大鼠肠系膜与肾被膜下移植胰岛治疗糖尿病的疗效对比
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摘要
目的:探讨经大鼠肠系膜与肾被膜下移植胰岛在糖尿病治疗中的疗效对比与新路径.方法:采用链脲佐菌素诱导SD大鼠制作糖尿病模型,随机分为肠系膜部胰岛移植组(肠系膜组)和肾被膜下胰岛移植组(肾被膜组),各18只. V型胶原酶消化供体大鼠胰腺,Ficoll密度梯度法分离、纯化胰岛,双硫腙和台盼蓝染色检测胰岛纯度和活性.移植术后于不同时间点监测两组大鼠血糖波动、血清C肽水平及腹腔糖耐量功能,记录胰岛存活时间并切取移植部位行HE染色观察组织形态学改变.结果:每只供体大鼠获取(347. 08±33. 22)胰岛当量,胰岛纯度为(88. 03±4. 94)%,存活率为(90. 55±3. 98)%.两组大鼠血糖于移植后3 d降至正常水平,与移植前相比明显下降(P <0. 05);高糖刺激实验表明,移植后10、31 d两组大鼠刺激后的血清C肽水平均较刺激前上升(P <0. 05);腹腔糖耐量结果提示,两移植组大鼠糖耐受功能均良好(P> 0. 05);肠系膜组与肾被膜组移植胰岛的中位存活时间分别为61 d与46 d; HE染色显示移植部位可见伴生血管的成团胰岛细胞.结论:经肠系膜移植胰岛能安全有效地发挥降糖作用,恢复糖尿病大鼠正常血糖水平,疗效优于肾被膜下移植.
Objective: To compare the effect of islet transplantation under mesentery and renal capsule on treatment of diabetes in rats and to find out new transplantation routes. Methods: Streptozotocininduced diabetes rat model was developed. The rats were randomly divided into two groups,mesentery islet transplantation group( mesentery group) and renal capsule islet transplantation group( renal capsule group),each group 18 rats. The pancreases of donator rats were digested by collagenase V. Ficoll density gradient method was used to isolate and purify the islets. The purity and viability were estimated by dithizone and trypan blue staining. The blood glucose fluctuation,serum C peptide level and intraperitoneal glucose tolerance were monitored at different times after transplantation. The survival time of the islets was recorded and the histomorphological change of transplantation site was observed by using HE staining. Results: Islet equivalent of( 347. 08 ± 33. 22) was obtained from each donator rat with a purity of( 88. 03 ± 4. 94) % and a survival rate of( 90. 55 ± 3. 98) %. The blood glucose level of all rats dropped to normal value 3 days after transplantation,which showed a marked fall compared to that before transplantation( P < 0. 05). In high glucose stimulation test,the rats were stimulated 10 days and 31 days respectively after transplantation. The results indicated that the serum C peptide level rose in both groups of rats after stimulation( P < 0. 05) when compared to that before stimulation. The rats of both groups demonstrated good intraperitoneal glucose tolerance( P > 0. 05). The median survival time of mesentery group and renal capsule group was 61 days and 46 days,respectively. With HE dyeing,agglomerate islet cells associated with blood vessels were observed at the transplantation site.Conclusion: Islet transplantation under mesentery can exert hypoglycemic effect safely and effectively to recover the blood glucose level of diabetic rats to normal. The therapeutic effect of islet transplantation under mesentery is superior than under renal capsule.
