水蛭素对缺氧诱导心脏微血管内皮细胞间质转分化的作用及机制研究  

Effect and mechanism of hirudin on hypoxia induced interstitial transdifferentiation of cardiac microvascular endothelial cells

作　　者：刘依 尹玉洁 韩宁馨 贾振华[1,2,3,4] Liu Yi;Yin Yujie;Han Ningxin;Jia Zhenhua(Graduate School of Hebei Medical University,Hebei Province,Shijiazhuang 050017,China;不详)

机构地区：[1]河北医科大学研究生学院,石家庄050017 [2]络病研究与创新中药国家重点实验室,石家庄 [3]国家中医药管理局重点研究室,石家庄 [4]河北省中西医结合医药研究院,石家庄

出　　处：《疑难病杂志》2024年第9期1120-1126,共7页Chinese Journal of Difficult and Complicated Cases

基　　金：国家自然科学基金资助项目(81973692)。

摘　　要：目的探讨通络药物水蛭素对缺氧诱导的人心脏微血管内皮细胞(HCMECs)间质转分化(EndMT)的作用及可能机制。方法取常规培养的HCMECs细胞,随机分为对照组、缺氧组、水蛭素组(包括0、20、40、80、100μg/ml 5个浓度)。对照组常规培养不做任何处理,缺氧组置入低氧培养箱72 h,水蛭素组预加入Hirudin工作液,4 h后置入低氧培养箱72 h。MTS比色法检测HCMECs增殖能力;倒置显微镜观察HCMECs形态;免疫荧光鉴定HCMECs间质转分化情况,Western-blot检测内皮间质转分化相关蛋白:包括内皮细胞标记血小板—内皮细胞黏附分子(PECAM-1/CD31)、血管内皮钙黏蛋白(VE-cadherin),间质细胞标记α-平滑肌肌动蛋白(α-SMA)、成纤维细胞特异性蛋白-1(FSP-1),以及低氧诱导因子1α(HIF-1α)、转化生长因子β1(TGF-β1)、Smad同源物2/3(Smad2/3)、锌指转录因子(snail)等相关信号通路的蛋白表达。结果MTS法检测显示,缺氧显著抑制细胞活性(P<0.01),水蛭素在20~100μg/ml浓度范围内可提高细胞活性,且呈现浓度依赖性,当浓度为100μg/ml时细胞活性最强(P<0.01)。各组细胞培养72 h后,倒置显微镜下观察发现:对照组细胞呈铺路石样或鹅卵石状结构,缺氧组细胞由鹅卵石状结构变为分散的长梭形,接近成纤维细胞形态,水蛭素组长梭形细胞形态明显改善,细胞恢复鹅卵石样。Western-blot与免疫荧光结果显示:与对照组比较,缺氧组CD31、VE-cadherin蛋白水平降低(P<0.01),且vWF表达减少,α-SMA、FSP-1蛋白水平升高(P<0.01),且vimentin表达增强;与缺氧组比较,水蛭素明显增加CD31、VE-cadherin蛋白表达(P<0.01),并增强vWF表达,下调α-SMA、FSP-1蛋白表达(P<0.01),并减弱vimentin表达;与对照组相比,缺氧组信号通路HIF-1α、TGF-β1、p-smad2/3、snail蛋白表达均升高(P<0.01),与缺氧组比较,水蛭素下调HIF-1α、TGF-β1、p-smad2/3、snail蛋白表达(P<0.01)。结论水蛭素可以改善缺氧诱导的HCMECs细�Objective To investigate the effect and possible mechanism of the drug hirudin on hypoxia induced human microvascular endothelial cells(HCMECs)mesenchymal transition(EndMT).Methods HCMECs cells cultured conventionally were randomly divided into control group,Hypoxia group and Hirudin group.The control group was routinely cultured without any treatment.The hypoxic group was placed in a hypoxic incubator for 72h,and the Hirudin group was pre-added with Hirudin working solution,and then placed in a hypoxic incubator for 72h after 4h.The proliferation capacity of HCMECs was detected by MTS colorimetry.The morphology of HCMECs was observed by inverted microscope.Immunofluorescence identification of HCMECs interstitial trans differentiation,Western Blot detection of endothelial interstitial trans differentiation related proteins:Endothelial cells labeled platelet endothelial cell adhesion molecule(PECAM-1/CD31)and vascular endothelial cadherin(VE-cadherin),and stromal cells labeledαsmooth muscle actin(α-SMA)and fibroblast specific protein 1(FSP-1).And the expression of hypoxia inducible factor-1α(HIF-1α),transforming growth factorβ1(TGF-β1),Smad homology 2/3(Smad2/3),Zinc finger transcription factor(snail)related signaling pathways.Results MTS showed that hypoxia significantly inhibited cell activity(P<0.01),and the cell activity was strongest when hirudin concentration was 100μg/ml(P<0.01).After 72h of cell culture in each group,the cells in the control group showed a pave-like or pebble-like structure under an inverted microscope,while the cells in the hypoxic group changed from pebble-like structure to dispersed long spindle shape,close to the shape of fibroblasts.Western Blot and immunofluorescence results showed:Compared with the normal group,the protein levels of CD31 and VE cadherin in hypoxia group were decreased(P<0.01),and the expression of vWF was decreased.α-SMA and FSP-1 protein levels increased(P<0.01)and vimentin expression was enhanced.Compared with hypoxia group,hirudin significantly increased

关 键 词：水蛭素 缺氧 人心脏微血管内皮细胞 内皮间质转分化 信号通路 作用机制 

分 类 号：R285.5[医药卫生—中药学]