• 四川大學(xué)華西醫(yī)院（成都，610041） 1 衛(wèi)生部移植工程與移植免疫重點實驗室，2 胰腺外科;

【摘要】　目的　 探討同種異基因骨髓間充質(zhì)干細(xì)胞（bone mesenchamal stem cells，BMSC）靜脈輸注對大鼠到小鼠胰島移植物的功能保護(hù)和小鼠糖尿病狀態(tài)改善。　方法　 全骨髓培養(yǎng)法獲得C57BL/6小鼠BMSC。不連續(xù)梯度離心法分離純化Sprague-Dawley（SD）大鼠胰島，將300胰島當(dāng)量的胰島單獨或與BMSC聯(lián)合移植入鏈脲菌素誘導(dǎo)的糖尿病BALB/c小鼠腎包膜下，并通過尾靜脈在移植后0、3和5 d注射CM-DiI標(biāo)記的BMSC 5×105/只，對照組給于磷酸鹽緩沖溶液。移植后監(jiān)測血糖，第9天處死小鼠，取肝、脾、胸腺、淋巴結(jié)和移植胰島的腎臟，冰凍切片，熒光顯微鏡觀察CM-DiI標(biāo)記細(xì)胞的組織分布；免疫熒光法觀察移植物中胰島素和胰高血糖素表達(dá)，評價胰島的功能。　結(jié)果　 BMSC靜脈輸注后主要分布于胸腺，其次是脾臟和淋巴結(jié)，腎和肝組織中未觀察到BMSC；BMSC聯(lián)合胰島移植組血糖控制水平優(yōu)于其他組，且在第7天的口服糖耐量實驗優(yōu)于單純胰島移植組。　結(jié)論　 與胰島聯(lián)合移植的BMSC對受者免疫器官和組織有明顯的趨向性，且對胰島細(xì)胞的體內(nèi)存活有一定保護(hù)作用。
【Abstract】　Objective　 To research on the protection function by the allogeneic rat bone mesenchymal stem cells (BMSC) on rat to mouse islet transplantation and the improvement of diabetic state in mouse. 　Methods　 BMSC were prepared from C57BL/6 mouse bone marrow cells and identified by flow cytometry (FCM). Islets were isolated from Sprague-Dawley (SD) rats with Ficoll discontinuous centrifugation. CM-DiI labeled BMSC at 5×105 for one mouse were intravenously infused into STZ induced diabetic BALB/c mice after rat to mouse islet transplantation at day 0, 3 and 5. Mice with PBS intravenously infused after islet transplantation were set as the negative controls. Blood glucose was monitored every day at the first 3 days after transplantation, and then monitored every two days. At day 9 after transplantation, spleen, thymus, lymph nods, liver and islets recipient kidney were harvested. Ice slices were prepared and CM-DiI labeled cells were investigated with fluorescence microscope. 　Results　 CM-DiI-labeled BMSC were mainly distributed in thymus followed by spleen and lymph nodes. In liver and kidney, there was no red fluorescence observed. The blood sugar control for combined BMSC infusion group was superior to other groups, and the control level of islet combined BMSC infusion group were better than single islet transplantation group in OGTT at day 7. 　Conclusion　 Allogeneic BMSC can sustain the insulin secretion of islets in vivo and tend to distribute in immune organs or adenoid tissues after infusion.

引用本文： 曾力,張爽,魏玲玲,田伯樂,麥剛,張杰,李勝富,陳又南,陸燕蓉. 骨髓間充質(zhì)干細(xì)胞對大鼠到小鼠胰島移植的保護(hù)作用. 華西醫(yī)學(xué), 2011, 26(5): 641-645. doi: 復(fù)制

