BMSCs對豬胰島缺氧再給氧損傷的保護作用_《中國修復重建外科雜志》

作者：

趙久茗 , 何斯榮 , 王成世 , 劉敬平 , 李勝富 , 陳又南 , 陸燕蓉 , 程驚秋

四川大學華西醫院再生醫學研究中心（成都，610041）;

關鍵詞：

BMSCs 胰島缺氧再給氧恒河猴豬

DOI：

10.7507/1002-1892.20130201

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目的研究恒河猴BMSCs在缺氧再給氧環境下對新生豬胰島活性和功能的保護作用。方法取健康成年恒河猴5只，體重6～10 kg，取骨髓采用骨髓單核細胞貼壁培養法篩選獲得BMSCs；取3～5日齡新生長白豬5 只，取胰腺以Ⅴ型膠原酶消化制備新生豬胰島。將實驗分為胰島正常組（A組）、胰島加BMSCs正常組（B組）、胰島缺氧再給氧組（C組）、胰島加BMSCs缺氧再給氧組（D組），觀察比較常規培養和缺氧（1%O2）12 h再給氧24 h或48 h條件下，胰島的功能活性變化：二乙酸熒光素/碘化丙啶（propidium iodide，PI）染色計算胰島成活率；細胞計數試劑盒8法檢測新生豬胰島的代謝活性；膜聯蛋白V-FITC/PI標記后流式細胞儀檢測胰島凋亡率；葡萄糖刺激法分析胰島功能糖刺激指數（stimulation index，SI）。結果C組較A、B組胰島團更為分散，死亡細胞增多；D組較C組胰島團完整，成活率更高。A、B、C、D組胰島成活率分別為90.2% ± 9.1%、88.3% ± 5.9%、52.3% ± 12.1%、71.4% ± 11.5%，C、D組顯著低于A、B組（P lt; 0.05），D組顯著高于C組（P lt; 0.05）。D組BMSCs以1∶10和1∶20比例與胰島缺氧再給氧共培養后，代謝活性顯著高于C組（P lt; 0.05）。A、B、C、D組胰島凋亡率分別為27.1% ± 3.2%、24.0% ± 1.0%、64.3% ± 1.8%、46.2% ± 1.4%，C、D組顯著高于A、B組（P lt; 0.05），但D組顯著低于C組（P lt; 0.05）。各時間點A、B組SI比較差異均無統計學意義（P gt; 0.05），C組顯著低于A、B組（P lt; 0.05），在24 h和72 h時D組顯著高于C組（P lt; 0.05）。結論BMSCs對缺氧再給氧誘導的新生豬胰島活性和功能損傷具有保護作用。

引用本文： 趙久茗,何斯榮,王成世,劉敬平,李勝富,陳又南,陸燕蓉,程驚秋. BMSCs對豬胰島缺氧再給氧損傷的保護作用. 中國修復重建外科雜志, 2013, 27(8): 910-915. doi: 10.7507/1002-1892.20130201 復制

