转化生长因子β1对小鼠失神经骨骼肌肌源性干细胞内结缔组织生长因子合成的影响
摘要
转化生长因子-β(TGF-β)是一种多功能细胞因子。近年发现,TGF-β在致组织和器官纤维化中起着重要作用,其致纤维化作用和调控机制已成为当前研究热点。肌源性干细胞(Muscle-derived Stem Cells, MDSC)是从骨骼肌组织中分离出的一类新型干细胞,表达造血干细胞的表型Sca-1和CD34,能向多种组织分化,自我更新能力强,在细胞治疗和基因工程研究中其表现明显优于肌卫星细胞。
本文旨在探讨转化生长因子β1(TGF-β1)对失神经骨骼肌肌源性干细胞内生成结缔组织生长因子(CTGF)的影响,为今后进一步探讨TGFβ1- CTGF通路在致失神经骨骼肌纤维化的作用及机制打下基础。
本实验分为两部分
1.失神经小鼠肌源性干细胞培养与鉴定:采用双酶(胶原酶XI和胰蛋白酶)消化法消化分离小鼠失神经骨骼肌组织,用差速贴壁法纯化,成功地获得原代肌源性干细胞,进行免疫组化染色证实为Sca-1+/CD34+细胞,然后在高血清条件下进行传代增殖,了解其细胞生物学特性。
2.将获得的肌源性干细胞,传代以便获得数量足够的细胞,然后在细胞培养基中加入TGF-β1,观察不同浓度培养相同时间和相同浓度培养不同时间下TGF-β1作用于体外培养小鼠失神经骨骼肌肌源性干细胞后,细胞内CTGF生成的变化。
实验结果表明:失神经小鼠MDSC能够在TGF-β1刺激下分泌CTGF,在10μg/L浓度范围内存在明显的剂量依赖关系,在3～12h时间范围内呈时间依赖关系。说明TGF-β1-CTGF通路可能通过对肌源性干细胞,也就是干细胞水平影响、参与了失神经骨骼肌组织纤维化进程。
The TGF-βis a factor of multiple functions. It is founded that TGF-βhad act key roles in the course of the fibrosis in the organs and tissue. So, the mechanism of the fibrosis caused by TGF-βis a popular issue in our research.
Muscle-derived stem cells are a new stem cell found in skeletal muscles, and express similar surface antigens, as observed in hematopoietic stem cells, Sca-1 and CD34. MDSC can differentiate to variant tissues and posses the strong power in self-renewal. In the study of cell therapy and gene engineering MDSC is better than skeletal muscle satellite cell.
The aim is to research the The influences of TGF-β1 on the denervated mouse MDSCs’producting the CTGF in vitro, and make the found for our following research in the mechanism of TGFβ1- CTGF pathway in the denervated muscular fibrosis. The experiment is composed of two parts
1. the identification of the cultured MDSC: we digested skeletal muscle tissues from new born mice using the dual-enzyme (collagenase XI and trypsin) digestion method, purified the cells by preplate technique and successfully cultivated muscle-derived stem cells in vitro. Immunohisochemistry staining showed that the muscle-derived stem cells expressed Sca-1 and CD34. Then the muscle-derived stem cells were cultivated and passaged in DMEM including 20% FBS. We observed their biological characteristic.
2.We obtain and proliferate the MDSC so that we have enough cells for research. The TGF-β1 was added into the DMEM including 20% FBS. We observed the variation of the CTGF’s production in MDSC treated with DMEM TGF-β1.
The results showed that: the MDSC from denervated mice can be helpful to the production of the CTGF in MDSC. The dose-dependent effect exist between the groups from 1ng/ml to 10ng/ml ,the time dependency exist between the groups from 3h to 12h.It is showed that the TGF-β1-CTGF pathway could act as an key role in the denervated muscular fibrosis by the influence to the MDSC.
