【摘 要】 目的 通過營養(yǎng)剝奪模擬體內(nèi)髓核細胞退變微環(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胞,將傳代后細
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、細胞免疫熒光染色及Western blot 檢測BNIP3 基因及蛋白表達,流式細胞儀檢測凋亡率及線粒體膜電位。 結(jié)果 實時熒光定量PCR、細胞免疫熒光染色及Western blot 檢測顯示對照組
細胞低表達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)計學意義(P lt; 0.05)。流式細胞儀檢測顯示,對照組細胞凋亡率較低,且細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
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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. 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.