血管生长素基因转染5-氮胞苷诱导后的骨髓基质细胞移植于缺血心肌的实验研究

目的　利用编码血管生长素基因的腺病毒 (Ad .ANG)转染 5 氮胞苷 (5 aza)诱导骨髓基质细胞 (BMSC) ,移植于大鼠缺血心肌后对心功能的保护和促血管新生作用。方法　体外培养Lewis大鼠的骨髓基质细胞 ,使用 3μmol/L的 5 aza体外强化诱导 2 4h ;然后在 5 0倍的转染倍数 (病毒数 /靶细胞 )下 ,用Ad .ANG转染诱导BMSC。通过ELISA方法检测其对ANG的表达和分泌。然后将其移植于结扎了冠状动脉前降支的Lewis大鼠缺血心肌内 (组Ⅰ )。同时设置单纯细胞移植组 (组Ⅱ )和基因治疗组 (组Ⅲ ) ,以注射无血清培养基的实验动物为对照组 (组Ⅳ )。通过心脏射血分数 (EF)、左室舒张末期容积等超声指标研究其对心功能的保护作用 ;另外通过免疫组化、电镜研究细胞存活、分化和血管新生情况。结果　Ad ANG转染BMSC后 ,在培养上清中ANG大量出现 ,4～ 7d时达到表达高峰 ,15d后仍可检测到蛋白质表达。移植于缺血心肌 4周后 ,组Ⅰ和组Ⅱ缺血区的移植细胞大部分分化为心肌样细胞 ,组ⅠEF改善程度明显好于组Ⅱ (P <0 0 5 )、组Ⅲ (P <0 0 1)和组Ⅳ (P <0 0 1) ,在促血管新生方面 ,组Ⅰ也明显优于以上 3组 (组Ⅰ 36 3个 /HPF ;组Ⅱ 10 5个 /HPF ;组Ⅲ 2 3 1个 /HPF ;组Ⅳ 0 9个 /HPF)。结论　转染Ad ANG后的BMSC移植于缺

Cell transplantation of 5-aza cytidine induced bone marrow stromal cells transfected by angiogenin gene ex vivo into infarcted myocardium,an experimental study

OBJECTIVE: To investigate the effect of transfection of 5-aza cytidine (5-aza) induced BMSCs that were transfected with Ad. ANG ex vivo into infarcted myocardium. METHODS: Lewis rat BMSCs were cultured in vitro and incubated together with 5-aza (3 micro mol/l) for 24 hours, and the induced BMSCs were transfected with Ad. ANG with a infection multiple of 50. ELISA method was applied to assay the expression and secretion of ANG in the medium. Then such BMSCs expressing ANG were transplanted into the ischemic myocardium of 14 Lewis rats whose left descending branch of coronary artery had been ligated 4 weeks ago (Group I). Eight rats with infarcted myocardium were transplanted with such BMSCs too (group II). Another eight rats received only Ad. ANG injection (group III). Twelve rats receiving serum-free medium injection were used as controls (Group IV). Four weeks after the ligation of LDA (for group I) and 4 weeks after the beginning of experiment (for all groups), the parameters of heart function, such as ejection fraction (EF) and end-diastole volume of left heart (EDLV), were examined by echocardiography. Four weeks after the beginning of experiment, the rats were killed, and the survival and differentiation of transplanted BMSCs and angiogenesis were observed by immunohistochemistry and transmission electron micrography. RESULTS: One day after the beginning of experiment, the concentration of ANG in the supernatant of medium was 162 +/- 10 pg/ml, significantly higher than that in the supernatant of medium in control group (86 +/- 5 pg/ml, P < 0.01). The concentration of ANG gradually increased and reached its peak at the 4th to 7th day; ANG could still be assayed 15 days later. 4 weeks after the transplantation, most of the 5-aza-induced BMSCs in group I and group II had differentiated into cardiomyogenic cells in the transplanted area. The EF value was 0.42 +/- 0.114 weeks after ligation of LAD, and was 0.69 +/- 0.034 weeks after the transplantation in group I. The EF value was 0.46 +/- 0.06 at the beginning of experiment and was 0.64 +/- 0.144 weeks after the transplantation in group II. The improvement of EF in Group I was significantly than that in other 3 groups (P < 0.05 or P < 0.01). The number of new vessels in group I was 36.3/high-power field (HPF), significantly higher than those in the other three groups (10.5/HPF in Group II, 23.1/HPF in Group III, and 0.9/HPF in Group IV). CONCLUSION: Induced by 5-aza, BMSCs differentiate into cardiomyogenic cells in infracted myocardium. ANG-expressing BMSCs transplantation is one of the safe and beneficial new strategies for repairing damaged myocardium and enhancing angiogenesis in ischemic myocardium.