nucleus pulposus. Methods Thirty-two New Zealand white rabbits, 2.0-2.5 kg in weight and 4 months in age, were used to establish the IDD models at L_{3, 4}, L_{4, 5}, and L_{5, 6} discs with annular puncture via transabdominal approach. Thirty rabbits with successful modeling were randomly divided into 5 groups, 6 rabbits every group. At 4 weeks after modeling, rabbits were injected with Lv-BMP-2 (group A), with Lv-BMP-2 and Lv-Id1 (group B), with Lv-Id1 (group C), with Lv-green fluorescent protein (group D), and with PBS (group E). At 2, 4, and 8 weeks after injection, T2-mapping MRI was performed on 2 rabbits each group to obtain the T2 values, and then subsequently the lumbar disc tissues were harvested to test the mRNA expressions and contents of collagen type II and proteoglycan by real-time fluorescent quantitative PCR and ELISA methods. Results T2-mapping MRI demonstrated that there was no significant difference in the T2 value between different groups at immediate and 2 weeks after injection (P>0.05). The T2 value of groups A and B was significantly higher than that of groups C, D, and E at 4 weeks after injection (P<0.05), but no significant difference was observed between group A and group B (P>0.05). The T2 value of group B was significantly higher than that of the other groups at 8 weeks after injection (P<0.05). The real-time fluorescent quantitative PCR and ELISA showed that the expressions and contents of collagen type II and proteoglycan in group B were significantly higher than those in the other groups at 2, 4, and 8 weeks after injection (P<0.05). Conclusion Combined application of Lv-BMP-2 and Lv-Id1 can delay IDD changes in rabbit IDD models.

Citation： LEITao, QUANZhengxue, ZHANGYuan, LUOXiaoji, YUChang, ZHOUQiang. Transfection of lentivirus-bone morphogenetic protein 2 and lentivirus-inhibitor of differentiation 1 into nucleus pulposus for delaying intervertebral disc degeneration in anin vivo rabbit model . Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(1): 73-79. doi: 10.7507/1002-1892.201609035 Copy

