猪胰岛细胞微囊化后腹腔移植治疗小鼠糖尿病

Transplantation of encapsulated adult pig islets in peritoneal cavity of diabetic mice

目的探讨猪胰岛细胞微囊化后腹腔移植治疗小鼠糖尿病的效果。方法采用胶原酶灌注消化法及不连续密度梯度离心法分离并纯化成体猪胰岛，并用海藻酸钠、多聚赖氨酸对猪胰岛进行微囊化。双硫腙（DTZ）染色观察分离胰岛纯度，葡萄糖刺激胰岛素释放实验检测纯化胰岛和微囊化胰岛体外功能。利用链脲佐菌素（STZ）200mg／kg腹腔内注射，制作BALB／c小鼠1型糖尿病动物模型。成模后动物随机分为3组：猪胰岛微囊组、空微囊组和生理盐水对照组。每只小鼠腹腔内分别移植500个相应胰岛，检测移植前后小鼠的血糖变化。采用单因素方差分析法进行统计分析。结果不连续密度梯度离心纯化后，得到胰岛的纯度为〉90％；碘化丙啶／双醋酸荧光素（PI／FDA）染色后，测定胰岛的活力为〉90％；葡萄糖刺激胰岛素分泌检测，分离纯化的胰岛和微囊化胰岛的刺激指数均〉2．0；将微囊化胰岛移植到STZ诱导的糖尿病小鼠腹腔内，能够使小鼠血糖恢复并维持正常超过60d〈9．3mmol／L）。与空囊移植组和生理盐水对照组比较，血糖为正常水平的控制时间明显延长（F=13．587，P〈0．05）。病理学检查发现，微囊内胰岛形态基本完整，胰岛素染色为阳性。结论微囊化胰岛腹腔移植后，能有效降低小鼠血糖，微囊具有较好的免疫隔离作用。

Objective To evaluate the effects of intraperitoneal transplantation of encapsulated adult pit islets on blood glucose of diabetic mice. Methods Panereata were digested with collagenase P using Rieordi chamber and purified with discontinued density gradient centrifugation and subsequently cultured. Islets were encapsulated using alginate-polylysine polymers. The quantity and purity of islets was determined by manual and counter machine after dithizone （DTZ） staining. Glucose stimulated insulin secretion test was performed to test the function of purified islets and encapsulated islets in vitro. BALB/c mice were intraperitoneal injected with streptozocin （STZ） 200 mg/kg body weight to induce type 1 diabetes model. Diabetic mice were randomly divided into 3 groups：encapsulated pig islet group, empty capsule group and saline control group. Encapsulated capsules （equivilent to 500 pig islets）, empty caplules and normal saline were respectively transplanted into peritoneal cavity of three groups. The blood glucose of all the mice were regularly monitored before and after transplantation. Results After purification of discontinued density gradient centrifugation, the mean purity of the islets was more than 90%. The viability of the islets was more than 90% by propidium iodide （PI） and fluorescein diaeetate （FDA） staining. Glucose-stimulated insulin secretion （GSIS） of purified islet and encapsulated islet was more than 2. 0 in vitro. In vivo, after transplantation, encapsulated pig islets group had a significantly improved survival and biochemical profile, compared to mice transplanted with empty capsule group and saline control group. Blood glucose of encapsulated pig islets group significantly reduced more than 60 days （ 〈 9. 3 mmol/L） , Duration time of normal blood glucose in encapsulated pig islets group was significantly longer, compared with empty capsulesgroup and saline control group （ F = 13. 587, P 〈 0. 05 ）. The pathologic structure of encapsulated islets was normal, which of insulin staining was positive. Con^luslons Encapsulated pig islet transplantation exerts good effects of reduction of blood glucose on diabetic mice and the capsules have good immuno-isolating function.