摘要
目的探讨壳聚糖对家兔动静脉内瘘（AVF）血管平滑肌细胞（VSMCs）增殖的抑制作用及机制研究。方法建立家兔颈总动脉-颈内静脉内瘘模型，1个月后取AVF处血管采用组织贴壁法培养VSMCs，成功培养的家兔VSMCs用于实验。细胞随机分组：①正常对照组。②高浓度血清组：分别不同浓度胎牛血清（FBS）（5％、10％、20％）作用于家兔VSMCs48h，建立家兔VSMCs增殖模型。③壳聚糖组：高浓度血清（20％FBS）作用于家兔VSMCs，不同浓度壳聚糖（10、100、500、1000、2000μg／ml）处理细胞48h；用1000μg／ml壳聚糖处理细胞不同时间（0、12、24、48h）。Western印迹方法检测VSMCs的PCNA和TLR4／NF-κB相关蛋白的表达水平。RT—PCR法检测PCNA和TLR4mRNA的表达。细胞免疫荧光检测TLR4和NF-κB表达及分布。结果与5％FBS组比较，20％FBS组VSMCs中PCNA和TLR4mRNA和蛋白表达增加，并呈浓度依赖性（均P〈0．05）。不同浓度壳聚糖干预后，20％FBS培养的VSMCs中PCNA和TLR4mRNA水平下调，同时PCNA、TLR4、MyD88和NF-κB蛋白表达明显下降，并呈浓度和时间依赖性（均P〈0．05）。家兔VSMCs中TLR4主要表达在胞浆；NF-κB主要表达在VSMCs的核内。与正常组比较，壳聚糖干预后VSMCs中TLR4及NF-κB免疫荧光明显减弱，蛋白表达明显下降（均P〈0．05）。结论高浓度血清可诱导VSMCs增殖。壳聚糖可抑制家兔VSMCs增殖，推测其作用机制可能与激活TLR4受体，降低下游因子MyD88和NF-κB的表达有关，提示用壳聚糖做血管外支架，可能成为防治动静脉内瘘血管内膜增生的新方法。
Objective To explore the effect of chitosan on vascular smooth muscle cells inhibited proliferation from rabbit arteriovenous fistula and its mechanisms. Methods Established rabbit fistula model on carotid artery- internal jugular vein. After 1 month cultured VSMCs with primary culture by tissue-pieces inoculation. Cultured VSMCs were divided into three groups ：①normal control group. ②FBS-treated group：cell were treated with 5% , 10% , 20% for 48 h ,respectively; established the model of rabbit VSMCs proliferation. ③chitosan-treated group： VSMCs cultured with 20% FBS were exposed to different doses of chitosan（ 10, 100, 500, 1000, 2000μg/ml） for 48 h. And VSMCs were treated for different time （0, 12, 24, 48 h） with Chitosan 1000 μg/ml. Expression levels of PCNA and TLR4/ NF- κB were detected by Western blotting. RT- PCR were applied to measure the mRNA expression of PCNA and TLR4. The protein levels of TLR4 and NF- κB were detected by immunofluorescence. Results Compared with low concentration serum group, FBS-treated VSMCs exhibited a increase in mRNA and protein expression of PCNA and TLR4. FBS-induced protein expression of PCNA and TLR4/NF-κB were reduced by chitosan. Also mRNA expression of PCNA and TLR4 were reduced. They were dependent on concentration and time. In rabbit VSMCs TLR4 was mainly expressed in the cytoplasm and NF-κB expressed mainly in the nucleus. Compared with normal control group, TLR4 and NF-κB protein expression were significantly decreased by chitosan. Conclusion High concentration serum induced VSMCs proliferation. Chitosan can inhibit the proliferation of rabbit VSMCs. It is speculated that themechanism may be related to the expression of TLR4 receptor activation, reducing expression of downstream factor MyD88 and NF-κB. It is suggest that chitosan can become potential new drugs of arteriovenous fistula prevention of intimal hyperplasia.
出处
《中华显微外科杂志》
CSCD
北大核心
2014年第5期475-479,共5页
Chinese Journal of Microsurgery
基金
江西省教育厅课题（GJJ13144）
