EFFECTS OF RECOMBINANT ADENOVIRUS VECTOR CARRYING HUMAN INSULIN-LIKE GROWTH FACTOR 1 GENE ON THE APOPTOSIS OF NUCLEUS PULPOSUS CELLS IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Changchun , CUI Guopeng , SHAO Chen , ZHOU Xinshe , XIAO Yuzhou , ZHOU Jiansheng.

Anhui Province Tissue Transplantation Key Laboratory, the First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui, 233004, P.R.China. Corresponding author: ZHOU Jiansheng, E-mail: jpylc@byyfy.com.cn;

Corresponding author：

Keywords：

Intervertebral disc degeneration; Insulin-like growth factor 1; Adenovirus vector; Nucleus pulposus cells; Apoptosis; Rabbit

DOI：

10.7507/1002-1892.20130298

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Objective To investigate the effects of human insulin-like growth factor 1 (hIGF-1) gene transfected by recombinant adenovirus vector (Ad-hIGF-1) on the apoptosis of rabbit nucleus pulposus cells induced by tumor necrosis factor α (TNF-α). Methods The intervertebral disc nucleus pulposus were harvested from 8 healthy adult domestic rabbits (male or female, weighing 2.0-2.5 kg). The nucleus pulposus cells were isolated with collagenase II digestion and the passage 2 cells were cultured to logarithm growing period, and then they were divided into 3 groups according to culture condition: DMEM/F12 medium containing 10% PBS, DMEM/F12 medium containing 10% PBS and 100 ng/mL TNF-α, and DMEM/ F12 medium containing 10% PBS, 100 ng/ mL TNF-α, and Ad-hIGF-1 (multiplicity of infection of 50) were used in control group, TNF-α group, and Ad-hIGF-1 group, respectively. The results of transfection by adenovirus vector carrying hIGF-1 gene were observed by fluorescent microscopy; the expression of hIGF-1 protein was detected by Western blot, hIGF-1 mRNA expression by RT-PCR, and the cell apoptosis rate by TUNEL and flow cytometry. Results Green fluorescence was observed by fluorescent microscopy in Ad-hIGF-1 group, indicating that successful cell transfection. The expressions of hIGF-1 protein and mRNA were detected in Ad-hIGF-1 group by Western blot and RT-PCR, while the control group and TNF-α group had no expression. The cell apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 34.24% ± 4.60%, 6.59% ± 1.03%, and 0.40% ± 0.15%, respectively. The early apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 22.16% ± 2.69%, 5.03% ± 0.96%, and 0.49% ± 0.05%, respectively; the late cell apoptosis rates were 13.96% ± 4.86%, 10.68% ± 3.42%, and 0.29% ± 0.06%, respectively. Compared with TNF-α group, the cell apoptosis rates of Ad-hIGF-1 group and control group were significantly reduced (P lt; 0.05); the cell apoptosis rate of Ad-hIGF-1 group was significantly higher than that of control group (P lt; 0.05). Conclusion Ad-hIGF-1 could inhibit the apoptosis of nucleus pulposus cells induced by TNF-α.

Citation： ZHANG Changchun,CUI Guopeng,SHAO Chen,ZHOU Xinshe,XIAO Yuzhou,ZHOU Jiansheng.. EFFECTS OF RECOMBINANT ADENOVIRUS VECTOR CARRYING HUMAN INSULIN-LIKE GROWTH FACTOR 1 GENE ON THE APOPTOSIS OF NUCLEUS PULPOSUS CELLS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(11): 1375-1379. doi: 10.7507/1002-1892.20130298 Copy

