毛乳頭細胞促進組織工程皮膚血管化的實驗研究_《中國修復重建外科雜志》

作者：

劉坡 , 祁少海 , 舒斌 , 謝舉臨 , 徐盈斌 , 劉旭盛

1 東莞市塘廈醫院外一科（廣東東莞，523721）;;
2 中山大學附屬第一醫院燒傷科;

關鍵詞：

組織工程皮膚，毛乳頭細胞，表皮干細胞，同種異體脫細胞真皮基質，血管化，裸鼠

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【摘要】目的探討毛乳頭細胞對表皮干細胞與同種異體脫細胞真皮基質構建的組織工程皮膚血管化的影響。方法取門診包皮環切術患者皮膚，用于表皮細胞培養；取孕19、20 周引產胎兒頭部皮膚，用于毛乳頭細胞培養；患者及家屬均知情同意。以中性蛋白酶聯合胰蛋白酶消化、分離表皮細胞，Ⅳ型膠原黏附法分選表皮干細胞；以Ⅰ型膠原酶消化法分離毛乳頭，常規成纖維細胞培養液培養。以同種異體脫細胞真皮基質為支架，在真皮基質的真皮乳頭側種植毛乳頭細胞，基底膜側種植表皮干細胞構建實驗組組織工程皮膚替代物；對照組組織工程皮膚替代物不種植毛乳頭細胞。取6 ～ 8 周齡BALB/C-nu 裸鼠60 只，隨機分成實驗組和對照組（n=30）；實驗動物背部制造1 cm × 1 cm 大小全層皮膚缺損模型，將兩組組織工程皮膚替代物移植修復創面，2 周后觀察移植皮膚成活率。術后2、4 周，取移植皮膚替代物標本，行HE 及免疫組織化學染色，觀察移植皮膚替代物CD31 表達水平，并計算兩組標本血管密度。結果 Ⅳ型膠原分選后表皮細胞同時表達角蛋白19、β1 整合素，提示為表皮干細胞。由毛乳頭培養出的細胞表達α 平滑肌肌動蛋白，提示為毛乳頭細胞。動物移植術后2 周，實驗組移植皮膚成活率為93.3%（28/30），對照組為80.0%（24/30），比較差異有統計學意義（χ2=2.31，P=0.00）。HE 染色觀察示實驗組表皮層達12 層，表皮細胞體積較大；對照組表皮層達4 ～ 6 層，表皮細胞體積較小。實驗組術后2、4 周，血管密度分別為（38.56 ± 2.49）個/mm2 和（49.12 ± 2.39）個/mm2，對照組分別為（25.16 ± 3.73）個 / mm2 和（36.26 ± 3.24）個 / mm2，兩組比較差異均有統計學意義（P lt; 0.01）。結論毛乳頭細胞可促進移植皮膚替代物血管形成，利于表皮層重建，提高組織工程皮膚移植成活率。

引用本文： 劉坡,祁少海,舒斌,謝舉臨,徐盈斌,劉旭盛. 毛乳頭細胞促進組織工程皮膚血管化的實驗研究. 中國修復重建外科雜志, 2012, 26(2): 135-140. doi: 復制

1.	Ehrlich HP. Understanding experimental biology of skin equivalent: from laboratory to clinical use in patients with burns and chronic wounds. Am J Surg, 2004, 187(5A): 29S-33S.
2.	Roh C, Tao Q, Lyle S. Dermal papilla-induced hair differentiation of adult epithelial stem cells from human skin. Physiol Genomics, 2004, 19(2): 207-217.
3.	Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in wound healing and induction. Exp Dermatol, 2003, 12(2): 126-136.
4.	劉坡, 祁少海, 舒斌, 等. Ⅳ型膠原黏附法分選表皮干細胞. 中國組織工程研究與臨床康復, 2007, 11(7): 1201-1204.
5.	劉坡, 祁少海, 徐盈斌, 等. 胎兒毛乳頭細胞的培養和鑒定及其體外誘導分化. 中華醫學美學美容雜志, 2006, 12(6): 354-357.
6.	祁少海, 沈銳, 謝舉臨, 等. 以血管內皮生長因子受體為靶點抑制瘢痕血管增生的研究. 中華實驗外科雜志, 2005, 22(4): 421-423.
7.	Wisser D, Steffes J. Skin replacement with a collagen based dermal substitute, autologous keratinocytes and fibroblasts in burn trauma. Burns, 2003, 29(4): 375-380.
8.	Halim AS, Khoo TL, Mohd Yussof SJ. Biologic and synthetic skin substitutes: An overview. Indian J Plast Surg, 2010, 43(Suppl): S23-28.
9.	Klingenberg JM, McFarland KL, Friedman AJ, et al. Engineered human skin substitutes undergo large-scale genomic reprogramming and normal skin-like maturation after transplantation to athymic mice. J Invest Dermatol, 2010, 130(2): 587-601.
10.	Toyozawa S, Yamamoto Y, Kishioka A, et al. Effective treatment of intractable skin ulcers using allogeneic cultured dermal substitutes in patients with systemic lupus erythematosus. Eur J Dermatol, 2009, 19(6): 594-596.
11.	Ehrenreich M, Ruszczak Z. Update on dermal substitutes. Acta Dermatovenerol Croat, 2006, 14(3): 172-187.
12.	Srivastava A, Jennings LJ, Hanumadass M, et al. Xenogeneic acellular dermal matrix as a dermal substitute in rats. J Burn Care Rehabil, 1999, 20(5): 382-390.
13.	Heimbach D, Luterman A, Burke J, et al. Artificial dermis for major burns. A multi-center randomized clinical trial. Ann Surg, 1988, 208(3): 313-320.
14.	汪道新, 張靈, 范錕铻, 等. 人臍靜脈血管內皮細胞移植對脫細胞豬真皮早期血管化的影響. 臨床和實驗醫學雜志, 2007, 6(4): 11-12.
15.	Fujie T, Katoh S, Oura H, et al. The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells. J Dermatol Sci, 2001, 25(3): 206-212.
16.	Hibberts NA, Messenger AG, Randall VA. Dermal papilla cells derived from beard hair follicles secrete more stem cell factor (SCF) in culture than scalp cells or dermal fibroblasts. Biochem Biophys Res Commun, 1996, 222(2): 401-405.
17.	Itami S, Kurata S, Takayasu S. Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth factor-I from dermal papilla cells. Biochem Biophys Res Commun, 1995, 212(3): 988-994.
18.	Bao P, Kodra A, Tomic-Canic M, et al. The role of vascular endothelial growth factor in wound healing. J Surg Res, 2009, 153(2): 347-358.
19.	Koolwijk P, van Erck MG, de Vree WJ, et al. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol, 1996, 132(6): 1177-1188.
20.	Bauer SM, Bauer RJ, Liu ZJ, et al. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg, 2005, 41(4): 699-707.

