外膜成纤维细胞在实验性脑血管痉挛中的作用

目的 在体外模拟血管壁外层结构,建立外膜成纤维细胞(AF)和平滑肌细胞(SMC)共培养,研究血管AF在蛛网膜下腔出血后脑血管痉挛中的作用.方法 (1)以多聚碳酸酯膜(PET膜)作为载体,将AF和SMC进行共培养.(2)模拟体内出血环境,在PET膜一侧接种AF细胞并加入含10-6 mol/L OxyHb的培养液,分别共培养24h、48h、72h,应用电镜观察SMC的长度.(3)提取各组AF缝隙连接蛋白Cx43进行RT-PCR半定量分析.结果 (1)扫描电镜测量发现,加入OxyHb处理的AF能使PET膜对侧未经OxyHb处理的SMC产生收缩(P＜0.001),且收缩程度与OxyHb处理AF细胞时间成正比.(2)AF缝隙连接蛋白Cx43半定量RT-PCR分析显示:24h、48h、72h组中Cx43mRNA平均相对含量较正常组上调(P＜0.01).结论 血管壁外层的AF受到OxyHb作用后可以引起未直接接触OxyHb的SMC发生持续收缩且缝隙连接蛋白Cx43mRNA表达上调,提示AF在蛛网膜下腔出血引起的脑血管痉挛中可能发挥重要作用.

The pathological role of the adventitial fibroblasts in the experimental oxyhemoglobin-induced cerebral vasospasm

Objective To study the pathological role of the adventitial fibroblasts in the experimenlal cerebral vasospasm through the co-culture of adventitial fibroblasts and smooth muscle cells in vitro,imitating the outer layer of vascular wall.Methods (1)Smooth muscle cells(SMC)and adventitial fibroblasts(AF)were grown each on opposite sides of polyethylene terephthalate(PET).(2)The AF were incubated in the medium containing 10-6 mol/L oxyhemoglobin.When the two types of the cells were cocuhured for 24h,48h and 72h.the length changes of the smooth muscle cells were examined with electron microscopy.(3)The total RNA of the adventitial fibroblasts in the co-culture system was extracted.Semiquantitative RT-PCR was performed to measure the relative contain of mRNA for connexins in those cells.Results (1)The AF incubated with oxyhemoglobin induced the contraction of the cocuhured smooth muscle cells,which incubated without oxyhemoglobin(P＜0.001).(2)The results of semiquantitative RT-PCR showed that the average relative contain of mRNA for Cx43 at 24h,48h and 72h groups was distinctly higher than that in the normal group(P＜0.01).Conclusion The AF incubated with oxyhemoglobin can produce the contraction of the cocuhured SMC,even the SMC is not derectly stimulated by oxyhemoglobin.The expression of mRNA for connexin43 in adventitial fibroblasts was upregulated in the cell model of cerebral vasospasm.This phenomenon suggests that the AF play an important role in cerebral vasospasm,the mechanism of the smooth muscle cells contraction.