本文旨在探讨转化生长因子β1(TGF-β1)对失神经骨骼肌肌源性干细胞内生成结缔组织生长因子(CTGF)的影响,为今后进一步探讨TGFβ1- CTGF通路在致失神经骨骼肌纤维化的作用及机制打下基础。
本实验分为两部分
1.失神经小鼠肌源性干细胞培养与鉴定:采用双酶(胶原酶XI和胰蛋白酶)消化法消化分离小鼠失神经骨骼肌组织,用差速贴壁法纯化,成功地获得原代肌源性干细胞,进行免疫组化染色证实为Sca-1+/CD34+细胞,然后在高血清条件下进行传代增殖,了解其细胞生物学特性。
2.将获得的肌源性干细胞,传代以便获得数量足够的细胞,然后在细胞培养基中加入TGF-β1,观察不同浓度培养相同时间和相同浓度培养不同时间下TGF-β1作用于体外培养小鼠失神经骨骼肌肌源性干细胞后,细胞内CTGF生成的变化。
实验结果表明:失神经小鼠MDSC能够在TGF-β1刺激下分泌CTGF,在10μg/L浓度范围内存在明显的剂量依赖关系,在3～12h时间范围内呈时间依赖关系。说明TGF-β1-CTGF通路可能通过对肌源性干细胞,也就是干细胞水平影响、参与了失神经骨骼肌组织纤维化进程。
The TGF-βis a factor of multiple functions. It is founded that TGF-βhad act key roles in the course of the fibrosis in the organs and tissue. So, the mechanism of the fibrosis caused by TGF-βis a popular issue in our research.
Muscle-derived stem cells are a new stem cell found in skeletal muscles, and express similar surface antigens, as observed in hematopoietic stem cells, Sca-1 and CD34. MDSC can differentiate to variant tissues and posses the strong power in self-renewal. In the study of cell therapy and gene engineering MDSC is better than skeletal muscle satellite cell.
The aim is to research the The influences of TGF-β1 on the denervated mouse MDSCs’producting the CTGF in vitro, and make the found for our following research in the mechanism of TGFβ1- CTGF pathway in the denervated muscular fibrosis. The experiment is composed of two parts
1. the identification of the cultured MDSC: we digested skeletal muscle tissues from new born mice using the dual-enzyme (collagenase XI and trypsin) digestion method, purified the cells by preplate technique and successfully cultivated muscle-derived stem cells in vitro. Immunohisochemistry staining showed that the muscle-derived stem cells expressed Sca-1 and CD34. Then the muscle-derived stem cells were cultivated and passaged in DMEM including 20% FBS. We observed their biological characteristic.
2.We obtain and proliferate the MDSC so that we have enough cells for research. The TGF-β1 was added into the DMEM including 20% FBS. We observed the variation of the CTGF’s production in MDSC treated with DMEM TGF-β1.
The results showed that: the MDSC from denervated mice can be helpful to the production of the CTGF in MDSC. The dose-dependent effect exist between the groups from 1ng/ml to 10ng/ml ,the time dependency exist between the groups from 3h to 12h.It is showed that the TGF-β1-CTGF pathway could act as an key role in the denervated muscular fibrosis by the influence to the MDSC.
引文
1. Lee J, Qu-Petersen B, Cao S, et al. Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J Cell Biol, 2000, 150:1085-1100.
2. Huard J, Cao B, Zhuqing QP. Muscle-derived stem cells : potential for muscle regeneration. Birth Defects Des, 2003, 69: 230-237.
3. Torrente Y, Tremblay JP, Pisati F, et al. Intraarterial injection of muscle-derived CD343(+)Scal-1(+)stem cell restores dystrophin in mdx mice. J Cell Biol, 2001, 152: 335-348.
4. Mina R, Shi YY, et al. Purinoceptor expression in regenerating skeletal muscle in the mdx mouse model of muscular dystrophy and in satellite cell cultures. FASEB J. 2004.18(2):1404-1406.
5. Gussoni E, Soneoka Y, Strickland CD, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature, 1999, 23, 401:390-294.
6. Seale P, Rudnicki MA. A new look at the origin, function, and“stem cell”status of muscle satellite cells. Dev Biol, 2000,218:115-124.
7. Baroffio A, Hamann M, Bernheim L, et al. Identification of self-renewing myoblasts in the progeny of single human muscle satellite cells. Differentiation, 1996, 60: 47-57.
8. Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol, 2002, 157: 851-864.
9. owski RJ, Haluszczak C, Trucco M, Huard J. Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells. Hum Gene Ther, 2001, 12: 619-628.
