胰岛素样生长因子-Ⅰ在大鼠骨胳肌运动性损伤再生中的作用
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摘要
研究背景：在日常的体育锻炼及军事训练中，适应与损伤是两个并存的重要方面，训练结果如何取决于两者的平衡状态。目前国内骨骼肌运动损伤的研究比较少，主要集中在运动对骨骼肌超微结构、生化及酶学等方面的影响，国外在细胞因子在运动性肌损伤中的表达也处于起步阶段。骨骼肌损伤后的再生，伴随着复杂的生物学调节机制，与大量的细胞因子相关。其中IGF参与骨骼肌损伤修复过程中的细胞增殖和分化，IGF-Ⅰ主要促进骨骼肌的增值，IGF-Ⅱ主要对增殖后的细胞分化产生作用；bFGF是调控骨骼肌胚胎发生与生长发育的主要生长因子。细胞凋亡是骨骼肌损伤后细胞发生坏死过程中的重要作用因素，如凋亡相关基因：Bcl-2、Bax等在骨骼肌细胞凋亡过程中起着非常重要的作用。
目的：通过研究胰岛素样生长因子-Ⅰ(IGF-Ⅰ)对骨骼肌运动损伤后卫星细胞再生的影响，探讨IGF-Ⅰ在骨骼肌运动损伤修复中的应用前景。
方法：使用SD大鼠1周力竭模型，首次力竭后第1、3、5天给予右侧腓肠肌中部的肌膜下分别注射外源性IGF-Ⅰ 300mg／ml(实验组)、生理盐水1ml(对照组)，分别在首次力竭后第7、9、11、17、24、30天取材，进行骨骼肌HE染色光镜观察，电镜超微结构观察和增殖细胞核抗原(PCNA)表达比较。
结果：HE染色光镜观察：首次力竭后第7天，腓肠肌局部充血、水肿、部分纤维断裂，炎性细胞浸润，聚集于肌损伤处(图4、5)。第11天，对照组(一周力竭训练+生理盐水组 C组)损伤处有大量的成纤维细胞出
现，细胞核排列充紊乱;实验组(一周力竭训练+I GF一I组D组)有大量
新生的肌管和肌纤维细胞，细胞核数目非常多，肌管的细胞核位于细胞
中央，肌纤维的细胞核位于细胞边缘(图6，7)。第17天，对照组(一
周力竭训练+生理盐水组C组)有较多胶原纤维，排列紊乱;实验组(一
周力竭训练+I GF一工组D组)新生肌细胞排列成串珠样，较整齐(图8，9)。
第30天，对照组(一周力竭训练+生理盐水组C组)有胶原纤维瘫痕形
成;实验组(一周力竭训练+I GF一I组D组)再生较完全，基本未见纤维
瘫痕(图10，11)。
超微结构电镜观察:首次力竭后第7天，对照组(一周力竭训练+生理盐
水组C组)可见肌浆网扩张，糖原数量增多，线粒体肿胀空腔化;实验
组(一周力竭训练+I GF一I组D组)线粒体数量增多，空腔化少，肌浆网
扩张少(图14，15)。首次力竭后第n天，对照组(一周力竭训练+生理
盐水组C组)可见结缔组织增生，胶原纤维多;实验组(一周力竭训练
+IGF一I组D组)可见新生肌纤维，卫星细胞(图16，17)。
PCNA指数实验组明显高于对照组(一周力竭训练十生理盐水组C组)
(P(0.05)。
结论:工GF一I与骨骼肌的损伤及修复相关，IGF一I对运动性骨骼肌损伤
后的卫星细胞再生有促进作用。
Background The training effect lies on the equilibrium of the two facets. Presently, the domestic study of skeletal muscle exercise damage is rarely done which is mainly localized in the study of exercise effect to the ultrastructure, bio-chemistry and enzymology in the skeletal muscle. Overseas study of cell factors expression in exercise-induced skeletal muscle damage is in the initial phase.The regeneration of skeletal muscle which is followed by complicated biological regulative mechanism is relative to many cell factors. Thereinto the cell factor insulin-like growth factor(IGF) is concerned with cell proliferation and differentiation in the rehabilitation for skeletal muscle damage. IGF-I chiefly promotes the cell proliferation of skeletal muscle, while IGF-II is vital in the differentication of the proliferated skeletal muscle cells. Otherwise, basic fibroblast growth factor (bFGF) is the primary growth factor in the regulation of skeletal muscle embryogenesis, growth and development which is potent re
gulator in skeletal muscle proliferation and differentiation. Cell apoptosis is an important factor in the cell necrose following the skeletal muscle damage. The apoptosis relative genes such as Bcl-2, Bax are crucial in skeletal muscle cell spoptosis.Adaptation and damage are two dichotomal facets in daily physical exercise and military training.
