EFFECT OF ADENOVIRUS HUMAN BONE MORPHOGENETIC PROTEIN 4 ON HUMAN DEGENERATIVE LUMBAR INTERVERTEBRAL DISC CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CAO Dejun , QUAN Zhengxue , JIANG Dianming , LUO Xiaoji , ZHONG Weiyang , LIU Gang.

Department of Orthopaedics, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, P.R.China. Corresponding author: QUAN Zhengxue, E-mail: quanzx18@126.com;

Corresponding author：

Keywords：

Bone morphogenetic protein 4; Intervertebral disc cell; Adenovirus expression vector; Gene transfection Intervertebral disc degeneration

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Objective To research the transfer of adenovirus human bone morphogenetic protein 4 (Ad-hBMP-4) to human degenerative lumbar intervertebral disc cells in vitro and analyze its effect on the proteoglycan, collagen type II, and Sox9 of intervertebral disc cells. Methods Identified Ad-hBMP-4 was amplified and detected. Degenerative lumbar intervertebral disc cells were aspirated from the degenerative lumbar intervertebral disc of patients with Modic III level disc protrusion (aged, 27-50 years). The expressing position of collagen type II was identified in the intervertebral disc cells through the laser confocal microscope. The intervertebral disc cells at passage 1 were transfected with Ad-hBMP-4 as experimental group. After 3 and 6 days of transfection, RT-PCR was used to detect the mRNA expressions of proteoglycan, collagen type II, and Sox9, and Western blot to detect the expressions of proteoglycan and collagen type II proteins. Non-transfected cells at passage 1 served as control group. Results The virus titer of Ad-hBMP-4 was 5 × 106 PFU/mL. No morphological changes in the cells after transfection by Ad-hBMP-4. Collagen type II mainly expressed in the cell cytoplasm. The mRNA expressions of the proteoglycan, collagen type II, and Sox9 in experimental group at 3 and 6 days after transfection were significantly higher than those in control group by RT-PCR (P lt; 0.05), and the expressions of proteoglycan and collagen type II proteins were significantly higher than those in contorl group by Western blot (P lt; 0.05). There were significant differences between 3 days and 6 days in experimental group (P lt; 0.05). Conclusion Ad-hBMP-4 could transfect human degenerative lumbar intervertebral cells with high efficiency and promote collagen type II, proteoglycan, and Sox9 expressions. hBMP-4 may play an important role in the repair process during early disc degeneration.

Citation： CAO Dejun,QUAN Zhengxue,JIANG Dianming,LUO Xiaoji,ZHONG Weiyang,LIU Gang.. EFFECT OF ADENOVIRUS HUMAN BONE MORPHOGENETIC PROTEIN 4 ON HUMAN DEGENERATIVE LUMBAR INTERVERTEBRAL DISC CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(12): 1442-1447. doi: Copy

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2.	Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
3.	Zhang Y, Anderson DG, Phillips FM, et al. Comparative effects of bone morphogenetic proteins and Sox9 overexpression on matrix accumulation by bovine anulus fibrosus cells: implications for anular repair. Spine (Phila Pa 1976), 2007, 32(23): 2515-2520.
4.	Luo J, Deng ZL, Luo X, et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc, 2007, 2(5): 1236-1247.
5.	曹洁玮, 李小鹰. HEK293T细胞扩增重组腺病毒研究. 军医进修学院学报, 2009, 30(6): 860-862.
6.	Modic MT, Masaryk TJ, Hoss JS, et al．Imaging of degenerative disk disease. Radiology, 1988, 168(1): 177-186.
7.	Le Visage C, Kim SW, Tateno K, et al. Interaction of human mesenchymal stem cells with disc cells: changes in extracellular matrix biosynthesis. Spine (Phila Pa 1976), 2006, 31(18): 2036-2042.
8.	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.
9.	余礼厚. 激光共聚焦显微镜样品制备方法(一)——细胞培养样品. 电子显微学报, 2010, 29(2): 185-188.
10.	Sive JI, Baird P, Jcziorsk M, et al. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol, 2002, 55(2): 91-97.
11.	Gruber HE, Norton HJ, Ingram JA, et al. The Sox9 transcription factor in the human disc: decreased immunolocalization with age and disc degeneration. Spine (Phila Pa 1976), 2005, 30(6): 625-630.
12.	Li Y, Tew SR, Russell AM, et al. Transduction of passaged human articular chondrocytcs with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Eng, 2004, 10(3-4): 575-584.
13.	顾海伦, 吕刚, 刘莉, 等. 人骨形成蛋白-2腺病毒表达载体转染体外培养兔腰椎间盘细胞的研究. 中国修复重建外科杂志, 2006, 20(8): 820-824.
14.	Fei QM, Jiang XX, Chen TY, et al. Changes with age and the effect of recombinant human BMP-2 on proteoglycan and collagen gene expression in rabbit anulus fibrosus cells. Acta Biochim Biophys Sin (Shanghai), 2006, 38(11): 773-779.
15.	何李明, 马迅, 张丽, 等. 骨形态发生蛋白-7对椎间盘细胞Sox9和Ⅱ型胶原基因的调节作用. 中国医疗前沿, 2010, 5(5): 18-10, 31.
16.	Imai Y, Miyamoto K, An HS, et al. Recombinant human osteogenic protein-1 upregulates proteoglycan metabolism of human anulus fibrosus and nucleus pulposus cells. Spine (Phila Pa 1976), 2007, 32(12): 1303-1309.
17.	Gilbertson L, Ahn SH, Teng PN, et al. The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells. Spine J, 2008, 8(3): 449-456.
18.	张虎林, 陈玉琴, 俞诗源, 等. 骨形态发生蛋白-4的研究进展. 解剖学杂志, 2007, 30(6): 810-812.
19.	Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation: molecular clones and activities. Science, 1998, 242(4885): 1528-1534.
20.	Hassel S, Schmitt S, Hartung A, et al. Initiation of Smad dependent and Smad-Independent signaling via distinct BMP receptor complexes. J Bone Joint Surg (Am), 2003, 85-A Suppl 3: 44-51.
21.	Cheng JC, Guo X, Law LP, et al. How dose recombinant bone morphogenefic protein-4 enhance posterior spinal fusion? Spine (Phila Pa 1976), 2002, 27(5): 467-474.

