異源性膀胱脫細胞基質移植修復兔膀胱缺損的局部和全身安全性評價_《中國修復重建外科雜志》

作者：

周六化 ¹ , 楊斌 ¹² , 王鵬基 ¹ , 夏佳東 ¹ , 章宜芬 ³ , 陳赟 ¹ , 戴玉田 ¹

南京大學醫學院附屬鼓樓醫院（南京，210008）1泌尿男科，3病理科;;
2上海市第十人民醫院（暨同濟大學附屬第十人民醫院）泌尿外科;

關鍵詞：

膀胱再生膀胱脫細胞基質異種移植安全性評價豬兔

DOI：

10.7507/1002-1892.20130205

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目的采用異源性膀胱脫細胞基質（bladder acellular matrix，BAM）移植修復兔膀胱缺損，檢測組織再生和機體的組織反應情況，評價其生物安全性。方法將完整新鮮豬膀胱通過一系列物理、化學和酶消化法進行脫細胞處理，得到豬BAM，通過HE染色和掃描電鏡觀察其脫細胞效果和超微結構。將18只新西蘭大白兔（體重2.5～3.0 kg）隨機分為實驗組（n=12）和對照組（n=6），行膀胱部分（約30%）切除術，實驗組采用與切除面積等大的豬BAM替代修補，對照組直接行膀胱切口原位縫合。術后觀察動物存活情況，分別于術后15 d，1、2、3、6個月行血常規及腎功能、電解質檢測。術后1、2、3、6個月行尿流動力學檢測最大膀胱容量、排尿點膀胱內壓力以及膀胱順應性；然后處死動物，大體觀察膀胱再生情況，并取再生膀胱組織行HE染色及免疫組織化學染色；同時取周圍臟器及淋巴組織，經大體觀察及HE染色觀察有無異常變化。結果制備的豬BAM完全去除了細胞成分，掃描電鏡觀察呈多孔樣結構。實驗動物均存活至取材各時間點，血常規檢測示術后15 d兩組白細胞計數升高，之后逐漸恢復至正常水平，兩組間差異無統計學意義（P gt; 0.05）。腎功能、電解質檢測提示兩組間差異無統計學意義（P gt; 0.05），其中肌酐有上升趨勢，但至術后6個月時仍維持在正常范圍內。術后實驗組最大膀胱容量及膀胱順應性與對照組相比明顯恢復，術后3、6個月兩組比較差異有統計學意義（P lt; 0.05）；兩組間各時間點排尿點膀胱內壓力比較差異均無統計學意義（P gt; 0.05）。組織學觀察可見兩組中均有尿路上皮層、平滑肌組織和血管的再生。兩組術后1個月均可見慢性炎性細胞浸潤，隨后慢性炎性細胞明顯減少（P lt; 0.05），直至基本消退；術后3、6個月，兩組間慢性炎性細胞浸潤程度評分比較差異無統計學意義（P gt; 0.05）。隨著時間推移，兩組α-平滑肌肌動蛋白（α-smooth muscle actin，α-SMA）表達量明顯增多（P lt; 0.05），對照組各時間點α-SMA表達量明顯高于實驗組（P lt; 0.05）。取檢全身其他器官及區域淋巴結經大體觀察及HE染色觀察均無異常改變。結論采用豬BAM進行異種移植修補兔膀胱缺損未發現明顯免疫排斥反應，具有相對安全性。

引用本文： 周六化,楊斌,王鵬基,夏佳東,章宜芬,陳赟,戴玉田. 異源性膀胱脫細胞基質移植修復兔膀胱缺損的局部和全身安全性評價. 中國修復重建外科雜志, 2013, 27(8): 935-944. doi: 10.7507/1002-1892.20130205 復制