Objective To study the effect of IGF-I on the regeneration of satellite cells after sports injury, and explore the applications of IGF-Iin the field of repair
of skeletal muscle after sports injury.
Methods Using SD rat 1 week exhausting model, on 1,3,5th day after the first exhausting,300mg/lml IGF-I was injected into the middle of right gastrocnemius muscle of the treatment group. The control groups were injected 1ml saline. The samples were taken on the 7,9,11,17,24,30th days after the first exhausting, the skeletal muscle was dyed with HE and observed with optical microscope and electric microscope. The expression of proliferating cell nuclear antigen (PCNA) in it were compared. Results On the 7,11,17th days after the first exhausting, the satellite cells decreased significantly in the treatment group, the collagen fibres increased in the control groups. On the 30th day, the muscle fibre's shape was in good condition for the treatment groups, but healed with scar tissue in the control groups. Observed by electric microscope, the biolast's shape is normal, new born muscle fibre can be found in the treatment groups, expanded sarcoplasmic reticulum, increased glycogen granule, engorged biolast and apop
totic cells can be found in the control groups. The PCNA exponent of the treatment group was obviously higher than the control group.
Conclusions IGF-I can accelerate the regeneration of skeletal muscle satellite cells after sports injury.
目的：通过研究胰岛素样生长因子-Ⅰ(IGF-Ⅰ)对骨骼肌运动损伤后卫星细胞再生的影响，探讨IGF-Ⅰ在骨骼肌运动损伤修复中的应用前景。
方法：使用SD大鼠1周力竭模型，首次力竭后第1、3、5天给予右侧腓肠肌中部的肌膜下分别注射外源性IGF-Ⅰ 300mg／ml(实验组)、生理盐水1ml(对照组)，分别在首次力竭后第7、9、11、17、24、30天取材，进行骨骼肌HE染色光镜观察，电镜超微结构观察和增殖细胞核抗原(PCNA)表达比较。
结果：HE染色光镜观察：首次力竭后第7天，腓肠肌局部充血、水肿、部分纤维断裂，炎性细胞浸润，聚集于肌损伤处(图4、5)。第11天，对照组(一周力竭训练+生理盐水组 C组)损伤处有大量的成纤维细胞出
现，细胞核排列充紊乱;实验组(一周力竭训练+I GF一I组D组)有大量
新生的肌管和肌纤维细胞，细胞核数目非常多，肌管的细胞核位于细胞
中央，肌纤维的细胞核位于细胞边缘(图6，7)。第17天，对照组(一
周力竭训练+生理盐水组C组)有较多胶原纤维，排列紊乱;实验组(一
周力竭训练+I GF一工组D组)新生肌细胞排列成串珠样，较整齐(图8，9)。
第30天，对照组(一周力竭训练+生理盐水组C组)有胶原纤维瘫痕形
成;实验组(一周力竭训练+I GF一I组D组)再生较完全，基本未见纤维
瘫痕(图10，11)。
超微结构电镜观察:首次力竭后第7天，对照组(一周力竭训练+生理盐
水组C组)可见肌浆网扩张，糖原数量增多，线粒体肿胀空腔化;实验
组(一周力竭训练+I GF一I组D组)线粒体数量增多，空腔化少，肌浆网
扩张少(图14，15)。首次力竭后第n天，对照组(一周力竭训练+生理
盐水组C组)可见结缔组织增生，胶原纤维多;实验组(一周力竭训练
+IGF一I组D组)可见新生肌纤维，卫星细胞(图16，17)。
PCNA指数实验组明显高于对照组(一周力竭训练十生理盐水组C组)
(P(0.05)。
结论:工GF一I与骨骼肌的损伤及修复相关，IGF一I对运动性骨骼肌损伤
后的卫星细胞再生有促进作用。
Background The training effect lies on the equilibrium of the two facets. Presently, the domestic study of skeletal muscle exercise damage is rarely done which is mainly localized in the study of exercise effect to the ultrastructure, bio-chemistry and enzymology in the skeletal muscle. Overseas study of cell factors expression in exercise-induced skeletal muscle damage is in the initial phase.The regeneration of skeletal muscle which is followed by complicated biological regulative mechanism is relative to many cell factors. Thereinto the cell factor insulin-like growth factor(IGF) is concerned with cell proliferation and differentiation in the rehabilitation for skeletal muscle damage. IGF-I chiefly promotes the cell proliferation of skeletal muscle, while IGF-II is vital in the differentication of the proliferated skeletal muscle cells. Otherwise, basic fibroblast growth factor (bFGF) is the primary growth factor in the regulation of skeletal muscle embryogenesis, growth and development which is potent re
