体外诱导hASC向胰岛样细胞团分化及其移植治疗Ⅰ型糖尿病的实验研究
|
摘要
第一部分人脂肪来源干细胞的分离、培养和鉴定
目的
分离、纯化人脂肪来源的干细胞(human adipose tissue-derived stem cell, hASC)并鉴定其生物学特性。
方法
采用zuk等建立的人脂肪间充质干细胞分离培养方法：借助普通外科手术获得脂肪组织；分离、纯化脂肪来源干细胞；流式细胞术分析第四代细胞表面标志和胞浆抗原表达；绘制细胞生长曲线；借助端粒酶活性检测,评价其体外增殖能力；借助吉姆萨染色,分析第4、8、12、16代细胞染色体核型,评价干细胞遗传稳定性；双层琼脂培养检测干细胞成瘤性。
结果
在培养第四代始,细胞出现均一的旋涡状生长,增殖速度较快。流式细胞术分析显示,第四代细胞表达CD73(99.1%)、CD90(97.9%)、CD105(97.2%),符合间充质干细胞特征。细胞生长曲线显示,细胞倍增时间约为36小时。染色体核型分析显示,hASCs为正常二倍体核型,G显带检测未见异常。体外双层琼脂检测未见成瘤性。
结论
hASC经分离、纯化,可在体外有效扩增,符合脂肪间充质干细胞表面标志物鉴定的国际标准和公认的干细胞特征。体外培养过程,hASC不表达胰腺相关标志物,染色体核型稳定且无变异,未见成瘤性。
第二部分胎胰蛋白/损伤胰腺蛋白复合物诱导hASC向胰腺方向的分化
目的
评价大鼠胚胎胰腺/损伤胰腺组织的可溶性蛋白提取物诱导hASC向胰岛素分泌细胞分化的能力。
方法
制备大鼠胚胎胰腺/损伤胰腺的蛋白匀浆,提取可溶性细胞因子复合物,与hASC共培养20天,在不同时间点检测胰腺发育相关基因表达水平和胰岛素/C肽分泌水平。
结果
胚胎胰腺/损伤胰腺的蛋白提取物,可诱导hASC表达与胰腺发育密切相关的基因产物(如PDX-1、Pax-4、Pax-6等),部分分化细胞可表达胰岛素和C肽。
结论
提示损伤胰腺和胚胎胰腺内富含胰腺发育相关蛋白,可诱导hASC向胰腺方向分化,并最终诱生胰岛素、C-肽阳性细胞。
第三部分胰岛新生相关蛋白肽方案促进hASC向胰腺方向的深度分化
目的
评价胰岛新生相关蛋白肽4步方案诱导hASC分化为胰岛样细胞团的效果。
方法
应用胰岛新生蛋白肽联合胰腺发育相关细胞因子及化学因子进行四步诱导,RT-PCR、实时定量PCR及细胞免疫荧光检测诱导后细胞胰腺发育及功能基因表达的改变,在不同浓度(5.6mM、25mM)葡萄糖刺激下,检测分化细胞胰岛素及C肽分泌量,评价细胞的葡萄糖应激能力。
结果
诱导过程中,hASC的干细胞相关基因Oct3/4表达降低至原始水平的1/20,而与胰腺系分化发育相关的基因(如ck19、nestin PDX-1、nkx2.2等)表达水平均显著升高。诱导分化后期,INGAP-pp诱导组出现直径约150-200μm的胰岛样细胞团,Scramble-pp诱导组出现直径约50-100μm的胰岛样细胞团,前者更接近天然胰岛的直径。免疫荧光检测显示,INGAP-pp所诱生的胰岛样细胞团内存在胰岛素和C肽双阳性细胞群,可对不同浓度葡萄糖刺激产生反应,分泌量约占天然胰岛细胞1/10-1/15。
结论
INGAP-pp诱导方案可经间质细胞向上皮细胞转化的途径,诱导hASC向胰腺系细胞分化,在体外形成具有胰岛素分泌功能的胰岛样细胞团。虽然与天然胰岛相比尚不完善,但提示在适当条件诱导下,hASC确可分化为具有葡萄糖应激能力的胰岛样细胞。
第四部分胰岛样细胞团移植治疗糖尿病大鼠的效果评价
目的
评价纯化INGAP-pp蛋白及INGAP-pp诱导分化产生的ILC移植治疗糖尿病大鼠的效果。
方法
STZ诱导大鼠Ⅰ型糖尿病模型,在体视镜下新鲜分离人胰岛及INGAP-pp诱导组所产生ILC各200个,分别移植于糖尿病大鼠模型左肾包膜下,比较正常胰岛移植组、ILC移植组、INGAP-pp蛋白注射组、糖尿病组及正常大鼠组在实验周期的血糖水平,每3天检测1次。对各组进行腹腔内葡萄糖耐受实验检测,统计各组存活率。
结果
移植正常人胰岛的糖尿病大鼠血糖水平趋于正常,与正常大鼠血糖接近；移植ILC的糖尿病大鼠组血糖水平在术后短暂上升,遂即下降,并在稍高于正常大鼠血糖水平保持稳定；注射INGAP-pp蛋白的糖尿病大鼠在注射过程中血糖下降,停止注射蛋白后缓慢回升；糖尿病大鼠组血糖持续异常,至实验结束。腹腔内葡萄糖应激实验中,移植第20天,移植正常胰岛和ILC的糖尿病大鼠均出现接近于正常大鼠的反应曲线,但ILC移植组血糖水平在各时间点均高于胰岛移植组。由于血糖持续波动,INGAP-pp蛋白注射组未进行葡萄糖应激实验。另外,整个实验过程中,糖尿病组大鼠逐渐死亡,至51天全部死亡；整个实验过程中胰岛移植组和ILC移植组大鼠无一死亡。
结论
ILC移植可降低糖尿病大鼠血糖水平,并延长糖尿病大鼠生存时间。虽然与天然胰岛组相比,降低血糖的程度尚不足,但确已具有葡萄糖应激能力。提示：INGAP-pp诱生的ILC具有治疗糖尿病的临床应用前景。
Part I:
Objective:To isolate, purify and characterize human adipose tissue-derived stem cells.
Methods:With slightly modified protocol demonstrated by zuk et al, human adipose tissue-derived stem cells (hASCs) were isolated and expanded in vitro. FACS analysis was performed to identify the cell surface and cytoplasm antigen.Tolemerase repeated amplification protocol (TRAP) and G-banding Karyotype analysis were performed to evaluate the safety of hASCs cultured in vitro. Results:Since passage 4, hASCs developed a uniform morphology resembling that of fibroblasts, FACS analysis of the fourth passage cells showed purified hASCs expressed high levels of cell surface markers, such as CD73(99.1%),CD90(97.9%), CD105(97.2%). These results are consistent with the definition that mesenchymal stem cells (MSCs) must express CD73, CD90, and CD105. Also, extremely low expression levels of CK-19 and Pdx-1, which supposed to be markers of pancreatic stem cells, were detected. Negative expression of CD45 means no contamination of hemopoiesis stem cells. TRAP test revealed P4, P8, P12 had nomal tolemerase activity and G-banding analysis showed P4, P8, P12 and P16 had normal katyotypes. Conclusions:hASCs could expand in vitro efficiently after being isolated and purified. The hASCs we cultured in the study were consistent to those reported previously. When cultured in vitro, hASCs have stable chromosome structure as devided in vitro and being safe for cytothrapy.
