CAJ | 학술논문

目的观察川芎嗪(TMP)对人脐静脉内皮细胞(HUVECs)缺氧相关因子表达的影响.方法体外培养HUVECs,取2～5代细胞进行实验.将HUVECs分为对照组、氯化钴(CoCl2)缺氧组及50、100、200 μmol/L TMP药物处理组.对照组不作任何处理.CoCl2缺氧组利用150 μmol/L的CoCl2干预HUVECs 4 h;50、100、200 μmol/L TMP药物处理组在CoCl2干预HUVECs 4 h后,再加入不同浓度TMP继续培养8h.分别提取各组细胞RNA和总蛋白,采用实时荧光定量聚合酶链反应(RT-PCR)和蛋白免疫印迹法(Western blot)检测脯氨酰羟化酶2(PHD2)、缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)mRNA和蛋白的表达.结果实时RT-PCR检测结果显示,与对照组比较,CoCl2缺氧组PHD2mRNA表达下降(t=3.734),HIF-1α和VEGF mRNA表达升高(t=4.589、3.926),差异均有统计学意义(P＜0.05).50、100、200 μmol/L TMP药物处理组分别与CoCl2缺氧组比较,PHD2 mRNA表达均升高(t=3.286、3.617、3.886),差异有统计学意义(P＜0.05)；HIF-1α mRNA表达均无明显变化(t=1.025、0.726、-1.386),差异无统计学意义(P＞0.05)；VEGF mRNA表达均有所下降,但50 μmol/L TMP药物处理组与之差异无统计学意义(t=1.696,P＞0.05),100、200 μmol/L TMP药物处理组与之差异有统计学意义(t=2.974、3.492,P＜0.05).Western blot检测结果显示,与对照组比较,CoCl2缺氧组PHD2蛋白表达下降,差异有统计学意义(t=3.122,P＜0.05)；HIF-1α及VEGF蛋白表达均有不同程度升高,差异有统计学意义(t=3.778、3.257,P＜0.05).50、100、200 μmol/L TMP药物处理组分别与CoCl2缺氧组比较,PHD2蛋白表达均升高(t=2.813、3.026、3.078),差异有统计学意义(P＜0.05)；HIF-1α、VEGF蛋白表达均有所下降,但50 μmol/L TMP药物处理组与之差异无统计学意义(t=2.056、1.986,P＞0.05),100 μmol/L TMP药物处理组(t=3.058、2.976)及200 μmol/L TMP药物处理组(t=3.828、3.136)与之差异有统计学意义(P＜0.05).结论 TMP能上调缺氧HUVECs中PHD2 mRNA和蛋白的表达,下调HIF-1α蛋白、VEGF mRNA和蛋白的表达. 目的觀察川芎嗪(TMP)對人臍靜脈內皮細胞(HUVECs)缺氧相關因子錶達的影響.方法體外培養HUVECs,取2～5代細胞進行實驗.將HUVECs分為對照組、氯化鈷(CoCl2)缺氧組及50、100、200 μmol/L TMP藥物處理組.對照組不作任何處理.CoCl2缺氧組利用150 μmol/L的CoCl2榦預HUVECs 4 h;50、100、200 μmol/L TMP藥物處理組在CoCl2榦預HUVECs 4 h後,再加入不同濃度TMP繼續培養8h.分彆提取各組細胞RNA和總蛋白,採用實時熒光定量聚閤酶鏈反應(RT-PCR)和蛋白免疫印跡法(Western blot)檢測脯氨酰羥化酶2(PHD2)、缺氧誘導因子-1α(HIF-1α)、血管內皮生長因子(VEGF)mRNA和蛋白的錶達.結果實時RT-PCR檢測結果顯示,與對照組比較,CoCl2缺氧組PHD2mRNA錶達下降(t=3.734),HIF-1α和VEGF mRNA錶達升高(t=4.589、3.926),差異均有統計學意義(P＜0.05).50、100、200 μmol/L TMP藥物處理組分彆與CoCl2缺氧組比較,PHD2 mRNA錶達均升高(t=3.286、3.617、3.886),差異有統計學意義(P＜0.05)；HIF-1α mRNA錶達均無明顯變化(t=1.025、0.726、-1.386),差異無統計學意義(P＞0.05)；VEGF mRNA錶達均有所下降,但50 μmol/L TMP藥物處理組與之差異無統計學意義(t=1.696,P＞0.05),100、200 μmol/L TMP藥物處理組與之差異有統計學意義(t=2.974、3.492,P＜0.05).Western blot檢測結果顯示,與對照組比較,CoCl2缺氧組PHD2蛋白錶達下降,差異有統計學意義(t=3.122,P＜0.05)；HIF-1α及VEGF蛋白錶達均有不同程度升高,差異有統計學意義(t=3.778、3.257,P＜0.05).50、100、200 μmol/L TMP藥物處理組分彆與CoCl2缺氧組比較,PHD2蛋白錶達均升高(t=2.813、3.026、3.078),差異有統計學意義(P＜0.05)；HIF-1α、VEGF蛋白錶達均有所下降,但50 μmol/L TMP藥物處理組與之差異無統計學意義(t=2.056、1.986,P＞0.05),100 μmol/L TMP藥物處理組(t=3.058、2.976)及200 μmol/L TMP藥物處理組(t=3.828、3.136)與之差異有統計學意義(P＜0.05).結論 TMP能上調缺氧HUVECs中PHD2 mRNA和蛋白的錶達,下調HIF-1α蛋白、VEGF mRNA和蛋白的錶達.
