中华显微外科杂志
中華顯微外科雜誌
중화현미외과잡지
Chinese Journal of Microsurgery
2014年
5期
475-479
,共5页
鄢艳%郑婕%谢建军%苏晓霞%吕金雷%肖俊%陈钦开
鄢豔%鄭婕%謝建軍%囌曉霞%呂金雷%肖俊%陳欽開
언염%정첩%사건군%소효하%려금뢰%초준%진흠개
壳聚糖%Toll样受体4%血管平滑肌细胞%增殖抑制%动静脉内痿
殼聚糖%Toll樣受體4%血管平滑肌細胞%增殖抑製%動靜脈內痿
각취당%Toll양수체4%혈관평활기세포%증식억제%동정맥내위
Chitosan%TLR4%Vascular smooth muscle cells (VSMCs)%Inhibiting proliferation%Arteriovenous fistula
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VSMCs 48 h,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、2000 μg/ml)处理细胞48 h;用1000 μg/ml壳聚糖处理细胞不同时间(0、12、24、48 h).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TLR4 mRNA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TLR4 mRNA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TLR4 mRNA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.
目的 探討殼聚糖對傢兔動靜脈內瘺(AVF)血管平滑肌細胞(VSMCs)增殖的抑製作用及機製研究. 方法 建立傢兔頸總動脈-頸內靜脈內瘺模型,1箇月後取AVF處血管採用組織貼壁法培養VSMCs,成功培養的傢兔VSMCs用于實驗.細胞隨機分組:①正常對照組.②高濃度血清組:分彆不同濃度胎牛血清(FBS)(5％、10％、20％)作用于傢兔VSMCs 48 h,建立傢兔VSMCs增殖模型.③殼聚糖組:高濃度血清(20％FBS)作用于傢兔VSMCs,不同濃度殼聚糖(10、100、500、1000、2000 μg/ml)處理細胞48 h;用1000 μg/ml殼聚糖處理細胞不同時間(0、12、24、48 h).Western印跡方法檢測VSMCs的PCNA和TLR4/NF-κB相關蛋白的錶達水平.RT-PCR法檢測PCNA和TLR4 mRNA的錶達.細胞免疫熒光檢測TLR4和NF-κB錶達及分佈.結果 與5％ FBS組比較,20％ FBS組VSMCs中PCNA和TLR4 mRNA和蛋白錶達增加,併呈濃度依賴性(均P＜0.05).不同濃度殼聚糖榦預後,20％ FBS培養的VSMCs中PCNA和TLR4 mRNA水平下調,同時PCNA、TLR4、MyD88和NF-κB蛋白錶達明顯下降,併呈濃度和時間依賴性(均P＜0.05).傢兔VSMCs中TLR4主要錶達在胞漿;NF-κB主要錶達在VSMCs的覈內.與正常組比較,殼聚糖榦預後VSMCs中TLR4及NF-κB免疫熒光明顯減弱,蛋白錶達明顯下降(均P＜0.05). 結論 高濃度血清可誘導VSMCs增殖.殼聚糖可抑製傢兔VSMCs增殖,推測其作用機製可能與激活TLR4受體,降低下遊因子MyD88和NF-κB的錶達有關,提示用殼聚糖做血管外支架,可能成為防治動靜脈內瘺血管內膜增生的新方法.
목적 탐토각취당대가토동정맥내루(AVF)혈관평활기세포(VSMCs)증식적억제작용급궤제연구. 방법 건립가토경총동맥-경내정맥내루모형,1개월후취AVF처혈관채용조직첩벽법배양VSMCs,성공배양적가토VSMCs용우실험.세포수궤분조:①정상대조조.②고농도혈청조:분별불동농도태우혈청(FBS)(5％、10％、20％)작용우가토VSMCs 48 h,건립가토VSMCs증식모형.③각취당조:고농도혈청(20％FBS)작용우가토VSMCs,불동농도각취당(10、100、500、1000、2000 μg/ml)처리세포48 h;용1000 μg/ml각취당처리세포불동시간(0、12、24、48 h).Western인적방법검측VSMCs적PCNA화TLR4/NF-κB상관단백적표체수평.RT-PCR법검측PCNA화TLR4 mRNA적표체.세포면역형광검측TLR4화NF-κB표체급분포.결과 여5％ FBS조비교,20％ FBS조VSMCs중PCNA화TLR4 mRNA화단백표체증가,병정농도의뢰성(균P＜0.05).불동농도각취당간예후,20％ FBS배양적VSMCs중PCNA화TLR4 mRNA수평하조,동시PCNA、TLR4、MyD88화NF-κB단백표체명현하강,병정농도화시간의뢰성(균P＜0.05).가토VSMCs중TLR4주요표체재포장;NF-κB주요표체재VSMCs적핵내.여정상조비교,각취당간예후VSMCs중TLR4급NF-κB면역형광명현감약,단백표체명현하강(균P＜0.05). 결론 고농도혈청가유도VSMCs증식.각취당가억제가토VSMCs증식,추측기작용궤제가능여격활TLR4수체,강저하유인자MyD88화NF-κB적표체유관,제시용각취당주혈관외지가,가능성위방치동정맥내루혈관내막증생적신방법.
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 arteryinternal 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 the mechanism 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.