Part II:
In Vitro differentiation of human adipose tissue-derived stem cells into Insulin/c-peptide positive cells by embryonic pancreas extract
Objective:To evaluate the effect of embryo pancreas extract (EPE) from pre-born Sprague-Dawley rats on induction of pancreatic differentiation of human adipose tissue-derived mesenchymal stem cells(hASCs).
Methods:By using real-time PCR and immunofluorescence technology, expression levels of genes related with pancreas development of hASCs cultured with EPE for 20 days were detected.
Results:undifferentiated hASCs expressed key markers of embryo stem cells. Genes involved in early pancreas development showed increased expression in EPE-treated culture. Immunocytochemical analysis showed C-peptide/insulin positive cells in EPE-treated cells but not in undifferentiated hASCs.
Conclusion:hASCs have the potential to differentiate into pancreas cell lineages with EPE induction, so could be substitution of embryo and bone-marrow derived stem cells for future cell therapy applications.
Part III:
IN Vitro Differentiation of human adipose-derived stem cells intoIslet-like cell clusters by INGAP-pp protocol
Objective:To establish a novel method for the differentiation of islet-like clusters from human adipose-derived stem cells with INGAP-related pentadecapeptide (INGAP-PP) combined with chemical molecules and cytokines.
Methods:the human adipose-derived stem cells were isolated, purified and expanded in vitro and differentiated using a four-step protocol that included TSA, INGAP-PP/Scrambled peptide (Scrambled-P), nicotinamide and exendin-4. By using real-time PCR and immunofluorescence technology, expression levels of genes related with pancreas development. Insulin and C-peptide released by differentiated cells in response to high/low glucose level were detected by ELISA.
Results Undifferentiated hASCs expressed oct3/4, key markers of embryo stem cells and at the end of induction, Oct3/4 expression level decreased to 1/20 of original. The expression level of genes involved in early pancreas development increased in both INGAP-PP/Scrambled peptide culture significantly. Islet-like clusters were obtained in both INGAP-PP/Scrambled peptide group, but in INGAP-pp group, the diameters of clusters were much closer to native islets, which were about 150-200μm. Immunocytochemical analysis showed C-peptide/insulin positive cells in INGAP-pp induced group but not in undifferentiated hMSCs.
Conclusions:By INGAP-pp protocol, hASCs could differentiate into islet-like clusters more efficiently through Mesenchymal/epithelium transition. It suggests hASCs have the potential to differentiate into pancreas cell lineages by INGAP-pp protocol; so could be substitution of embryo or bone-marrow derived stem cells for future cell therapy applications.
Part IV:
Objective:To evaluate the effects of transplantation of ILCs induced by INGAP-pp and INGAP-pp injection to cure diabetic rats.
Methods:Diabetes was induced in 8-week-old male Sprague-Dawley rats by streptozocin (Sigma,55 mg/kg body weight in citrate buffer, pH 4.4, i.p).48 hours after injection, animals with blood glucose levels≥16.65 mmol/L for two consecutive days were transplanted with ILCs induced by INGAP-pp (200IEQ/l00μ1 High glucose DMEM) into the left subrenal capsule (n=10). As controls, fresh isolated human islets (200IEQ/100μ1 High glucose DMEM) and saline without cells were transplanted respectively. Two transplantation group were administered acrolimus (50ng/kg.d,p.o.) to avoid of immune rejection. To evaluate the effects of INGAP-pp protein on remission of diabetes, we also injected purified INGAP-PP (250μg/0.5mL, i.p.) to diabetic rats twice a day for twenty days. Blood glucose levels of all groups were measured every three days. Glucose tolerance tests were performed after injection of glucose (2g/kg bodywight, i.p.). Diabetic rats transplanted with normal islets and normal rats served as positive controls. Diabetic rats were used as negative control.
Results:Blood glucose levels of rats transplanted with INGAP-induced ILCs fell to 7.8±0.8 mmol/L and kept this level for the whole experimental process while diabetic rats transplanted with normal islets, as a control, showed blood glucose levels similar to normal rats after 1-2 weeks. In addition, non-transplanted diabetic rats remained hyperglycemic and died consecutively. Interestingly, the group treated with INGAP-pp protein injection also showed a decrease of blood glucose level after 5-day injections, but never fell to normal level and re-raised 2 weeks later since the day INGAP-PP injection stopped. Diabetic rats transplanted with INGAP-induced ILCs or normal islets survived the whole 69-day process while diabetic rats without treatment suffered from hyperglycemic and died off within 51 days. Diabetic rats undergone INGAP-pp injections also all survived until the end of experiment. Intraperitoneal glucose tolerance tests showed SD rats transplanted with ILCs induced by INGAP-pp had the similar kinetics of the glucose clearance to the group transplanted with normal islets, although the blood glucose levels were higher at all time points. In contrast, untreated diabetic rats revealed no reaction to the high glucose concentration
Conclusions:The results of transplantation of ILCs and normal islets to diabetic rats showed ILCs did decrease the blood glucose levels and extended the life-span of diabetic rats. ILCs transplantation reversed diabetes partially in animal models.