gulator in skeletal muscle proliferation and differentiation. Cell apoptosis is an important factor in the cell necrose following the skeletal muscle damage. The apoptosis relative genes such as Bcl-2, Bax are crucial in skeletal muscle cell spoptosis.Adaptation and damage are two dichotomal facets in daily physical exercise and military training.
Objective To study the effect of IGF-I on the regeneration of satellite cells after sports injury, and explore the applications of IGF-Iin the field of repair
of skeletal muscle after sports injury.
Methods Using SD rat 1 week exhausting model, on 1,3,5th day after the first exhausting,300mg/lml IGF-I was injected into the middle of right gastrocnemius muscle of the treatment group. The control groups were injected 1ml saline. The samples were taken on the 7,9,11,17,24,30th days after the first exhausting, the skeletal muscle was dyed with HE and observed with optical microscope and electric microscope. The expression of proliferating cell nuclear antigen (PCNA) in it were compared. Results On the 7,11,17th days after the first exhausting, the satellite cells decreased significantly in the treatment group, the collagen fibres increased in the control groups. On the 30th day, the muscle fibre's shape was in good condition for the treatment groups, but healed with scar tissue in the control groups. Observed by electric microscope, the biolast's shape is normal, new born muscle fibre can be found in the treatment groups, expanded sarcoplasmic reticulum, increased glycogen granule, engorged biolast and apop
totic cells can be found in the control groups. The PCNA exponent of the treatment group was obviously higher than the control group.
Conclusions IGF-I can accelerate the regeneration of skeletal muscle satellite cells after sports injury.
引文
1. Lehto MU, Jarvinen MJ. Muscle injuries their healing process and treatment. Ann Clair Gynaecol 1991, 80: 102-108.
2. Daughaday WH, Rotwein P. Insulin-like growth factors 1 and 2: Peptide, messenger ribonucleic acid and gene structure, serum and tissue concerntration.Endo Rev 1989, 10(1): 68-91.
3. Ferrari G. Cusella D, Angelis G. Coletta,-M; Muscle regeneration by bone marrow-derived myogenic progenitors. J-Cell-Biol. 1999 Nov 15, 147(4): 869-878.
4. Allen DL, Linderman JK, Roy RR, et al. Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweghting. Am J physiol, 1997, 273: c579-587.
5. Svanberg E, Zachrisson H, Ohlsson C, et al. Role of insulin and IGF-Ⅰ in activation of muscle protein synthesis after oral feeding. Am J Physiol, 1996, 81: 2509-2516.
6. Adams GR, Haddad E The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy J-Appl-Physiol. 1996 Dec; 81 (6): 2509-2516.
7. Adams GR, Mccue SA. Localized infusion of IGF-1 results in skeletal muscle hypertrophy in rats. J Appl Physiol, 1998, 84: 1716-1722.
8. Levinovitz A, Jennisehe E, Oldfors A. Activation of insulin-like growth factor Ⅱ expression during skeletal muscle regeneration in the rot: correlation with myotube formation. Mol-Endoerinol, 1992 Aug; 6(8): 1227-1234.