1.	?Luoma K, Riihim?ki H, Luukkonen R, et al. Low back pain in relation to lumbar disc degenerationc. Spine (Phila Pa 1976), 2000, 25(4): 487-492.
2.	Hoy D, March L, Brooks P, et al. The global burden of low back pain: estimates from the Global Burden of Disease 2010 study. Ann Rheum Dis, 2014, 73(6): 968-974.
3.	Than KD, Rahman SU, Vanaman MJ, et al. Bone morphogenetic proteins and degenerative disk disease. Neurosurgery, 2012, 70(4): 996-1002.
4.	Kim DJ, Moon SH, Kim H, et al. Bone morphogenetic protein-2 facilitates expression of chondrogenic, not osteogenic, phenotype of human intervertebral disc cells. Spine (Phila Pa 1976), 2003, 28(24): 2679-2684.
5.	Kim H, Lee JU, Moon SH, et al. Zonal responsiveness of the human intervertebral disc to bone morphogenetic protein-2. Spine (Phila Pa 1976), 2009, 34(17): 1834-1838.
6.	Du Y, Yip H. Effects of bone morphogenetic protein 2 on Id expression and neuroblastoma cell differentiation. Differentiation, 2010, 79(2): 84-92.
7.	周強, 權正學, 羅小輯, 等. 分化抑制因子1基因對BMP-2促進兔椎間盤軟骨終板細胞軟骨特性基因表達的影響. 中國修復重建外科雜志, 2015, 29(12): 1547-1552.
8.	Ren S, Liu Y, Ma J, et al. Treatment of rabbit intervertebral disc degeneration with co-transfection by adeno-associated virus-mediated SOX9 and osteogenic protein-1 double genes in vivo. IntJMolMed, 2013, 32(5): 1063-1068.
9.	Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
10.	Nerlich AG, Schleicher ED, Boos N. 1997 Volvo Award winner in basic science studies. Immunohistologic markers for age-related changes of human lumbar intervertebral discs. Spine (Phila Pa 1976), 1997, 22(24): 2781-2795.
11.	Kalson NS, Richardson S, Hoyland JA. Strategies for regeneration of the intervertebral disc. Regen Med, 2008, 3(5): 717-729.
12.	Li J, Yoon ST, Hutton WC. Effect of bone morphogenetic protein-2 (BMP-2) on matrix production, other BMPs, and BMP receptors in rat intervertebral disc cells. J Spinal Disord Tech, 2004, 17(5): 423-428.
13.	Wallach CJ, Sobajima S, Watanabe Y, et al. Gene transfer of the catabolic inhibitor TIMP-1 increases measured proteoglycans in cells from degenerated human intervertebral discs. Spine (Phila Pa 1976), 2003, 28(20): 2331-2337.
14.	Wang H, Kroeber M, Hanke M, et al. Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells. J Mol Med (Berl), 2004, 82(2): 126-134.
15.	Yoon ST, Park JS, Kim KS, et al. LMP-1 upregulates intervertebral disc cell production of proteoglycans and BMPsin vitro andin vivo. Spine (Phila Pa 1976), 2004, 29(23): 2603-2611.
16.	Than KD, Rahman SU, Vanaman MJ, et al. Bone morphogenetic proteins and degenerative disk disease. Neurosurgery, 2012, 70(4): 996-1002.
17.	Norton JD. ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis. J Cell Sci, 2000, 113(Pt 22): 3897-3905.
18.	Perk J, Iavarone A, Benezra R. Id family of helix-loop-helix proteins in cancer. Nat Rev Cancer, 2005, 5(8): 603-614.
19.	Moon SH, Nishida K, Gilbertson LG, et al. Biologic response of human intervertebral disc cells to gene therapy cocktail. Spine (Phila Pa 1976), 2008, 33(17): 1850-1855.
20.	Ren S, Liu Y, Ma J, et al. Treatment of rabbit intervertebral disc degeneration with co-transfection by adeno-associated virus-mediated SOX9 and osteogenic protein-1 double genesin vivo. IntJMolMed, 2013, 32(5): 1063-1068.
21.	Ogon I, Takebayashi T, Takashima H, et al. Analysis of chronic low back pain with magnetic resonance imaging T2 mapping of lumbar intervertebral disc. J Orthop Sci, 2015, 20(2): 295-301.
22.	Detiger SE, Holewijn RM, Hoogendoorn RJ, et al. MRI T2 mapping correlates with biochemistry and histology in intervertebral disc degeneration in a large animal model. Eur Spine J, 2015, 24(9): 1935-1943.
23.	Inoue H, Montgomery SR, Aghdasi B, et al. The effect of bone morphogenetic protein-2 injection at different time points on intervertebral disk degeneration in a rat tail model. J Spinal Disord Tech, 2015, 28(1): E35-44.
24.	馮剛. 椎間盤退行性變疾病不同臨床期生物治療策略. 西部醫學, 2013, 25(8): 1121-1124.
25.	Kurooka H, Nakahiro T, Mori K, et al. BMP signaling is responsible for serum-induced Id2 expression. Biochem Biophys Res Commun, 2012, 420(2): 281-287.
26.	Nakahiro T, Kurooka H, Mori K, et al. Identification of BMP-responsive elements in the mouse Id2 gene. Biochem Biophys Res Commun, 2010, 399(3): 416-421.
27.	Somia N, Verma IM. Gene therapy: trials and tribulations. Nat RevGenet, 2000, 1(2): 91-99.
28.	Wallach CJ, Kim JS, Sobajima S, et al. Safety assessment of intradiscal gene transfer: a pilot study. Spine J, 2006, 6(2): 107-112.