目的
分离、纯化人脂肪来源的干细胞(human adipose tissue-derived stem cell, hASC)并鉴定其生物学特性。
方法
采用zuk等建立的人脂肪间充质干细胞分离培养方法：借助普通外科手术获得脂肪组织；分离、纯化脂肪来源干细胞；流式细胞术分析第四代细胞表面标志和胞浆抗原表达；绘制细胞生长曲线；借助端粒酶活性检测,评价其体外增殖能力；借助吉姆萨染色,分析第4、8、12、16代细胞染色体核型,评价干细胞遗传稳定性；双层琼脂培养检测干细胞成瘤性。
结果
在培养第四代始,细胞出现均一的旋涡状生长,增殖速度较快。流式细胞术分析显示,第四代细胞表达CD73(99.1%)、CD90(97.9%)、CD105(97.2%),符合间充质干细胞特征。细胞生长曲线显示,细胞倍增时间约为36小时。染色体核型分析显示,hASCs为正常二倍体核型,G显带检测未见异常。体外双层琼脂检测未见成瘤性。
结论
hASC经分离、纯化,可在体外有效扩增,符合脂肪间充质干细胞表面标志物鉴定的国际标准和公认的干细胞特征。体外培养过程,hASC不表达胰腺相关标志物,染色体核型稳定且无变异,未见成瘤性。
第二部分胎胰蛋白/损伤胰腺蛋白复合物诱导hASC向胰腺方向的分化
目的
评价大鼠胚胎胰腺/损伤胰腺组织的可溶性蛋白提取物诱导hASC向胰岛素分泌细胞分化的能力。
方法
制备大鼠胚胎胰腺/损伤胰腺的蛋白匀浆,提取可溶性细胞因子复合物,与hASC共培养20天,在不同时间点检测胰腺发育相关基因表达水平和胰岛素/C肽分泌水平。
结果
胚胎胰腺/损伤胰腺的蛋白提取物,可诱导hASC表达与胰腺发育密切相关的基因产物(如PDX-1、Pax-4、Pax-6等),部分分化细胞可表达胰岛素和C肽。
结论
提示损伤胰腺和胚胎胰腺内富含胰腺发育相关蛋白,可诱导hASC向胰腺方向分化,并最终诱生胰岛素、C-肽阳性细胞。
第三部分胰岛新生相关蛋白肽方案促进hASC向胰腺方向的深度分化
目的
评价胰岛新生相关蛋白肽4步方案诱导hASC分化为胰岛样细胞团的效果。
方法
应用胰岛新生蛋白肽联合胰腺发育相关细胞因子及化学因子进行四步诱导,RT-PCR、实时定量PCR及细胞免疫荧光检测诱导后细胞胰腺发育及功能基因表达的改变,在不同浓度(5.6mM、25mM)葡萄糖刺激下,检测分化细胞胰岛素及C肽分泌量,评价细胞的葡萄糖应激能力。
结果
诱导过程中,hASC的干细胞相关基因Oct3/4表达降低至原始水平的1/20,而与胰腺系分化发育相关的基因(如ck19、nestin PDX-1、nkx2.2等)表达水平均显著升高。诱导分化后期,INGAP-pp诱导组出现直径约150-200μm的胰岛样细胞团,Scramble-pp诱导组出现直径约50-100μm的胰岛样细胞团,前者更接近天然胰岛的直径。免疫荧光检测显示,INGAP-pp所诱生的胰岛样细胞团内存在胰岛素和C肽双阳性细胞群,可对不同浓度葡萄糖刺激产生反应,分泌量约占天然胰岛细胞1/10-1/15。
结论
INGAP-pp诱导方案可经间质细胞向上皮细胞转化的途径,诱导hASC向胰腺系细胞分化,在体外形成具有胰岛素分泌功能的胰岛样细胞团。虽然与天然胰岛相比尚不完善,但提示在适当条件诱导下,hASC确可分化为具有葡萄糖应激能力的胰岛样细胞。
第四部分胰岛样细胞团移植治疗糖尿病大鼠的效果评价
目的
评价纯化INGAP-pp蛋白及INGAP-pp诱导分化产生的ILC移植治疗糖尿病大鼠的效果。
方法
STZ诱导大鼠Ⅰ型糖尿病模型,在体视镜下新鲜分离人胰岛及INGAP-pp诱导组所产生ILC各200个,分别移植于糖尿病大鼠模型左肾包膜下,比较正常胰岛移植组、ILC移植组、INGAP-pp蛋白注射组、糖尿病组及正常大鼠组在实验周期的血糖水平,每3天检测1次。对各组进行腹腔内葡萄糖耐受实验检测,统计各组存活率。
结果
移植正常人胰岛的糖尿病大鼠血糖水平趋于正常,与正常大鼠血糖接近；移植ILC的糖尿病大鼠组血糖水平在术后短暂上升,遂即下降,并在稍高于正常大鼠血糖水平保持稳定；注射INGAP-pp蛋白的糖尿病大鼠在注射过程中血糖下降,停止注射蛋白后缓慢回升；糖尿病大鼠组血糖持续异常,至实验结束。腹腔内葡萄糖应激实验中,移植第20天,移植正常胰岛和ILC的糖尿病大鼠均出现接近于正常大鼠的反应曲线,但ILC移植组血糖水平在各时间点均高于胰岛移植组。由于血糖持续波动,INGAP-pp蛋白注射组未进行葡萄糖应激实验。另外,整个实验过程中,糖尿病组大鼠逐渐死亡,至51天全部死亡；整个实验过程中胰岛移植组和ILC移植组大鼠无一死亡。
结论
ILC移植可降低糖尿病大鼠血糖水平,并延长糖尿病大鼠生存时间。虽然与天然胰岛组相比,降低血糖的程度尚不足,但确已具有葡萄糖应激能力。提示：INGAP-pp诱生的ILC具有治疗糖尿病的临床应用前景。
Part I:
Objective:To isolate, purify and characterize human adipose tissue-derived stem cells.
Methods:With slightly modified protocol demonstrated by zuk et al, human adipose tissue-derived stem cells (hASCs) were isolated and expanded in vitro. FACS analysis was performed to identify the cell surface and cytoplasm antigen.Tolemerase repeated amplification protocol (TRAP) and G-banding Karyotype analysis were performed to evaluate the safety of hASCs cultured in vitro. Results:Since passage 4, hASCs developed a uniform morphology resembling that of fibroblasts, FACS analysis of the fourth passage cells showed purified hASCs expressed high levels of cell surface markers, such as CD73(99.1%),CD90(97.9%), CD105(97.2%). These results are consistent with the definition that mesenchymal stem cells (MSCs) must express CD73, CD90, and CD105. Also, extremely low expression levels of CK-19 and Pdx-1, which supposed to be markers of pancreatic stem cells, were detected. Negative expression of CD45 means no contamination of hemopoiesis stem cells. TRAP test revealed P4, P8, P12 had nomal tolemerase activity and G-banding analysis showed P4, P8, P12 and P16 had normal katyotypes. Conclusions:hASCs could expand in vitro efficiently after being isolated and purified. The hASCs we cultured in the study were consistent to those reported previously. When cultured in vitro, hASCs have stable chromosome structure as devided in vitro and being safe for cytothrapy.
Part II:
In Vitro differentiation of human adipose tissue-derived stem cells into Insulin/c-peptide positive cells by embryonic pancreas extract
Objective:To evaluate the effect of embryo pancreas extract (EPE) from pre-born Sprague-Dawley rats on induction of pancreatic differentiation of human adipose tissue-derived mesenchymal stem cells(hASCs).
Methods:By using real-time PCR and immunofluorescence technology, expression levels of genes related with pancreas development of hASCs cultured with EPE for 20 days were detected.
Results:undifferentiated hASCs expressed key markers of embryo stem cells. Genes involved in early pancreas development showed increased expression in EPE-treated culture. Immunocytochemical analysis showed C-peptide/insulin positive cells in EPE-treated cells but not in undifferentiated hASCs.
Conclusion:hASCs have the potential to differentiate into pancreas cell lineages with EPE induction, so could be substitution of embryo and bone-marrow derived stem cells for future cell therapy applications.
Part III:
IN Vitro Differentiation of human adipose-derived stem cells intoIslet-like cell clusters by INGAP-pp protocol
Objective:To establish a novel method for the differentiation of islet-like clusters from human adipose-derived stem cells with INGAP-related pentadecapeptide (INGAP-PP) combined with chemical molecules and cytokines.
Methods:the human adipose-derived stem cells were isolated, purified and expanded in vitro and differentiated using a four-step protocol that included TSA, INGAP-PP/Scrambled peptide (Scrambled-P), nicotinamide and exendin-4. By using real-time PCR and immunofluorescence technology, expression levels of genes related with pancreas development. Insulin and C-peptide released by differentiated cells in response to high/low glucose level were detected by ELISA.
Results Undifferentiated hASCs expressed oct3/4, key markers of embryo stem cells and at the end of induction, Oct3/4 expression level decreased to 1/20 of original. The expression level of genes involved in early pancreas development increased in both INGAP-PP/Scrambled peptide culture significantly. Islet-like clusters were obtained in both INGAP-PP/Scrambled peptide group, but in INGAP-pp group, the diameters of clusters were much closer to native islets, which were about 150-200μm. Immunocytochemical analysis showed C-peptide/insulin positive cells in INGAP-pp induced group but not in undifferentiated hMSCs.
