• 四川大學(xué)華西口腔醫(yī)院（成都，610041）1頭頸腫瘤外科，2口腔疾病研究國(guó)家重點(diǎn)實(shí)驗(yàn)室;

【摘 要】 目的  構(gòu)建高表達(dá)膠質(zhì)源性神經(jīng)營(yíng)養(yǎng)因子（glial cell derived neurotrophic factor，GDNF）的成肌細(xì)胞（myoblast，Mb）并作為種子細(xì)胞，以去細(xì)胞膠原海綿為支架，體內(nèi)構(gòu)建組織工程骨骼肌，觀察構(gòu)建組織能否與舌下神經(jīng)斷端發(fā)生連接。 方法  取7只2日齡雄性L(fǎng)ewis大鼠四肢肌肉體外分離培養(yǎng)Mb，采用攜帶GDNF基因的重組腺病毒Ad-GDNF轉(zhuǎn)染第3代Mb（MbGDNF）。將Mb及MbGDNF與去細(xì)胞膠原海綿支架體外復(fù)合培養(yǎng)構(gòu)建細(xì)胞支架復(fù)合物，24 h后掃描電鏡觀察細(xì)胞黏附情況。取8周齡雌性L(fǎng)ewis大鼠54只，解剖分離舌下神經(jīng)，取1.0～1.5 cm舌下神經(jīng)離斷其遠(yuǎn)心端，用Mb支架復(fù)合物（Mb組，n=27）和MbGDNF支架復(fù)合物（MbGDNF組，n=27）包裹并固定其近心端，術(shù)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(xué)染色檢測(cè)植入物生長(zhǎng)情況，并行霍亂毒素B標(biāo)記的過(guò)氧化物酶逆行示蹤染色檢測(cè)損傷后舌下神經(jīng)核運(yùn)動(dòng)神經(jīng)元存活情況。 結(jié)果 構(gòu)建的MbGDNF能高表達(dá)轉(zhuǎn)染基因。Mb及MbGDNF在支架上黏附和生長(zhǎng)狀態(tài)良好。HE染色：術(shù)后12周，兩組植入物與周?chē)M織緊密連接，有新生肌纖維從周?chē)＜〗M織長(zhǎng)入，與正常組織分界漸不明顯。免疫組織化學(xué)染色：術(shù)后1、6及12周均可檢測(cè)到細(xì)胞質(zhì)呈myogenin及slow skeletal myosin陽(yáng)性的肌源性細(xì)胞，以及AchRα1呈彌散性陽(yáng)性細(xì)胞。Y染色體染色陽(yáng)性細(xì)胞隨時(shí)間延長(zhǎng)而減少。術(shù)后1、6及12周，MbGDNF組陽(yáng)性標(biāo)記的神經(jīng)元數(shù)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(shù)后1、6周MbGDNF組神經(jīng)元數(shù)量多于Mb組（P  lt; 0.05），術(shù)后12周兩組差異無(wú)統(tǒng)計(jì)學(xué)意義（P  gt; 0.05）。 結(jié)論  構(gòu)建的組織工程骨骼肌與舌下神經(jīng)斷端發(fā)生了直接物質(zhì)聯(lián)系，MbGDNF產(chǎn)生的重組GDNF能通過(guò)逆行運(yùn)輸方式保護(hù)損傷后舌下神經(jīng)核團(tuán)內(nèi)運(yùn)動(dòng)神經(jīng)元的存活。

引用本文： 唐休發(fā) ,張富貴,馮揚(yáng),周偉,劉濟(jì)遠(yuǎn),華成舸. 舌下神經(jīng)植入組織工程骨骼肌的體內(nèi)實(shí)驗(yàn)研究. 中國(guó)修復(fù)重建外科雜志, 2012, 26(3): 359-364. doi: 復(fù)制