1.	Koh A, Senior P, Salam A, et al. Insulin-heparin infusions peritransplant substantially improve single-donor clinical islet transplant success. Transplantation, 2010, 89(4): 465-471.
2.	Langer RM. Islet transplantation: lessons learned since the Edmonton breakthrough. Transplant Proc, 2010, 42(5): 1421-1424.
3.	Ichii H, Ricordi C. Current status of islet cell transplantation. J Hepatobiliary Pancreat Surg, 2009, 16(2): 101-112.
4.	Kobayashi N. The current status of islet transplantation and its perspectives. Rev Diabet Stud, 2008, 5(3): 136-143.
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8.	Vardanyan M, Parkin E, Gruessner C, et al. Pancreas vs. islet transplantation: a call on the future. Curr Opin Organ Transplant, 2010, 15(1): 124-130.
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13.	Korbutt GS, Elliott JF, Ao Z, et al. Large scale isolation, growth, and function of porcine neonatal islet cells. J Clin Invest, 1996, 97(9): 2119-2129.
14.	Ma X, Ye B, Gao F, et al. Tissue factor knockdown in porcine islets: an effective approach to suppressing the instant blood-mediated inflammatory reaction. Cell Transplant, 2012, 21(1): 61-71.
15.	Karaoz E, Genc ZS, Demircan PC, et al. Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells. Cell Death Dis, 2010, 1: e36.
16.	朱海濤, 于良, 王博. 豬胰島細胞異種移植治療糖尿病的研究進展. 世界華人消化雜志, 2012, 20(9): 737-742.
17.	Skiles ML, Wilder NB, Sahai S, et al. Identifying HIF activity in three-dimensional cultures of islet-like clusters. Int J Artif Organs, 2013, 36(3): 175-183.
18.	Padmasekar M, Lingwal N, Samikannu B, et al. Exendin-4 protects hypoxic islets from oxidative stress and improves islet transplantation outcome. Endocrinology, 2013, 154(4): 1424-1433.
19.	Yook S, Jeong JH, Jung YS, et al. Molecularly engineered islet cell clusters for diabetes mellitus treatment. Cell Transplant, 2012, 21(8): 1775-1789.
20.	魯亞成, 任甫. 胚胎干細胞體外定向誘導分化為胰島細胞的相關研究. 中國組織工程研究與臨床康復, 2007, 11(11): 2156-2159.
21.	李富榮, 龍愛梅, 齊暉, 等. 胰島干細胞轉分化胰島樣細胞與天然胰島功能的比較. 中國組織工程研究與臨床康復, 2008, 12(25): 4833-4836.
22.	孟林燕, 劉暢, 王欣燕, 等. 肌源性干細胞移植修復糖尿病胰島功能的旁分泌機制研究. 中國醫學工程, 2011, 19(5): 35-36, 39.
23.	周靜, 李進, 林戈, 等. 誘導人孤雌胚胎干細胞分化為胰島樣細胞團. 中國組織工程研究與臨床康復, 2010, 14(27): 5001-5005.
24.	張妍, 郭希民, 王常勇, 等. 硒促進小鼠胚胎干細胞分化為胰島細胞的實驗研究. 軍事醫學科學院院刊, 2004, 28(2): 126-129, 139.
25.	Wang JC, Xia L, Song XB, et al. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells improves survival of ultra-long random skin flap. Chin Med J (Engl), 2011, 124(16): 2507-2511.
26.	Lennon DP, Caplan AI. Isolation of rat marrow-derived mesenchymal stem cells. Exp Hematol, 2006, 34(11): 1606-1607.
27.	Fehrer C, Brunauer R, Laschober G, et al. Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell, 2007, 6(6): 745-757.
28.	Lu Y, Jin X, Chen Y, et al. Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury. Cell Biochem Funct, 2010, 28(8): 637-643.
29.	Grayson WL, Zhao F, Izadpanah R, et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol, 2006, 207(2): 331-339.
30.	Xu YX, Chen L, Wang R, et al. Mesenchymal stem cell therapy for diabetes through paracrine mechanisms. Med Hypotheses, 2008, 71(3): 390-393.
31.	Figliuzzi M, Cornolti R, Perico N, et al. Bone marrow-derived mesenchymal stem cells improve islet graft function in diabetic rats. Transplant Proc, 2009, 41(5): 1797-1800.
32.	Li YY, Liu HH, Chen HL, et al. Adipose-derived mesenchymal stem cells ameliorate STZ-induced pancreas damage in type 1 diabetes. Biomed Mater Eng, 2012, 22(1-3): 97-103.
33.	Luo L, Badiavas E, Luo JZ, et al. Allogeneic bone marrow supports human islet beta cell survival and function over six months. Biochem Biophys Res Commun, 2007, 361(4): 859-864.
34.	Chao KC, Chao KF, Chen CF, et al. A novel human stem cell coculture system that maintains the survival and function of culture islet-like cell clusters. Cell Transplant, 2008, 17(6): 657-664.
35.	Cavallari G, Olivi E, Bianchi F, et al. Mesenchymal stem cells and islet cotransplantation in diabetic rats: improved islet graft revascularization and function by human adipose tissue-derived stem cells preconditioned with natural molecules. Cell Transplant, 2012, 21(12): 2771-2781.
36.	Clarkin CE, King AJ, Dhadda P, et al. Activin Receptor-like kinase 5 Inhibition Reverses Impairment of Endothelial Cell Viability by Endogenous Islet Mesenchymal Stromal Cells. Stem Cells, 2013, 31(3): 547-559.

