RESTORING PHENOTYPE OF DEDIFFERENTIATED NORMAL NUCLEUS PULPOSUS CELLS BY RESVERATROL_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

SHEN Jieliang , HU Zhenming , ZHONG Xiaoming , WANG Dawu , XU Linfei.

Department of Orthopaedics, First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, P.R.China. Corresponding author: HU Zhenming, E-mail: zhenminghu62@yahoo.com.cn;

Corresponding author：

Keywords：

Nucleus pulposus cells; Cell phenotype; Monolayer culture; Three-dimensional alginate microsphere culture; Resveratrol

DOI：

10.7507/1002-1892.20130122

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To investigate the effects of in-vitro monolayer culture and three-dimensional (3-D) alginate microsphere culture on the differentiation of normal human nucleus pulposus cells (NPCs), and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol (RES) in 3-D alginate microsphere. Methods Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group (group A), RES group (group B), silent mating type information regulation 2 homolog 1 (SIRT1)- small interfering RNA (siRNA) + RES group (group C), and negative control-siRNA + RES group (group D); and NPCs in the in-vitro monolayer culture was monolayer control group (group E). After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression. Results The cultured cells were identified to be NPCs. Morphological observation, senescence-associated β-galactosidase (SA-β-gal) staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture (P lt; 0.05), but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference between groups E and A (P lt; 0.05). The protein expressions of SIRT1, collagen type II, and Aggrecan in group B were significantly improved when compared with that in group A (P lt; 0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expressions of SIRT1 mRNA and proteins in group C were significantly inhibited after transfected with SIRT1-siRNA when compared with those in groups B and D (P lt; 0.05), and the protein expressions of collagen type II and Aggrecan in group C were significantly lower than those in groups B and D (P lt; 0.05). Conclusion Continuous in-vitro monolayer culture could efficiently cultivate numerous seeding NPCs, but it is liable to dedifferentiate. In 3-D alginate microsphere culture, RES could restore the phenotype of dedifferentiated NPCs and synthesize more extracellular matrix, which is related to the regulation of SIRT1.

Citation： SHEN Jieliang,HU Zhenming,ZHONG Xiaoming,WANG Dawu,XU Linfei.. RESTORING PHENOTYPE OF DEDIFFERENTIATED NORMAL NUCLEUS PULPOSUS CELLS BY RESVERATROL. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(5): 547-553. doi: 10.7507/1002-1892.20130122 Copy