1. Ehrlich HP. Understanding experimental biology of skin equivalent: from laboratory to clinical use in patients with burns and chronic wounds. Am J Surg, 2004, 187(5A): 29S-33S.
2. Roh C, Tao Q, Lyle S. Dermal papilla-induced hair differentiation of adult epithelial stem cells from human skin. Physiol Genomics, 2004, 19(2): 207-217.
3. Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in wound healing and induction. Exp Dermatol, 2003, 12(2): 126-136.
4. 劉坡, 祁少海, 舒斌, 等. Ⅳ型膠原黏附法分選表皮干細胞. 中國組織工程研究與臨床康復, 2007, 11(7): 1201-1204.
5. 劉坡, 祁少海, 徐盈斌, 等. 胎兒毛乳頭細胞的培養和鑒定及其體外誘導分化. 中華醫學美學美容雜志, 2006, 12(6): 354-357.
6. 祁少海, 沈銳, 謝舉臨, 等. 以血管內皮生長因子受體為靶點抑制瘢痕血管增生的研究. 中華實驗外科雜志, 2005, 22(4): 421-423.
7. Wisser D, Steffes J. Skin replacement with a collagen based dermal substitute, autologous keratinocytes and fibroblasts in burn trauma. Burns, 2003, 29(4): 375-380.
8. Halim AS, Khoo TL, Mohd Yussof SJ. Biologic and synthetic skin substitutes: An overview. Indian J Plast Surg, 2010, 43(Suppl): S23-28.
9. Klingenberg JM, McFarland KL, Friedman AJ, et al. Engineered human skin substitutes undergo large-scale genomic reprogramming and normal skin-like maturation after transplantation to athymic mice. J Invest Dermatol, 2010, 130(2): 587-601.
10. Toyozawa S, Yamamoto Y, Kishioka A, et al. Effective treatment of intractable skin ulcers using allogeneic cultured dermal substitutes in patients with systemic lupus erythematosus. Eur J Dermatol, 2009, 19(6): 594-596.
11. Ehrenreich M, Ruszczak Z. Update on dermal substitutes. Acta Dermatovenerol Croat, 2006, 14(3): 172-187.
12. Srivastava A, Jennings LJ, Hanumadass M, et al. Xenogeneic acellular dermal matrix as a dermal substitute in rats. J Burn Care Rehabil, 1999, 20(5): 382-390.
13. Heimbach D, Luterman A, Burke J, et al. Artificial dermis for major burns. A multi-center randomized clinical trial. Ann Surg, 1988, 208(3): 313-320.
14. 汪道新, 張靈, 范錕铻, 等. 人臍靜脈血管內皮細胞移植對脫細胞豬真皮早期血管化的影響. 臨床和實驗醫學雜志, 2007, 6(4): 11-12.
15. Fujie T, Katoh S, Oura H, et al. The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells. J Dermatol Sci, 2001, 25(3): 206-212.
16. Hibberts NA, Messenger AG, Randall VA. Dermal papilla cells derived from beard hair follicles secrete more stem cell factor (SCF) in culture than scalp cells or dermal fibroblasts. Biochem Biophys Res Commun, 1996, 222(2): 401-405.
17. Itami S, Kurata S, Takayasu S. Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth factor-I from dermal papilla cells. Biochem Biophys Res Commun, 1995, 212(3): 988-994.
18. Bao P, Kodra A, Tomic-Canic M, et al. The role of vascular endothelial growth factor in wound healing. J Surg Res, 2009, 153(2): 347-358.
19. Koolwijk P, van Erck MG, de Vree WJ, et al. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol, 1996, 132(6): 1177-1188.
20. Bauer SM, Bauer RJ, Liu ZJ, et al. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg, 2005, 41(4): 699-707.

《中國修復重建外科雜志》

毛乳頭細胞促進組織工程皮膚血管化的實驗研究

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《中國修復重建外科雜志》

毛乳頭細胞促進組織工程皮膚血管化的實驗研究

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