1.	Kokubun S, Sakurai M, Tanaka Y. Cartilaginous endplate in cervical disc herniation. Spine (Phila Pa 1976), 1999, 21(2): 190-195.
2.	Valdes AM, Hassett G, Hart DJ, et al. Radiographic progression of lumbar spine disc degeneration is influenced by variation at inflammatory genes: A candidate SNP association study in the chingford cohort. Spine (Phila Pa 1976), 2005, 30(21): 2445-2451.
3.	Kawaguchi S, Yamashita T, Katahira G, et al. Chemokine profile of herniated intervertebal discs infliltrated with monocytes and macrophages. Spine (Phila Pa 1976), 2002, 27(14): 1511-1516.
4.	Gruber HE, Norton HJ, Hanley EN Jr. Anti-apoptotic effects of IGF- 1 and PDGF on human intervertebal disc cells in vitro. Spine (Phila Pa 1976), 2000, 25(17): 2153-2157.
5.	Wang YJ, Shi Q, Sun P, et al. Insulin-like growth factor-1 treatment prenvents anti-fas antibody-induced apoptosis in endplate chondrocytes. Spine (Phlia Pa 1976), 2006, 31(7): 736-741.
6.	刘巍, 唐天驷, 李涛, 等. 腺病毒介导的基因在人退变髓核细胞的表达. 苏州大学学报: 医学版, 2004, 24(6): 826-828.
7.	Nam V, Christopher S, Paulo C, et al. Identification of novel TNF-α regulated extracellular matrix and cell-matrix adhesion genes in intervertebral disc cells: A microarray gene profiling of human nucleus pulposus cells. Spine J, 2008, 8(5): 238-242.
8.	Séguin CA, Phillar RM, Roughley PJ, et al. Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue. Spine (Phila Pa 1976), 2005, 30 (17): 1940-1948.
9.	Zhang CC, Zhou JS, Hu JG, et al. Effects of IGF-1 on IL-1β-induced apoptosis in rabbit nucleus pulposus cells in vitro. Mol Med Rep, 2013, 7(2): 441-444.
10.	Wei A, Brisby H, Chung SA, et al. Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis. Spine J, 2008, 8(3): 466-474.
11.	Murata Y, Rydevik B, Nannmark U, et al. Local application of interleukin-6 to the dorsal root ganglion induces tumor necrosis factor-alpha in the dorsal root ganglion and results in apoptosis of the dorsal root ganglion cells. Spine (Phila Pa 1976), 2011, 36(12): 926-932.
12.	Jane MS, Patrick WC, Anthony JF, et al. Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration. Arthritis Research & Therapy, 2009, 11(3): 65-75.
13.	Pratsinis H, Constantinou V, Pavlakis K, et al. Exogenous and autocrine growth factors stimulate human intervertebral disc cell proliferationvia the ERK and Akt pathways. J Orthop Res, 2012, 30(6): 958-964.
14.	Mavrogonatou E, Kletsas D. Effect of varying osmotic conditions on the response of bovine nucleus pulposus cells togrowth factors and the activation of the ERK and Akt pathways. J Orthop Res, 2010, 28(10): 1276-1282.
15.	Zhang R, Ruan D, Zhang C. Effects of TGF-beta1 and IGF-1 on proliferation of human nucleus pulposus cells in medium with different serum concentrations. J Orthop Surg Res, 2006, 1: 9.
16.	Casolari DA, Pereira MC, de Bessa Garcia SA, et al. Insulin-like growth factor-1 and 17β-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells. Int J Mol Med, 2011, 28(3): 337-342.
17.	金贞爱, 赵以坤, 金正勇, 等. 胰岛素样生长因子-1对高氧诱导的A549细胞凋亡的影响. 中国当代儿科杂志, 2013, 15(6): 490-493.
18.	Hayashi Y, Yamamoto N, Nakagawa T, et al. Insulin-like growth factor 1 inhibits hair cell apoptosis and promotes the cell cycle of supporting cells by activating different downstream cascades after pharmacological hair cell injury in neonatal mice. Mol Cell Neurosci, 2013, 56C: 29-38.
19.	Yi C, Ma C, Xie Z, et al. Down-regulation of programmed cell death 5 by insulin-like growth factor 1 in osteoarthritis chondrocytes. Int Orthop, 2013, 37(5): 937-943.
20.	Li Y, Shelat H, Geng YJ. IGF-1 prevents oxidative stress induced-apoptosis in induced pluripotent stem cells which is mediated by microRNA-1. Biochem Biophys Res Commun, 2012, 426(4): 615-619.
21.	Kim Y, Li E, Park S. Insulin-like growth factor-1 inhibits 6-hydroxydopamine-mediated endoplasmic reticulum stress-induced apoptosis via regulation of heme oxygenase-1 and Nrf2 expression in PC12 cells. Int J Neurosci, 2012, 122(11): 641-649.
22.	Hao CN, Geng YJ, Li F, et al. Insulin-like growth factor-1 receptor activation prevents hydrogen peroxide-induced oxidative stress, mitochondrial dysfunction and apoptosis. Apoptosis, 2011, 16(11): 1118-1127.
23.	Paul R, Haydon RC, Cheng H, et al. Potential use of Sox9 gene therapy for intervertebral degenerative disc disease. Spine (Phila Pa 1976), 2003, 28(8): 755-763.
24.	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.
25.	曹德军, 权正学, 蒋电明, 等. 人BMP-4腺病毒表达载体对人退变腰椎间盘细胞的影响. 中国修复重建外科杂志, 2012, 26(12): 1442-1147.