10. Yotoku M, Grossberg AL, Pressman D. A cell surface antigenic determinant presents on mouse plasmacytes and only half of mouse thymocytes. J Immunol, 1974, 112: 1774-1781.
11. Caryn YI, Carol YJ, Bernstein A, et al. Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A null mice. Blood, 2003,101: 517-523.
12. Tindle RW, Nichol RA, Chan L, et al. A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow. J Leuk Res, 1985, 9: 1-9.
1. Liu Y. Renal fibrosis: new insights into the pathogenesis and therapeutics. Kidney Int 2006;69(2):213-217
2. Gressner AM, Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med 2006; 10(1):76-99
3. Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002;157(5): 851-864
4. Li Y, Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol 2002;161(3):895-907
5. Yamaai T, Nakanishi T, Asano M, et al. Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development.J Bone Miner Metab 2005; 23(4):280-288
6. Li JH, Zhu HJ, Huang XR, et al. Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation. J Am Soc Nephrol 2002; 13(6):1464-1472
7. Heusinger-Ribeiro J, Eberlein M, Wahab NA, et al. Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP.J Am Soc Nephrol 2001; 12(9):1853-1861
8. Utsugi M, Dobashi K, Ishizuka T, et al. C-Jun-NH2-terminal kinase mediates expression of connective tissue growth factor induced by transforming growth factor -beta1 in human lung fibroblasts. Am J Respir Cell Mol Biol 2003; 28(6): 754 -761
9. Xie S, Sukkar MB, Issa R, et al. Regulation of TGF-beta 1-induced connective tissue growth factor expression in airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2005; 288(1):L68-L76
10. Khan R, Sheppard R. Fibrosis in heart disease: understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia.Immunology 2006; 118(1):10-24
11. Okada H, Kikuta T, Kobayashi T, et al. Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis.J Am Soc Nephrol 2005; 16(1):133-143
12. Qi W, Twigg S, Chen X, et al. Integrated actions of transforming growth factor-beta1 and connective tissue growth factor in renal fibrosis. Am J Physiol Renal Physiol 2005: 288(4):F800-F809
13. Dean RG, Balding LC, Candido R, et al. Connective tissue growth factor and cardiac fibrosis after myocardial infarction.J Histochem Cytochem 2005:53(10):1245-1256
14. Watts KL, Cottrell E, Hoban PR,et al. RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis.Respir Res 2006; 7:88
15. Andreetta F , Bernasconi P, Baggi F, et al. Immunomodulation of TGF-beta 1 in mdx mouse inhibits connective tissue proliferation in diaphragm but increases inflammatory response: implications for antifibrotic therapy. J Neuroimmunol 2006; 175(1-2):77-86
16. Foster W, Li Y, Usas A, et al. Gamma interferon as an antifibrosis agent in skeletal muscle. J Orthop Res 2003; 21(5):798-804
17. Chan YS, Li Y, Foster W, et al. Antifibrotic effects of suramin in injured skeletal muscle after laceration.J Appl Physiol 2003; 95(2):771-780
18. Li Y, Foster W, Deasy BM, et al. Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis. Am J Pathol 2004; 164(3):1007-1019
1. Holzer U, Rieck M, Buckner JH. J Autoimmun, 2006; 26(4): 241-251
