劉杰 1,2 , 彭娜 3 , 曾順福 1 , 陸焱 4 , 王建 1 , 周躍 1
  • 第三軍醫(yī)大學(xué)新橋醫(yī)院骨科（重慶，400037）1 骨科，3 手術(shù)室;;
  • 2 解放軍93197 部隊衛(wèi)生所;;
  • 4 解放軍第187 醫(yī)院骨科;

【摘 要】 目的 通過營養(yǎng)剝奪模擬體內(nèi)髓核細(xì)胞退變微環(huán)境，檢測Bcl-2/ 腺病毒干擾蛋白3（Bcl-2/adenovirus
E1B 19-kDa-interacting protein 3，BNIP3）表達及線粒體轉(zhuǎn)位情況，為進一步探索髓核細(xì)胞退變死亡機制提供實驗依
據(jù)。 方法 成年清潔級SD 大鼠2 只，雌雄不限，體重150 ～ 200 g。體外分離獲取鼠尾椎間盤髓核細(xì)胞，將傳代后細(xì)
胞分別置入正常環(huán)境（對照組：L-DMEM 培養(yǎng)基、10%FBS、21%O2）和營養(yǎng)剝奪環(huán)境（實驗組：DMEM 無糖無血清培養(yǎng)
基、 1% O2）培養(yǎng)24、48、72 h 后，實時熒光定量PCR、細(xì)胞免疫熒光染色及Western blot 檢測BNIP3 基因及蛋白表達，流式細(xì)胞儀檢測凋亡率及線粒體膜電位。 結(jié)果 實時熒光定量PCR、細(xì)胞免疫熒光染色及Western blot 檢測顯示對照組
細(xì)胞低表達BNIP3；實驗組隨培養(yǎng)時間延長，BNIP3 表達呈上升趨勢，且BNIP3 與線粒體相結(jié)合；除培養(yǎng)后24 h 實驗組
BNIP3 基因表達與對照組比較差異無統(tǒng)計學(xué)意義（P  gt; 0.05）外，其余各時間點實驗組BNIP3 基因及蛋白表達與對照組比
較差異均有統(tǒng)計學(xué)意義（P  lt; 0.05）。流式細(xì)胞儀檢測顯示，對照組細(xì)胞凋亡率較低，且細(xì)胞保持較高的線粒體膜電位；而
實驗組隨培養(yǎng)時間延長細(xì)胞凋亡率增加、線粒體膜電位降低，與對照組比較差異均有統(tǒng)計學(xué)意義（P  lt; 0.05）。 結(jié)論 營
養(yǎng)剝奪可能通過誘導(dǎo)BNIP3 表達增加并結(jié)合線粒體導(dǎo)致線粒體功能障礙，最終導(dǎo)致髓核細(xì)胞死亡。

引用本文： 劉杰,彭娜,曾順福,陸焱,王建,周躍. 營養(yǎng)剝奪誘導(dǎo)髓核細(xì)胞Bcl-2/ 腺病毒干擾蛋白3 表達及線粒體轉(zhuǎn)位的實驗研究. 中國修復(fù)重建外科雜志, 2012, 26(2): 166-171. doi: 復(fù)制

1. Crock HV, Goldwasser M, Yoshizawa H. Vascular anatomy related to the intervertebral disc//Ghosh P. The biology of the intervertebral disc. Boca Raton (FL): CRC Press, 1988: 109-133.
2. Urban JP, Holm S, Maroudas A, et al. Nutrition of the intervertebral disk. An in vivo study of solute transport. Clin Orthop Relat Res, 1977, (129): 101-114.
3. Holm S, Maroudas A, Urban JP, et al. Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res, 1981, 8(2): 101-119.
4. Stairmand J, Holm S, Urban J. Factors influencing oxygen concentration gradients in the intervertebral disc: a theoretical analysis. Spine (Phila Pa 1976), 1991, 16(4): 444-449.
5. Thompson JP, Pearce RH, Schechter MT, et al. Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine (Phila Pa 1976), 1990, 15(5): 411-415.
