同种胰岛细胞移植的最佳移植数量研究

Study on optimized quantity of islet cells in homogeneous transplantation of type-Ⅰ diabetes rats

目的探讨同种胰岛移植最低胰岛细胞有效数量,为临床高效利用有限的胰岛细胞提供实验依据。方法同种胰岛来自SD大鼠,原位灌注切取胰腺,用胶原酶Ⅴ消化胰腺组织,Ficoll400密度梯度离心纯化得到胰岛细胞,DTZ染色进行胰岛细胞计数及纯度鉴定、AO-PI荧光染色进行活性鉴定,采用体外胰岛素释放试验鉴定胰岛功能。大鼠一次性静脉注射STZ60mg/kg诱导Ⅰ型糖尿病模型,然后按照移植的胰岛细胞数量不同随机分为:A组(6000 IEQ/kg)、B组(9000 IEQ/kg)、C组(12000 IEQ/kg)、D组(15000 IEQ/kg)。经门静脉途径进行胰岛移植。观察移植后连续10d的血糖变化,移植3d后血液中胰岛素分泌水平的变化。结果纯化后胰岛细胞收获量为(3.49±0.23)×105 IEQ/kg,纯度为86%,活性为90%。体外胰岛素释放实验显示胰岛功能良好,胰岛细胞移植后,A组、B组血糖未降至正常。C、D组血糖降至正常范围,最早出现时间分别为22h、25h。胰岛移植3d后,随着胰岛数量增多,受体大鼠血液中的胰岛素量随之升高。结论每次以12000 IEQ/kg的数量进行胰岛移植可以作为大鼠胰岛细胞移植的最低有效浓度,可以达到胰岛移植的最佳效果也可以最大程度地利用有限的胰岛细胞。

Objective To optimize the quantity of islet cells for homogeneous transplantation in type Ⅰ diabetes rats. Methods Homogeneous islet grafts were isolated from SD rats. The pancreas was gained by in situ perfusion, and then was digested by collagenase V and cleared by Ficoll400 for islet cells. Islet cells were confirmed by DTZ staining and then calculated. AO-PI fluorescent staining was adopted to detect the activity of islet cells. Insulin release test was used to evaluate the function of islet cells. Type- I diabetes rat models were induced by intravenous injection of STZ（60 mg/kg）. The model rats were randomly divided into four groups according to the concentration of islet grafts ： group A （ 6 000 IEQ/kg）, group B （ 9 000 IEQ/kg）, group C （ 12 000 IEQ/kg）, group D （ 15 000 IEQ/ kg）. The levels of blood glucose and insulin were monitored at different time points. Results In group A and group B,blood glucose did not drop to the normal level after transplantation. In group C and group D, blood glucose reduced to the normal level after transplantation for 22 h,25 h, respectively. After islet transplantation for 3 d, the levels of insulin stepped up with the increased quantity of islet cells. Conclusion The concentration of 12 000 IEQ/kg is the optimized concentration for homogeneous transplantation of islet in rats.