Objective: To compare the effect of islet transplantation under mesentery and renal capsule on treatment of diabetes in rats and to find out new transplantation routes. Methods: Streptozotocininduced diabetes rat model was developed. The rats were randomly divided into two groups,mesentery islet transplantation group( mesentery group) and renal capsule islet transplantation group( renal capsule group),each group 18 rats. The pancreases of donator rats were digested by collagenase V. Ficoll density gradient method was used to isolate and purify the islets. The purity and viability were estimated by dithizone and trypan blue staining. The blood glucose fluctuation,serum C peptide level and intraperitoneal glucose tolerance were monitored at different times after transplantation. The survival time of the islets was recorded and the histomorphological change of transplantation site was observed by using HE staining. Results: Islet equivalent of( 347. 08 ± 33. 22) was obtained from each donator rat with a purity of( 88. 03 ± 4. 94) % and a survival rate of( 90. 55 ± 3. 98) %. The blood glucose level of all rats dropped to normal value 3 days after transplantation,which showed a marked fall compared to that before transplantation( P < 0. 05). In high glucose stimulation test,the rats were stimulated 10 days and 31 days respectively after transplantation. The results indicated that the serum C peptide level rose in both groups of rats after stimulation( P < 0. 05) when compared to that before stimulation. The rats of both groups demonstrated good intraperitoneal glucose tolerance( P > 0. 05). The median survival time of mesentery group and renal capsule group was 61 days and 46 days,respectively. With HE dyeing,agglomerate islet cells associated with blood vessels were observed at the transplantation site.Conclusion: Islet transplantation under mesentery can exert hypoglycemic effect safely and effectively to recover the blood glucose level of diabetic rats to normal. The therapeutic effect of islet transplantation under mesentery is superior than under renal capsule.
引文
[1] GVAZAVA I G,ROGOVAYA O S,BORISOV M A,et al. Pathogenesis of type 1 diabetes mellitus and rodent experimental models[J]. Acta Naturae,2018,10(1):24-33.
[2] AGARWAL A,BRAYMAN K L. Update on islet cell transplantation for type 1 diabetes[J]. Semin Intervent Radiol,2012,29(2):90-98.
[3] VANTYGHEM M C, DEFRANCE F, QUINTIN D.Treating diabetes with islet transplantation:Lessons from the past decade in Lille[J]. Diabetes&Metabolism,2014,40(2):108-119.
[4] LIU X,ZHANG J,LI Y,et al. Gastric subserous space islet transplantation:techniques and initial results in diabetic inbred Lewis rats[J]. Annals of Transplantation,2014,19:331-336.
[5] MATSUMOTO S,TAKITA M,SHIMODA M,et al.Impact of tissue volume and purification on clinical autologous islet transplantation for the treatment of chronic pancreatitis[J]. Cell Transplantation,2012,21(4):625-532.
[6]李峰,李晓航,张佳林.胰岛移植部位研究进展[J].中华器官移植杂志,2018,39(3):182-186.LI F,LI X H,ZHANG J L. Advances in the site of islet transplantation[J]. Chin J Organ Transplant,2018,39(3):182-186.
[7]胡倩,傅红兴,蒋煊,等.经脾实质胰岛移植治疗糖尿病小鼠的实验研究[J].肝胆胰外科杂志,2017,29(6):486-489.HU Q,FU H X,JIANG X,et al. An experimental study on the treatment of diabetes by islets allotransplantation into spleen in mice[J]. Journal of Hepatopancreatobiliary Surgery,2017,29(6):486-489.
[8] STOKES R A, CHENG K, LALWANI A, et al.Transplantation sites for human and murine islets[J].Diabetologia,2017,60(10):1961-1971.
[9] ESPES D,LAU J,QUACH M,et al. Rapid restoration of vascularity and oxygenation in mouse and human islets transplanted to omentum may contribute to their superior function compared to intraportally transplanted islets[J].American Journal of Transplantation,2016,16(11):3246-3254.
[10] DING X,WANG X,XUE W,et al. Blockade of the nuclear factor kappa B pathway prolonged islet allograft survival[J]. Artificial Organs,2012,36(3):21-27.
[11]刘定志,罗诗樵,杜成友,等.移植部位对移植胰岛长期存活的影响[J].上海交通大学学报(医学版),2012,32(3):257-262.LIU D Z,LUO S Q,DU C Y,et al. Influence of graft site on long-term survival of transplanted pancreatic islet[J]. Journal of Shanghai Jiaotong University(Medical Science Edition),2012,32(3):257-262.
[12] LEMBERT N,WESCHE J,PETERSEN P,et al. Areal density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets[J]. Cell Transplantation,2003,12(1):33-41.
[13]焦自钊,薛武军,孙元竹,等.联合移植的血管内皮细胞对胰岛移植物血管重建及移植效果的影响[J]中华内分泌代谢杂志,2018,34(6):516-521.JIAO Z Z,XUE W J,SUN Y Z,et al. The revascular and transplanted effects of vascular endothelial cells on islets grafts[J]. Chinese Journal of Endocrinology and Metabolism,2018,34(6):516-521.