2. Chae HJ, Ha MS, Yun DH, et al. J Dent Res, 2006; 85(6):515-519
3. Khan R, Sheppard R. Immunology, 2006;118(1):10-24
4. Liu Y. Kidney Int, 2006;69(2):213-217
5. Gressner AM, Weiskirchen R. J Cell Mol Med, 2006;10(1):76-99
6. Yamaai T, Nakanishi T, Asano M, et al. J Bone Miner Metab, 2005; 23(4):280-288
7. Okada H, Kikuta T, Kobayashi T, et al. J Am Soc Nephrol, 2005; 16(1):133-143
8. Qi W, Twigg S, Chen X, et al. Am J Physiol Renal Physiol, 2005; 288(4):F800-F809
9. Dean RG, Balding LC, Candido R, et al. J Histochem Cytochem, 2005; 53(10): 1245-1256
10. Watts KL, Cottrell E, Hoban PR, et al. Respir Res,2006; 7:88
11. Ji C, Eickelberg O, McCarthy TL, et al. Endocrinology, 2001;142(9):3873-3879
12. Li JH, Zhu HJ, Huang XR, et al. J Am Soc Nephrol, 2002; 13(6):1464-1472
13. Heusinger-Ribeiro J, Eberlein M, Wahab NA ,et al. J Am Soc Nephrol, 2001;
12(9):1853-1861
14. Utsugi M, Dobashi K, Ishizuka T, et al. Am J Respir Cell Mol Biol, 2003;28(6): 754 -761
15. Xie S, Sukkar MB,Issa R, et al. Am J Physiol Lung Cell Mol Physiol, 2005;288(1):L68-L76
16. Andreetta F, Bernasconi P, Baggi F, et al. J Neuroimmunol, 2006;175(1-2):77-86
17. Li Y, Foster W, Deasy BM, et al. Am J Pathol, 2004;164(3):1007-1019
18. Li Y, Huard J. Am J Pathol, 2002;161(3):895-907
19. Foster W, Li Y, Usas A, et al. J Orthop Res, 2003;21(5):798-804
20. Chan YS, Li Y, Foster W, et al. J Appl Physiol, 2003;95(2):771-780
21. Obreo J, Diez-Marques L, Lamas S, et al. Cell Physiol Biochem, 2004;14(4-6): 301-310
22. Garrett Q, Khaw PT, Blalock TD, et al. Invest Ophthalmol Vis Sci, 2004;45(4): 1109 -1116
2. Huard J, Cao B, Zhuqing QP. Muscle-derived stem cells : potential for muscle regeneration. Birth Defects Des, 2003, 69: 230-237.
3. Torrente Y, Tremblay JP, Pisati F, et al. Intraarterial injection of muscle-derived CD343(+)Scal-1(+)stem cell restores dystrophin in mdx mice. J Cell Biol, 2001, 152: 335-348.
4. Mina R, Shi YY, et al. Purinoceptor expression in regenerating skeletal muscle in the mdx mouse model of muscular dystrophy and in satellite cell cultures. FASEB J. 2004.18(2):1404-1406.
5. Gussoni E, Soneoka Y, Strickland CD, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature, 1999, 23, 401:390-294.
6. Seale P, Rudnicki MA. A new look at the origin, function, and“stem cell”status of muscle satellite cells. Dev Biol, 2000,218:115-124.
7. Baroffio A, Hamann M, Bernheim L, et al. Identification of self-renewing myoblasts in the progeny of single human muscle satellite cells. Differentiation, 1996, 60: 47-57.
8. Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol, 2002, 157: 851-864.
9. owski RJ, Haluszczak C, Trucco M, Huard J. Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells. Hum Gene Ther, 2001, 12: 619-628.
10. Yotoku M, Grossberg AL, Pressman D. A cell surface antigenic determinant presents on mouse plasmacytes and only half of mouse thymocytes. J Immunol, 1974, 112: 1774-1781.
11. Caryn YI, Carol YJ, Bernstein A, et al. Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A null mice. Blood, 2003,101: 517-523.
12. Tindle RW, Nichol RA, Chan L, et al. A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow. J Leuk Res, 1985, 9: 1-9.