1. Koh A, Senior P, Salam A, et al. Insulin-heparin infusions peritransplant substantially improve single-donor clinical islet transplant success. Transplantation, 2010, 89(4): 465-471.
2. Langer RM. Islet transplantation: lessons learned since the Edmonton breakthrough. Transplant Proc, 2010, 42(5): 1421-1424.
3. Ichii H, Ricordi C. Current status of islet cell transplantation. J Hepatobiliary Pancreat Surg, 2009, 16(2): 101-112.
4. Kobayashi N. The current status of islet transplantation and its perspectives. Rev Diabet Stud, 2008, 5(3): 136-143.
5. Lakey JR, Mirbolooki M, Shapiro AM. Current status of clinical islet cell transplantation. Methods Mol Biol, 2006, 333: 47-104.
6. Korsgren O, Nilsson B, Berne C, et al. Current status of clinical islet transplantation. Transplantation, 2005, 79(10): 1289-1293.
7. Markmann JF, Deng S, Desai NM, et al. The use of non-heart-beating donors for isolated pancreatic islet transplantation. Transplantation, 2003, 75(9): 1423-1429.
8. Vardanyan M, Parkin E, Gruessner C, et al. Pancreas vs. islet transplantation: a call on the future. Curr Opin Organ Transplant, 2010, 15(1): 124-130.
9. Carlsson PO, Palm F, Mattsson G. Low revascularization of experimentally transplanted human pancreatic islets. J Clin Endocrinol Metab, 2002, 87(12): 5418-5423.
10. Steinert AF, Rackwitz L, Gilbert F, et al. Concise review: the clinical application of mesenchymal stem cells for musculoskeletal regeneration: current status and perspectives. Stem Cells Transl Med, 2012, 1(3): 237-247.
11. Pozzobon M, Piccoli M, De Coppi P. Sources of mesenchymal stem cells: current and future clinical use. Adv Biochem Eng Biotechnol, 2012. [Epub ahead of print].
12. Gregory CA, Prockop DJ, Spees JL. Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation. Exp Cell Res, 2005, 306(2): 330-335.
13. Korbutt GS, Elliott JF, Ao Z, et al. Large scale isolation, growth, and function of porcine neonatal islet cells. J Clin Invest, 1996, 97(9): 2119-2129.
14. Ma X, Ye B, Gao F, et al. Tissue factor knockdown in porcine islets: an effective approach to suppressing the instant blood-mediated inflammatory reaction. Cell Transplant, 2012, 21(1): 61-71.
15. Karaoz E, Genc ZS, Demircan PC, et al. Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells. Cell Death Dis, 2010, 1: e36.
16. 朱海濤, 于良, 王博. 豬胰島細胞異種移植治療糖尿病的研究進展. 世界華人消化雜志, 2012, 20(9): 737-742.
17. Skiles ML, Wilder NB, Sahai S, et al. Identifying HIF activity in three-dimensional cultures of islet-like clusters. Int J Artif Organs, 2013, 36(3): 175-183.
18. Padmasekar M, Lingwal N, Samikannu B, et al. Exendin-4 protects hypoxic islets from oxidative stress and improves islet transplantation outcome. Endocrinology, 2013, 154(4): 1424-1433.