Conclusions:By INGAP-pp protocol, hASCs could differentiate into islet-like clusters more efficiently through Mesenchymal/epithelium transition. It suggests hASCs have the potential to differentiate into pancreas cell lineages by INGAP-pp protocol; so could be substitution of embryo or bone-marrow derived stem cells for future cell therapy applications.
Part IV:
Objective:To evaluate the effects of transplantation of ILCs induced by INGAP-pp and INGAP-pp injection to cure diabetic rats.
Methods:Diabetes was induced in 8-week-old male Sprague-Dawley rats by streptozocin (Sigma,55 mg/kg body weight in citrate buffer, pH 4.4, i.p).48 hours after injection, animals with blood glucose levels≥16.65 mmol/L for two consecutive days were transplanted with ILCs induced by INGAP-pp (200IEQ/l00μ1 High glucose DMEM) into the left subrenal capsule (n=10). As controls, fresh isolated human islets (200IEQ/100μ1 High glucose DMEM) and saline without cells were transplanted respectively. Two transplantation group were administered acrolimus (50ng/kg.d,p.o.) to avoid of immune rejection. To evaluate the effects of INGAP-pp protein on remission of diabetes, we also injected purified INGAP-PP (250μg/0.5mL, i.p.) to diabetic rats twice a day for twenty days. Blood glucose levels of all groups were measured every three days. Glucose tolerance tests were performed after injection of glucose (2g/kg bodywight, i.p.). Diabetic rats transplanted with normal islets and normal rats served as positive controls. Diabetic rats were used as negative control.
Results:Blood glucose levels of rats transplanted with INGAP-induced ILCs fell to 7.8±0.8 mmol/L and kept this level for the whole experimental process while diabetic rats transplanted with normal islets, as a control, showed blood glucose levels similar to normal rats after 1-2 weeks. In addition, non-transplanted diabetic rats remained hyperglycemic and died consecutively. Interestingly, the group treated with INGAP-pp protein injection also showed a decrease of blood glucose level after 5-day injections, but never fell to normal level and re-raised 2 weeks later since the day INGAP-PP injection stopped. Diabetic rats transplanted with INGAP-induced ILCs or normal islets survived the whole 69-day process while diabetic rats without treatment suffered from hyperglycemic and died off within 51 days. Diabetic rats undergone INGAP-pp injections also all survived until the end of experiment. Intraperitoneal glucose tolerance tests showed SD rats transplanted with ILCs induced by INGAP-pp had the similar kinetics of the glucose clearance to the group transplanted with normal islets, although the blood glucose levels were higher at all time points. In contrast, untreated diabetic rats revealed no reaction to the high glucose concentration
Conclusions:The results of transplantation of ILCs and normal islets to diabetic rats showed ILCs did decrease the blood glucose levels and extended the life-span of diabetic rats. ILCs transplantation reversed diabetes partially in animal models.
引文
1. Atkinson M.A., Eisenbarth G.S. Type 1 diabetes:new perspectives on disease pathogenesis and treatment. Lancet 2001,358:221-229.
2. Casanova D. Pancreatic islets transplantation in the treatment of diabetes mellitus: present and future. Cir Esp.2009 85(2):76-83.
3. Choudhury A. The pancreas as a transplantable organ. Ann R Coll Surg Engl. 1973 53(4):218-36.
4. Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006; 28; 355(13):1318-30.
5. Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54(7):2060-2069.
6. Soria, B., Bedoya, F.J., Tejedo, J.R., et al. Cell Therapy for Diabetes Mellitus:An Opportunity for Stem Cells? Cells, tissues, organs.2008188(1-2):70-77.
7. Choi K.S., Shin J.S., Lee J.J., et al. In vitro transdifferentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract. Biochem. Biophys. Res. Commun.2005 330:1299-1305.
8. Fujikawa T, Oh SH, Pi L.et al. Teratoma formation leads to failure of treatment for type Ⅰ diabetes using embryonic stem cell-derived insulin-producing cells.Am J Pathol.2005 166:1781-1791.
9. Pomplun D, Florian S, Schulz T. et al. Alterations of pancreatic beta-cell mass and islet number due to Ins2-controlled expression of Cre recombinase:RIP-Cre revisited; part 2. Horm Metab Res.2007 39(5):336-40.
10. Bernal-Mizrachi E, Cras-Meneur C, Ye BR,et al. Transgenic overexpression of active calcineurin in beta-cells results in decreased beta-cell mass and hyperglycemia. PLoS One.2010 5(8):el 1969.
11. Russ HA, Bar Y, Ravassard P, et al. In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing. Diabetes.2008 57(6):1575-1583.
12. Parnaud G, Bosco D, Berney T, et al. Proliferation of sorted human and rat beta cells. Diabetologia.2008 51(1):91-100.
13. Li L, Lili R, Hui Q, et al. Combination of GLP-1 and sodium butyrate promote differentiation of pancreatic progenitor cells into insulin-producing cells. Tissue Cell.2008 40(6):437-45.
14. Zuk P.A, Zhu N, Mizuno H,et al. Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.2001 7:211-228.
15. Zuk P.A, Zhu M., Ashjian P., et al. Human adipose tissue is a source of multipotent stem cells. Mol.Biol.Cell.2002 13:4279-4295.
16. Sen A, Lea-Currie YR, Sujkowska D, et al. Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous. J Cell Biochem,2001 81(2):312-319.
17. B. Fang, Y. Song, Q. Lin, et al. Human adipose tissue-derived mesenchymal stromal cells as salvage therapy for treatment of severe refractory acute graft-vs.-host disease in two children. Pediatr. Transplant.2007 11:814-817.
18. Verseijden F, Jahr H, Posthumus-van Sluijs SJ, et al. Angiogenic capacity of human adipose-derived stromal cells during adipogenic differentiation:an in vitro study.Tissue Eng Part A.2009 15(2):445-52.
19. Puissant B., Barreau C., Bourin P., et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells:comparison with bone marrow mesenchymal stem cells. Br. J. Haematol.2005 129:118-129.
20. K.Timper, D. Seboek, M. Eberhardt, et al. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagons expressing cells, Biochem. Biophys. Res. Commun.2006 341:1135-1140.
21. Kabelitz D, Geissler EK, Soria B, et al. Toward cell-based therapy of type Ⅰ diabetes. Trends Immunol.2008 29(2):68-74.
22. Lee J, Han DJ, Kim SC. et al. In vitro differentiation of human adipose tissue-derived stem cells into cells with pancreatic phenotype by regenerating pancreas extract. Biochem Biophys Res Commun.2008 375(4):547-51.
23. Taylor-Fishwick DA, Rittman S,Kendall H, et al. Cloning genomic INGAP:a Reg-related family member with distinct transcriptional regulation sites. Biochim Biophys Acta.2003 1638:83-89.
24. Rosenberg L, Lipsett M, Yoon JW, et al. A pentadecapeptide fragment of islet neogenesis-associated protein increases beta-cell mass and reverses diabetes in C57BL/6J mice. Ann Surg.2004 240:875-884.
