切换至 "中华医学电子期刊资源库"

中华肝脏外科手术学电子杂志 ›› 2019, Vol. 08 ›› Issue (06) : 557 -560. doi: 10.3877/cma.j.issn.2095-3232.2019.06.020

所属专题: 文献

基础研究

人胰岛细胞消化与提纯改良方法研究
谢炬平1, 蒋小峰1, 黄镇辉1, 张大伟1, 梁浩1, 薛平1,()   
  1. 1. 510260 广州医科大学附属第二医院肝胆外科
  • 收稿日期:2019-08-18 出版日期:2019-12-10
  • 通信作者: 薛平
  • 基金资助:
    广州市科技计划项目(201607010033); 广州医科大学附属第二医院新技术新业务临床研究与应用项目(Y-03)

Study of modified methods of digestion and purification of human islet cells

Juping Xie1, Xiaofeng Jiang1, Zhenhui Huang1, Dawei Zhang1, Hao Liang1, Ping Xue1,()   

  1. 1. Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China
  • Received:2019-08-18 Published:2019-12-10
  • Corresponding author: Ping Xue
  • About author:
    Corresponding author: Xue Ping, Email:
引用本文:

谢炬平, 蒋小峰, 黄镇辉, 张大伟, 梁浩, 薛平. 人胰岛细胞消化与提纯改良方法研究[J]. 中华肝脏外科手术学电子杂志, 2019, 08(06): 557-560.

Juping Xie, Xiaofeng Jiang, Zhenhui Huang, Dawei Zhang, Hao Liang, Ping Xue. Study of modified methods of digestion and purification of human islet cells[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2019, 08(06): 557-560.

目的

探讨改良人胰岛细胞消化与提纯方法。

方法

胰腺供体来源于20例成人公民逝世后器官捐献(DCD)供者捐献,由广州医科大学附属第二医院器官移植中心提供,且通过医院伦理委员会审查。供者男13例,女7例;平均年龄(45±7)岁。4℃的UW液腹主动脉灌注后迅速将胰腺切除,胰腺组织4℃ UW液保存,胰管修剪后灌注胶原酶V,放入新型自制改良胰腺消化罐,38℃的胶原酶消化液恒温消化30 min,通过不连续密度梯度法分离提纯胰岛细胞,通过双硫腙(DTZ)染色显示胰岛细胞,计算胰岛当量。吖啶橙(AO)染色法测定存活率。

结果

共消化20例成人胰腺,胰腺组织消化率为92.7%。单次消化的胶原酶使用量为400 ml。胰岛细胞经过DTZ染色后呈猩红色的类圆形胰岛细胞团。每条胰腺消化后的胰岛细胞当量为(69±28)×103 IEQ/g,提纯后为(53±29)×103 IEQ/g ,纯度为79%。胰岛存活率为(94.1±2.3)%。

结论

采用我院新型自制改良胰腺消化罐可获得良好的人胰岛素细胞消化和提纯效果,且可明显减少胶原酶的使用量。

Objective

To explore the modified methods of digestion and purification of human islet cells.

Methods

Donor pancreases from 20 donors after cardiac death (DCD) were provided by the Organ Transplantation Center of the Second Affiliated Hospital of Guangzhou Medical University and the local ethical committee approval was received. Among 20 donors, 13 cases were male and 7 female, aged (45±7) years on average. The pancreas was resected immediately after perfusion of 4℃ UW solution in the abdominal aorta and the pancreatic tissues were stored in 4℃ UW solution. The trimmed pancreatic duct was injected with collagenase V and was digested with collagenase digestion juice at 38℃ for 30 min in a self-designed pancreatic digestion tank. The islet cells were isolated and purified by discontinuous density gradient centrifugation method and were stained with dithizone (DTZ) staining. The islet equivalent quantity was calculated. The survival rate of islet cells was determined by acridine orange (AO) staining.

Results

A total of 20 samples of adult pancreases were digested with a digestive rate of 92.7%. The amount of collagenase for single digestion was 400 ml. After DTZ staining, the islet cells were observed in scarlet round-like cell clusters. The islet equivalent quantity of each pancreas after digestion was (69±28)×103 IEQ/g, and (53±29)×103 IEQ/g after purification with a purity of 79%. The survival rate of islet was (94.1±2.3)%.