1. 　Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the edmonton protocol for islet transplantation[J]. N Engl J Med, 2006, 355(13): 1318-1330.
2. 　Nanji SA, Shapiro AM. Advances in pancreatic islet transplantation in humans[J]. Diabetes Obes Meta, 2006, 8(1): 15-25.
3. 　Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation[J]. Diabetes, 2005, 54(7): 2060-2069.
4. 　Frank A, Deng S, Huang X, et al. Transplantation for type 1 diabetes comparison of vascularized whole-organ pancreas with isolated pancreatic islets[J]. Ann Surg, 2004, 240(4): 631-643.
5. 　Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immuno-suppressive regimen[J]. N Engl J Med, 2000, 343(3): 230-238.
6. 　D′Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAM I cells[J]. Bone, 2006, 39(3): 513-522.
7. 　Grayson WL, Zhao F, Izadpanah R, et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs[J]. J Cell Physiol, 2006, 207(2): 331-339.
8. 　Mclntosh K, Bartholomew A. Stromal cell modulation of the immune system[J]. Graft, 2000, 3(6): 324-328.
9. 　Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo[J]. Exp Hematol, 2002, 30(1): 42-48.
10. 　周光炎, 孫方臻. 異種移植[M]. 上海: 上?？茖W(xué)技術(shù)出版社, 2006: 214-218.
11. 　袁宇, 叢聰, 張靜, 等. 大鼠胰島的分離純化方法改進(jìn)與功能鑒定[J]. 中國修復(fù)重建外科雜志, 2008, 22(1): 75-79.
12. 　Garcia R, Aguiar J, Alberti E, et al. Bone marrow stromal cells pruduce nerve growth factor and glial cell line-derived neurotrophic factors[J]. Biochem Biophys Res Commun, 2004, 316(3): 753-754.
13. 　Machado AF, Zimmerman EF, Hovland DN Jr, et al. Diabetic embryo-pathy in C57BL/6J mice. Altered fetal sex ratio and impact of the splotch allele[J]. Diabetes, 2001, 50(5): 1193-1199.
14. 　Lu Y, Jin X, Chen Y, et al. Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury[J]. Cell Biochem Funct, 2010, 28(8): 637-643.
15. 　Roh D, Nelson GN, Udelsman BV, et al. Centrifugal seeding increases seeding efficiency and cellular distribution of bone marrow stromal cells in porous biodegradable scaffolds[J]. Tissue Eng, 2007, 13(11): 2743-2749.
16. 　Nauta AJ, Westerhuis G, Kruisselbrink AB, et al. Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting[J]. Blood, 2006, 108(6): 2114-2120.
  1. 1. 　Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the edmonton protocol for islet transplantation[J]. N Engl J Med, 2006, 355(13): 1318-1330.
  2. 2. 　Nanji SA, Shapiro AM. Advances in pancreatic islet transplantation in humans[J]. Diabetes Obes Meta, 2006, 8(1): 15-25.
  3. 3. 　Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation[J]. Diabetes, 2005, 54(7): 2060-2069.
  4. 4. 　Frank A, Deng S, Huang X, et al. Transplantation for type 1 diabetes comparison of vascularized whole-organ pancreas with isolated pancreatic islets[J]. Ann Surg, 2004, 240(4): 631-643.
  5. 5. 　Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immuno-suppressive regimen[J]. N Engl J Med, 2000, 343(3): 230-238.
  6. 6. 　D′Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAM I cells[J]. Bone, 2006, 39(3): 513-522.
  7. 7. 　Grayson WL, Zhao F, Izadpanah R, et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs[J]. J Cell Physiol, 2006, 207(2): 331-339.
  8. 8. 　Mclntosh K, Bartholomew A. Stromal cell modulation of the immune system[J]. Graft, 2000, 3(6): 324-328.
  9. 9. 　Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo[J]. Exp Hematol, 2002, 30(1): 42-48.
  10. 10. 　周光炎, 孫方臻. 異種移植[M]. 上海: 上?？茖W(xué)技術(shù)出版社, 2006: 214-218.
  11. 11. 　袁宇, 叢聰, 張靜, 等. 大鼠胰島的分離純化方法改進(jìn)與功能鑒定[J]. 中國修復(fù)重建外科雜志, 2008, 22(1): 75-79.
  12. 12. 　Garcia R, Aguiar J, Alberti E, et al. Bone marrow stromal cells pruduce nerve growth factor and glial cell line-derived neurotrophic factors[J]. Biochem Biophys Res Commun, 2004, 316(3): 753-754.
  13. 13. 　Machado AF, Zimmerman EF, Hovland DN Jr, et al. Diabetic embryo-pathy in C57BL/6J mice. Altered fetal sex ratio and impact of the splotch allele[J]. Diabetes, 2001, 50(5): 1193-1199.
  14. 14. 　Lu Y, Jin X, Chen Y, et al. Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury[J]. Cell Biochem Funct, 2010, 28(8): 637-643.
  15. 15. 　Roh D, Nelson GN, Udelsman BV, et al. Centrifugal seeding increases seeding efficiency and cellular distribution of bone marrow stromal cells in porous biodegradable scaffolds[J]. Tissue Eng, 2007, 13(11): 2743-2749.
  16. 16. 　Nauta AJ, Westerhuis G, Kruisselbrink AB, et al. Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting[J]. Blood, 2006, 108(6): 2114-2120.