1.	Oberpenning F, Meng J, Yoo JJ, et al. De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol, 1999, 17(2): 149-155.
2.	Atala A, Bauer SB, Soker S, et al. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet, 2006, 367(9518): 1241-1246.
3.	Atala A. Tissue engineering of human bladder. Br Med Bull, 2011, 97: 81-104.
4.	Brown AL, Farhat W, Merguerian PA, et al. 22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model. Biomaterials, 2002, 23(10): 2179-2190.
5.	Zhu WD, Xu YM, Feng C, et al. Bladder reconstruction with adipose-derived stem cell-seeded bladder acellular matrix grafts improve morphology composition. World J Urol, 2010, 28(4): 493-498.
6.	袁銘, 李漢忠, 張玉石, 等. 豬源性膀胱無細胞基質在異種移植中的生物安全性. 中國組織工程研究與臨床康復, 2008, 12(40): 7845-7849.
7.	Yang B, Zhang Y, Zhou L, et al. Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering. Tissue Eng Part C Methods, 2010, 16(5): 1201-1211.
8.	楊斌, 孫則禹, 周六化, 等. 多孔的保留有生物活性因子的膀胱無細胞基質及制備方法: 中國, ZL 2008 1 0020808.0. 2012-7-25.
9.	Zhou L, Yang B, Sun C, et al. Coadministration of platelet-derived growth factor-BB and vascular endothelial growth factor with bladder acellular matrix enhances smooth muscle regeneration and vascularization for bladder augmentation in a rabbit model. Tissue Eng Part A, 2013, 19(1-2): 264-276.
10.	陶亮, 李強, 任昊楨, 等. 緩釋bFGF膠原膜修復豬膽管缺損的實驗研究. 中國修復重建外科雜志, 2013, 27(2): 212-218.
11.	Duan Y, Learoyd J, Meliton AY, et al. Inhibition of Pyk2 blocks airway inflammation and hyperresponsiveness in a mouse model of asthma. Am J Respir Cell Mol Biol, 2010, 42(4): 491-497.
12.	吳葆菁, 朱軍, 檀衛平, 等. 地塞米松對急性過敏性哮喘小鼠肺AQP5表達的影響. 南方醫科大學學報, 2008, 28(9): 1670-1673.
13.	Atala A. Tissue engineering, stem cells, and cloning for the regeneration of urologic organs. Clin Plast Surg, 2003, 30(4): 649-667.
14.	Atala A. Bioengineered tissues for urogenital repair in children. Pediatr Res, 2008, 63(5): 569-575.
15.	Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials, 2006, 27(19): 3675-3683.
16.	Chun SY, Lim GJ, Kwon TG, et al. Identification and characterization of bioactive factors in bladder submucosa matrix. Biomaterials, 2007, 28(29): 4251-4256.
17.	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.
18.	Yang B, Zhou L, Sun Z, et al. In vitro evaluation of the bioactive factors preserved in porcine small intestinal submucosa through cellular biological approaches. J Biomed Mater Res A, 2010, 93(3): 1100-1109.
19.	Bolland F, Korossis S, Wilshaw SP, et al. Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering. Biomaterials, 2007, 28(6): 1061-1070.
20.	Merguerian PA, Reddy PP, Barrieras DJ, et al. Acellular bladder matrix allografts in the regeneration of functional bladders: evaluation of large-segment (> 24 cm) substitution in a porcine model. BJU Int, 2000, 85(7): 894-898.
21.	Chen BS, Zhang SL, Geng H, et al. Ex vivo functional evaluation of isolated strips in BAMG tissue-engineered bladders. Int J Artif Organs, 2009, 32(3): 159-165.
22.	Piechota HJ, Dahms SE, Probst M, et al. Functional rat bladder regeneration through xenotransplantation of the bladder acellular matrix graft. Br J Urol, 1998, 81(4): 548-559.
23.	Ayyildiz A, Nuhoglu B, Huri E, et al. Using porcine acellular collagen matrix (Pelvicol) in bladder augmentation: experimental study. Int Braz J Urol, 2006, 32(1): 88-93.
24.	Kwon TG, Yoo JJ, Atala A. Local and systemic effects of a tissue engineered neobladder in a canine cystoplasty model. J Urol, 2008, 179(5): 2035-2041.
25.	Frangogiannis NG. Regulation of the inflammatory response in cardiac repair. Circ Res, 2012, 110(1): 159-173.
26.	Kim MS, Ahn HH, Shin YN, et al. An in vivo study of the host tissue response to subcutaneous implantation of PLGA- and/or porcine small intestinal submucosa-based scaffolds. Biomaterials, 2007, 28(34): 5137-5143.
27.	Horst M, Madduri S, Milleret V, et al. A bilayered hybrid microfibrous PLGA—acellular matrix scaffold for hollow organ tissue engineering. Biomaterials, 2013, 34(5): 1537-1545.
28.	Ceonzo K, Gaynor A, Shaffer L, et al. Polyglycolic acid-induced inflammation: role of hydrolysis and resulting complement activation. Tissue Eng, 2006, 12(2): 301-308.

