雪旺細胞純化培養及體外復合豬小腸黏膜下層的實驗研究_《中國修復重建外科雜志》

作者：

劉宏銀 , 馮喆 , 柏琳 , 侯江龍 , 羅靜聰 , 楊志明 , 解慧琪

四川大學華西醫院再生醫學研究中心干細胞與組織工程研究室（成都，610041）;

關鍵詞：

組織工程雪旺細胞小腸黏膜下層 NGF 生物相容性神經修復

DOI：

10.7507/1002-1892.20130165

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目的改進雪旺細胞（Schwann cells，SCs）原代培養方法，以獲得大量高純度SCs；研究豬小腸黏膜下層（small intestinal submucosa，SIS）與SCs的生物相容性；通過復合SCs，使SIS負載NGF。方法取2～3日齡SD乳鼠雙側坐骨神經，以胰蛋白酶、Ⅱ型膠原酶分步消化，差速貼壁20 min，G418處理48 h，含2.5%FBS的H-DMEM培養基培養；MTT法檢測SCs增殖情況，抗S-100免疫熒光染色檢測SCs純度。將第3代SCs和SIS復合培養，行HE染色和掃描電鏡觀察兩者復合生長情況，復合培養1、2、3、4、5、7 d ELISA法檢測培養上清中NGF含量。復合培養3、5、7、10、13、15 d后反復凍融脫細胞處理，ELISA法檢測脫細胞后SIS中NGF含量。結果純化后的SCs純度達98%以上；MTT檢測示SCs傳代后3 d進入對數生長期，7 d后進入平臺期。HE染色和掃描電鏡觀察均顯示SCs在SIS上黏附良好，細胞形態呈紡錘形，有明顯突起，分泌較多細胞外基質。ELISA檢測發現SCs與SIS復合培養后，培養上清中的NGF含量隨時間延長而逐漸升高，與同時間點對照組比較差異均無統計學意義（P gt; 0.05）。通過與SCs復合后反復凍融脫細胞，SIS中NGF含量在培養10 d達峰值（414.29 ± 20.87）pg/cm2，顯著高于未復合細胞的單純SIS材料中NGF含量（4.92 ± 2.06） pg/ cm2，比較差異有統計學意義（P lt; 0.05）。結論聯合雙酶消化、差速貼壁、G418處理、低濃度酶消化和低濃度血清培養，可在較短時間內獲得大量高純度SCs。SCs與SIS復合生長相容性好，不影響SCs的NGF分泌。通過與SCs的復合、反復凍融脫細胞后，SIS中負載了大量NGF，有望成為良好的神經修復材料。

引用本文： 劉宏銀,馮喆,柏琳,侯江龍,羅靜聰,楊志明,解慧琪. 雪旺細胞純化培養及體外復合豬小腸黏膜下層的實驗研究. 中國修復重建外科雜志, 2013, 27(6): 743-747. doi: 10.7507/1002-1892.20130165 復制

