内皮祖细胞灌注供心延长心脏移植物存活时间的实验研究

 目的 研究供体内皮祖细胞（endotherial progenitor cell， EPC）灌注供心对同种异体心脏移植物生存时间的影响。方法 获取Sprague Dawley （SD）大鼠的骨髓细胞，通过密度梯度离心法分离培养单个核细胞，取贴壁细胞选择性诱导培养2周以上，获取内皮祖细胞（EPC）。以Dil-ac-LDL、FITC-UEA-1双荧光染色法鉴定EPC。20只体重200～250g的雄性SD大鼠为供体，20只体重200～250g的雌性Wistar大鼠为受体，分为4组，每组5对。组I：术后不使用环胞霉素；组II：术后7d每天使用环胞霉素5mg/kg；组III：术中供心经冠脉灌注内皮祖细胞1×106个，术后不使用环胞霉素；组IV：术中供心经冠脉灌注内皮祖细胞，术后7d每天使用环胞霉素5mg/kg。观察各组供心存活时间。结果 贴壁细胞经诱导培养后3d开始伸展，5d形成集落，7~10d增殖加速并出现条索状结构，2周大部分细胞呈多角形。Dil-ac-LDL、FITC-UEA-1双染，免疫荧光染色阳性率大于70％。组I、II、III、IV各组供心平均存活时间分别为6.2±0.8、11.6±1.1、9.4±1.1和18.8±1.6d。组II、III、IV移植物的平均存活时间较组I显著延长（P<0.05）。结论 内皮祖细胞灌注合并环胞霉素运用可以延长大鼠心脏移植物的存活时间。

Perfusion of donor endothelial progenitor cell perfusion prolongs the survival of rat cardiac allograft

Objective To study the effect of endotherial progenitor cell (EPC) perfusion on survival time of cardiac allograft and acute rejection in heart transplantation. Methods The mononuclear cells were isolated from bone marrow of Sprague Dawley (SD) rats by density gradient centrifugation and cultured for 48 hours. Then the wall attached cells were cultured in the selective culture medium (with the supplement of VEGF, bFGF, IFG-1 and EGF) for 2 weeks. The cells were stained with Dil-ac-LDL and FITC-UEA-1 for identification. Twenty pairs of Wistar rats and SD rats were randomizely divided into four groups (5 pairs in each group). The animal transplantation model was used. Group I was set up as the control group. In Group II, the rats were fed with CsA after transplantation for 7 days. In Group III, the donor heart were perfused with 1×106 EPCs before implantation. In Group IV, the donor hearts were perfused with 1×106 EPCs before implantation and the rats were fed with CsA after transplantation for 7 days. Results The attached cells stretched, differentiated, and exhibited the colonal morphology after inducing culture for 3 to 5 days. The cells proliferated faster after 7-10-day incubation when cord-like structure was observed. After 2-week induction, most of the cells exhibited multi-angular morphology. More than 70% cells were stained positively with Dil-ac-LDL and FITC-UEA-1 (double positive fluorescence). The average donor heart survival time was (6.2±0.8) days in Group I，(11.6±1.1) days in Group II，(9.4±1.1) days in Group III and (18.8±1.6) days in Group IV. The survival times in Group II, III and IV were prolonged significantly than that of the Group I (P<0.01). The survival time in Group IV was the longest among these four groups (P<0.01). Conclusion EPC perfusion with CsA administration prolongs the survival time of the allograft heart.