同种异体骨髓间充质干细胞移植治疗糖尿病大鼠的实验研究

目的：观察同种异体骨髓间充质干细胞（BMSC）移植对糖尿病大鼠的作用。方法：通过贴壁法分离培养同种异体SD大鼠的BMSC，应用重组腺病毒-绿色荧光蛋白（Ad-GFP）病毒感染传2代的BMSC，并经筛选获得稳定表达GFP的BMSC用于移植。将链脲佐菌素（STZ）诱导的糖尿病大鼠（血糖≥16．7mmol/L）随机分为实验对照组（22．27±2．23）mmol／L，n=6）和移植组（2218±194）mmol／L,n=9）]。移植组大鼠尾静脉注射BMSC悬液04ml，细胞数lxlO^6xlO^7／ml；在实验对照组及造模前随机抽取的5只正常对照组大鼠尾静脉注射生理盐水。移植2周后采血测各组血糖、糖化血红蛋白（HbAc）、胰岛素，并处死大鼠剖取胰腺，制作石蜡切片进行胰岛素免疫组化及冰冻切片荧光倒置显微镜直接观察GFP表达。结果：实验2周，实验对照组糖尿病大鼠血糖（27．96±1．87）mmol／L，n=6）]、HbAlc（22．51±2．99，n=6）明显高于正常对照组（7．97±1.00）mmol／L；（9．15±0．82，n=5）]，胰岛素（11．57±3．25）mU／L，n=6）]明显低于正常对照组（33．68±6．21）mUL，n=5）]（P〈0．001）。移植组血糖（23．63±6．42）mmol／L，n=9）]、HbA1c（18．26±3．96，n=9）低于实验对照组（尸〈O．05），胰岛素（13．89±7．89）mU／L，n=9）1高于实验对照组（尸〉0．05）。免疫组化结果显示，实验对照组胰岛及胰岛中心细胞数明显减少。胰岛素反应基本阴性。仅见个别胰岛细胞内出现少量微弱淡黄色的细颗粒。移植组大鼠胰岛中心部细胞增多，大部分胰岛素反应阳性或强阳性呈黄色或褐色颗粒。胰腺外分泌组织可见表达EGFP的细胞，胰岛中未发现其表达。结论：糖尿病大鼠经同种异体BMSC移植后，血糖、HbA，c均下降，胰岛素水平升高，可能通过内源性胰岛细胞增生发挥治疗作用。

An experimental study on treatment of diabetic rats through transplantation of allogeneic bone marrow mesenchymal stem cells

Objective： To observe the curative effectiveness of allogeneic bone marrow mesenchymal stem cells （BMSC） transplantation in diabetic rats and the possible mechanism. Methods： BMSC were isolated from allogeneic bone marrow of SD rat by adhesive screening method and cultured in vitro. The second passage of BMSC was transfected with Ad GFP. BMSC that stably expressed GFP were obtained before transplantation. Streptozotocin induced diabetic rats （plasma glucose t〉 16.7 mmol/L） were divided at random into untreated group （UTG） and BMSC transplantation group （TPG）. BMSC were trans planted into allogeneic diabetic rats of TPG through vena caudalis （0.4 ml, lxlO^6-lxlO7/ml）. Another 5 normal＇ rats picked out at random （normal control group） and the diabetic rats of UTG were injected via tail vein with the same quantity of physiological saline. After the 2 weeks transplantation, the levels of plasma glucose, HbAlc and insulin were determined. The pancreas was dissected out for pathological sections. Insulin reaction with immunohistochemistry of rats was observed. Fluorescent microscopy was used to observe the expression of GFP labeled cells in the pancreas. Results： The result showed that levels of plasma glucose and HbAjc were higher obviously in UTG than in normal control group （NCG）, and in sulin level lower （P〈0.001）. The levels of plasma glucose and HbAlc were lower obviously in TPG than in UTG （P〈O.05）, and insulin level higher （P〉O.05）. The numbers of beta cells of the UTG decreased and the insulin reaction showed negative results. The numbers of beta cells of the TPG increased and the insulin reaction showed positive and strong positive re suits. The cells of GFP were observed in the excrine tissues of pancreas, but not observed in the islet. Conclusion： It is concluded that BMSC transplantation by tail vein injection could engraft into pancreas tissues of diabetic rats, depress the levels of plasma glucose and HbA1c, accelerate the synthesis of the insulin. BMSC transplantation might promote endoge- nous regeneration of islets in diabetic rats.