25. Barbosa H, Bordin S, Stoppiglia L, et al. Islet Neogenesis Associated Protein (INGAP)modulates gene expression in cultured neonatal rat islets. Regul Pept 2006136:78-84.
26. Borelli MI, Stoppiglia LF, Rezende LF, et al. INGAP-related pentadecapeptide:its modulatory effect upon insulin secretion. Regul Pept.2005 131:97-102.
27. Hanley S, Rosenberg L. Islet-Derived Progenitors as a Source of In Vitro Islet Regeneration. Methods Mol Biol.2009 482:371-385.
28. Pittenger GL, Taylor-Fishwick DA, Johns RH, et al. Intramuscular injection of islet neogenesis-associated protein peptide stimulates pancreatic islet neogenesis in healthy dogs. Pancreas.2007 34:103-111.
29. Meenal Banerjeea, Anil Kumarb, Ramesh R. Bhonde, et al. Reversal of experimental diabetes by multiple bone marrow transplantation. Biochemical and Biophysical Research Communications.2005,328:318-324.
30. Rangappa S, Fen C, Lee EH, et al. Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes. Ann Thorac Surg.2003 75(3):775-779.
31. Vija L, Farge D, Gautier JF, et al. Mesenchymal stem cells:Stem cell therapy perspectives for type 1 diabetes. Diabetes Metab.2009 35(2):85-93.
32. Vidal MA, Robinson SO, Lopez MJ, et al.Comparison of chondrogenic potential in equine mesenchymal stromal cells derived from adipose tissue and bone marrow.Vet Surg.2008 37(8):713-24.
33. Zhang DZ, Gai LY, Liu HW. Differences between adipose-derived stem cells and mesenchymal stem cells in differentiation into cardiomyocytes. Sheng Li Xue Bao.2008 60(3):341-7.
34. Hardikar A.A., Bhonde R.R., Modulating experimental diabetes by treatment with cytosolic extract from the regenerating pancreas, Diabetes Res. Clin. Pract. 46(1999)203-211.
35. Chang C, Wang X, Niu D, et al Mesenchymal stem cells adopt beta-cell fate upon diabetic pancreatic microenvironment.Pancreas.2009 38(3):275-81
36. Masamune A, Shimosegawa T. Signal transduction in pancreatic stellate cells.J Gastroenterol.2009 44(4):249-60.
37. Tayaramma T, Ma B, Rohde M, et al. Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells. Stem Cells, 2006,24(12):2858-2867.
38. De Vos P, Hamel AF, Tatarkiewicz K. et al. Considerations for successful transplantation of encapsulated pancreatic islets. Diabetologia.2002,45 (2):159-173.
39. Roche E, Sepulcre P, Reig JA, et al. Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells. Faseb J.2005, 19(10):1341-1343.
40. Yue F, Cui L, Johkura K, et al. Glucagon-like peptide-1 differentiation of primate embryonic stem cells into insulin-producing cells. Tissue Eng.2006,12 (8):2105-2116.
41. Bonner-Weir, S., Weir, G.C. New sources of pancreatic beta-cells. Nature biotechnology.2005 23:857-861.
42. Ramiya V.K., Maraist M., Arfors K.E., et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat.Med.2000 6:278-282.
43. Oh S.H., Muzzonigro T.M. Bae S.H. et al. Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes. Lab.Invest.2004 84:607-617.
44. Lumelsky N, Blondel O, Laeng P. et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science.2001 292:1389-1394.
45.刘霆,张桂珍,卜丽莎,等.STZ小剂量多次注射诱导大鼠胰岛素依赖性糖尿病动物模型探讨[J].白求恩医科大学学报.2001,27(6)：578.
46.董叫云,陈志龙,青春,等.糖尿病动物模型制作及进展[J].中华国际医学杂志,2001,1(2)：148.
47. Linetsky E, Bottino R, Lehmann R, et al. Improved human islet isolation using a new enzyme blend, liberase. Diabetes.1997 46:1120-1123.
48. Ricordi C, Lacy PE, Finke EH, et al. Automated method for isolation of human pancreatic islets. Diabetes.1988 37:413-420.
49. Nanji SA, Shapiro A M. Advances in pancreatic islet transplantation in humans. Diabetes Obes Metab.20068(1):15-25.
50. Butler AE, Janson J, Soeller WC, et al. Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes:evidence for role of islet amyloid formation rather than direct action of amyloid. Diabetes 2005 52:2304-2314.
51. Kendall WF Jr, Collins BH, Opara EC. Islet cell transplantation for the treatment of diabetes mellitus. Expert Opin Biol Ther 2001 1(1):109-19.
52. Ianus,A.,Holz,G.G.,Theise,N.D.et al. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion.The Journal of clinical investigation 111,843-850.
53. Ryan EA, Lakey JRT, Paty BW, et al. Successful islet transplantation:continued insulin reserve provides long-term glycemic control. Diabetes.2002 21(7):2148-2157.
54. Bogdani M, Suenens K, Bock T, et al. Growth and functional maturation of beta-cells in implants of endocrine cells purified from prenatal porcine pancreas. Diabetes.200554(12):3387-94.
55. Jun HS, Yoon JW. Approaches for the cure of type 1 diabetes by cellular and gene therapy. Curr Gene Ther.2005 5(2):249-62.
56. Baharvand H, Jafary H, Massumi M,et al. Generation of insulin-secreting cells from human embryonic stem cells. The potential for stem cell therapy in diabetes. Pediatr Res.2006 59(4 Pt 2):65R-73R.
57. Kobayashi N, Okitsu T, Lakey JR, et al. The current situation in human pancreatic islet transplantation:problems and prospects. J Artif Organs 2004 7(1):1-8.
58. van Schilfgaarde R, de Vos P. Aspects of immune protection of islets. Transplant Proc.1991 28(6):3516-3517.
59. de Groot M, Keizer PP, de Haan BJ, et al. Micro-capsules and their ability to protect islets against cytokine-mediated dysfunction. Transplant Proc.2003 3(1-2):1711-1712.
60. Scharp DW, Swanson CJ, Olack BJ, et al. Protection of encapsulated human islets implanted without immunosuppression in patients with type I or type II diabetes and in non diabetic control subjects. Diabetes.1994 43(9):1167-70.
61. Elliott RB, Escobar L, Tan PL, et al. Intraperitoneal alginateen capsulated neonatal porcine islets in a placebo controlled study with 16 diabetic cynomolgus primmates. Transplant Proc.2005 37(8):3505-8.
1. Casanova D. Pancreatic islets transplantation in the treatment of diabetes mellitus:present and future. Cir Esp.2009 85(2):76-83.
2. Choudhury A. The pancreas as a transplantable organ. Ann R Coll Surg Engl.1973 53(4): 218-236.
3. Shapiro A M, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006; 28; 355(13):1318-1330.
4. Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54(7):2060-2069.
5. Bernal-Mizrachi E, Cras-Meneur C, Ye BR,et al. Transgenic overexpression of active calcineurin in beta-cells results in decreased beta-cell mass and hyperglycemia. PLoS One. 2010 5(8):el 1969.
6. Russ HA, Bar Y, Ravassard P, et al. In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing. Diabetes.2008 57(6):1575-1583.
7. Parnaud G, Bosco D, Berney T, et al. Proliferation of sorted human and rat beta cells. Diabetologia.2008 51(1):91-100.