1. ?Luoma K, Riihim?ki H, Luukkonen R, et al. Low back pain in relation to lumbar disc degenerationc. Spine (Phila Pa 1976), 2000, 25(4): 487-492.
2. Hoy D, March L, Brooks P, et al. The global burden of low back pain: estimates from the Global Burden of Disease 2010 study. Ann Rheum Dis, 2014, 73(6): 968-974.
3. Than KD, Rahman SU, Vanaman MJ, et al. Bone morphogenetic proteins and degenerative disk disease. Neurosurgery, 2012, 70(4): 996-1002.
4. Kim DJ, Moon SH, Kim H, et al. Bone morphogenetic protein-2 facilitates expression of chondrogenic, not osteogenic, phenotype of human intervertebral disc cells. Spine (Phila Pa 1976), 2003, 28(24): 2679-2684.
5. Kim H, Lee JU, Moon SH, et al. Zonal responsiveness of the human intervertebral disc to bone morphogenetic protein-2. Spine (Phila Pa 1976), 2009, 34(17): 1834-1838.
6. Du Y, Yip H. Effects of bone morphogenetic protein 2 on Id expression and neuroblastoma cell differentiation. Differentiation, 2010, 79(2): 84-92.
7. 周強, 權正學, 羅小輯, 等. 分化抑制因子1基因對BMP-2促進兔椎間盤軟骨終板細胞軟骨特性基因表達的影響. 中國修復重建外科雜志, 2015, 29(12): 1547-1552.
8. Ren S, Liu Y, Ma J, et al. Treatment of rabbit intervertebral disc degeneration with co-transfection by adeno-associated virus-mediated SOX9 and osteogenic protein-1 double genes in vivo. IntJMolMed, 2013, 32(5): 1063-1068.
9. Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
10. Nerlich AG, Schleicher ED, Boos N. 1997 Volvo Award winner in basic science studies. Immunohistologic markers for age-related changes of human lumbar intervertebral discs. Spine (Phila Pa 1976), 1997, 22(24): 2781-2795.
11. Kalson NS, Richardson S, Hoyland JA. Strategies for regeneration of the intervertebral disc. Regen Med, 2008, 3(5): 717-729.
12. Li J, Yoon ST, Hutton WC. Effect of bone morphogenetic protein-2 (BMP-2) on matrix production, other BMPs, and BMP receptors in rat intervertebral disc cells. J Spinal Disord Tech, 2004, 17(5): 423-428.
13. Wallach CJ, Sobajima S, Watanabe Y, et al. Gene transfer of the catabolic inhibitor TIMP-1 increases measured proteoglycans in cells from degenerated human intervertebral discs. Spine (Phila Pa 1976), 2003, 28(20): 2331-2337.
14. Wang H, Kroeber M, Hanke M, et al. Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells. J Mol Med (Berl), 2004, 82(2): 126-134.
15. Yoon ST, Park JS, Kim KS, et al. LMP-1 upregulates intervertebral disc cell production of proteoglycans and BMPsin vitro andin vivo. Spine (Phila Pa 1976), 2004, 29(23): 2603-2611.
16. Than KD, Rahman SU, Vanaman MJ, et al. Bone morphogenetic proteins and degenerative disk disease. Neurosurgery, 2012, 70(4): 996-1002.
17. Norton JD. ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis. J Cell Sci, 2000, 113(Pt 22): 3897-3905.
18. Perk J, Iavarone A, Benezra R. Id family of helix-loop-helix proteins in cancer. Nat Rev Cancer, 2005, 5(8): 603-614.
19. Moon SH, Nishida K, Gilbertson LG, et al. Biologic response of human intervertebral disc cells to gene therapy cocktail. Spine (Phila Pa 1976), 2008, 33(17): 1850-1855.
20. Ren S, Liu Y, Ma J, et al. Treatment of rabbit intervertebral disc degeneration with co-transfection by adeno-associated virus-mediated SOX9 and osteogenic protein-1 double genesin vivo. IntJMolMed, 2013, 32(5): 1063-1068.
21. Ogon I, Takebayashi T, Takashima H, et al. Analysis of chronic low back pain with magnetic resonance imaging T2 mapping of lumbar intervertebral disc. J Orthop Sci, 2015, 20(2): 295-301.
22. Detiger SE, Holewijn RM, Hoogendoorn RJ, et al. MRI T2 mapping correlates with biochemistry and histology in intervertebral disc degeneration in a large animal model. Eur Spine J, 2015, 24(9): 1935-1943.
23. Inoue H, Montgomery SR, Aghdasi B, et al. The effect of bone morphogenetic protein-2 injection at different time points on intervertebral disk degeneration in a rat tail model. J Spinal Disord Tech, 2015, 28(1): E35-44.
24. 馮剛. 椎間盤退行性變疾病不同臨床期生物治療策略. 西部醫學, 2013, 25(8): 1121-1124.
25. Kurooka H, Nakahiro T, Mori K, et al. BMP signaling is responsible for serum-induced Id2 expression. Biochem Biophys Res Commun, 2012, 420(2): 281-287.
26. Nakahiro T, Kurooka H, Mori K, et al. Identification of BMP-responsive elements in the mouse Id2 gene. Biochem Biophys Res Commun, 2010, 399(3): 416-421.
27. Somia N, Verma IM. Gene therapy: trials and tribulations. Nat RevGenet, 2000, 1(2): 91-99.
28. Wallach CJ, Kim JS, Sobajima S, et al. Safety assessment of intradiscal gene transfer: a pilot study. Spine J, 2006, 6(2): 107-112.

Chinese Journal of Reparative and Reconstructive Surgery

Transfection of lentivirus-bone morphogenetic protein 2 and lentivirus-inhibitor of differentiation 1 into nucleus pulposus for delaying intervertebral disc degeneration in anin vivo rabbit model

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