[14] KIM H I,YU J E,PARK C G,et al. Comparison of four pancreatic islet implantation sites[J]. Journal of Korean Medical Science,2010,25(2):203-210.
[15] AGARWAL A,BRAYMAN K L. Update on islet cell transplantation for type 1 diabetes[J]. Semin Intervent Radiol,2012,29(2):90-98.
[16] WINDT D J,ECHEVERRI G J,IJZERMANS J N,et al.The choice of anatomical site for islet transplantation[J].Cell Transplantation,2008,17(9):1005-1014.
[17]杨洁.糖化血红蛋白与血清C肽联合检验对糖尿病的诊断价值[J].中国实用医刊,2018,9(45):31-36.YANG JIE. Diagnosis value of combined test of glycosylated hemoglobin and serum C peptide on diagnosis diabetes[J]. Chinese Journal of Practical Medicine,2018,9(45):31-36
[18] STICE M J,DUNN T B,BELLIN M D,et al. Omental pouch technique for combined site islet autotransplantation following total pancreatectomy[J]. Cell transplantation,2018,27(10):1561-1568.
[19] BERMAN D M,MOLANO R D,FOTINO C,et al.Bioengineering the endocrine pancreas:intraomental islet transplantation within a biologic resorbable scaffold[J].Diabetes,2016,65(5):1350-1361.
[20] LITBARG N O,GUDEHITHLU K P,SETHUPATHI P,et al. Activated omentum becomes rich in factors that promote healing and tissue regeneration[J]. Cell&Tissue Research,2007,328(3):487-497.
[21] YIN Z Z,WANG S S,LI Q,et al. Gastric submucosa is inferior to the liver as transplant site for autologous islet transplantation in pancreatectomized diabetic beagles[J].Huazhong Univ Sci Technolog Med Sci,2016,36(4):529-533.
[22] ESPES D,LAU J,QUACH M,et al. Rapid restoration of vascularity and oxygenation in mouse and human islets transplanted to omentum may contribute to their superior function compared to intraportally transplanted islets[J].American Journal of Transplantation,2016,16(11):3246-3254.
[23] BERMAN D M,MOLANO R D,FOTINO C,et al.Bioengineering the endocrine pancreas:intraomental islet transplantation within a biologic resorbable scaffold[J].Diabetes,2016,65(5):1350-1361.
[24] STOKES R A,SIMOND D M,BURNS H,et al.Transplantation sites for porcine islets[J]. Diabetologia,2017,60(10):1972-1976.
[25] BAIDAL D A,RICORDI C,BERMAN D M,et al.Bioengineering of an intraabdominal endocrine pancreas[J]. New England Journal of Medicine,2017,376(19):1887-1889.
[2] AGARWAL A,BRAYMAN K L. Update on islet cell transplantation for type 1 diabetes[J]. Semin Intervent Radiol,2012,29(2):90-98.
[3] VANTYGHEM M C, DEFRANCE F, QUINTIN D.Treating diabetes with islet transplantation:Lessons from the past decade in Lille[J]. Diabetes&Metabolism,2014,40(2):108-119.
[4] LIU X,ZHANG J,LI Y,et al. Gastric subserous space islet transplantation:techniques and initial results in diabetic inbred Lewis rats[J]. Annals of Transplantation,2014,19:331-336.
[5] MATSUMOTO S,TAKITA M,SHIMODA M,et al.Impact of tissue volume and purification on clinical autologous islet transplantation for the treatment of chronic pancreatitis[J]. Cell Transplantation,2012,21(4):625-532.
[6]李峰,李晓航,张佳林.胰岛移植部位研究进展[J].中华器官移植杂志,2018,39(3):182-186.LI F,LI X H,ZHANG J L. Advances in the site of islet transplantation[J]. Chin J Organ Transplant,2018,39(3):182-186.
[7]胡倩,傅红兴,蒋煊,等.经脾实质胰岛移植治疗糖尿病小鼠的实验研究[J].肝胆胰外科杂志,2017,29(6):486-489.HU Q,FU H X,JIANG X,et al. An experimental study on the treatment of diabetes by islets allotransplantation into spleen in mice[J]. Journal of Hepatopancreatobiliary Surgery,2017,29(6):486-489.