1. 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.
2. Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
3. Zhang Y, Anderson DG, Phillips FM, et al. Comparative effects of bone morphogenetic proteins and Sox9 overexpression on matrix accumulation by bovine anulus fibrosus cells: implications for anular repair. Spine (Phila Pa 1976), 2007, 32(23): 2515-2520.
4. Luo J, Deng ZL, Luo X, et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc, 2007, 2(5): 1236-1247.
5. 曹洁玮, 李小鹰. HEK293T细胞扩增重组腺病毒研究. 军医进修学院学报, 2009, 30(6): 860-862.
6. Modic MT, Masaryk TJ, Hoss JS, et al．Imaging of degenerative disk disease. Radiology, 1988, 168(1): 177-186.
7. Le Visage C, Kim SW, Tateno K, et al. Interaction of human mesenchymal stem cells with disc cells: changes in extracellular matrix biosynthesis. Spine (Phila Pa 1976), 2006, 31(18): 2036-2042.
8. 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.
9. 余礼厚. 激光共聚焦显微镜样品制备方法(一)——细胞培养样品. 电子显微学报, 2010, 29(2): 185-188.
10. Sive JI, Baird P, Jcziorsk M, et al. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol, 2002, 55(2): 91-97.
11. Gruber HE, Norton HJ, Ingram JA, et al. The Sox9 transcription factor in the human disc: decreased immunolocalization with age and disc degeneration. Spine (Phila Pa 1976), 2005, 30(6): 625-630.
12. Li Y, Tew SR, Russell AM, et al. Transduction of passaged human articular chondrocytcs with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Eng, 2004, 10(3-4): 575-584.
13. 顾海伦, 吕刚, 刘莉, 等. 人骨形成蛋白-2腺病毒表达载体转染体外培养兔腰椎间盘细胞的研究. 中国修复重建外科杂志, 2006, 20(8): 820-824.
14. Fei QM, Jiang XX, Chen TY, et al. Changes with age and the effect of recombinant human BMP-2 on proteoglycan and collagen gene expression in rabbit anulus fibrosus cells. Acta Biochim Biophys Sin (Shanghai), 2006, 38(11): 773-779.
15. 何李明, 马迅, 张丽, 等. 骨形态发生蛋白-7对椎间盘细胞Sox9和Ⅱ型胶原基因的调节作用. 中国医疗前沿, 2010, 5(5): 18-10, 31.
16. Imai Y, Miyamoto K, An HS, et al. Recombinant human osteogenic protein-1 upregulates proteoglycan metabolism of human anulus fibrosus and nucleus pulposus cells. Spine (Phila Pa 1976), 2007, 32(12): 1303-1309.
17. Gilbertson L, Ahn SH, Teng PN, et al. The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells. Spine J, 2008, 8(3): 449-456.
18. 张虎林, 陈玉琴, 俞诗源, 等. 骨形态发生蛋白-4的研究进展. 解剖学杂志, 2007, 30(6): 810-812.
19. Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation: molecular clones and activities. Science, 1998, 242(4885): 1528-1534.
20. Hassel S, Schmitt S, Hartung A, et al. Initiation of Smad dependent and Smad-Independent signaling via distinct BMP receptor complexes. J Bone Joint Surg (Am), 2003, 85-A Suppl 3: 44-51.
21. Cheng JC, Guo X, Law LP, et al. How dose recombinant bone morphogenefic protein-4 enhance posterior spinal fusion? Spine (Phila Pa 1976), 2002, 27(5): 467-474.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF ADENOVIRUS HUMAN BONE MORPHOGENETIC PROTEIN 4 ON HUMAN DEGENERATIVE LUMBAR INTERVERTEBRAL DISC CELLS

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