舌下神經植入組織工程骨骼肌的體內實驗研究_《中國修復重建外科雜志》

作者：

唐休發 ¹ , 張富貴 ² , 馮揚 ² , 周偉 ² , 劉濟遠 ² , 華成舸 ¹

四川大學華西口腔醫院（成都，610041）1頭頸腫瘤外科，2口腔疾病研究國家重點實驗室;

關鍵詞：

組織工程骨骼肌膠質源性神經營養因子去細胞膠原海綿舌下神經成肌細胞大鼠

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【摘要】目的 構建高表達膠質源性神經營養因子（glial cell derived neurotrophic factor，GDNF）的成肌細胞（myoblast，Mb）并作為種子細胞，以去細胞膠原海綿為支架，體內構建組織工程骨骼肌，觀察構建組織能否與舌下神經斷端發生連接。方法取7只2日齡雄性Lewis大鼠四肢肌肉體外分離培養Mb，采用攜帶GDNF基因的重組腺病毒Ad-GDNF轉染第3代Mb（MbGDNF）。將Mb及MbGDNF與去細胞膠原海綿支架體外復合培養構建細胞支架復合物，24 h后掃描電鏡觀察細胞黏附情況。取8周齡雌性Lewis大鼠54只，解剖分離舌下神經，取1.0～1.5 cm舌下神經離斷其遠心端，用Mb支架復合物（Mb組，n=27）和MbGDNF支架復合物（MbGDNF組，n=27）包裹并固定其近心端，術后1、6及12周分別行HE染色，myogenin、slow skeletal myosin及乙酰膽堿受體α1（actylcholine receptor α1，AchRα1）免疫組織化學染色檢測植入物生長情況，并行霍亂毒素B標記的過氧化物酶逆行示蹤染色檢測損傷后舌下神經核運動神經元存活情況。結果構建的MbGDNF能高表達轉染基因。Mb及MbGDNF在支架上黏附和生長狀態良好。HE染色：術后12周，兩組植入物與周圍組織緊密連接，有新生肌纖維從周圍正常肌組織長入，與正常組織分界漸不明顯。免疫組織化學染色：術后1、6及12周均可檢測到細胞質呈myogenin及slow skeletal myosin陽性的肌源性細胞，以及AchRα1呈彌散性陽性細胞。Y染色體染色陽性細胞隨時間延長而減少。術后1、6及12周，MbGDNF組陽性標記的神經元數量分別為261.0 ± 6.6、227.3 ± 8.5及173.3 ± 9.1；Mb組分別為234.7 ± 5.5、196.0 ± 13.5及166.7 ± 11.7；術后1、6周MbGDNF組神經元數量多于Mb組（P lt; 0.05），術后12周兩組差異無統計學意義（P gt; 0.05）。結論構建的組織工程骨骼肌與舌下神經斷端發生了直接物質聯系，MbGDNF產生的重組GDNF能通過逆行運輸方式保護損傷后舌下神經核團內運動神經元的存活。

引用本文： 唐休發 ,張富貴,馮揚,周偉,劉濟遠,華成舸. 舌下神經植入組織工程骨骼肌的體內實驗研究. 中國修復重建外科雜志, 2012, 26(3): 359-364. doi: 復制