1. Liu Y. Renal fibrosis: new insights into the pathogenesis and therapeutics. Kidney Int 2006;69(2):213-217
2. Gressner AM, Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med 2006; 10(1):76-99
3. Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002;157(5): 851-864
4. Li Y, Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol 2002;161(3):895-907
5. Yamaai T, Nakanishi T, Asano M, et al. Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development.J Bone Miner Metab 2005; 23(4):280-288
6. Li JH, Zhu HJ, Huang XR, et al. Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation. J Am Soc Nephrol 2002; 13(6):1464-1472
7. Heusinger-Ribeiro J, Eberlein M, Wahab NA, et al. Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP.J Am Soc Nephrol 2001; 12(9):1853-1861
8. Utsugi M, Dobashi K, Ishizuka T, et al. C-Jun-NH2-terminal kinase mediates expression of connective tissue growth factor induced by transforming growth factor -beta1 in human lung fibroblasts. Am J Respir Cell Mol Biol 2003; 28(6): 754 -761
9. Xie S, Sukkar MB, Issa R, et al. Regulation of TGF-beta 1-induced connective tissue growth factor expression in airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2005; 288(1):L68-L76
10. Khan R, Sheppard R. Fibrosis in heart disease: understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia.Immunology 2006; 118(1):10-24
11. Okada H, Kikuta T, Kobayashi T, et al. Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis.J Am Soc Nephrol 2005; 16(1):133-143
12. Qi W, Twigg S, Chen X, et al. Integrated actions of transforming growth factor-beta1 and connective tissue growth factor in renal fibrosis. Am J Physiol Renal Physiol 2005: 288(4):F800-F809
13. Dean RG, Balding LC, Candido R, et al. Connective tissue growth factor and cardiac fibrosis after myocardial infarction.J Histochem Cytochem 2005:53(10):1245-1256
14. Watts KL, Cottrell E, Hoban PR,et al. RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis.Respir Res 2006; 7:88
15. Andreetta F , Bernasconi P, Baggi F, et al. Immunomodulation of TGF-beta 1 in mdx mouse inhibits connective tissue proliferation in diaphragm but increases inflammatory response: implications for antifibrotic therapy. J Neuroimmunol 2006; 175(1-2):77-86
16. Foster W, Li Y, Usas A, et al. Gamma interferon as an antifibrosis agent in skeletal muscle. J Orthop Res 2003; 21(5):798-804
17. Chan YS, Li Y, Foster W, et al. Antifibrotic effects of suramin in injured skeletal muscle after laceration.J Appl Physiol 2003; 95(2):771-780
18. Li Y, Foster W, Deasy BM, et al. Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis. Am J Pathol 2004; 164(3):1007-1019
1. Holzer U, Rieck M, Buckner JH. J Autoimmun, 2006; 26(4): 241-251
2. Chae HJ, Ha MS, Yun DH, et al. J Dent Res, 2006; 85(6):515-519
3. Khan R, Sheppard R. Immunology, 2006;118(1):10-24
4. Liu Y. Kidney Int, 2006;69(2):213-217
5. Gressner AM, Weiskirchen R. J Cell Mol Med, 2006;10(1):76-99
6. Yamaai T, Nakanishi T, Asano M, et al. J Bone Miner Metab, 2005; 23(4):280-288
7. Okada H, Kikuta T, Kobayashi T, et al. J Am Soc Nephrol, 2005; 16(1):133-143
8. Qi W, Twigg S, Chen X, et al. Am J Physiol Renal Physiol, 2005; 288(4):F800-F809
9. Dean RG, Balding LC, Candido R, et al. J Histochem Cytochem, 2005; 53(10): 1245-1256
10. Watts KL, Cottrell E, Hoban PR, et al. Respir Res,2006; 7:88
11. Ji C, Eickelberg O, McCarthy TL, et al. Endocrinology, 2001;142(9):3873-3879
12. Li JH, Zhu HJ, Huang XR, et al. J Am Soc Nephrol, 2002; 13(6):1464-1472
13. Heusinger-Ribeiro J, Eberlein M, Wahab NA ,et al. J Am Soc Nephrol, 2001;
12(9):1853-1861
14. Utsugi M, Dobashi K, Ishizuka T, et al. Am J Respir Cell Mol Biol, 2003;28(6): 754 -761
15. Xie S, Sukkar MB,Issa R, et al. Am J Physiol Lung Cell Mol Physiol, 2005;288(1):L68-L76
16. Andreetta F, Bernasconi P, Baggi F, et al. J Neuroimmunol, 2006;175(1-2):77-86
17. Li Y, Foster W, Deasy BM, et al. Am J Pathol, 2004;164(3):1007-1019
18. Li Y, Huard J. Am J Pathol, 2002;161(3):895-907
19. Foster W, Li Y, Usas A, et al. J Orthop Res, 2003;21(5):798-804
20. Chan YS, Li Y, Foster W, et al. J Appl Physiol, 2003;95(2):771-780
21. Obreo J, Diez-Marques L, Lamas S, et al. Cell Physiol Biochem, 2004;14(4-6): 301-310
22. Garrett Q, Khaw PT, Blalock TD, et al. Invest Ophthalmol Vis Sci, 2004;45(4): 1109 -1116