8. Li L, Lili R, Hui Q, et al. Combination of GLP-1 and sodium butyrate promote differentiation of pancreatic progenitor cells into insulin-producing cells. Tissue Cell.2008 40(6):437-445.
9. Lacy PE, Kostianovsky M. A method for isolation of intact islets of Langerhans from the rat pancreas. Diabetes.1967 16:35-39.
10. Ricordi C. The automated method for islet isolation. In Pancreatic Islet Cell Transplantation 1892-1991:One Century of Transplantation for Diabetes. Ricordi C, Ed. Austin, TX, R.G. Landes Company 1992 p.99-112.
11. Ricordi C. Islet transplantation:a brave new world. Diabetes 2003 52(7):1595-1603.
12. Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes 1988 37:413-420.
13. Lakey JR, Warnock GL, Shapiro AM, et al. Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. Cell Transplant 1999 8:285-292.
14. Linetsky E, Bottino R, Lehmann R, et al. Improved human islet isolation using a new enzyme blend, liberase. Diabetes 199746:1120-1123.
15. Robertson GS, Chadwick DR, Contractor H, et al. The optimization of largescale density gradient isolation of human islets. Acta Diabetol 1993 30:93-98.
16. Robertson RP. Islet transplantation:travels up the learning curve. Curr Diab Rep 2002 2(4):365-370.
17. Onaca N, Klintmalm GB, Levy MF. Pancreatic islet cell transplantation:a treatment strategy for type I diabetes mellitus. Nutr Clin Pract.2004 19(2):154-164.
18. Binette TM, Seeberger KL, Lyon JG, et al. Porcine endogenous retroviral nucleic acid in peripheral tissues is associated with migration of porcine cells post islet transplant. Am J Transplant.2004 4(7):1051-1060.
19. Patience C, Takeuchi Y, Weiss RA. Infection of human cells by an endogenous retrovirus of pigs. Nat Med.1997 3:282-286.
20. Zhang YH, Wang HF, Liu W,et al. Insulin-producing cells derived from rat bone marrow and their autologous transplantation in the duodenal wall for treating diabetes. Anat Rec (Hoboken).2009 292(5):728-735.
21. Vija L, Farge D, Gautier JF, et al. Mesenchymal stem cells:Stem cell therapy perspectives for type 1 diabetes. Diabetes Metab.2009 35(2):85-93.
22. Lin HY, Tsai CC, Chen LL, et al. Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK. J Biomed Sci.2010 17:56.
23. Zuk P.A, Zhu N, Mizuno H,et al. Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.20017:211-228.
24. Zuk P.A, Zhu M., Ashjian P., et al. Human adipose tissue is a source of multipotent stem cells. Mol.Biol.Cell.200213:4279-4295.
25. B. Fang, Y. Song, Q. Lin, et al. Human adipose tissue-derived mesenchymal stromal cells as salvage therapy for treatment of severe refractory acute graft-vs.-host disease in two children. Pediatr. Transplant.2007 11:814-817.
26. Verseijden F, Jahr H, Posthumus-van Sluijs SJ, et al. Angiogenic capacity of human adipose-derived stromal cells during adipogenic differentiation:an in vitro study.Tissue Eng PartA.2009 15(2):445-452.
27. Puissant B., Barreau C., Bourin P., et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells:comparison with bone marrow mesenchymal stem cells. Br. J. Haematol.2005 129:118-129.
28. K.Timper, D. Seboek, M. Eberhardt, et al. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagons expressing cells, Biochem. Biophys. Res. Commun.2006 341:1135-1140.
29. Rosenberg L, Lipsett M, Yoon JW, et al. A pentadecapeptide fragment of islet neogenesis-associated protein increases beta-cell mass and reverses diabetes in C57BL/6J mice. Ann Surg.2004 240:875-884.
30. Bonner-Weir, S., Weir, G.C. New sources of pancreatic beta-cells. Nature biotechnology.2005 23:857-861.
31. Li J, Wang Y, Yu X, et al. Islet neogenesis-associated protein-related pentadecapeptide enhances the differentiation of islet-like clusters from human pancreatic duct cells. Peptides. 200930(12):2242-2249.
32. Kemp CB, Knight MJ, Scharp DW, et al. Transplantation of isolated pancreatic islets into the portal vein of diabetic rats. Nature.1973 244(5416):447.
33. Andersson A, Korsgren O, Jansson L. Intrapportally transplanted pancreatic islets revascullarized from hepatic arterial system. Diabetes.1989 38 Suppl 1:192-195.
34. van Suylichem PT, Strubbe JH, Houwing H, et al. Rat islet isograft function. Effect of graft volume and transplantation site. Transplantation.1994 57(7):1010-1017.
35. Kendall WF Jr, Collins BH, Opara EC. Islet cell transplantation for the treatment of diabetes mellitus. Expert Opin Biol Ther.2001 1(1):109-119.
36. Maki T, Lodge JP, Carretta M, Ohzato H, Borlland KM, Sullivan SJ, et al. Treatment of severe diabetes mellitus for more than one year using a vascularized hybrid artificial pancreas. Transplantation.1993 55(4):713-717.
37. Uludag H, De Vos P, Tresco PA. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev.2000 42(1-2):29-64.
2. Casanova D. Pancreatic islets transplantation in the treatment of diabetes mellitus: present and future. Cir Esp.2009 85(2):76-83.
3. Choudhury A. The pancreas as a transplantable organ. Ann R Coll Surg Engl. 1973 53(4):218-36.
4. Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006; 28; 355(13):1318-30.
5. Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54(7):2060-2069.
6. Soria, B., Bedoya, F.J., Tejedo, J.R., et al. Cell Therapy for Diabetes Mellitus:An Opportunity for Stem Cells? Cells, tissues, organs.2008188(1-2):70-77.
7. Choi K.S., Shin J.S., Lee J.J., et al. In vitro transdifferentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract. Biochem. Biophys. Res. Commun.2005 330:1299-1305.
8. Fujikawa T, Oh SH, Pi L.et al. Teratoma formation leads to failure of treatment for type Ⅰ diabetes using embryonic stem cell-derived insulin-producing cells.Am J Pathol.2005 166:1781-1791.
9. Pomplun D, Florian S, Schulz T. et al. Alterations of pancreatic beta-cell mass and islet number due to Ins2-controlled expression of Cre recombinase:RIP-Cre revisited; part 2. Horm Metab Res.2007 39(5):336-40.
10. Bernal-Mizrachi E, Cras-Meneur C, Ye BR,et al. Transgenic overexpression of active calcineurin in beta-cells results in decreased beta-cell mass and hyperglycemia. PLoS One.2010 5(8):el 1969.
11. Russ HA, Bar Y, Ravassard P, et al. In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing. Diabetes.2008 57(6):1575-1583.
12. Parnaud G, Bosco D, Berney T, et al. Proliferation of sorted human and rat beta cells. Diabetologia.2008 51(1):91-100.
13. Li L, Lili R, Hui Q, et al. Combination of GLP-1 and sodium butyrate promote differentiation of pancreatic progenitor cells into insulin-producing cells. Tissue Cell.2008 40(6):437-45.