1. Kokubun S, Sakurai M, Tanaka Y. Cartilaginous endplate in cervical disc herniation. Spine (Phila Pa 1976), 1999, 21(2): 190-195.
2. Valdes AM, Hassett G, Hart DJ, et al. Radiographic progression of lumbar spine disc degeneration is influenced by variation at inflammatory genes: A candidate SNP association study in the chingford cohort. Spine (Phila Pa 1976), 2005, 30(21): 2445-2451.
3. Kawaguchi S, Yamashita T, Katahira G, et al. Chemokine profile of herniated intervertebal discs infliltrated with monocytes and macrophages. Spine (Phila Pa 1976), 2002, 27(14): 1511-1516.
4. Gruber HE, Norton HJ, Hanley EN Jr. Anti-apoptotic effects of IGF- 1 and PDGF on human intervertebal disc cells in vitro. Spine (Phila Pa 1976), 2000, 25(17): 2153-2157.
5. Wang YJ, Shi Q, Sun P, et al. Insulin-like growth factor-1 treatment prenvents anti-fas antibody-induced apoptosis in endplate chondrocytes. Spine (Phlia Pa 1976), 2006, 31(7): 736-741.
6. 刘巍, 唐天驷, 李涛, 等. 腺病毒介导的基因在人退变髓核细胞的表达. 苏州大学学报: 医学版, 2004, 24(6): 826-828.
7. Nam V, Christopher S, Paulo C, et al. Identification of novel TNF-α regulated extracellular matrix and cell-matrix adhesion genes in intervertebral disc cells: A microarray gene profiling of human nucleus pulposus cells. Spine J, 2008, 8(5): 238-242.
8. Séguin CA, Phillar RM, Roughley PJ, et al. Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue. Spine (Phila Pa 1976), 2005, 30 (17): 1940-1948.
9. Zhang CC, Zhou JS, Hu JG, et al. Effects of IGF-1 on IL-1β-induced apoptosis in rabbit nucleus pulposus cells in vitro. Mol Med Rep, 2013, 7(2): 441-444.
10. Wei A, Brisby H, Chung SA, et al. Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis. Spine J, 2008, 8(3): 466-474.
11. Murata Y, Rydevik B, Nannmark U, et al. Local application of interleukin-6 to the dorsal root ganglion induces tumor necrosis factor-alpha in the dorsal root ganglion and results in apoptosis of the dorsal root ganglion cells. Spine (Phila Pa 1976), 2011, 36(12): 926-932.
12. Jane MS, Patrick WC, Anthony JF, et al. Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration. Arthritis Research & Therapy, 2009, 11(3): 65-75.
13. Pratsinis H, Constantinou V, Pavlakis K, et al. Exogenous and autocrine growth factors stimulate human intervertebral disc cell proliferationvia the ERK and Akt pathways. J Orthop Res, 2012, 30(6): 958-964.
14. Mavrogonatou E, Kletsas D. Effect of varying osmotic conditions on the response of bovine nucleus pulposus cells togrowth factors and the activation of the ERK and Akt pathways. J Orthop Res, 2010, 28(10): 1276-1282.
15. Zhang R, Ruan D, Zhang C. Effects of TGF-beta1 and IGF-1 on proliferation of human nucleus pulposus cells in medium with different serum concentrations. J Orthop Surg Res, 2006, 1: 9.
16. Casolari DA, Pereira MC, de Bessa Garcia SA, et al. Insulin-like growth factor-1 and 17β-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells. Int J Mol Med, 2011, 28(3): 337-342.
17. 金贞爱, 赵以坤, 金正勇, 等. 胰岛素样生长因子-1对高氧诱导的A549细胞凋亡的影响. 中国当代儿科杂志, 2013, 15(6): 490-493.
18. Hayashi Y, Yamamoto N, Nakagawa T, et al. Insulin-like growth factor 1 inhibits hair cell apoptosis and promotes the cell cycle of supporting cells by activating different downstream cascades after pharmacological hair cell injury in neonatal mice. Mol Cell Neurosci, 2013, 56C: 29-38.
19. Yi C, Ma C, Xie Z, et al. Down-regulation of programmed cell death 5 by insulin-like growth factor 1 in osteoarthritis chondrocytes. Int Orthop, 2013, 37(5): 937-943.
20. Li Y, Shelat H, Geng YJ. IGF-1 prevents oxidative stress induced-apoptosis in induced pluripotent stem cells which is mediated by microRNA-1. Biochem Biophys Res Commun, 2012, 426(4): 615-619.
21. Kim Y, Li E, Park S. Insulin-like growth factor-1 inhibits 6-hydroxydopamine-mediated endoplasmic reticulum stress-induced apoptosis via regulation of heme oxygenase-1 and Nrf2 expression in PC12 cells. Int J Neurosci, 2012, 122(11): 641-649.
22. Hao CN, Geng YJ, Li F, et al. Insulin-like growth factor-1 receptor activation prevents hydrogen peroxide-induced oxidative stress, mitochondrial dysfunction and apoptosis. Apoptosis, 2011, 16(11): 1118-1127.
23. Paul R, Haydon RC, Cheng H, et al. Potential use of Sox9 gene therapy for intervertebral degenerative disc disease. Spine (Phila Pa 1976), 2003, 28(8): 755-763.
24. 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.
25. 曹德军, 权正学, 蒋电明, 等. 人BMP-4腺病毒表达载体对人退变腰椎间盘细胞的影响. 中国修复重建外科杂志, 2012, 26(12): 1442-1147.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF RECOMBINANT ADENOVIRUS VECTOR CARRYING HUMAN INSULIN-LIKE GROWTH FACTOR 1 GENE ON THE APOPTOSIS OF NUCLEUS PULPOSUS CELLS IN VITRO

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