超声微泡联合双特异性抗体对骨髓间充质干细胞干预心肌纤维化的实验研究

Bone marrow mesenchymai stem cells with support of ultrasound-mediated microbubbles and bispecific antibody prevent myocardial fibrosis via HSP-70

目的探讨骨髓间充质干细胞（MSCs）在超声辐照微泡（MB）和双特异抗体（BiAb）的辅助下干预心肌纤维化的效果及可能机制。方法在MB的辅助下，将雄性小鼠MsCs与BiAb输入异丙肾性心肌纤维化雌性小鼠（MSCs＋BiAb＋MB组）。BiAb由能识别间充质干细胞的anti—CD29和能特异性结合损伤心肌的抗肌凝蛋白轻链抗体（AMLcA）制备。另设未治疗组、单纯MSCs组、单纯BiAb组、单纯MB组、MSCs＋BiAb组和正常对照组。5周后处死小鼠，实时荧光定量PCR检测心肌Y染色体鉴别基因（SRY）的表达。天狼猩红染色对比心脏胶原纤维含量。免疫组织化学染色法观察心脏热休克蛋白-70（HSP-70）表达。结果MSCs归巢数最多的是MSCs＋BiAb＋MB组，其次为MSCs＋BiAb组，MSCs组归巢数最少。与未治疗组相比，MSCs＋BiAb＋MB组、MSCs＋BiAb组和单纯MCSs组的心肌胶原纤维含量下降（P〈0．05），HSP-70表达下调（P〈0．05）。其中，与单纯MSCs组相比，MSCs＋BiAb＋MB组的心肌胶原沉积量更低（P〈0．05）。单纯MB组、单纯BlAb组与未治疗组的胶原沉积量、HSP-70表达差异无统计学意义（P〉0．05）。结论MB可促进骨髓间充质于细胞的归巢，联合BiAb，可进一步增加干细胞的归巢率和修复效果。MSCs归巢后能干预心肌纤维化，其机制可能与HSP-70的介导有关。

Objective To explore the effects of transplantated bone marrow mesenchymal stem cells （MSCs） on myocardial fibrosis with the aid of ultrasound-mediated microbubbles （MB） and bispecific antibody（BlAb） combination. Methods With the aid of MB,isolated MSCs from male mice and the BlAb were transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein （MSCs ＋ BlAb ＋ MB group）. BiAb was producted with anti-CD29 which can recognize MSCs and anti-myosin light chain antibody （AMLCA） which can specifically bind to injured myocardium. There were six groups investigated： MSC ＋ BlAb ＋ MB, MSC, BiAb, MB, MSC ＋ BlAb, untreated, and control groups. Five weeks after treatment administration,the expressions of sex-determining region of Y-chromosome （SRY） in myocardium were detected by fluorescent quantitative PCR. The distribution of collagen was observed by sirius red staining. Heat shock protein-70 （HSP-70） in myocardium was detected by immunohistochemistry. Results The highest homing number of MSCs was in the MSCs ＋ BlAb ＋ MB group, MSCs ＋ BlAb group ranked the second, and lowest in MSCs group. Compared to the untreated group, the MSCs ＋ BlAb ＋ MB, MSCs ＋ BlAb and MSCs groups had less collagen deposition （ P〈 0.05）, and decreased level of HSP-70. Compared to those of the MSCs group,the collagen deposition were decreased in MSCs ＋ BlAb ＋ MB group （ P〈0.05）. Conclusions MB and BlAb can promote the homing number of MSCs in mice. MB can further the homing rate and the repairing efficacy of MSCs. The homing MSCs can prevent the process of myocardial fibrosis. And HSP-70 was involved in the internal mechanism.