1. Oberpenning F, Meng J, Yoo JJ, et al. De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol, 1999, 17(2): 149-155.
2. Atala A, Bauer SB, Soker S, et al. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet, 2006, 367(9518): 1241-1246.
3. Atala A. Tissue engineering of human bladder. Br Med Bull, 2011, 97: 81-104.
4. Brown AL, Farhat W, Merguerian PA, et al. 22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model. Biomaterials, 2002, 23(10): 2179-2190.
5. Zhu WD, Xu YM, Feng C, et al. Bladder reconstruction with adipose-derived stem cell-seeded bladder acellular matrix grafts improve morphology composition. World J Urol, 2010, 28(4): 493-498.
6. 袁銘, 李漢忠, 張玉石, 等. 豬源性膀胱無細胞基質在異種移植中的生物安全性. 中國組織工程研究與臨床康復, 2008, 12(40): 7845-7849.
7. Yang B, Zhang Y, Zhou L, et al. Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering. Tissue Eng Part C Methods, 2010, 16(5): 1201-1211.
8. 楊斌, 孫則禹, 周六化, 等. 多孔的保留有生物活性因子的膀胱無細胞基質及制備方法: 中國, ZL 2008 1 0020808.0. 2012-7-25.
9. Zhou L, Yang B, Sun C, et al. Coadministration of platelet-derived growth factor-BB and vascular endothelial growth factor with bladder acellular matrix enhances smooth muscle regeneration and vascularization for bladder augmentation in a rabbit model. Tissue Eng Part A, 2013, 19(1-2): 264-276.
10. 陶亮, 李強, 任昊楨, 等. 緩釋bFGF膠原膜修復豬膽管缺損的實驗研究. 中國修復重建外科雜志, 2013, 27(2): 212-218.
11. Duan Y, Learoyd J, Meliton AY, et al. Inhibition of Pyk2 blocks airway inflammation and hyperresponsiveness in a mouse model of asthma. Am J Respir Cell Mol Biol, 2010, 42(4): 491-497.
12. 吳葆菁, 朱軍, 檀衛平, 等. 地塞米松對急性過敏性哮喘小鼠肺AQP5表達的影響. 南方醫科大學學報, 2008, 28(9): 1670-1673.
13. Atala A. Tissue engineering, stem cells, and cloning for the regeneration of urologic organs. Clin Plast Surg, 2003, 30(4): 649-667.
14. Atala A. Bioengineered tissues for urogenital repair in children. Pediatr Res, 2008, 63(5): 569-575.
15. Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials, 2006, 27(19): 3675-3683.
16. Chun SY, Lim GJ, Kwon TG, et al. Identification and characterization of bioactive factors in bladder submucosa matrix. Biomaterials, 2007, 28(29): 4251-4256.
17. 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.
18. Yang B, Zhou L, Sun Z, et al. In vitro evaluation of the bioactive factors preserved in porcine small intestinal submucosa through cellular biological approaches. J Biomed Mater Res A, 2010, 93(3): 1100-1109.
19. Bolland F, Korossis S, Wilshaw SP, et al. Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering. Biomaterials, 2007, 28(6): 1061-1070.
20. Merguerian PA, Reddy PP, Barrieras DJ, et al. Acellular bladder matrix allografts in the regeneration of functional bladders: evaluation of large-segment (> 24 cm) substitution in a porcine model. BJU Int, 2000, 85(7): 894-898.
21. Chen BS, Zhang SL, Geng H, et al. Ex vivo functional evaluation of isolated strips in BAMG tissue-engineered bladders. Int J Artif Organs, 2009, 32(3): 159-165.
22. Piechota HJ, Dahms SE, Probst M, et al. Functional rat bladder regeneration through xenotransplantation of the bladder acellular matrix graft. Br J Urol, 1998, 81(4): 548-559.
23. Ayyildiz A, Nuhoglu B, Huri E, et al. Using porcine acellular collagen matrix (Pelvicol) in bladder augmentation: experimental study. Int Braz J Urol, 2006, 32(1): 88-93.
24. Kwon TG, Yoo JJ, Atala A. Local and systemic effects of a tissue engineered neobladder in a canine cystoplasty model. J Urol, 2008, 179(5): 2035-2041.
25. Frangogiannis NG. Regulation of the inflammatory response in cardiac repair. Circ Res, 2012, 110(1): 159-173.
26. Kim MS, Ahn HH, Shin YN, et al. An in vivo study of the host tissue response to subcutaneous implantation of PLGA- and/or porcine small intestinal submucosa-based scaffolds. Biomaterials, 2007, 28(34): 5137-5143.
27. Horst M, Madduri S, Milleret V, et al. A bilayered hybrid microfibrous PLGA—acellular matrix scaffold for hollow organ tissue engineering. Biomaterials, 2013, 34(5): 1537-1545.
28. Ceonzo K, Gaynor A, Shaffer L, et al. Polyglycolic acid-induced inflammation: role of hydrolysis and resulting complement activation. Tissue Eng, 2006, 12(2): 301-308.

《中國修復重建外科雜志》

異源性膀胱脫細胞基質移植修復兔膀胱缺損的局部和全身安全性評價

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《中國修復重建外科雜志》

異源性膀胱脫細胞基質移植修復兔膀胱缺損的局部和全身安全性評價

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