목적 관찰천궁진(TMP)대인제정맥내피세포(HUVECs)결양상관인자표체적영향.방법 체외배양HUVECs,취2～5대세포진행실험.장HUVECs분위대조조、록화고(CoCl2)결양조급50、100、200 μmol/L TMP약물처리조.대조조불작임하처리.CoCl2결양조이용150 μmol/L적CoCl2간예HUVECs 4 h;50、100、200 μmol/L TMP약물처리조재CoCl2간예HUVECs 4 h후,재가입불동농도TMP계속배양8h.분별제취각조세포RNA화총단백,채용실시형광정량취합매련반응(RT-PCR)화단백면역인적법(Western blot)검측포안선간화매2(PHD2)、결양유도인자-1α(HIF-1α)、혈관내피생장인자(VEGF)mRNA화단백적표체.결과 실시RT-PCR검측결과현시,여대조조비교,CoCl2결양조PHD2mRNA표체하강(t=3.734),HIF-1α화VEGF mRNA표체승고(t=4.589、3.926),차이균유통계학의의(P＜0.05).50、100、200 μmol/L TMP약물처리조분별여CoCl2결양조비교,PHD2 mRNA표체균승고(t=3.286、3.617、3.886),차이유통계학의의(P＜0.05)；HIF-1α mRNA표체균무명현변화(t=1.025、0.726、-1.386),차이무통계학의의(P＞0.05)；VEGF mRNA표체균유소하강,단50 μmol/L TMP약물처리조여지차이무통계학의의(t=1.696,P＞0.05),100、200 μmol/L TMP약물처리조여지차이유통계학의의(t=2.974、3.492,P＜0.05).Western blot검측결과현시,여대조조비교,CoCl2결양조PHD2단백표체하강,차이유통계학의의(t=3.122,P＜0.05)；HIF-1α급VEGF단백표체균유불동정도승고,차이유통계학의의(t=3.778、3.257,P＜0.05).50、100、200 μmol/L TMP약물처리조분별여CoCl2결양조비교,PHD2단백표체균승고(t=2.813、3.026、3.078),차이유통계학의의(P＜0.05)；HIF-1α、VEGF단백표체균유소하강,단50 μmol/L TMP약물처리조여지차이무통계학의의(t=2.056、1.986,P＞0.05),100 μmol/L TMP약물처리조(t=3.058、2.976)급200 μmol/L TMP약물처리조(t=3.828、3.136)여지차이유통계학의의(P＜0.05).결론 TMP능상조결양HUVECs중PHD2 mRNA화단백적표체,하조HIF-1α단백、VEGF mRNA화단백적표체.
Objective To observe the effect of tetramethypyrazine (TMP) on the expression of hypoxia-related factors in human umbilical vein endothelial cells (HUVECs).Methods The second to fifth passage cultured HUVECs were divided into five groups:control group,CoCl2-induced hypoxic group and 50,100,200 μmol/L TMP treatment groups.HUVECs in control group were not treated.HUVECs in CoCl2-induced hypoxic group were treated with 150 μmol/L CoCl2 for four hours.HUVECs in 50,100,200 μmol/L TMP treated groups were pretreated with 150 μmol/L CoCl2 for four hours,followed by treatment with 50,100,200 μmol/L TMP for eight hours.Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of prolyl hydroxylase 2 (PHD2),hypoxia-induced factor-1α(HIF-1α) and vascular endothelial growth factor (VEGF).Protein levels of PHD2,HIF-1α,and VEGF were detected using Western blot.Results Compared with the control group,the CoCl2-induced hypoxic group showed decreased mRNA and protein levels of PHD2 (t=3.734,3.122; P＜0.05),while those of HIF-1αand VEGF increased (HIF-1α mRNA:t=4.589,P＜0.05; HIF-1α protein:t=3.778,P＜0.05.VEGF mRNA:t=3.926,P＜0.05; VEGF protein:t=3.257,P＜0.05).Compared with the CoCl2-induced hypoxic group,50,100,200 μmol/L TMP treated groups showed increased mRNA and protein levels of PHD2 (PHD2 mRNA:t=3.286,3.617,3.886; P＜0.05.PHD2 protein:t=2.813,3.026,3.078; P＜0.05); while those of VEGF decreased (VEGF mRNA:50 μmol/L TMP:t=1.696,P＞0.05; 100 μmol/LTMP:t =2.974,P＜0.05; 200 μmol/L TMP:t=3.492,P＜0.05; VEGFprotein:50 μmol/LTMP:t=1.986,P＞0.05; 100 μmol/L TMP:t=2.976,P＜0.05; 200 μmol/L TMP:t=3.136,P＜0.05); although changes in HIF-1αmRNA levels were not statistically significant (t=1.025,0.726,-1.386; P＞ 0.05),showed a decrease in HIF 1αprotein levels (50 μmol/L TMP:t=2.056,P＞0.05; 100 μmol/L TMP:t=3.058,P＜0.05; 200 μmol/L TMP:t=3.828,P＜0.05).Conclusion In HUVECs,TMP can up regulate the mRNA and protein expression of PHD2,while down-regulating HIF-1α protein expression and VEGF mRNA and protein expression under acute hypoxic conditions.