骨髓间充质干细胞在小肠移植大鼠体内的定植研究

Location of bone marrow mesenchymal stem ceils in rats after small intestinal transplantation

目的观察骨髓间充质干细胞（MSC）在异位小肠移植大鼠体内的定植情况。方法骨髓细胞取白1月龄的雄性Lewis大鼠，通过密度梯度离心法结合贴壁法分离和培养骨髓MSC；通过流式细胞术对MSC表面抗原（CD29、CD90、CD34和CD45）进行鉴定；应用终浓度为5Ixmol／L的5（6）-羧基二乙酸荧光素琥珀酰亚胺酯（CFSE）对生长良好的第3代MSC进行标记。以成年雄性近交系F344大鼠作为供体、成年雄性Lewis大鼠作为受体，建立大鼠异位小肠移植模型。术后经阴茎背静脉将CFSE标记的MSC注入受体体内，术后第7天收集冰冻病理标本，在荧光显微镜下观察MSC的定位情况。结果成功分离培养卅骨骼MSC，其表面抗原CD29、CD90、CD34和CD45的平均阳性表达率分别为96．48％、99．77％、2．41％和1．39％。CFSE能有效标记MSC，术后7d在移植小肠的间质、脾脏和胸腺可观察到大量绿色荧光，自体小肠组织仅见微量荧光，而心、肝和肺组织基本未见绿色荧光。结论骨骼MSC能成功定植于大鼠小肠移植术后的移植小肠中。

Objective To study the bone marrow mesenchymal stem ceils （MSC） settled in rats after small intestinal transplantation. Methods Bone marrow MSCs were taken from 1-month male Lewis rats, isolated and cuhured by density gradient centrifugation and differential adherent culture. The surface antigens （CD29, CD90, CD34 and CD45） of MSC were identified by flow cytometry. Final concentration of 5 μg/L CFSE was used to mark the third generation of MSCs. Adult male inbred line F344 rats were used as donor and adult male Lewis rats as acceptor. A heterotopic intestinal transplant rat model was established by F344 to Lewis. Labeled MSCs were injected into model rats through vena dorsalis penis after operation. Tissues at postoperative 7-day were collected for frozen pathology to reveal the location of transplanted MSCs under fluorescence microscope. Results MSCs were successfully isolated from rat bone marrow. The average positive expression rates of surface antigens CD29, CD90, CD34 and CD45 were 96.48%, 99.77%, 2.41% and 1.39% respectively. MSCs were successfully and effectively marked with CFSE. Seven days after operation, a large number of green fluorescence could be observed in transplanted intestine, spleen and thymus. Autograft intestinal tissues only showed trace fluorescence, and the heart, liver and lung tissue basically did not present the green fluorescence. Conclusion Bone marrow MSCs can settle in transplanted small intestine of rat.