骨髓细胞参与移植物动脉硬化的作用研究

Effect of Bone Marrow Cells on Arteriosclerosis of Aortic Allograft in Rats

目的研究大鼠移植动脉新生内膜平滑肌细胞Sry基因的表达,探讨骨髓细胞在移植物动脉硬化中的作用。方法采集雄性Wistar大鼠的骨髓细胞,将其移植到经γ射线照射预处理的雌性Wistar大鼠体内,制备嵌合体大鼠;4周后建立大鼠腹主动脉移植慢性排斥反应模型,分为4组:雌性同系移植组、雌性异系移植组、雄性异系移植组、嵌合体异系移植组。术后8周,制备移植动脉组织标本进行病理组织学观察,分析移植动脉硬化情况;用抗平滑肌α-肌动蛋白(α-SMA)单抗进行SABC法免疫组化染色,检测移植动脉新生内膜细胞α-SMA的表达情况;采用显微切割技术收集移植动脉冰冻切片新生内膜平滑肌细胞,用PCR检测其Sry基因的表达,分析移植动脉新生内膜平滑肌细胞是否来源于骨髓细胞。结果异系移植组移植动脉新生内膜中α-SMA表达阳性平滑肌细胞大量增生,致动脉内膜显著增厚,血管管腔明显狭窄;动脉壁新生内膜面积及新生内膜/中膜面积比均显著高于同系移植组(P<0.01),而异系移植组之间差异无显著性意义(P>0.05)。PCR扩增显示,嵌合体异系移植组和雄性异系移植组一致,在相对分子量为225bp处均有一条特异性扩增条带,而雌性异系移植组则无相应的核酸扩增条带;结果提示移植物动脉新生内膜平滑肌细胞来源于骨髓细胞。结论在动脉移植慢性排斥反应中,骨髓细胞分化为血管新生内膜平滑肌细胞,参与移植物动脉硬化的发生和发展。

Objective To study the expression of Sry gene in neointimal smooth muscle cells and investigate the effect of bone marrow cells on transplant arteriosclerosis of aortic allograft in rats.Methods Sex-mismatched bone marrow transplantations were performed from male Wistar rats to female Wistar rats.Four weeks after transplantation,the aortic transplant model was constituted by means of micro-surgery in rats.The recipients were divided into 4 groups：female-female aortic isografts,female-female aortic allografts,male-male aortic allografts,female-chimera aortic allografts.Eight weeks after transplantation,aortic grafts were removed at autopsy and processed for histological evaluation,and the expression of α-SMA protein of neointimal cells in grafted aorta was detected by immunohistochemistry.Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method.The Sry gene was amplified from the genome of neointimal smooth muscle cells by PCR to analyze whether neointimal smooth muscle cells of aortic allograft originated from the bone marrow cells.ResultsExcessive accumulation of α-SMA-positive smooth muscle cells resulted in a significant neointima formation in rat aortic allografts 8 weeks after transplantation.The neointimal area and NIA/MA ratio of grafted aorta were significantly increased in all of aortic allograft groups as compared with those in aortic isograft group（P〈0.01）,and there was no significant difference between aortic allograft groups（P〉0.05）.PCR assay indicated that a distinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively,but not in the female-female aortic allograft group.It was suggested that neointimal smooth muscle cells of aortic allograft originated from the bone marrow cells.Conclusion In arteriosclerosis of chronic allograft rejection,bone marrow cells could differentiate into neointimal smooth muscle cells that participate in the pathogenesis of transplant arteriosclerosis.