體外轉染人IGF-1基因重組腺病毒載體對兔椎間盤髓核細胞凋亡的影響_《中國修復重建外科雜志》

作者：

張長春 , 崔國鵬 , 邵晨 , 周新社 , 肖玉周 , 周建生

蚌埠醫學院第一附屬醫院安徽省組織移植重點實驗室（安徽蚌埠，233004）;

關鍵詞：

椎間盤退變 IGF-1 腺病毒載體髓核細胞細胞凋亡兔

DOI：

10.7507/1002-1892.20130298

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目的探討體外轉染人IGF-1（human IGF-1，hIGF-1）基因腺病毒載體（Ad-hIGF-1）對TNF-α誘導的兔椎間盤髓核細胞凋亡的影響。方法取8只健康成年家兔（雌雄不限，體重2.0～2.5 kg）椎間盤髓核，以Ⅱ型膠原酶消化法分離培養髓核細胞。取第2代對數生長期髓核細胞，根據培養條件不同分為3組。空白組：以含10%PBS的DMEM/F12培養基培養；TNF-α組：在空白組培養基中加入100 ng/mL TNF-α；Ad-hIGF-1組：在TNF-α組培養基中加入感染復數為50的Ad-hIGF-1。熒光顯微鏡下觀察Ad-hIGF-1組病毒轉染情況。各組培養48 h后，行RT-PCR及Western blot檢測hIGF-1 mRNA和蛋白表達，TUNEL法及流式細胞儀檢測細胞凋亡情況。結果熒光顯微鏡下觀察示，Ad-hIGF-1組細胞發出綠色熒光，提示轉染成功。RT-PCR及Western blot檢測示，Ad-hIGF-1組可見hIGF-1 mRNA及蛋白表達條帶，而空白組和TNF-α組均未見。TUNEL法檢測示，TNF-α組、Ad-hIGF-1組及空白組細胞凋亡率分別為34.24% ± 4.60%、6.59% ± 1.03%、0.40% ± 0.15%；流式細胞儀檢測示，TNF-α組、Ad-hIGF-1組、空白組早期凋亡率分別為22.16% ± 2.69%、5.03% ± 0.96%、0.49% ± 0.05%，晚期凋亡率分別為13.96% ± 4.86%、10.68% ± 3.42%、0.29% ± 0.06%；以上檢測指標TNF-α組均顯著高于空白組、Ad-hIGF-1組（P lt; 0.05），Ad-hIGF-1組高于空白組，差異均有統計學意義（P lt; 0.05）。結論hIGF-1基因對TNF-α體外誘導的兔髓核細胞凋亡有抑制作用。

引用本文： 張長春,崔國鵬,邵晨,周新社,肖玉周,周建生. 體外轉染人IGF-1基因重組腺病毒載體對兔椎間盤髓核細胞凋亡的影響. 中國修復重建外科雜志, 2013, 27(11): 1375-1379. doi: 10.7507/1002-1892.20130298 復制

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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.	劉巍, 唐天駟, 李濤, 等. 腺病毒介導的基因在人退變髓核細胞的表達. 蘇州大學學報: 醫學版, 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.
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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.	金貞愛, 趙以坤, 金正勇, 等. 胰島素樣生長因子-1對高氧誘導的A549細胞凋亡的影響. 中國當代兒科雜志, 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.	曹德軍, 權正學, 蔣電明, 等. 人BMP-4腺病毒表達載體對人退變腰椎間盤細胞的影響. 中國修復重建外科雜志, 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. 劉巍, 唐天駟, 李濤, 等. 腺病毒介導的基因在人退變髓核細胞的表達. 蘇州大學學報: 醫學版, 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. 金貞愛, 趙以坤, 金正勇, 等. 胰島素樣生長因子-1對高氧誘導的A549細胞凋亡的影響. 中國當代兒科雜志, 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. 曹德軍, 權正學, 蔣電明, 等. 人BMP-4腺病毒表達載體對人退變腰椎間盤細胞的影響. 中國修復重建外科雜志, 2012, 26(12): 1442-1147.

《中國修復重建外科雜志》

體外轉染人IGF-1基因重組腺病毒載體對兔椎間盤髓核細胞凋亡的影響

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《中國修復重建外科雜志》

體外轉染人IGF-1基因重組腺病毒載體對兔椎間盤髓核細胞凋亡的影響

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