9. Stewart CE, Rotwein P. Insulin-like growth factor-Ⅱ is an autocrine survival factor for differentiating myoblasts. J Biol Chem, 1996, 271: 11330-11338.
10. Florini JR, Magri KA, Ewton DZ, et al. "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-Ⅱ. Cancer-Res. 1991 Nov 1; 51(21):
5997-6000.
11. Kawaguchi H, Nakamura K, Tabata Y. Acceleration of fracture healing in nonhuman primates by fibroblast growth factor-2. J-Clin-Endocrinol-Metab. 2001 Feb; 86(2): 875-880.
12. Raschke M, Kolbeck S, Bail H. Homologous growth hormone accelerates healing of segmental bone defects. Bone. 2001 Oct; 29(4): 368-373.
13. Franchimont N, Crangji V, Durant D. Interleukin-6 with its soluble receptor enhances the expression of insulin-like growth factor-Ⅰ in osteoblasts. Endocrinology. 1997 Dec; 138(12): 5248-5255.
14. Shida J I, Jingushi S, Izumi T. Basic fibroblast growth factor regulates expression of growth factors in rat epiphyseal chondrocytes. J-Orthop-Res. 2001 Mar; 19(2): 259-264.
15．周京旭，杨希山，周殿元．肿瘤，1998，71-75．．
16. Chen C. J Biol Chem, 1997, 272: 12862.
17. Glansbeek HL. Cytokine, 1997, 9: 347.
18．苏佳灿，张春长，任可。转化生长因子与骨折修复研究进展〔J〕．现代康复，2001，12(4)：121-123．
19. Jiao Y, Wang D, Han WL, et al. Effects of various growth factors on human mandibular condylar cartilage cell proliferation Zhonghua-Kou-Qiang-Yi-Xue-Za-Zhi. 2000 Sep; 35(5): 346-349.
20. Raj DA, Booker TS, Beleastro AN. Striated muscle calcium-stimulated cysteine protease (calpain-like) activity promotes myeloperoxidase activity with exercise. Pflugers Arch, 1998,435: 804-809.
21．林文驶主编．运动生物化学．北京：人民体育出版社，1999：100-101．
22．林文弢主编．运动生物化学．北京：人民体育出版社，1999：137．
23. Daniel A. Raj. Timothy S.Booker Angelo N.BelCastro. Striated muscle Calcium-stimulated eysteine protease (Calpain-like) activity promotes myeloperoxidase activity with exercise. Pflügers Areh-Eur J Physiol. 1997; 435: 804-809.
24. Jyrki Komulainen. Timo E. S. Takala. Harm Kuipers Matthijs K. C. Hesselink. The disruption of myofibre structures in rat skeletal muscle after forced lengthening contractions. Pflügers Arch-Eur J Physiol. 1998; 436: 735-741.
25．程时主编．生物膜与医学．北京：北京医科大学出版社，2000．11．
26．罗吉伟，余斌，覃承诃．反复力竭运动后大鼠骨骼肌线粒体超微结构 改变及维生素E的保护作用．第一军医大学学报，2003，12：1326—1330．
27. Grounds MD. Towards understanding skeletal muscle regeneration. Path Res Pratt, 1991, 187: 1-22.
28. Allbrook D. Skeletal muscle regeneration. Muscle Nerve, 1981, 4: 234.
29. Hurme T, Kalimo H. Activation of myogenic precursor cells after muscle injury. Med Sci Sports Exere, 1992, 24: 197-205.
30. Robertson TA, Padadimitriou JM, Grounds MD, et al. Fusion of myogenic cells to the newly sealed region of damaged myofibers in skeletal muscle regeneration. Neuropathol Appl Neurobiol, 1993, 19: 350-358.
31. Hurme T, Kalimo H, Lehto M, et al. Healing of skeletal muscle injury: an ultrastruetural and immunohistochemical study. Med Sci Sports Exere, 1991, 23: 801-810.
32. Russell B, Dix DJ, Hailer DL, et al. Repair of injured skeletal musele: a molecular approach. Med Sci Sports Exert,1992, 24: 189.