1.	Hoy D, Brooks P, Blyth F, et al. The Epidemiology of low back pain. Best Pract Res Clin Rheumatol, 2010, 24(6): 769-781.
2.	Hoy D, March L, Brooks P, et al. Measuring the global burden of low back pain. Best Pract Res Clin Rheumatol, 2010, 24(2): 155-165.
3.	Cheung KM, Karppinen J, Chan D, et al. Prevalence and pattern of lumbar magnetic resonance imaging changes in a population study of one thousand forty-three individuals. Spine (Phila Pa 1976), 2009, 34(9): 934-940.
4.	Clouet J, Vinatier C, Merceron C, et al. The intervertebral disc: from pathophysiology to tissue engineering. Joint Bone Spine, 2009, 76(6): 614-618.
5.	Videman T, Gibbons LE, Battien MC. Age- and pathology-specific measures of disc degeneration. Spine (Phila Pa 1976), 2008, 33(25): 2781-2788.
6.	Hanley EN Jr, Herkowitz HN, Kirkpatrick JS, et al. Debating the value of spine surgery. J Bone Joint Surg (Am), 2010, 92(5): 1293-1304.
7.	Levin PE. Disc surgery in nonradicular lower back pain. J Bone Joint Surg (Am), 2010, 92(15): 2617.
8.	Hohaus C, Ganey TM, Minkus Y, et al. Cell transplantation in lumbar spine disc degeneration disease. Eur Spine J, 2008, 17 Suppl 4: 492-503.
9.	Jang M, Cai L, Udeani GO, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science, 1997, 275 (5297): 218-220.
10.	Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov, 2006, 5(6): 493-506.
11.	Wuertz K, Quero L, Sekiguchi M, et al. The red wine polyphenol resveratrol shows promising potential for the treatment of nucleus pulposus-mediated pain in vitro and in vivo. Spine (Phila Pa 1976), 2011, 36 (21): E1373-1384.
12.	Dvir-Ginzberg M, Gagarina V, Lee EJ, et al. Regulation of cartilage-specific gene expression in human chondrocytes by SirT1 and nicotinamide phosphoribosyltransferase. J Biol Chem, 2008, 283(52): 36300-36310.
13.	Griffith JF, Wang YX, Antonio GE, et al. Modified Pfirrmann grading system for lumbar intervertebral disc degeneration. Spine (Phila Pa 1976), 2007, 32(24): E708-712.
14.	张丽, 马迅, 关晓明, 等. 应用流式细胞术测定CD24表达鉴定人髓核细胞. 中国组织工程研究与临床康复, 2011, 15(46): 8583-8586.
15.	Dinri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A, 1995, 92(20): 9363-9367.
16.	Xia XP, Chen HL, Cheng HB. Prevalence of adjacent segment degeneration after spine surgery: a systematic review and Meta-analysis. Spine (Phila Pa 1976), 2013, 38(7): 597-608.
17.	Sebrastine IM, Williams DJ. Current developments in tissue engineering of nucleus pulposus for the treatment of intervertebral disc degeneration//Proceedings of the 29th Annual International Conference of the IEEE EMBS. Lyon: s.l., 2007.
18.	Kluba T, Niemeyer T, Gaissmaier C, et al. Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc: effect of degeneration and culture system on cell phenotype. Spine (Phila Pa 1976), 2005, 30(24): 2743-2478.
19.	郭尚春, 袁霆, 芮碧宇, 等. 联合单层培养和三维培养的两步法培养软骨细胞. 第二军医大学学报, 2010, 31(9): 970-974.
20.	Bron JL, Vonk LA, Smit TH, et al. Engineering alginate for intervertebral disc repair. J Mech Behav Biomed Mater, 2011, 4(7): 1196-1205.
21.	Schuurman W, Gawlitta D, Klein TJ, et al. Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in vitro redifferentiation. Am J Sports Med, 2009, 37 Suppl 1: 97S-104S.
22.	Chung S, Yao H, Caito S, et al. Regulation of SIRT1 in cellular functions: role of polyphenols. Arch Biochem Biophys, 2010, 501(1): 79-90.
23.	钟小明, 胡侦明, 沈皆亮, 等. 白藜芦醇通过调节SIRT1活性促进退变椎间盘终板软骨细胞合成细胞外基质. 中国老年学杂志, 2012, 32(9): 1846-1849.
24.	王大武, 胡侦明, 郝杰, 等. 沉默信息调节因子2同源蛋白1通过Akt/PKB通路抑制人退变椎间盘髓核细胞凋亡. 生物化学与生物物理进展, 2012, 39(6): 563-573.