19. Yook S, Jeong JH, Jung YS, et al. Molecularly engineered islet cell clusters for diabetes mellitus treatment. Cell Transplant, 2012, 21(8): 1775-1789.
20. 魯亞成, 任甫. 胚胎干細胞體外定向誘導分化為胰島細胞的相關研究. 中國組織工程研究與臨床康復, 2007, 11(11): 2156-2159.
21. 李富榮, 龍愛梅, 齊暉, 等. 胰島干細胞轉分化胰島樣細胞與天然胰島功能的比較. 中國組織工程研究與臨床康復, 2008, 12(25): 4833-4836.
22. 孟林燕, 劉暢, 王欣燕, 等. 肌源性干細胞移植修復糖尿病胰島功能的旁分泌機制研究. 中國醫學工程, 2011, 19(5): 35-36, 39.
23. 周靜, 李進, 林戈, 等. 誘導人孤雌胚胎干細胞分化為胰島樣細胞團. 中國組織工程研究與臨床康復, 2010, 14(27): 5001-5005.
24. 張妍, 郭希民, 王常勇, 等. 硒促進小鼠胚胎干細胞分化為胰島細胞的實驗研究. 軍事醫學科學院院刊, 2004, 28(2): 126-129, 139.
25. Wang JC, Xia L, Song XB, et al. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells improves survival of ultra-long random skin flap. Chin Med J (Engl), 2011, 124(16): 2507-2511.
26. Lennon DP, Caplan AI. Isolation of rat marrow-derived mesenchymal stem cells. Exp Hematol, 2006, 34(11): 1606-1607.
27. Fehrer C, Brunauer R, Laschober G, et al. Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell, 2007, 6(6): 745-757.
28. Lu Y, Jin X, Chen Y, et al. Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury. Cell Biochem Funct, 2010, 28(8): 637-643.
29. Grayson WL, Zhao F, Izadpanah R, et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol, 2006, 207(2): 331-339.
30. Xu YX, Chen L, Wang R, et al. Mesenchymal stem cell therapy for diabetes through paracrine mechanisms. Med Hypotheses, 2008, 71(3): 390-393.
31. Figliuzzi M, Cornolti R, Perico N, et al. Bone marrow-derived mesenchymal stem cells improve islet graft function in diabetic rats. Transplant Proc, 2009, 41(5): 1797-1800.
32. Li YY, Liu HH, Chen HL, et al. Adipose-derived mesenchymal stem cells ameliorate STZ-induced pancreas damage in type 1 diabetes. Biomed Mater Eng, 2012, 22(1-3): 97-103.
33. Luo L, Badiavas E, Luo JZ, et al. Allogeneic bone marrow supports human islet beta cell survival and function over six months. Biochem Biophys Res Commun, 2007, 361(4): 859-864.
34. Chao KC, Chao KF, Chen CF, et al. A novel human stem cell coculture system that maintains the survival and function of culture islet-like cell clusters. Cell Transplant, 2008, 17(6): 657-664.
35. Cavallari G, Olivi E, Bianchi F, et al. Mesenchymal stem cells and islet cotransplantation in diabetic rats: improved islet graft revascularization and function by human adipose tissue-derived stem cells preconditioned with natural molecules. Cell Transplant, 2012, 21(12): 2771-2781.
36. Clarkin CE, King AJ, Dhadda P, et al. Activin Receptor-like kinase 5 Inhibition Reverses Impairment of Endothelial Cell Viability by Endogenous Islet Mesenchymal Stromal Cells. Stem Cells, 2013, 31(3): 547-559.

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BMSCs對豬胰島缺氧再給氧損傷的保護作用

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《中國修復重建外科雜志》

BMSCs對豬胰島缺氧再給氧損傷的保護作用

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