33．李云霞，陈世益．胰岛素样生长因子与骨骼肌再生．中华创伤杂志，2001，17(11)：701—703．
34．李云霞，陈世益，马昕，等．骨骼肌损伤修复过程中组织胰岛素样生长因子的表达．中国运动医学杂志，2001，20：171—173．
35. Magri KA, Benedict MR, Ewton DZ, et al. Negative feedback regulation of insulin-like growth factor-Ⅱ gene expression in differentiating myoblas in vitro. Endoerinol, 1994, 135: 53-62.
36. Ewton DZ, Roof SL,Magri KA, et al. IGF-Ⅱ is more active than IGF-Ⅰ in stimulating L6A1 myogenesis: Greater mitogenic action of
IGF-Ⅰ delay differentiation. J Cell Physiol, 1994, 16: 277-284.
37. Florini JR, Magri KA. Effects of growth factors on myogenic differentiation. Am J Physoil, 1989, 256: C701-C711.
38. Levinovitz A, Jennische E, Oldfors A, et al. Activation of insulin-like growth factor-Ⅱ expression during skeletal muscle regeneration in the rat: correlation with myotube formation. Mol Endocrinol, 1992, 6: 1227-1234.
39. Engert DC, Berglund EB, Rosenthal N. Proliferation precedes differentiation in IGF-Ⅰ-stimulated myogenesis. J Cell Biology, 1996, 135: 431-440.
40. Lo HC, Ney DM. GH and IGF-Ⅰ differentially increase protein synthesis in skeletal muscle and jejunum of parenterally fed rats. Am J Physiol, 1996, 271(Endoerinol Metab 34): E872-E878.
41. Zdanowicz MM, Moyse J, Wingertzahn MA, et al. Effects of insulin-like growth factor Ⅰ in murine muscular dystrophy. Endocrinol, 1995, 136: 4880-4886.
42. Svanberg E, Zachrisson H, Ohlsson C,et al. Role of insulin and IGF-Ⅰ in activation of muscle protein synthesis after oral feeding. Am J Physiol, 1996, 270(Endocrinol Metab 33): E614-E620.
43. Adams GR, Haddad F. Insulin-like growth factor Ⅰ(IGF-Ⅰ) peptide levels have been shown to increase in overloaded skeletal muscles. J Appl Physiol, 1996, 81: 2509-2516.
44. Adams GR, Mccue SA. Localized infusion of IGF-Ⅰ results in skeletal muscle hypertrophy in rats. J Appl Physiol, 1998, 84: 1716-1722.
45. Menetrey J, Kasemkilwattana C, Day CS, et al. Growth factors improve muscle healing in vivo. J Bone Joint Surg(Br), 2000, 82: 131-137.
2. Daughaday WH, Rotwein P. Insulin-like growth factors 1 and 2: Peptide, messenger ribonucleic acid and gene structure, serum and tissue concerntration.Endo Rev 1989, 10(1): 68-91.
3. Ferrari G. Cusella D, Angelis G. Coletta,-M; Muscle regeneration by bone marrow-derived myogenic progenitors. J-Cell-Biol. 1999 Nov 15, 147(4): 869-878.
4. Allen DL, Linderman JK, Roy RR, et al. Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweghting. Am J physiol, 1997, 273: c579-587.
5. Svanberg E, Zachrisson H, Ohlsson C, et al. Role of insulin and IGF-Ⅰ in activation of muscle protein synthesis after oral feeding. Am J Physiol, 1996, 81: 2509-2516.
6. Adams GR, Haddad E The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy J-Appl-Physiol. 1996 Dec; 81 (6): 2509-2516.
7. Adams GR, Mccue SA. Localized infusion of IGF-1 results in skeletal muscle hypertrophy in rats. J Appl Physiol, 1998, 84: 1716-1722.
8. Levinovitz A, Jennisehe E, Oldfors A. Activation of insulin-like growth factor Ⅱ expression during skeletal muscle regeneration in the rot: correlation with myotube formation. Mol-Endoerinol, 1992 Aug; 6(8): 1227-1234.
9. Stewart CE, Rotwein P. Insulin-like growth factor-Ⅱ is an autocrine survival factor for differentiating myoblasts. J Biol Chem, 1996, 271: 11330-11338.