6. Yasuma T, Koh S, Okamura T, et al. Histological changes in aging lumbar intervertebral discs. Their role in protrusions and prolapses. J Bone Joint Surg (Am), 1990, 72(2): 220-229.
7. Taylor JR, Twomey LT. The role of the notochord and blood vessels in vertebral column development and in the aetiology of Schmorl’s nodes//Grieve J. Modern manual therapy. Edinburgh: Churchill Livingstone, 1986: 21-29.
8. Battié MC, Videman T, Gill K, et al. Smoking and lumbar intervertebral disc degeneration: an MRI study of identical twins. Spine (Phila Pa 1976), 1991, 16(9): 1015-1021.
9. Kauppila LI, Penttilä A, Karhunen PJ, et al. Lumbar disc degeneration and atherosclerosis of the abdominal aorta. Spine (Phila Pa 1976), 1994, 19(8): 923-929.
10. McFadden KD, Taylor JR. End-plate lesions of the lumbar spine. Spine (Phila Pa 1976), 1989, 14(8): 867-869.
11. Roberts S, Menage J, Eisenstein SM. The cartilage end-plate and intervertebral disc in scoliosis: calcification and other sequelae. J Orthop Res, 1993, 11(5): 747-757.
12. Aoki J, Yamamoto I, Kitamura N, et al. End plate of the discovertebral joint: degenerative change in the elderly adult. Radiology, 1987, 164(2): 411-414.
13. Brown MD, Tsaltas T. Studies on the permeability of the intervertebral disc during skeletal maturation. Spine (Phila Pa 1976), 1986, 1: 240-244.
14. Hassler O. The human intervertebral disc. A micro-angiographical study of its vascular supply at various ages. Acta Orthop Scand, 1970, 40(6): 765-772.
15. Roberts S, Urban JPG, Evans H, et al. Transport properties of the human cartilage endplate in relation to its composition and calcification. Spine (Phila Pa 1976), 1996, 21(4): 415-420.
16. Urban MR, Fairbank JC, Etherington PJ, et al. Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer. Spine (Phila Pa 1976), 2001, 26(8): 984-990.
17. Nachemson A, Lewin T, Maroudas A, et al. In vitro diffusion of dye through the end-plates and annulus fibrosus of human lumbar inter-vertebral discs. Acta Orthop Scand, 1970, 41(6): 589-607.
18. Lee H, Paik SG. Regulation of BNIP3 in normal and cancer cells. Mol Cells, 2006, 21(2): 1-6.
19. Vande Velde C, Cizeau J, Dubik D, et al. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol Cell Biol, 2000, 20(15): 5454-5468.
20. Ray R, Chen G, Vande Velde C, et al. BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology3 (BH3) domain at both mitochondrial and nonmitochondrial sites. J Biol Chem, 2000, 275(2): 1439-1448.
21. Chen G, Ray R, Dubik D, et al. The E1B 19K/Bcl-2 binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. J Exp Med, 1997, 186(12): 1975-1983.
22. Yan J, Yun H, Yang Y, et al. Upregulation of BNIP3 promotes apoptosis of lung cancer cells that were induced by p53. Biochem Biophys Res Commun, 2006, 346(2): 501-507.
23. Twomey LT, Taylor JR. Age changes in lumbar vertebrae and intervertebral discs. Clin Orthop Relat Res, 1987, (224): 97-104.
24. Roberts S, Menage J, Urban JP. Biochemical and structural properties of the cartilage end-plate and its relation to the intervertebral disc. Spine (Phila Pa 1976), 1989, 14(2): 166-174.
25. Maroudas A, Stockwell RA, Nachemson A, et al. Factors involved in the nutrition of the intervertebral disc: cellularity and diffusion of glucose in vitro. J Anat, 1975, 120(Pt 1): 113-130.
26. Trout JJ, Buckwalter JA, Moore KC. Ultrastructure of the human intervertebral disc: II. Cells of the nucleus pulposus. Anat Rec, 1982, 204(4): 307-314.
27. Gruber HE, Hanley EN. Analysis of aging and degeneration of the human intervertebral disc—comparison of surgical specimens with normal controls. Spine (Phila Pa 1976), 1998, 23(7): 751-757.
28. Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
29. Webster KA, Graham RM, Bishopric NH. BNIP3 and signalspecific programmed death in the heart. J Mol Cell Cardiol, 2005, 38(1): 35-45.
30. Kubasiak LA, Hernandez OM, Bishopric NH, et al. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNip3. Proc Natl Acad Sci U S A, 2002, 99(20): 12825-12830.
31. Yasuda M, Han JW, Dionne CA, et al. BNIP3alpha: a human homolog of mitochondrial proapoptotic protein BNIP3. Cancer Res, 1999, 59(3): 533-537.
32. Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res, 2002, 91(3): 226-231.
33. Zhang Z, Yang X, Zhang S, et al. BNIP3 upregulation and EndoG translocation in delayed neuronal death in stroke and in hypoxia. Stroke, 2007, 38(5): 1606-1613.
34. Goping IS, Gross A, Lavoie JN, et al. Regulated targeting of BAX to mitochondria. J Cell Biol, 1998, 143(1): 207-215.
35. Shimizu S, Konishi A, Kodama T, et al. BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc Natl Acad Sci U S A, 2000, 97(7): 3100-3105.
36. Zhang L, Li L, Liu H, et al. HIF-1alpha activation by a redox-sensitive pathway mediates cyanide-induced BNIP3 upregulation and mitochondrial-dependent cell death. Free Radic Biol Med, 2007, 43(1): 117-127.
  1. 1. Crock HV, Goldwasser M, Yoshizawa H. Vascular anatomy related to the intervertebral disc//Ghosh P. The biology of the intervertebral disc. Boca Raton (FL): CRC Press, 1988: 109-133.
  2. 2. Urban JP, Holm S, Maroudas A, et al. Nutrition of the intervertebral disk. An in vivo study of solute transport. Clin Orthop Relat Res, 1977, (129): 101-114.
  3. 3. Holm S, Maroudas A, Urban JP, et al. Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res, 1981, 8(2): 101-119.
  4. 4. Stairmand J, Holm S, Urban J. Factors influencing oxygen concentration gradients in the intervertebral disc: a theoretical analysis. Spine (Phila Pa 1976), 1991, 16(4): 444-449.
  5. 5. Thompson JP, Pearce RH, Schechter MT, et al. Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine (Phila Pa 1976), 1990, 15(5): 411-415.
  6. 6. Yasuma T, Koh S, Okamura T, et al. Histological changes in aging lumbar intervertebral discs. Their role in protrusions and prolapses. J Bone Joint Surg (Am), 1990, 72(2): 220-229.
  7. 7. Taylor JR, Twomey LT. The role of the notochord and blood vessels in vertebral column development and in the aetiology of Schmorl’s nodes//Grieve J. Modern manual therapy. Edinburgh: Churchill Livingstone, 1986: 21-29.
  8. 8. Battié MC, Videman T, Gill K, et al. Smoking and lumbar intervertebral disc degeneration: an MRI study of identical twins. Spine (Phila Pa 1976), 1991, 16(9): 1015-1021.
  9. 9. Kauppila LI, Penttilä A, Karhunen PJ, et al. Lumbar disc degeneration and atherosclerosis of the abdominal aorta. Spine (Phila Pa 1976), 1994, 19(8): 923-929.
  10. 10. McFadden KD, Taylor JR. End-plate lesions of the lumbar spine. Spine (Phila Pa 1976), 1989, 14(8): 867-869.
  11. 11. Roberts S, Menage J, Eisenstein SM. The cartilage end-plate and intervertebral disc in scoliosis: calcification and other sequelae. J Orthop Res, 1993, 11(5): 747-757.
  12. 12. Aoki J, Yamamoto I, Kitamura N, et al. End plate of the discovertebral joint: degenerative change in the elderly adult. Radiology, 1987, 164(2): 411-414.
  13. 13. Brown MD, Tsaltas T. Studies on the permeability of the intervertebral disc during skeletal maturation. Spine (Phila Pa 1976), 1986, 1: 240-244.
  14. 14. Hassler O. The human intervertebral disc. A micro-angiographical study of its vascular supply at various ages. Acta Orthop Scand, 1970, 40(6): 765-772.