[8] STOKES R A, CHENG K, LALWANI A, et al.Transplantation sites for human and murine islets[J].Diabetologia,2017,60(10):1961-1971.
[9] ESPES D,LAU J,QUACH M,et al. Rapid restoration of vascularity and oxygenation in mouse and human islets transplanted to omentum may contribute to their superior function compared to intraportally transplanted islets[J].American Journal of Transplantation,2016,16(11):3246-3254.
[10] DING X,WANG X,XUE W,et al. Blockade of the nuclear factor kappa B pathway prolonged islet allograft survival[J]. Artificial Organs,2012,36(3):21-27.
[11]刘定志,罗诗樵,杜成友,等.移植部位对移植胰岛长期存活的影响[J].上海交通大学学报(医学版),2012,32(3):257-262.LIU D Z,LUO S Q,DU C Y,et al. Influence of graft site on long-term survival of transplanted pancreatic islet[J]. Journal of Shanghai Jiaotong University(Medical Science Edition),2012,32(3):257-262.
[12] LEMBERT N,WESCHE J,PETERSEN P,et al. Areal density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets[J]. Cell Transplantation,2003,12(1):33-41.
[13]焦自钊,薛武军,孙元竹,等.联合移植的血管内皮细胞对胰岛移植物血管重建及移植效果的影响[J]中华内分泌代谢杂志,2018,34(6):516-521.JIAO Z Z,XUE W J,SUN Y Z,et al. The revascular and transplanted effects of vascular endothelial cells on islets grafts[J]. Chinese Journal of Endocrinology and Metabolism,2018,34(6):516-521.
[14] KIM H I,YU J E,PARK C G,et al. Comparison of four pancreatic islet implantation sites[J]. Journal of Korean Medical Science,2010,25(2):203-210.
[15] AGARWAL A,BRAYMAN K L. Update on islet cell transplantation for type 1 diabetes[J]. Semin Intervent Radiol,2012,29(2):90-98.
[16] WINDT D J,ECHEVERRI G J,IJZERMANS J N,et al.The choice of anatomical site for islet transplantation[J].Cell Transplantation,2008,17(9):1005-1014.
[17]杨洁.糖化血红蛋白与血清C肽联合检验对糖尿病的诊断价值[J].中国实用医刊,2018,9(45):31-36.YANG JIE. Diagnosis value of combined test of glycosylated hemoglobin and serum C peptide on diagnosis diabetes[J]. Chinese Journal of Practical Medicine,2018,9(45):31-36
[18] STICE M J,DUNN T B,BELLIN M D,et al. Omental pouch technique for combined site islet autotransplantation following total pancreatectomy[J]. Cell transplantation,2018,27(10):1561-1568.
[19] BERMAN D M,MOLANO R D,FOTINO C,et al.Bioengineering the endocrine pancreas:intraomental islet transplantation within a biologic resorbable scaffold[J].Diabetes,2016,65(5):1350-1361.
[20] LITBARG N O,GUDEHITHLU K P,SETHUPATHI P,et al. Activated omentum becomes rich in factors that promote healing and tissue regeneration[J]. Cell&Tissue Research,2007,328(3):487-497.
[21] YIN Z Z,WANG S S,LI Q,et al. Gastric submucosa is inferior to the liver as transplant site for autologous islet transplantation in pancreatectomized diabetic beagles[J].Huazhong Univ Sci Technolog Med Sci,2016,36(4):529-533.
[22] ESPES D,LAU J,QUACH M,et al. Rapid restoration of vascularity and oxygenation in mouse and human islets transplanted to omentum may contribute to their superior function compared to intraportally transplanted islets[J].American Journal of Transplantation,2016,16(11):3246-3254.
[23] BERMAN D M,MOLANO R D,FOTINO C,et al.Bioengineering the endocrine pancreas:intraomental islet transplantation within a biologic resorbable scaffold[J].Diabetes,2016,65(5):1350-1361.
[24] STOKES R A,SIMOND D M,BURNS H,et al.Transplantation sites for porcine islets[J]. Diabetologia,2017,60(10):1972-1976.
[25] BAIDAL D A,RICORDI C,BERMAN D M,et al.Bioengineering of an intraabdominal endocrine pancreas[J]. New England Journal of Medicine,2017,376(19):1887-1889.