14. Zuk P.A, Zhu N, Mizuno H,et al. Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.2001 7:211-228.
15. Zuk P.A, Zhu M., Ashjian P., et al. Human adipose tissue is a source of multipotent stem cells. Mol.Biol.Cell.2002 13:4279-4295.
16. Sen A, Lea-Currie YR, Sujkowska D, et al. Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous. J Cell Biochem,2001 81(2):312-319.
17. B. Fang, Y. Song, Q. Lin, et al. Human adipose tissue-derived mesenchymal stromal cells as salvage therapy for treatment of severe refractory acute graft-vs.-host disease in two children. Pediatr. Transplant.2007 11:814-817.
18. Verseijden F, Jahr H, Posthumus-van Sluijs SJ, et al. Angiogenic capacity of human adipose-derived stromal cells during adipogenic differentiation:an in vitro study.Tissue Eng Part A.2009 15(2):445-52.
19. Puissant B., Barreau C., Bourin P., et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells:comparison with bone marrow mesenchymal stem cells. Br. J. Haematol.2005 129:118-129.
20. K.Timper, D. Seboek, M. Eberhardt, et al. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagons expressing cells, Biochem. Biophys. Res. Commun.2006 341:1135-1140.
21. Kabelitz D, Geissler EK, Soria B, et al. Toward cell-based therapy of type Ⅰ diabetes. Trends Immunol.2008 29(2):68-74.
22. Lee J, Han DJ, Kim SC. et al. In vitro differentiation of human adipose tissue-derived stem cells into cells with pancreatic phenotype by regenerating pancreas extract. Biochem Biophys Res Commun.2008 375(4):547-51.
23. Taylor-Fishwick DA, Rittman S,Kendall H, et al. Cloning genomic INGAP:a Reg-related family member with distinct transcriptional regulation sites. Biochim Biophys Acta.2003 1638:83-89.
24. Rosenberg L, Lipsett M, Yoon JW, et al. A pentadecapeptide fragment of islet neogenesis-associated protein increases beta-cell mass and reverses diabetes in C57BL/6J mice. Ann Surg.2004 240:875-884.
25. Barbosa H, Bordin S, Stoppiglia L, et al. Islet Neogenesis Associated Protein (INGAP)modulates gene expression in cultured neonatal rat islets. Regul Pept 2006136:78-84.
26. Borelli MI, Stoppiglia LF, Rezende LF, et al. INGAP-related pentadecapeptide:its modulatory effect upon insulin secretion. Regul Pept.2005 131:97-102.
27. Hanley S, Rosenberg L. Islet-Derived Progenitors as a Source of In Vitro Islet Regeneration. Methods Mol Biol.2009 482:371-385.
28. Pittenger GL, Taylor-Fishwick DA, Johns RH, et al. Intramuscular injection of islet neogenesis-associated protein peptide stimulates pancreatic islet neogenesis in healthy dogs. Pancreas.2007 34:103-111.
29. Meenal Banerjeea, Anil Kumarb, Ramesh R. Bhonde, et al. Reversal of experimental diabetes by multiple bone marrow transplantation. Biochemical and Biophysical Research Communications.2005,328:318-324.
30. Rangappa S, Fen C, Lee EH, et al. Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes. Ann Thorac Surg.2003 75(3):775-779.
31. Vija L, Farge D, Gautier JF, et al. Mesenchymal stem cells:Stem cell therapy perspectives for type 1 diabetes. Diabetes Metab.2009 35(2):85-93.
32. Vidal MA, Robinson SO, Lopez MJ, et al.Comparison of chondrogenic potential in equine mesenchymal stromal cells derived from adipose tissue and bone marrow.Vet Surg.2008 37(8):713-24.
33. Zhang DZ, Gai LY, Liu HW. Differences between adipose-derived stem cells and mesenchymal stem cells in differentiation into cardiomyocytes. Sheng Li Xue Bao.2008 60(3):341-7.
34. Hardikar A.A., Bhonde R.R., Modulating experimental diabetes by treatment with cytosolic extract from the regenerating pancreas, Diabetes Res. Clin. Pract. 46(1999)203-211.
35. Chang C, Wang X, Niu D, et al Mesenchymal stem cells adopt beta-cell fate upon diabetic pancreatic microenvironment.Pancreas.2009 38(3):275-81
36. Masamune A, Shimosegawa T. Signal transduction in pancreatic stellate cells.J Gastroenterol.2009 44(4):249-60.
37. Tayaramma T, Ma B, Rohde M, et al. Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells. Stem Cells, 2006,24(12):2858-2867.
38. De Vos P, Hamel AF, Tatarkiewicz K. et al. Considerations for successful transplantation of encapsulated pancreatic islets. Diabetologia.2002,45 (2):159-173.
39. Roche E, Sepulcre P, Reig JA, et al. Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells. Faseb J.2005, 19(10):1341-1343.
40. Yue F, Cui L, Johkura K, et al. Glucagon-like peptide-1 differentiation of primate embryonic stem cells into insulin-producing cells. Tissue Eng.2006,12 (8):2105-2116.
41. Bonner-Weir, S., Weir, G.C. New sources of pancreatic beta-cells. Nature biotechnology.2005 23:857-861.
42. Ramiya V.K., Maraist M., Arfors K.E., et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat.Med.2000 6:278-282.
43. Oh S.H., Muzzonigro T.M. Bae S.H. et al. Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes. Lab.Invest.2004 84:607-617.
44. Lumelsky N, Blondel O, Laeng P. et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science.2001 292:1389-1394.
45.刘霆,张桂珍,卜丽莎,等.STZ小剂量多次注射诱导大鼠胰岛素依赖性糖尿病动物模型探讨[J].白求恩医科大学学报.2001,27(6)：578.
46.董叫云,陈志龙,青春,等.糖尿病动物模型制作及进展[J].中华国际医学杂志,2001,1(2)：148.
47. Linetsky E, Bottino R, Lehmann R, et al. Improved human islet isolation using a new enzyme blend, liberase. Diabetes.1997 46:1120-1123.
48. Ricordi C, Lacy PE, Finke EH, et al. Automated method for isolation of human pancreatic islets. Diabetes.1988 37:413-420.
49. Nanji SA, Shapiro A M. Advances in pancreatic islet transplantation in humans. Diabetes Obes Metab.20068(1):15-25.
50. Butler AE, Janson J, Soeller WC, et al. Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes:evidence for role of islet amyloid formation rather than direct action of amyloid. Diabetes 2005 52:2304-2314.
51. Kendall WF Jr, Collins BH, Opara EC. Islet cell transplantation for the treatment of diabetes mellitus. Expert Opin Biol Ther 2001 1(1):109-19.
52. Ianus,A.,Holz,G.G.,Theise,N.D.et al. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion.The Journal of clinical investigation 111,843-850.
53. Ryan EA, Lakey JRT, Paty BW, et al. Successful islet transplantation:continued insulin reserve provides long-term glycemic control. Diabetes.2002 21(7):2148-2157.
54. Bogdani M, Suenens K, Bock T, et al. Growth and functional maturation of beta-cells in implants of endocrine cells purified from prenatal porcine pancreas. Diabetes.200554(12):3387-94.
55. Jun HS, Yoon JW. Approaches for the cure of type 1 diabetes by cellular and gene therapy. Curr Gene Ther.2005 5(2):249-62.