  15. 15. Roberts S, Urban JPG, Evans H, et al. Transport properties of the human cartilage endplate in relation to its composition and calcification. Spine (Phila Pa 1976), 1996, 21(4): 415-420.
  16. 16. Urban MR, Fairbank JC, Etherington PJ, et al. Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer. Spine (Phila Pa 1976), 2001, 26(8): 984-990.
  17. 17. Nachemson A, Lewin T, Maroudas A, et al. In vitro diffusion of dye through the end-plates and annulus fibrosus of human lumbar inter-vertebral discs. Acta Orthop Scand, 1970, 41(6): 589-607.
  18. 18. Lee H, Paik SG. Regulation of BNIP3 in normal and cancer cells. Mol Cells, 2006, 21(2): 1-6.
  19. 19. Vande Velde C, Cizeau J, Dubik D, et al. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol Cell Biol, 2000, 20(15): 5454-5468.
  20. 20. Ray R, Chen G, Vande Velde C, et al. BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology3 (BH3) domain at both mitochondrial and nonmitochondrial sites. J Biol Chem, 2000, 275(2): 1439-1448.
  21. 21. Chen G, Ray R, Dubik D, et al. The E1B 19K/Bcl-2 binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. J Exp Med, 1997, 186(12): 1975-1983.
  22. 22. Yan J, Yun H, Yang Y, et al. Upregulation of BNIP3 promotes apoptosis of lung cancer cells that were induced by p53. Biochem Biophys Res Commun, 2006, 346(2): 501-507.
  23. 23. Twomey LT, Taylor JR. Age changes in lumbar vertebrae and intervertebral discs. Clin Orthop Relat Res, 1987, (224): 97-104.
  24. 24. Roberts S, Menage J, Urban JP. Biochemical and structural properties of the cartilage end-plate and its relation to the intervertebral disc. Spine (Phila Pa 1976), 1989, 14(2): 166-174.
  25. 25. Maroudas A, Stockwell RA, Nachemson A, et al. Factors involved in the nutrition of the intervertebral disc: cellularity and diffusion of glucose in vitro. J Anat, 1975, 120(Pt 1): 113-130.
  26. 26. Trout JJ, Buckwalter JA, Moore KC. Ultrastructure of the human intervertebral disc: II. Cells of the nucleus pulposus. Anat Rec, 1982, 204(4): 307-314.
  27. 27. Gruber HE, Hanley EN. Analysis of aging and degeneration of the human intervertebral disc—comparison of surgical specimens with normal controls. Spine (Phila Pa 1976), 1998, 23(7): 751-757.
  28. 28. Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
  29. 29. Webster KA, Graham RM, Bishopric NH. BNIP3 and signalspecific programmed death in the heart. J Mol Cell Cardiol, 2005, 38(1): 35-45.
  30. 30. Kubasiak LA, Hernandez OM, Bishopric NH, et al. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNip3. Proc Natl Acad Sci U S A, 2002, 99(20): 12825-12830.
  31. 31. Yasuda M, Han JW, Dionne CA, et al. BNIP3alpha: a human homolog of mitochondrial proapoptotic protein BNIP3. Cancer Res, 1999, 59(3): 533-537.
  32. 32. Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res, 2002, 91(3): 226-231.
  33. 33. Zhang Z, Yang X, Zhang S, et al. BNIP3 upregulation and EndoG translocation in delayed neuronal death in stroke and in hypoxia. Stroke, 2007, 38(5): 1606-1613.
  34. 34. Goping IS, Gross A, Lavoie JN, et al. Regulated targeting of BAX to mitochondria. J Cell Biol, 1998, 143(1): 207-215.
  35. 35. Shimizu S, Konishi A, Kodama T, et al. BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc Natl Acad Sci U S A, 2000, 97(7): 3100-3105.
  36. 36. Zhang L, Li L, Liu H, et al. HIF-1alpha activation by a redox-sensitive pathway mediates cyanide-induced BNIP3 upregulation and mitochondrial-dependent cell death. Free Radic Biol Med, 2007, 43(1): 117-127.