Co-transplantation of fetal rat pancreatic stem cells and islets of langerhans to preserve viability of islets

doi:10.3969/j.issn.2095-4344.2014.19.017

Abstract

Abstract:

BACKGROUND: Pancreatic stem cells can maintain the islet structure in vitro, reduce necrosis and apoptosis of islet cells, in vitro prolong islet survival, and keep islet activity.
OBJECTIVE: To observe the possibility of preserving viability of islets in vivo by co-transplanting fetal rat pancreatic stem cells and islets so as to improve the outcome of islet transplantation.
METHODS: Thirty-five adult rats were randomly divided into five groups, including co-transplantation, islet transplantation alone, pancreatic stem cell transplantation alone, diabetes control and normal control groups. Diabetic models were established in the former four groups by intraperitoneal injection of streptozotocin-citrate buffer. Pancreatic stem cells from the fetal rats at pregnant 16 days or islets from adult Sprague-Dawley rats.
RESULTS AND CONCLUSION: In the co-transplantation group, the levels of blood glucose and plasma insulin returned to the normal after 5 days of co-transplantation, and the survival time of islets was (18.2±2.4) days. In the islet transplantation alone group, the level of blood glucose was reduced to normal after 1 week of transplantation, and the survival time of islets was (14.4±2.1) days. Significant different was found between the survival time of islets between this two groups (P < 0.05). However, the level of blood glucose was still abnormal in the other groups. These findings indicate that the co-transplantation of fetal rat pancreatic stem cells and islets can prolong the survival time of islets in vivo, protect viability of islets and improve the outcome of islet transplantation.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: islets of langerhans, islets of langerhans transplantation, pancreas, insulin

CLC Number:

R394.2

Zhang Yu-sen, Bao Shi-yun, Zhang Yue, Li Fu-rong. Co-transplantation of fetal rat pancreatic stem cells and islets of langerhans to preserve viability of islets[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(19): 3054-3059.

Figures/Tables 1

2.1 培养的胎鼠胰腺干细胞胎鼠胰腺接种于培养板中，可见细胞自组织块中爬出、贴壁生长，细胞呈长梭形，类似成纤维细胞，胞核大近椭圆形，胞核胞浆比例大致相等，随着培养时间延长，细胞可增殖生长并爬满瓶底；取第3代细胞经免疫细胞化学染色可见巢蛋白阳性细胞，证实为胰腺干细胞；经计数可见巢蛋白阳性细胞约占80%。经流式细胞术鉴定巢蛋白阳性细胞约占74.1%，纯度满足进一步实验要求。见图1A，B。 2.2 分离纯化的胰岛分离纯化后经双硫腙染色，胰岛呈猩红色，圆形或椭圆形，大小不等，包膜完整。15只SD大鼠中，每只胰腺消化后可获得(784±51)个胰岛，Ficoll-400纯化后获得(361±32)个胰岛；回收率平均为46.1%，纯度达90%以上。见图1C，D。 2.3 实验动物数量分析所有大鼠均进入结果分析。糖尿病模型大鼠腹腔注射40 mg/kg 链脲霉素后，均于3 d内出现多饮症状，3 d后随机血糖均大于16.7 mmol/L，并一直维持高血糖状态。胰腺干细胞和胰岛移植过程顺利，未出现手术相关死亡(术后1周内死亡)，模型成功率100%，见图2A。 2.4 胰腺干细胞和胰岛移植后糖尿病大鼠血糖及血胰岛素水平的变化联合移植组大鼠均于术后第3天起血糖开始下降，血浆胰岛素水平逐渐升高，术后5 d内血糖可降至正常，血清胰岛素升高至正常水平；单独胰岛移植组大鼠血糖可于术后1周内降至正常，同时血清胰岛素达到正常水平；而观察期间单纯胰腺干细胞移植组大鼠的血糖及胰岛素一直未达到正常。经重复测量设计的方差分析表明各组间血糖及血胰岛素随时间变化的趋势不同(P < 0.05)，联合移植组大鼠血糖降至正常及血胰岛素升至正常时间均快于单独胰岛移植组，且维持时间更长(P < 0.05)；联合移植组胰岛存活时间明显长于单独胰岛移植组(P < 0.05)。而单纯胰腺干细胞移植组大鼠观察期内移植处于高血糖状态，且血胰岛素水平低(图3)。 2.5 胰腺干细胞和胰岛移植后糖尿病大鼠肾脏组织的病理变化苏木精-伊红染色显示，联合移植组及单独胰岛移植组大鼠肾下极包膜下均可见残存胰岛，周围可见多量炎症细胞浸润，提示存在排斥反应，部分胰岛已基本破坏，但联合移植组大鼠肾脏组织中的炎症反应较单独胰岛移植组稍轻，且残存胰岛较单独胰岛移植组稍多；而单纯胰腺干细胞移植组大鼠肾下极包膜下可见少量炎症细胞浸润，未见明显胰岛样细胞团形成(图2B)。"