Conclusions

Excellent digestion and purification effect of human islet cells can be obtained by using the novel self-designed modified pancreatic digestion tank, which can significantly reduce the dosage of collagenase.

图1 倒置显微镜下观察提纯后胰岛细胞(×200)
[1]
Yang SH, Dou KF, Song WJ. Prevalence of diabetes among men and women in China[J]. N Engl J Med, 2010, 362(25):2425-2426.
[2]
刘颖,肖平,陈焕伟.胰岛移植治疗1型糖尿病的现状和未来[J]. 器官移植,2019, 10(1):91-93.
[3]
Livingstone SJ, Levin D, Looker HC, et al. Estimated life expectancy in a Scottish cohort with type 1 diabetes, 2008-2010[J]. JAMA, 2015, 313(1):37-44.
[4]
Brennan DC, Kopetskie HA, Sayre PH, et al. Long-term follow-up of the Edmonton Protocol of islet transplantation in the United States[J]. Am J Transplant, 2016, 16(2):509-517.
[5]
姚豫桐,魏玲玲,杨卯竹,等.同种异体胰岛移植治疗1型糖尿病10例及随访观察3年的临床研究[J].中华器官移植杂志,2016, 37(2):90-94.
[6]
NIH CIT Consortium Chemistry Manufacturing Controls Monitoring Committee, NIH CIT Consortium. Purified human pancreatic islet: qualitative and quantitative assessment of islets using dithizone (DTZ): standard operating procedure of the NIH clinical islet transplantation consortium[J]. CellR4 Repair Replace Regen Reprogram, 2015, 3(1):e1369.
[7]
Street CN, Lakey JR, Shapiro AM, et al. Islet graft assessment in the Edmonton Protocol: implications for predicting long-term clinical outcome[J]. Diabetes, 2004, 53(12):3107-3114.
[8]
Thompson DM, Meloche M, Ao Z, et al. Reduced progression of diabetic microvascular complications with islet cell transplantation compared with intensive medical therapy[J]. Transplantation, 2011, 91(3):373-378.
[9]
Shapiro AM. State of the art of clinical islet transplantation and novel protocols of immunosuppression[J]. Curr Diab Rep, 2011, 11(5):345-354.
[10]
Anazawa T, Okajima H, Masui T, et al. Current state and future evolution of pancreatic islet transplantation[J]. Ann Gastroenterol Surg, 2018, 3(1):34-42.
[11]
Chang CA, Lawrence MC, Naziruddin B. Current issues in allogeneic islet transplantation[J]. Curr Opin Organ Transplant, 2017, 22(5):437-443.
[12]
Bottino R, Knoll MF, Knoll CA, et al. The future of islet transplantation is now[J]. Front Med, 2018(5):202.
[13]
Girman P, Saudek F. The IKEM pancreas and islet transplant program as part of healthcare for type 1 diabetes patients: retrospective analysis of outcome from 1983 to 2010[J]. Rev Diabet Stud, 2011, 8(1):35-43.
[14]
Gürol AO, Yıllar G, Kurşun AO, et al. A modified automated method for isolation of viable pancreatic islets in laboratory animals[J]. Transplant Proc, 2004, 36(5):1526-1527.
[15]
Shapiro AM, Pokrywczynska M, Ricordi C. Clinical pancreatic islet transplantation[J]. Nat Rev Endocrinol, 2017, 13(5):268-277.