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2.	Slaughter K, Li H, Sokoloff AJ. Neuromuscular organization of the superior longitudinalis muscle in the human tongue. Cells Tissues Organs, 2005, 181(1): 51-64.
3.	Pfrieger FW. Roles of glial cells in synapse development. Cell Mol Life Sci, 2009, 66(13): 2037-2047.
4.	Witzemann V. Development of the neuromuscular junction. Cell Tissue Res, 2006, 326(2): 263-271.
5.	唐休發, 溫玉明, 王大章. 舌下神經頦舌肌蒂植入失神經胸大肌瓣的實驗研究. 實用口腔醫學雜志, 2001, 17(5): 377-380.
6.	Ohishi M, Harada F, Rahman F, et al. GDNF expression in terminal schwann cells associated with the periodontal ruffini endings of the rat incisors during nerve regeneration. Anat Rec (Hoboken), 2009, 292(8): 1185-1191.
7.	Zhang L, Ma Z, Smith GM, et al. GDNF-enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons. Glia, 2009, 57(11): 1178-1191.
8.	Schmidt CE, Leach JB. Neural tissue engineering: Strategies for repair and regeneration. Annu Rev Biomed Eng, 2003, 5: 293-347.
9.	Wang H, Hughes I, Planer W, et al. The timing and location of glial cell line-derived neurotrophic factor expression determine enteric nervous system structure and function. J Neurosci, 2010, 30(4): 1523-1538.
10.	蔡煒嵩, 趙紅宇, 袁正偉. 膠質細胞源性神經營養因子對運動神經元發育及運動神經元性疾病的作用. 中國修復重建外科雜志, 2005, 19(6): 490-493.
11.	Li W, Brakefield D, Pan Y, et al. Muscle-derived but not centrally derived transgene GDNF is neuroprotective in G93A-SOD1 mouse model of ALS. Exp Neurol, 2007, 203(2): 457-471.
12.	Henderson CE, Phillips HS, Pollock RA, et al. GDNF: a potent survival factor for motoneurons present in periphery nerve and muscle. Science, 1994, 266(5187): 1062-1064.
13.	李軍, 張洹, 劉革修. 小鼠胎盤組織細胞移植的抗衰老作用研究. 生物醫學工程學雜志, 2010, 27(6): 1312-1316.
14.	Paxinos G, Waston C. 大鼠腦立體定位圖譜. 諸葛啟釧, 譯. 3版. 北京: 人民衛生出版社, 2005: 1-26.
15.	趙碩, 張彩順. 辣根過氧化物酶逆行示蹤評價無細胞神經移植物復合骨髓間充質干細胞對坐骨神經缺損大鼠運動神經元的作用. 中國組織工程研究與臨床康復, 2010, 14(19): 3495-3498.
16.	張斌, 黃富國. 骨骼肌成肌細胞移植研究. 中國修復重建外科雜志, 2006, 20(8): 849-853.
17.	Macfelda K, Kapeller B, Wilbacher I, et al. Behavior of cardiomyocytes and skeletal muscle cells on different extracellular matrix components—relevance for cardiac tissue engineering. Artif Organs, 2007, 31(1): 4-12.
18.	Toba T, Nakamura T, Shimizu Y, et al. Regeneration of canine peroneal nerve with the use of a polyglycolic acid-collagen tube filled with laminin-soaked collagen sponge: a comparative study of collagen sponge and collagen fibers as filling materials for nerve conduits. J Biomed Mater Res, 2001, 58(6): 622-630.
19.	徐林海, 焦向陽, 季正倫. 以膠原海綿為載體培養的人表皮細胞移植. 中國修復重建外科雜志, 2001, 15(2): 118-121.
20.	Kroehne V, Heschel I, Schugner F, et al. Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts. J Cell Mol Med, 2008, 12(5A): 1640-1648.
21.	Beier JP, Klumpp D, Rudisile M, et al. Collagen matrices from sponge to nano: new perspectives for tissue engineering of skeletal muscle. BMC Biotechnol, 2009, 9: 34.
22.	岑石強, 楊志明. 成肌細胞在基因治療和組織工程中的應用綜述. 中國修復重建外科雜志, 1999, 13(3): 173-177.
23.	Rich KM, Luszczynski JR, Osborne PA, et al. Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury. J Neurocyctol, 1987, 16(2): 261-268.
24.	Paratcha G, Ledda F. GDNF and GFRα: a versatile molecular complex for developing neurons. Trend Neurosci, 2008, 31(8): 384-391.

