白藜蘆醇恢復去分化正常髓核細胞表型的實驗研究_《中國修復重建外科雜志》

作者：

沈皆亮 , 胡偵明 , 鐘小明 , 王大武 , 徐林飛

重慶醫科大學附屬第一醫院骨科（重慶，400016）;

關鍵詞：

髓核細胞細胞表型單層培養藻酸鹽微球立體培養白藜蘆醇

DOI：

10.7507/1002-1892.20130122

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目的研究體外單層培養與藻酸鹽微球立體培養對人正常髓核細胞分化的影響，并探討白藜蘆醇（resveratrol，RES）恢復藻酸鹽微球立體培養下去分化髓核細胞表型的調節機制。方法取6例腰椎爆裂骨折患者自愿捐贈的正常髓核組織分離培養髓核細胞，并行細胞鑒定；取單層培養的第1、3、5、7代髓核細胞分別行形態學觀察、β-半乳糖苷酶（senescence-associated β-galactosidase，SA-β-gal）染色觀察細胞衰老情況及甲苯胺藍染色觀察蛋白多糖表達。分別取單層培養和藻酸鹽微球立體培養的第1代髓核細胞檢測細胞增殖情況。取立體培養1周的第7代髓核細胞隨機分為立體培養空白組（A組）、RES組（B組）、沉默交配型信息調節因子2同源蛋白1（silent mating type information regulation 2 homolog 1，SIRT1）-小干擾RNA（small interfering RNA，siRNA）+ RES組（C組）、陰性對照-siRNA+RES組（D組），另設置髓核細胞單層培養空白組（E組），行相應培養后采用Western blot檢測SIRT1、聚蛋白多糖（Aggrecan）和Ⅱ型膠原蛋白表達，實時熒光定量PCR檢測SIRT1 mRNA表達水平。結果細胞鑒定提示培養細胞為髓核細胞。形態學觀察及SA-β-gal、甲苯胺藍染色示，在體外連續單層培養條件下，正常髓核細胞易發生去分化，且隨著傳代次數增加趨勢愈明顯。藻酸鹽微球立體培養48 h內髓核細胞增殖顯著低于單層培養（P lt; 0.05），但能顯著提高去分化髓核細胞SIRT1、Ⅱ型膠原與Aggrecan蛋白的表達，E組與A組比較差異有統計學意義（P lt; 0.05）；與A組相比，B組3種蛋白表達顯著提高（P lt; 0.05）。C組SIRT1 mRNA和蛋白表達均受明顯抑制，顯著低于B、D組（P lt; 0.05），Ⅱ型膠原與Aggrecan蛋白表達也顯著低于B、D組（P lt; 0.05）。結論連續體外單層培養條件下，髓核細胞增殖速度較快，但在培養過程中易發生去分化現象；而在藻酸鹽微球立體培養條件下，RES可以恢復去分化髓核細胞表型，合成更多細胞外基質，這一機制與SIRT1的調節有關。

引用本文： 沈皆亮,胡偵明,鐘小明,王大武,徐林飛. 白藜蘆醇恢復去分化正常髓核細胞表型的實驗研究. 中國修復重建外科雜志, 2013, 27(5): 547-553. doi: 10.7507/1002-1892.20130122 復制

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14.	張麗, 馬迅, 關曉明, 等. 應用流式細胞術測定CD24表達鑒定人髓核細胞. 中國組織工程研究與臨床康復, 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. 聯合單層培養和三維培養的兩步法培養軟骨細胞. 第二軍醫大學學報, 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.	鐘小明, 胡偵明, 沈皆亮, 等. 白藜蘆醇通過調節SIRT1活性促進退變椎間盤終板軟骨細胞合成細胞外基質. 中國老年學雜志, 2012, 32(9): 1846-1849.
24.	王大武, 胡偵明, 郝杰, 等. 沉默信息調節因子2同源蛋白1通過Akt/PKB通路抑制人退變椎間盤髓核細胞凋亡. 生物化學與生物物理進展, 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. 張麗, 馬迅, 關曉明, 等. 應用流式細胞術測定CD24表達鑒定人髓核細胞. 中國組織工程研究與臨床康復, 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. 聯合單層培養和三維培養的兩步法培養軟骨細胞. 第二軍醫大學學報, 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. 鐘小明, 胡偵明, 沈皆亮, 等. 白藜蘆醇通過調節SIRT1活性促進退變椎間盤終板軟骨細胞合成細胞外基質. 中國老年學雜志, 2012, 32(9): 1846-1849.
24. 王大武, 胡偵明, 郝杰, 等. 沉默信息調節因子2同源蛋白1通過Akt/PKB通路抑制人退變椎間盤髓核細胞凋亡. 生物化學與生物物理進展, 2012, 39(6): 563-573.

《中國修復重建外科雜志》

白藜蘆醇恢復去分化正常髓核細胞表型的實驗研究

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《中國修復重建外科雜志》

白藜蘆醇恢復去分化正常髓核細胞表型的實驗研究

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