References

[1] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 2000;343(4):230-238.
[2] Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med. 2006;355(13):1318-1330.
[3] 刘宝林,刘世庆,曹献馗.胰岛移植的临床与科研现状及展望[J].世界华人消化杂志,2011,20(33):3186-3190.
[4] 唐成佳,杜成友,刘定志,等.新鲜与短期培养胰岛细胞移植对移植胰岛存活的影响[J].上海交通大学学报:医学版, 2013,33(6): 763-767.
[5] 刘定志,杜成友,罗诗樵,等.三种消化酶分离胰岛对移植胰岛存活的影响[J].上海交通大学学报:医学版,2012,32(5):567-571.
[6] Prockop DJ, Gregory CA, Spees JL. One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues. Proc Natl Acad Sci U S A. 2003;100 Suppl 1: 11917-11923.
[7] Munoz JR, Stoutenger BR, Robinson AP, et al. Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. Proc Natl Acad Sci U S A. 2005; 102(50):18171-18176.
[8] Spees JL, Olson SD, Ylostalo J, et al. Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci U S A. 2003;100(5):2397-2402.
[9] Spees JL, Olson SD, Whitney MJ, et al. Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci U S A. 2006;103(5):1283-1288.
[10] Lee RH, Seo MJ, Reger RL, et al. Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice. Proc Natl Acad Sci U S A. 2006;103(46):17438-17443.
[11] Scuteri A, Cassetti A, Tredici G. Adult mesenchymal stem cells rescue dorsal root ganglia neurons from dying. Brain Res. 2006;1116(1):75-81.
[12] Baffour R, Pakala R, Hellinga D, et al. Bone marrow-derived stem cell interactions with adult cardiomyocytes and skeletal myoblasts in vitro. Cardiovasc Revasc Med. 2006;7(4): 222-230.
[13] Chang Liu Z, Chang TM. Coencapsulation of hepatocytes and bone marrow cells: in vitro and in vivo studies. Biotechnol Annu Rev. 2006;12:137-151.
[14] Lacy PE, Kostianovsky M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes. 1967; 16(1):35-39.
[15] Fraidenraich D, Stillwell E, Romero E, et al. Rescue of cardiac defects in id knockout embryos by injection of embryonic stem cells. Science. 2004;306(5694):247-252.
[16] Kerr DA, Lladó J, Shamblott MJ, et al. Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron injury. J Neurosci. 2003;23(12): 5131-5140.
[17] Lladó J, Haenggeli C, Maragakis NJ, et al. Neural stem cells protect against glutamate-induced excitotoxicity and promote survival of injured motor neurons through the secretion of neurotrophic factors. Mol Cell Neurosci. 2004;27(3):322-331.
[18] Johansson U, Elgue G, Nilsson B, et al. Composite islet-endothelial cell grafts: a novel approach to counteract innate immunity in islet transplantation. Am J Transplant. 2005;5(11):2632-2639.
[19] 贺德,詹文华,彭翔,等.Sertoli细胞诱导大鼠肝内胰岛移植物免疫豁免的实验研究[J].中华实验外科杂志,2004,21(9):1056-1057.
[20] Soto-Montenegro L, Zugasti-Murillo A, Rollán E, et al. Influence of hepatocyte-rich liver cell mixture and liver fibroblasts on prolonging graft islet survival in rats without immunosuppressive drugs. Exp Clin Endocrinol Diabetes. 2004;112(10):580-586.
[21] Yin Z, Wu W, Fung JJ, et al. Cotransplanted hepatic stellate cells enhance vascularization of islet allografts. Microsurgery. 2007;27(4):324-327.
[22] Oh BJ, Oh SH, Jin SM, et al. Co-transplantation of bone marrow-derived endothelial progenitor cells improves revascularization and organization in islet grafts. Am J Transplant. 2013;13(6):1429-1440.
[23] Rackham CL, Dhadda PK, Chagastelles PC, et al. Pre-culturing islets with mesenchymal stromal cells using a direct contact configuration is beneficial for transplantation outcome in diabetic mice. Cytotherapy. 2013;15(4):449-459.
[24] Rackham CL, Jones PM, King AJ. Maintenance of islet morphology is beneficial for transplantation outcome in diabetic mice. PLoS One. 2013;8(2):e57844.
[25] Kerby A, Jones ES, Jones PM, et al. Co-transplantation of islets with mesenchymal stem cells in microcapsules demonstrates graft outcome can be improved in an isolated-graft model of islet transplantation in mice. Cytotherapy. 2013;15(2):192-200.
[26] Olerud J, Kanaykina N, Vasylovska S, et al. Neural crest stem cells increase beta cell proliferation and improve islet function in co-transplanted murine pancreatic islets. Diabetologia. 2009; 52(12):2594-2601.
[27] 张育森,周汉新,张悦,等.与胎鼠胰腺干细胞共培养保护大鼠胰岛活性[J].中国组织工程研究与临床康复,2008,12(12): 2313-2316.
[28] 周淑艳,张毅,齐晖,等.保护胰岛β细胞的体外研究进展[J].生命科学,2012,24(10):1207-1210.
[29] Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005; 105(4):1815-1822.
[30] Bonde S, Zavazava N. Immunogenicity and engraftment of mouse embryonic stem cells in allogeneic recipients. Stem Cells. 2006;24(10):2192-2201..
[31] Einstein O, Fainstein N, Vaknin I, et al. Neural precursors attenuate autoimmune encephalomyelitis by peripheral immunosuppression. Ann Neurol. 2007;61(3):209-218.
[32] 张剑,张琪,张英才,等.骨髓间充质干细胞对异种胰岛移植排斥反应的影响[J].器官移植,2011,2(6):350-353.
[33] Perez-Basterrechea M, Obaya AJ, Meana A, et al. Cooperation by fibroblasts and bone marrow-mesenchymal stem cells to improve pancreatic rat-to-mouse islet xenotransplantation. PLoS One. 2013;8(8):e73526.