[16]
Li N, Zhang Y, Liu Y, et al. Improved islet purity by the hypertonic-hypotonic method[J]. Int J Artif Organs, 2014, 37(6):477-485.
[17]
Ricordi C, Goldstein JS, Balamurugan AN, et al. National institutes of health-sponsored clinical islet transplantation consortium phase 3 trial: manufacture of a complex cellular product at eight processing facilities[J]. Diabetes, 2016, 65(11):3418-3428.
[18]
Foster ED, Bridges ND, Feurer ID, et al. Improved health-related quality of life in a phase 3 islet transplantation trial in type 1 diabetes complicated by severe hypoglycemia[J]. Diabetes Care, 2018, 41(5): 1001-1008.
[19]
Holmes-Walker DJ, Gunton JE, Hawthorne W, et al. Islet transplantation provides superior glycemic control with less hypoglycemia compared to continuous subcutaneous insulin infusion or multiple daily insulin injections[J]. Transplantation, 2017, 101(6):1268-1275.
[20]
Choudhary P, Rickels MR, Senior PA, et al. Evidence-informed clinical practice recommendations for treatment of type 1 diabetes complicated by problematic hypoglycemia[J]. Diabetes Care, 2015, 38(6): 1016-1029.
[1] 谢迎东, 孙帼, 徐超丽, 杨斌, 孙晖, 戴云. 超声造影定量评价不同生存期移植肾血流灌注的临床价值[J]. 中华医学超声杂志(电子版), 2023, 20(07): 749-754.
[2] 刘林峰, 王增涛, 王云鹏, 钟硕, 郝丽文, 仇申强, 陈超. 足底内侧皮瓣联合甲骨皮瓣在手指V度缺损再造中的临床应用[J]. 中华损伤与修复杂志(电子版), 2023, 18(06): 480-484.
[3] 陈永沛, 仲海燕, 陈勇, 王慜, 王倩, 邹鸣立, 袁斯明. 数字减影血管造影在腓动脉穿支皮瓣移植中的应用[J]. 中华损伤与修复杂志(电子版), 2023, 18(06): 507-510.
[4] 韩李念, 王君. 放射性皮肤损伤治疗的研究进展[J]. 中华损伤与修复杂志(电子版), 2023, 18(06): 533-537.
[5] 刘竹影, 周年苟, 李泳祺, 周丽斌. 空心环钻联合手术导板用于自体牙移植牙槽窝备洞[J]. 中华口腔医学研究杂志(电子版), 2023, 17(06): 418-423.
[6] 周岩冰, 刘晓东. 腹腔镜右半结肠癌D3根治术消化道吻合重建方式的选择[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 9-13.
[7] 吴方园, 孙霞, 林昌锋, 张震生. HBV相关肝硬化合并急性上消化道出血的危险因素分析[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 45-47.
[8] 逄世江, 黄艳艳, 朱冠烈. 改良π形吻合在腹腔镜全胃切除消化道重建中的安全性和有效性研究[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 66-69.
[9] 严庆, 刘颖, 邓斐文, 陈焕伟. 微血管侵犯对肝癌肝移植患者生存预后的影响[J]. 中华肝脏外科手术学电子杂志, 2023, 12(06): 624-629.
[10] 廖梅, 张红君, 金洁玚, 吕艳, 任杰. 床旁超声造影对肝移植术后早期肝动脉血栓的诊断价值[J]. 中华肝脏外科手术学电子杂志, 2023, 12(06): 630-634.
[11] 何吉鑫, 杨燕妮, 王继伟, 李建国, 谢铭. 肠道菌群及肠道代谢产物参与慢性便秘发生机制的研究进展[J]. 中华结直肠疾病电子杂志, 2023, 12(06): 495-499.
[12] 郭晓磊, 李晓云, 孙嘉怿, 金乐, 郭亚娟, 史新立. 含生长因子骨移植材料的研究进展和监管现状[J]. 中华老年骨科与康复电子杂志, 2023, 09(06): 373-378.
[13] 高显奎, 赵太云, 陆兴俊, 张洪领, 房修罗, 闫碧春, 王胤, 王永翠, 刘苗苗, 冉若男. 内镜电凝止血与组织胶注射治疗上消化道溃疡伴出血的疗效观察[J]. 中华消化病与影像杂志(电子版), 2023, 13(06): 452-455.
[14] 卓少宏, 林秀玲, 周翠梅, 熊卫莲, 马兴灶. CD64指数、SAA/CRP、PCT联合检测在小儿消化道感染性疾病鉴别诊断中的应用[J]. 中华消化病与影像杂志(电子版), 2023, 13(06): 505-509.
[15] 王家圆, 王晓东. 消化系统恶性肿瘤相关肌少症的研究进展[J]. 中华临床医师杂志(电子版), 2023, 17(07): 823-827.
阅读次数
全文


摘要