1. Kimata Y, Sakuraba M, Namba Y, et al. Functional reconstruction with free flaps following ablation of oropharyngeal cancer. Int J Clin Oncal, 2005, 10(4): 229-233.
2. Slaughter K, Li H, Sokoloff AJ. Neuromuscular organization of the superior longitudinalis muscle in the human tongue. Cells Tissues Organs, 2005, 181(1): 51-64.
3. Pfrieger FW. Roles of glial cells in synapse development. Cell Mol Life Sci, 2009, 66(13): 2037-2047.
4. Witzemann V. Development of the neuromuscular junction. Cell Tissue Res, 2006, 326(2): 263-271.
5. 唐休發, 溫玉明, 王大章. 舌下神經頦舌肌蒂植入失神經胸大肌瓣的實驗研究. 實用口腔醫學雜志, 2001, 17(5): 377-380.
6. Ohishi M, Harada F, Rahman F, et al. GDNF expression in terminal schwann cells associated with the periodontal ruffini endings of the rat incisors during nerve regeneration. Anat Rec (Hoboken), 2009, 292(8): 1185-1191.
7. Zhang L, Ma Z, Smith GM, et al. GDNF-enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons. Glia, 2009, 57(11): 1178-1191.
8. Schmidt CE, Leach JB. Neural tissue engineering: Strategies for repair and regeneration. Annu Rev Biomed Eng, 2003, 5: 293-347.
9. Wang H, Hughes I, Planer W, et al. The timing and location of glial cell line-derived neurotrophic factor expression determine enteric nervous system structure and function. J Neurosci, 2010, 30(4): 1523-1538.
10. 蔡煒嵩, 趙紅宇, 袁正偉. 膠質細胞源性神經營養因子對運動神經元發育及運動神經元性疾病的作用. 中國修復重建外科雜志, 2005, 19(6): 490-493.
11. Li W, Brakefield D, Pan Y, et al. Muscle-derived but not centrally derived transgene GDNF is neuroprotective in G93A-SOD1 mouse model of ALS. Exp Neurol, 2007, 203(2): 457-471.
12. Henderson CE, Phillips HS, Pollock RA, et al. GDNF: a potent survival factor for motoneurons present in periphery nerve and muscle. Science, 1994, 266(5187): 1062-1064.
13. 李軍, 張洹, 劉革修. 小鼠胎盤組織細胞移植的抗衰老作用研究. 生物醫學工程學雜志, 2010, 27(6): 1312-1316.
14. Paxinos G, Waston C. 大鼠腦立體定位圖譜. 諸葛啟釧, 譯. 3版. 北京: 人民衛生出版社, 2005: 1-26.
15. 趙碩, 張彩順. 辣根過氧化物酶逆行示蹤評價無細胞神經移植物復合骨髓間充質干細胞對坐骨神經缺損大鼠運動神經元的作用. 中國組織工程研究與臨床康復, 2010, 14(19): 3495-3498.
16. 張斌, 黃富國. 骨骼肌成肌細胞移植研究. 中國修復重建外科雜志, 2006, 20(8): 849-853.
17. Macfelda K, Kapeller B, Wilbacher I, et al. Behavior of cardiomyocytes and skeletal muscle cells on different extracellular matrix components—relevance for cardiac tissue engineering. Artif Organs, 2007, 31(1): 4-12.
18. Toba T, Nakamura T, Shimizu Y, et al. Regeneration of canine peroneal nerve with the use of a polyglycolic acid-collagen tube filled with laminin-soaked collagen sponge: a comparative study of collagen sponge and collagen fibers as filling materials for nerve conduits. J Biomed Mater Res, 2001, 58(6): 622-630.
19. 徐林海, 焦向陽, 季正倫. 以膠原海綿為載體培養的人表皮細胞移植. 中國修復重建外科雜志, 2001, 15(2): 118-121.
20. Kroehne V, Heschel I, Schugner F, et al. Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts. J Cell Mol Med, 2008, 12(5A): 1640-1648.
21. Beier JP, Klumpp D, Rudisile M, et al. Collagen matrices from sponge to nano: new perspectives for tissue engineering of skeletal muscle. BMC Biotechnol, 2009, 9: 34.
22. 岑石強, 楊志明. 成肌細胞在基因治療和組織工程中的應用綜述. 中國修復重建外科雜志, 1999, 13(3): 173-177.
23. Rich KM, Luszczynski JR, Osborne PA, et al. Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury. J Neurocyctol, 1987, 16(2): 261-268.
24. Paratcha G, Ledda F. GDNF and GFRα: a versatile molecular complex for developing neurons. Trend Neurosci, 2008, 31(8): 384-391.

《中國修復重建外科雜志》

舌下神經植入組織工程骨骼肌的體內實驗研究

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《中國修復重建外科雜志》

舌下神經植入組織工程骨骼肌的體內實驗研究

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