1.	Terenghi G, Calder JS, Birch R, et al. A morphological study of Schwann cells and axonal regeneration in chronically transected human peripheral nerves. J Hand Surg (Br), 1998, 23(5): 583-587.
2.	Wei RQ, Tan B, Tan MY, et al. Grafts of porcine small intestinal submucosa with cultured autologous oral mucosal epithelial cells for esophageal repair in a canine model. Exp Biol Med (Maywood), 2009, 234(4): 453-461.
3.	Caione P, Capozza N, Zavaglia D, et al. In vivo bladder regeneration using small intestinal submucosa: experimental study. Pediatr Surg Int, 2006, 22(7): 593-599.
4.	Lee EW, Shindel AW, Brandes SB. Small intestinal submucosa for patch grafting after plaque incision in the treatmentof Peyronie’s disease. Int Braz J Urol, 2008, 34(2): 191-197.
5.	Fiala R, Vidlar A, Vrtal R, et al. Porcine small intestinal submucosa graft for repair of anterior urethral strictures. Eur Urol, 2007, 51(6): 1702-1708.
6.	Voytik-Harbin SL, Brightman AO, Kraine MR, et al. Identification of extractable growth factors from small intestinal submucosa. J Cell Biochem, 1997, 67(4): 478-491.
7.	Rich KM, Alexander TD, Pryor JC, et al. Nerve growth factor enhances regeneration through silicone chambers. Exp Neurol, 1989, 105(2): 162-170.
8.	Whitworth IH, Brown RA, Doré CJ, et al. Nerve growth factor enhances nerve regeneration through fibronectin grafts. J Hand Surg (Br), 1996, 21(4): 514-522.
9.	Santos X, Rodrigo J, Hontanilla B, et al. Evaluation of peripheral nerve regeneration by nerve growth factor locally administered with a novel system. J Neurosci Methods, 1998, 85(1): 119-127.
10.	Piotrowicz A, Shoichet MS. Nerve guidance channels as drug delivery vehicles. Biomaterials, 2006, 27(9): 2018-2027.
11.	Luo JC, Chen W, Chen XH, et al. A multi-step method for preparation of porcine small intestinal submucosa (SIS). Biomaterials, 2011, 32(3): 706-713.
12.	Millesi H. Techniques for nerve grafting. Hand Clin, 2000, 16(1): 73-91.
13.	Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol, 1997, 14(1-2): 67-116.
14.	Terenghi G. Peripheral nerve regeneration and neurotrophic factors. J Anat, 1999, 194(Pt 1): 1-14.
15.	Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv, 2008, 26(1): 1-21.
16.	Gu X, Ding F, Yang Y, et al. Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol, 2011, 93(2): 204-230.
17.	Hart A, Terenghi G, Wiberg M. Tissue engineering for peripheral nerve regeneration//Tissue Engineering. Berlin: Springer, 2011: 245-262.
18.	Ansselin AD, Fink T, Davey DF. Peripheral nerve regeneration through nerve guides seeded with adult Schwann cells. Neuropathol Appl Neurobiol, 1997, 23(5): 387-398.
19.	Kim DH, Connolly SE, Kline DG, et al. Labeled Schwann cell transplants versus sural nerve grafts in nerve repair. J Neurosurg, 1994, 80(2): 254-260.
20.	Guénard V, Kleitman N, Morrissey TK, et al. Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration. J Neurosci, 1992, 12(9): 3310-3320.
21.	黃小強, 羅卓荊, 李明全. 新生SD大鼠坐骨神經雪旺細胞培養與純化的方法學研究. 中華創傷骨科雜志, 2005, 7(4): 341-343, 348.
22.	Jin YQ, Liu W, Hong TH, et al. Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment. J Neurosci Methods, 2008, 170(1): 140-148.
23.	Pannunzio ME, Jou IM, Long A, et al. A new method of selecting Schwann cells from adult mouse sciatic nerve. J Neurosci Methods, 2005, 149(1): 74-81.
24.	Vroemen M, Weidner N. Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve. J Neurosci Methods, 2003, 124(2): 135-143.
25.	Kreider BQ, Messing A, Doan H, et al. Enrichment of Schwann cells cultures from neonatal rat sciatic nerve by differential adhesion. Brain Res, 1981, 207(2): 433-444.
26.	張勇杰, 金巖, 聶鑫, 等. 多步法分離和純化雪旺細胞的實驗研究. 中華神經外科疾病研究雜志, 2002, 1(4): 347-350．.
27.	Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduct. J Neurosci Methods, 2003, 122(2): 195-200.
28.	蘇琰, 張長青, 孫魯源, 等. 小腸粘膜下層橋接周圍神經缺損的實驗研究. 中華創傷骨科雜志, 2004, 6(10): 1136-1139.
29.	Lu H, Hoshiba T, Kawazoe N, et al. Autologous extracellular matrix scaffolds for tissue engineering. Biomaterials, 2011, 32 (10): 2489-2499.