10. Florini JR, Magri KA, Ewton DZ, et al. "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-Ⅱ. Cancer-Res. 1991 Nov 1; 51(21):
5997-6000.
11. Kawaguchi H, Nakamura K, Tabata Y. Acceleration of fracture healing in nonhuman primates by fibroblast growth factor-2. J-Clin-Endocrinol-Metab. 2001 Feb; 86(2): 875-880.
12. Raschke M, Kolbeck S, Bail H. Homologous growth hormone accelerates healing of segmental bone defects. Bone. 2001 Oct; 29(4): 368-373.
13. Franchimont N, Crangji V, Durant D. Interleukin-6 with its soluble receptor enhances the expression of insulin-like growth factor-Ⅰ in osteoblasts. Endocrinology. 1997 Dec; 138(12): 5248-5255.
14. Shida J I, Jingushi S, Izumi T. Basic fibroblast growth factor regulates expression of growth factors in rat epiphyseal chondrocytes. J-Orthop-Res. 2001 Mar; 19(2): 259-264.
15．周京旭，杨希山，周殿元．肿瘤，1998，71-75．．
16. Chen C. J Biol Chem, 1997, 272: 12862.
17. Glansbeek HL. Cytokine, 1997, 9: 347.
18．苏佳灿，张春长，任可。转化生长因子与骨折修复研究进展〔J〕．现代康复，2001，12(4)：121-123．
19. Jiao Y, Wang D, Han WL, et al. Effects of various growth factors on human mandibular condylar cartilage cell proliferation Zhonghua-Kou-Qiang-Yi-Xue-Za-Zhi. 2000 Sep; 35(5): 346-349.
20. Raj DA, Booker TS, Beleastro AN. Striated muscle calcium-stimulated cysteine protease (calpain-like) activity promotes myeloperoxidase activity with exercise. Pflugers Arch, 1998,435: 804-809.
21．林文驶主编．运动生物化学．北京：人民体育出版社，1999：100-101．
22．林文弢主编．运动生物化学．北京：人民体育出版社，1999：137．
23. Daniel A. Raj. Timothy S.Booker Angelo N.BelCastro. Striated muscle Calcium-stimulated eysteine protease (Calpain-like) activity promotes myeloperoxidase activity with exercise. Pflügers Areh-Eur J Physiol. 1997; 435: 804-809.
24. Jyrki Komulainen. Timo E. S. Takala. Harm Kuipers Matthijs K. C. Hesselink. The disruption of myofibre structures in rat skeletal muscle after forced lengthening contractions. Pflügers Arch-Eur J Physiol. 1998; 436: 735-741.
25．程时主编．生物膜与医学．北京：北京医科大学出版社，2000．11．
26．罗吉伟，余斌，覃承诃．反复力竭运动后大鼠骨骼肌线粒体超微结构 改变及维生素E的保护作用．第一军医大学学报，2003，12：1326—1330．
27. Grounds MD. Towards understanding skeletal muscle regeneration. Path Res Pratt, 1991, 187: 1-22.
28. Allbrook D. Skeletal muscle regeneration. Muscle Nerve, 1981, 4: 234.
29. Hurme T, Kalimo H. Activation of myogenic precursor cells after muscle injury. Med Sci Sports Exere, 1992, 24: 197-205.
30. Robertson TA, Padadimitriou JM, Grounds MD, et al. Fusion of myogenic cells to the newly sealed region of damaged myofibers in skeletal muscle regeneration. Neuropathol Appl Neurobiol, 1993, 19: 350-358.
31. Hurme T, Kalimo H, Lehto M, et al. Healing of skeletal muscle injury: an ultrastruetural and immunohistochemical study. Med Sci Sports Exere, 1991, 23: 801-810.
32. Russell B, Dix DJ, Hailer DL, et al. Repair of injured skeletal musele: a molecular approach. Med Sci Sports Exert,1992, 24: 189.
33．李云霞，陈世益．胰岛素样生长因子与骨骼肌再生．中华创伤杂志，2001，17(11)：701—703．
34．李云霞，陈世益，马昕，等．骨骼肌损伤修复过程中组织胰岛素样生长因子的表达．中国运动医学杂志，2001，20：171—173．
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