小鼠血管内皮细胞表达人DAF和CD59对其心脏在人血清灌注下做功的影响

Effects of human DAF and CD59 co-expression in vascular endothelia in transgenic mice on heart work during human plasma peffasion

目的探讨联合转入衰变加速因子(DAF)和CD59基因对小鼠心脏在人血清灌注下做功及组织结构的影响。方法采用受精卵显微注射技术,将人DAF和CD59基因注入昆明小鼠受精卵的原核中,选取注射后仍健康的受精卵植入假孕母鼠的输卵管中待分娩。提取足月产小鼠(G_0代)基因组DNA,以聚合酶链反应(PCR)及Southern印迹杂交确定外源基因整合情况,应用流式细胞术检测人DAF和CD59在转基因小鼠白细胞上的表达,免疫组织化学染色检测人DAF和CD59在转基因小鼠心脏、肝脏及肾脏组织中的表达。取出转基因成功小鼠的心脏,在体外以人血清进行灌注,测定其做功能力,HE染色及免疫组化染色观察灌注后心脏的组织学变化以及补体C3c的沉积情况。以单一转DAF基因的小鼠为对照。结果共获得G_0代小鼠135只,经PCR及Southern印迹杂交分析,11只(8.1%,11/135)整合有人DAF和CD59外源基因,其中6只的白细胞膜表面人DAF和CD59表达阳性,强度分别为(86±6)%和(85±8)%,单转人DA F基因者(87±7)%,差异无统计学意义。人DAF及CD59仅表达于转基因小鼠心脏、肝脏及肾脏组织中的血管内皮细胞上。在人血清灌注后60min内,普通小鼠的心脏做功急剧下降,接近40min时心脏停止搏动;转DAF基因小鼠的心脏做功也下降,但仍维持在最大值的20%以上;转DAF和CD59双基因小鼠的心脏做功维持在最大值的40%以上。在人血清灌注的10～60min,转基因小鼠心脏做功能力明显强于普通小鼠心脏(P<0.05);灌注20～60min时,转双基因小鼠的心脏做功能力明显强于转DAF基因小鼠心脏(P<0.05)。普通小鼠心脏在人血清灌注后组织裂解较多见,肿胀严重,而转基因小鼠心脏组织的病理改变明显轻于普通小鼠,转双基因者的病理变化又明显轻于单一转DAF者,心肌血管中人C3c的沉积也明显少于单一转DAF者。结论血管内皮细胞特异性表达人DAF和CD59,可明显阻抑其心脏在异种血清灌注下的做功能力下降及C3c在血管中的沉积,这可能对抵御超急性排斥反应有一定帮助。

Objective To construct transgenic mice tissue-specifically co-expressing human DAF/CD59 in the vascular endothelia and to investigate the ability to protect against human complement-mediated attack. Methods Transgenic mice were generated by co-microinjection of hDAF/CD59 expression constructs driven by the human intercellular adhesion molecule-2 （ICAM-2） promoter. PCR and Southern blot of genomic DNA were used to assess the presence of hDAF/CD59 in the genome of the founders,and the expression at protein level was measured by flow eytometry. Immunohistochemistry was used to detect the distribution of hDAF/CD59. An ex vivo perfusion model was used to compare hearts from these hDAF/CD59 transgenic mice with hDAF hearts. Results After microinjection of genes,11 of 135 mice born were shown to be double-transgenic,and human DAF/CD59 were expressed on the surface of leucocytes in 6 of the 11 DAF/CD59-integrated mice. Expression levels of 6 founders ranged from 80% to 95% of that in human leucocytes. Human DAF/CD59 were strongly expressed in the vascular endothelia of heart, kidney,with little or no positive staining observed in non-endothelial cells. Compared to hDAF hearts that maintained approximately 20% maximum work during perfusion with 20 % human plasma, these endothelial-specific hDAF/CD59 hearts were further protected with work maintained at 40 % of the maximum level during 60 min. Conclusion The introduced hDAF/CD59 genes have been integrated and specifically expressed in the vascular endothelia. The endothelial co-expression of hDAF/CD59 can provide greater protection against human complement-mediated attack than the expression of hDAF alone.