1. Terenghi G, Calder JS, Birch R, et al. A morphological study of Schwann cells and axonal regeneration in chronically transected human peripheral nerves. J Hand Surg (Br), 1998, 23(5): 583-587.
2. Wei RQ, Tan B, Tan MY, et al. Grafts of porcine small intestinal submucosa with cultured autologous oral mucosal epithelial cells for esophageal repair in a canine model. Exp Biol Med (Maywood), 2009, 234(4): 453-461.
3. Caione P, Capozza N, Zavaglia D, et al. In vivo bladder regeneration using small intestinal submucosa: experimental study. Pediatr Surg Int, 2006, 22(7): 593-599.
4. Lee EW, Shindel AW, Brandes SB. Small intestinal submucosa for patch grafting after plaque incision in the treatmentof Peyronie’s disease. Int Braz J Urol, 2008, 34(2): 191-197.
5. Fiala R, Vidlar A, Vrtal R, et al. Porcine small intestinal submucosa graft for repair of anterior urethral strictures. Eur Urol, 2007, 51(6): 1702-1708.
6. Voytik-Harbin SL, Brightman AO, Kraine MR, et al. Identification of extractable growth factors from small intestinal submucosa. J Cell Biochem, 1997, 67(4): 478-491.
7. Rich KM, Alexander TD, Pryor JC, et al. Nerve growth factor enhances regeneration through silicone chambers. Exp Neurol, 1989, 105(2): 162-170.
8. Whitworth IH, Brown RA, Doré CJ, et al. Nerve growth factor enhances nerve regeneration through fibronectin grafts. J Hand Surg (Br), 1996, 21(4): 514-522.
9. Santos X, Rodrigo J, Hontanilla B, et al. Evaluation of peripheral nerve regeneration by nerve growth factor locally administered with a novel system. J Neurosci Methods, 1998, 85(1): 119-127.
10. Piotrowicz A, Shoichet MS. Nerve guidance channels as drug delivery vehicles. Biomaterials, 2006, 27(9): 2018-2027.
11. Luo JC, Chen W, Chen XH, et al. A multi-step method for preparation of porcine small intestinal submucosa (SIS). Biomaterials, 2011, 32(3): 706-713.
12. Millesi H. Techniques for nerve grafting. Hand Clin, 2000, 16(1): 73-91.
13. Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol, 1997, 14(1-2): 67-116.
14. Terenghi G. Peripheral nerve regeneration and neurotrophic factors. J Anat, 1999, 194(Pt 1): 1-14.
15. Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv, 2008, 26(1): 1-21.
16. Gu X, Ding F, Yang Y, et al. Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol, 2011, 93(2): 204-230.
17. Hart A, Terenghi G, Wiberg M. Tissue engineering for peripheral nerve regeneration//Tissue Engineering. Berlin: Springer, 2011: 245-262.
18. Ansselin AD, Fink T, Davey DF. Peripheral nerve regeneration through nerve guides seeded with adult Schwann cells. Neuropathol Appl Neurobiol, 1997, 23(5): 387-398.
19. Kim DH, Connolly SE, Kline DG, et al. Labeled Schwann cell transplants versus sural nerve grafts in nerve repair. J Neurosurg, 1994, 80(2): 254-260.
20. Guénard V, Kleitman N, Morrissey TK, et al. Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration. J Neurosci, 1992, 12(9): 3310-3320.
21. 黃小強, 羅卓荊, 李明全. 新生SD大鼠坐骨神經雪旺細胞培養與純化的方法學研究. 中華創傷骨科雜志, 2005, 7(4): 341-343, 348.
22. Jin YQ, Liu W, Hong TH, et al. Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment. J Neurosci Methods, 2008, 170(1): 140-148.
23. Pannunzio ME, Jou IM, Long A, et al. A new method of selecting Schwann cells from adult mouse sciatic nerve. J Neurosci Methods, 2005, 149(1): 74-81.
24. Vroemen M, Weidner N. Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve. J Neurosci Methods, 2003, 124(2): 135-143.
25. Kreider BQ, Messing A, Doan H, et al. Enrichment of Schwann cells cultures from neonatal rat sciatic nerve by differential adhesion. Brain Res, 1981, 207(2): 433-444.
26. 張勇杰, 金巖, 聶鑫, 等. 多步法分離和純化雪旺細胞的實驗研究. 中華神經外科疾病研究雜志, 2002, 1(4): 347-350．.
27. Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduct. J Neurosci Methods, 2003, 122(2): 195-200.
28. 蘇琰, 張長青, 孫魯源, 等. 小腸粘膜下層橋接周圍神經缺損的實驗研究. 中華創傷骨科雜志, 2004, 6(10): 1136-1139.
29. Lu H, Hoshiba T, Kawazoe N, et al. Autologous extracellular matrix scaffolds for tissue engineering. Biomaterials, 2011, 32 (10): 2489-2499.

《中國修復重建外科雜志》

雪旺細胞純化培養及體外復合豬小腸黏膜下層的實驗研究

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《中國修復重建外科雜志》

雪旺細胞純化培養及體外復合豬小腸黏膜下層的實驗研究

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