56. Baharvand H, Jafary H, Massumi M,et al. Generation of insulin-secreting cells from human embryonic stem cells. The potential for stem cell therapy in diabetes. Pediatr Res.2006 59(4 Pt 2):65R-73R.
57. Kobayashi N, Okitsu T, Lakey JR, et al. The current situation in human pancreatic islet transplantation:problems and prospects. J Artif Organs 2004 7(1):1-8.
58. van Schilfgaarde R, de Vos P. Aspects of immune protection of islets. Transplant Proc.1991 28(6):3516-3517.
59. de Groot M, Keizer PP, de Haan BJ, et al. Micro-capsules and their ability to protect islets against cytokine-mediated dysfunction. Transplant Proc.2003 3(1-2):1711-1712.
60. Scharp DW, Swanson CJ, Olack BJ, et al. Protection of encapsulated human islets implanted without immunosuppression in patients with type I or type II diabetes and in non diabetic control subjects. Diabetes.1994 43(9):1167-70.
61. Elliott RB, Escobar L, Tan PL, et al. Intraperitoneal alginateen capsulated neonatal porcine islets in a placebo controlled study with 16 diabetic cynomolgus primmates. Transplant Proc.2005 37(8):3505-8.
1. Casanova D. Pancreatic islets transplantation in the treatment of diabetes mellitus:present and future. Cir Esp.2009 85(2):76-83.
2. Choudhury A. The pancreas as a transplantable organ. Ann R Coll Surg Engl.1973 53(4): 218-236.
3. Shapiro A M, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006; 28; 355(13):1318-1330.
4. Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54(7):2060-2069.
5. Bernal-Mizrachi E, Cras-Meneur C, Ye BR,et al. Transgenic overexpression of active calcineurin in beta-cells results in decreased beta-cell mass and hyperglycemia. PLoS One. 2010 5(8):el 1969.
6. Russ HA, Bar Y, Ravassard P, et al. In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing. Diabetes.2008 57(6):1575-1583.
7. Parnaud G, Bosco D, Berney T, et al. Proliferation of sorted human and rat beta cells. Diabetologia.2008 51(1):91-100.
8. Li L, Lili R, Hui Q, et al. Combination of GLP-1 and sodium butyrate promote differentiation of pancreatic progenitor cells into insulin-producing cells. Tissue Cell.2008 40(6):437-445.
9. Lacy PE, Kostianovsky M. A method for isolation of intact islets of Langerhans from the rat pancreas. Diabetes.1967 16:35-39.
10. Ricordi C. The automated method for islet isolation. In Pancreatic Islet Cell Transplantation 1892-1991:One Century of Transplantation for Diabetes. Ricordi C, Ed. Austin, TX, R.G. Landes Company 1992 p.99-112.
11. Ricordi C. Islet transplantation:a brave new world. Diabetes 2003 52(7):1595-1603.
12. Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes 1988 37:413-420.
13. Lakey JR, Warnock GL, Shapiro AM, et al. Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. Cell Transplant 1999 8:285-292.
14. Linetsky E, Bottino R, Lehmann R, et al. Improved human islet isolation using a new enzyme blend, liberase. Diabetes 199746:1120-1123.
15. Robertson GS, Chadwick DR, Contractor H, et al. The optimization of largescale density gradient isolation of human islets. Acta Diabetol 1993 30:93-98.
16. Robertson RP. Islet transplantation:travels up the learning curve. Curr Diab Rep 2002 2(4):365-370.
17. Onaca N, Klintmalm GB, Levy MF. Pancreatic islet cell transplantation:a treatment strategy for type I diabetes mellitus. Nutr Clin Pract.2004 19(2):154-164.
18. Binette TM, Seeberger KL, Lyon JG, et al. Porcine endogenous retroviral nucleic acid in peripheral tissues is associated with migration of porcine cells post islet transplant. Am J Transplant.2004 4(7):1051-1060.
19. Patience C, Takeuchi Y, Weiss RA. Infection of human cells by an endogenous retrovirus of pigs. Nat Med.1997 3:282-286.
20. Zhang YH, Wang HF, Liu W,et al. Insulin-producing cells derived from rat bone marrow and their autologous transplantation in the duodenal wall for treating diabetes. Anat Rec (Hoboken).2009 292(5):728-735.
21. Vija L, Farge D, Gautier JF, et al. Mesenchymal stem cells:Stem cell therapy perspectives for type 1 diabetes. Diabetes Metab.2009 35(2):85-93.
22. Lin HY, Tsai CC, Chen LL, et al. Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK. J Biomed Sci.2010 17:56.
23. Zuk P.A, Zhu N, Mizuno H,et al. Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.20017:211-228.
24. Zuk P.A, Zhu M., Ashjian P., et al. Human adipose tissue is a source of multipotent stem cells. Mol.Biol.Cell.200213:4279-4295.
25. B. Fang, Y. Song, Q. Lin, et al. Human adipose tissue-derived mesenchymal stromal cells as salvage therapy for treatment of severe refractory acute graft-vs.-host disease in two children. Pediatr. Transplant.2007 11:814-817.
26. Verseijden F, Jahr H, Posthumus-van Sluijs SJ, et al. Angiogenic capacity of human adipose-derived stromal cells during adipogenic differentiation:an in vitro study.Tissue Eng PartA.2009 15(2):445-452.
27. Puissant B., Barreau C., Bourin P., et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells:comparison with bone marrow mesenchymal stem cells. Br. J. Haematol.2005 129:118-129.
28. K.Timper, D. Seboek, M. Eberhardt, et al. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagons expressing cells, Biochem. Biophys. Res. Commun.2006 341:1135-1140.
29. Rosenberg L, Lipsett M, Yoon JW, et al. A pentadecapeptide fragment of islet neogenesis-associated protein increases beta-cell mass and reverses diabetes in C57BL/6J mice. Ann Surg.2004 240:875-884.
30. Bonner-Weir, S., Weir, G.C. New sources of pancreatic beta-cells. Nature biotechnology.2005 23:857-861.
31. Li J, Wang Y, Yu X, et al. Islet neogenesis-associated protein-related pentadecapeptide enhances the differentiation of islet-like clusters from human pancreatic duct cells. Peptides. 200930(12):2242-2249.
32. Kemp CB, Knight MJ, Scharp DW, et al. Transplantation of isolated pancreatic islets into the portal vein of diabetic rats. Nature.1973 244(5416):447.
33. Andersson A, Korsgren O, Jansson L. Intrapportally transplanted pancreatic islets revascullarized from hepatic arterial system. Diabetes.1989 38 Suppl 1:192-195.
34. van Suylichem PT, Strubbe JH, Houwing H, et al. Rat islet isograft function. Effect of graft volume and transplantation site. Transplantation.1994 57(7):1010-1017.
35. Kendall WF Jr, Collins BH, Opara EC. Islet cell transplantation for the treatment of diabetes mellitus. Expert Opin Biol Ther.2001 1(1):109-119.
36. Maki T, Lodge JP, Carretta M, Ohzato H, Borlland KM, Sullivan SJ, et al. Treatment of severe diabetes mellitus for more than one year using a vascularized hybrid artificial pancreas. Transplantation.1993 55(4):713-717.
37. Uludag H, De Vos P, Tresco PA. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev.2000 42(1-2):29-64.