低氧条件下BAVM动物模型VSMC中TGF-β表达及细胞功能改变

Changes of TGF-β expression and cellular function in BAVM animal models under low oxygen condition

目的探讨分析缺氧对脑动静脉畸形(BAVM)血管内膜平滑肌细胞(VSMC)中转化生长因子-β(TGF-β)表达及VSMC细胞功能改变。方法建立稳定的BAVM实验用猪动物模型,分离脑底微血管网(RM)的VSMC后行原代培养。分组:1对照组采用正常实验用猪VSMC:(A组)21%O_2浓度;2实验组采用BAVM模型实验用猪VSMC:(B组)21%O_2浓度、(C组)1%O_2浓度。细胞免疫荧光检测VSMC密度、实时定量-多聚酶链反应(RT-PCR)和蛋白印记(Western blot)法验证VSMC中TGF-β mRNA及蛋白差异、末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口末端标记(TUNEL)法检测VSMC的调亡、Transwell测定VSMC的侵袭能力。结果 VSMC对缺氧敏感,实验组与对照组比较及实验组组间比较,TGF-β的mRNA及蛋白差异有统计学意义(P<0.01)、72 h VSMC凋亡及细胞侵袭数目差异有统计学意义(P<0.01)。结论缺氧对动物模型VSMC中TGF-β表达有显著的影响且促使VSMC凋亡及侵袭能力增加,加速畸形血管团的形成。在细胞分子生物学水平分析能为BAVM临床治疗提供一种有益的思路。

Objective The effect of hypoxia on endothelial growth factor （TGF-β） expression and functions of VSMC cells in brain arteriovenous malformations （BAVM） vascular intimal smooth muscle cells （VSMC） were studied. Methods The BAVM experimental pigs were well established and the separation of cerebral microvascular network （RM） was cultured by VSMC. There were three groups： the control group with normal experimental porcine VSMC （Group A） under 21%O2 concentration; the experimental group using porcine VSMC （Group B） under 21% O2concentration, and 1% O2 concentration （Group C）. VSMC density was detected by immunofluorescence, TGF-β mRNA and protein of VSMC were detected by quantitative real-time polymerase chain reaction and Western blot, respectively. Terminal deoxyribonucleic transferase mediated dUTP nick labeling （TUNEL） method was used to detect the difference of apoptosis of VSMC, and Transwell was used for the determination of VSMC invasion. Results VSMC was sensitive to hypoxia. There were statistically significant differences in mRNA and protein of TGF-β between control and experimental groups and among the experiments （P 〈0. 01 ）. There was statistically significant difference in VSMC apoptosis and cell invasion number at 72 h （P 〈0. 01）. Conclusion Hypoxia has a significant effect on expression of TGF-β in VSMC of animal model, and it improves the apoptosis and invasion of VSMC and accelerates the formation of the vascular malformation. The analysis of molecular biology can provide a useful idea for the clinical treatment of BAVM.