1. Hoy D, Brooks P, Blyth F, et al. The Epidemiology of low back pain. Best Pract Res Clin Rheumatol, 2010, 24(6): 769-781.
2. Hoy D, March L, Brooks P, et al. Measuring the global burden of low back pain. Best Pract Res Clin Rheumatol, 2010, 24(2): 155-165.
3. Cheung KM, Karppinen J, Chan D, et al. Prevalence and pattern of lumbar magnetic resonance imaging changes in a population study of one thousand forty-three individuals. Spine (Phila Pa 1976), 2009, 34(9): 934-940.
4. Clouet J, Vinatier C, Merceron C, et al. The intervertebral disc: from pathophysiology to tissue engineering. Joint Bone Spine, 2009, 76(6): 614-618.
5. Videman T, Gibbons LE, Battien MC. Age- and pathology-specific measures of disc degeneration. Spine (Phila Pa 1976), 2008, 33(25): 2781-2788.
6. Hanley EN Jr, Herkowitz HN, Kirkpatrick JS, et al. Debating the value of spine surgery. J Bone Joint Surg (Am), 2010, 92(5): 1293-1304.
7. Levin PE. Disc surgery in nonradicular lower back pain. J Bone Joint Surg (Am), 2010, 92(15): 2617.
8. Hohaus C, Ganey TM, Minkus Y, et al. Cell transplantation in lumbar spine disc degeneration disease. Eur Spine J, 2008, 17 Suppl 4: 492-503.
9. Jang M, Cai L, Udeani GO, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science, 1997, 275 (5297): 218-220.
10. Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov, 2006, 5(6): 493-506.
11. Wuertz K, Quero L, Sekiguchi M, et al. The red wine polyphenol resveratrol shows promising potential for the treatment of nucleus pulposus-mediated pain in vitro and in vivo. Spine (Phila Pa 1976), 2011, 36 (21): E1373-1384.
12. Dvir-Ginzberg M, Gagarina V, Lee EJ, et al. Regulation of cartilage-specific gene expression in human chondrocytes by SirT1 and nicotinamide phosphoribosyltransferase. J Biol Chem, 2008, 283(52): 36300-36310.
13. Griffith JF, Wang YX, Antonio GE, et al. Modified Pfirrmann grading system for lumbar intervertebral disc degeneration. Spine (Phila Pa 1976), 2007, 32(24): E708-712.
14. 张丽, 马迅, 关晓明, 等. 应用流式细胞术测定CD24表达鉴定人髓核细胞. 中国组织工程研究与临床康复, 2011, 15(46): 8583-8586.
15. Dinri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A, 1995, 92(20): 9363-9367.
16. Xia XP, Chen HL, Cheng HB. Prevalence of adjacent segment degeneration after spine surgery: a systematic review and Meta-analysis. Spine (Phila Pa 1976), 2013, 38(7): 597-608.
17. Sebrastine IM, Williams DJ. Current developments in tissue engineering of nucleus pulposus for the treatment of intervertebral disc degeneration//Proceedings of the 29th Annual International Conference of the IEEE EMBS. Lyon: s.l., 2007.
18. Kluba T, Niemeyer T, Gaissmaier C, et al. Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc: effect of degeneration and culture system on cell phenotype. Spine (Phila Pa 1976), 2005, 30(24): 2743-2478.
19. 郭尚春, 袁霆, 芮碧宇, 等. 联合单层培养和三维培养的两步法培养软骨细胞. 第二军医大学学报, 2010, 31(9): 970-974.
20. Bron JL, Vonk LA, Smit TH, et al. Engineering alginate for intervertebral disc repair. J Mech Behav Biomed Mater, 2011, 4(7): 1196-1205.
21. Schuurman W, Gawlitta D, Klein TJ, et al. Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in vitro redifferentiation. Am J Sports Med, 2009, 37 Suppl 1: 97S-104S.
22. Chung S, Yao H, Caito S, et al. Regulation of SIRT1 in cellular functions: role of polyphenols. Arch Biochem Biophys, 2010, 501(1): 79-90.
23. 钟小明, 胡侦明, 沈皆亮, 等. 白藜芦醇通过调节SIRT1活性促进退变椎间盘终板软骨细胞合成细胞外基质. 中国老年学杂志, 2012, 32(9): 1846-1849.
24. 王大武, 胡侦明, 郝杰, 等. 沉默信息调节因子2同源蛋白1通过Akt/PKB通路抑制人退变椎间盘髓核细胞凋亡. 生物化学与生物物理进展, 2012, 39(6): 563-573.

Chinese Journal of Reparative and Reconstructive Surgery

RESTORING PHENOTYPE OF DEDIFFERENTIATED NORMAL NUCLEUS PULPOSUS CELLS BY RESVERATROL

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content