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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(fā), 溫玉明, 王大章. 舌下神經(jīng)頦舌肌蒂植入失神經(jīng)胸大肌瓣的實(shí)驗(yàn)研究. 實(shí)用口腔醫(yī)學(xué)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(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子對(duì)運(yùn)動(dòng)神經(jīng)元發(fā)育及運(yùn)動(dòng)神經(jīng)元性疾病的作用. 中國(guó)修復(fù)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(pán)組織細(xì)胞移植的抗衰老作用研究. 生物醫(yī)學(xué)工程學(xué)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(wèi)生出版社, 2005: 1-26.
15. 趙碩, 張彩順. 辣根過(guò)氧化物酶逆行示蹤評(píng)價(jià)無(wú)細(xì)胞神經(jīng)移植物復(fù)合骨髓間充質(zhì)干細(xì)胞對(duì)坐骨神經(jīng)缺損大鼠運(yùn)動(dòng)神經(jīng)元的作用. 中國(guó)組織工程研究與臨床康復(fù), 2010, 14(19): 3495-3498.
16. 張斌, 黃富國(guó). 骨骼肌成肌細(xì)胞移植研究. 中國(guó)修復(fù)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(yáng), 季正倫. 以膠原海綿為載體培養(yǎng)的人表皮細(xì)胞移植. 中國(guó)修復(fù)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(qiáng), 楊志明. 成肌細(xì)胞在基因治療和組織工程中的應(yīng)用綜述. 中國(guó)修復(fù)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. 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. 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. 3. Pfrieger FW. Roles of glial cells in synapse development. Cell Mol Life Sci, 2009, 66(13): 2037-2047.
  4. 4. Witzemann V. Development of the neuromuscular junction. Cell Tissue Res, 2006, 326(2): 263-271.
  5. 5. 唐休發(fā), 溫玉明, 王大章. 舌下神經(jīng)頦舌肌蒂植入失神經(jīng)胸大肌瓣的實(shí)驗(yàn)研究. 實(shí)用口腔醫(yī)學(xué)雜志, 2001, 17(5): 377-380.
  6. 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. 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. 8. Schmidt CE, Leach JB. Neural tissue engineering: Strategies for repair and regeneration. Annu Rev Biomed Eng, 2003, 5: 293-347.
  9. 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. 10. 蔡煒嵩, 趙紅宇, 袁正偉. 膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子對(duì)運(yùn)動(dòng)神經(jīng)元發(fā)育及運(yùn)動(dòng)神經(jīng)元性疾病的作用. 中國(guó)修復(fù)重建外科雜志, 2005, 19(6): 490-493.
  11. 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. 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. 13. 李軍, 張洹, 劉革修. 小鼠胎盤(pán)組織細(xì)胞移植的抗衰老作用研究. 生物醫(yī)學(xué)工程學(xué)雜志, 2010, 27(6): 1312-1316.
  14. 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(wèi)生出版社, 2005: 1-26.
  15. 15. 趙碩, 張彩順. 辣根過(guò)氧化物酶逆行示蹤評(píng)價(jià)無(wú)細(xì)胞神經(jīng)移植物復(fù)合骨髓間充質(zhì)干細(xì)胞對(duì)坐骨神經(jīng)缺損大鼠運(yùn)動(dòng)神經(jīng)元的作用. 中國(guó)組織工程研究與臨床康復(fù), 2010, 14(19): 3495-3498.
  16. 16. 張斌, 黃富國(guó). 骨骼肌成肌細(xì)胞移植研究. 中國(guó)修復(fù)重建外科雜志, 2006, 20(8): 849-853.
  17. 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. 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. 19. 徐林海, 焦向陽(yáng), 季正倫. 以膠原海綿為載體培養(yǎng)的人表皮細(xì)胞移植. 中國(guó)修復(fù)重建外科雜志, 2001, 15(2): 118-121.
  20. 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. 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. 22. 岑石強(qiáng), 楊志明. 成肌細(xì)胞在基因治療和組織工程中的應(yīng)用綜述. 中國(guó)修復(fù)重建外科雜志, 1999, 13(3): 173-177.
  23. 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. 24. Paratcha G, Ledda F. GDNF and GFRα: a versatile molecular complex for developing neurons. Trend Neurosci, 2008, 31(8): 384-391.