生物素-亲和素技术锚定肝素于胰岛表面

Heparin anchored to the surface of islet by avidin-biotin technique

背景:胰岛微血管在胰岛分离过程中被破坏,进而影响移植后胰岛的营养供应。肝素对于血管的再生具有非常重要的意义;同时,临床胰岛移植多应用肝素来抑制血栓形成,但全身肝素化增加了出血的风险。而亲和素同时具备生物素和肝素2个较强的结合位点。目的:利用亲和素这一特性,应用生物素-亲和素技术,将肝素锚定于胰岛的表面,促进胰岛再血管化,同时达到避免全身应用大剂量肝素带来出血风险的目的。方法:根据Ricordi自动化法分离纯化成人胰腺,然后将成人胰岛依次和0,0.5,1,1.5,2g/L生物素(包括生物素N-羟基丁二酰亚胺酯、长臂活化生物素、肼化生物胞素、生物素酰肼、TFP-biotin),1g/L亲和素素,0.5,1.0,1.5,2.0g/L肝素孵育培养,观察肝素化变化。结果与结论:TFP-biotin介导胰岛表面肝素化效果最好,且其肝素化前后胰岛活力率变化的差异无显著性意义(P>0.05),同时肝素化和未肝素化胰岛具备相似的胰岛素释放反应(P>0.05)。提示生物素-亲和素技术是一种安全、有效的胰岛表面肝素化处理方法。

BACKGRUND：Islet capillaries are damaged in the process of islet isolation, thereby affecting the nutrient supply of islets after transplantation. Heparin has a very important significance for the regeneration of blood vessels; meanwhile, heparin is commonly used in the clinical islet transplantation to inhibit thrombosis. But systemic heparin can increase the risk of bleeding. The avidin has two strong binding sites of biotin and heparin respectively. .-, To improve islet revascularization and decrease risk of bleeding resulting from heparin systemic application through anchoring the heparin on the surface of islet with avidin-biotin technique based Adult human pancreas were isolated and purified with Ricordi automation method, then the islets were incubated and cultured with 0, 0.5, 1, 1.5, 2 g/L biotin （including biotin-N-hydroxysuccinimide ester, N-hydroxy-succinimido-6-biotinyl amido hexanoate, biocytin hydrazine, biotin hydrazide and TFP-biotin）, 1 g/L avidin, and 0.5, 1.0, 1.5 and 2.0 g/L heparin, the change of heparin was observed. TFP-biotin had the best effect to mediate the islet surface heparinization, and there was no significant difference in the activity of islet before and after heparinization （P 〉 0.05）; the heparinized and unheparinized heparin islets had the similar insulin release reaction （P 〉 0.05）. Biotin-avidin technique is a safe and effective islet surface heparinization treatment method.