阿帕替尼和5-氟尿嘧啶单药及联合对肠癌细胞系HT-29的促调亡作用

目的观察阿帕替尼(Apatinib,Apa)和5-氟尿嘧啶(5-Fluorouracil,5-Fu)分别在单药、联合情况下对肠癌细胞系HT-29的增殖抑制及促凋亡作用,并研究其作用机制是否与信号通路磷脂酰肌醇3-激酶(phospholipid inositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)相关。方法将细胞分为对照组(加入二甲基亚砜,dimethyl sulfoxide,DMSO)、阿帕替尼组(80μmol·L^-1 Apa)、5-Fu组(100μmol·L^-15-Fu),联合组(80μmol·L^-1 Apa+100μmol·L^-15-Fu)。给药48 h后,用四甲基偶氮唑蓝(Methyl Thiazolyl Tetrazolium,MTT)法检测不同浓度Apa及5-Fu对细胞增殖的影响;用流式细胞术检测Apa及5-Fu对细胞凋亡的影响;用克隆形成实验检测Apa及5-Fu对细胞克隆形成能力的影响;用蛋白质印迹法(Western blot)检测Apa及5-Fu对PI3K、磷酸化磷脂酰肌醇3-激酶(phosphorylated phospholipid inositol 3-kinase,p-PI3K)、Akt、磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)蛋白表达的影响。结果Apatinib 80μmol·L^-1+5-Fu 100μmol·L^-1联合组细胞增殖抑制率提高最显著,此时对照组、阿帕替尼组、5-Fu组和联合组细胞增殖抑制率分别为0%,(0.25±0.08)%,(0.40±0.04)%,(0.74±0.02)%;细胞凋亡率分别为(3.00±1.20)%,(41.60±2.15)%,(43.90±1.80)%,(56.10±1.50)%;细胞克隆数分别为(88.33±3.06),(61.71±1.72),(57.75±4.21),(36.12±2.16)个。阿帕替尼组、5-Fu组、联合组的上述指标与对照组比较,差异均有统计学意义(P<0.05),联合组的上述指标与阿帕替尼组、5-Fu组比较,差异均有统计学意义(P<0.05)。对照组、阿帕替尼组、5-Fu组、联合组细胞p-Akt蛋白表达量分别为1.05±0.10,0.10±0.01,0.80±0.08,0.11±0.01;p-PI3K蛋白表达量分别为0.92±0.06,0.61±0.10,1.01±0.11,0.80±0.05。阿帕替尼组、联合组的上述指标与对照组、5-Fu组比较,差异均有统计学意义(均P<0.05)。结论Apa及5-Fu单药及联合均能以浓度依赖的方式抑制肠癌HT-29细胞系增殖。Apa可通过抑制PI3K/Akt的信号通路传导,诱导肠癌细胞凋亡。Apa联合5-Fu具有协同促进肠癌细胞凋亡的作用。

RNA干扰ATG14抑制肝癌细胞自噬并促进索拉菲尼诱导细胞凋亡机制研究

目的探讨RNA干扰自噬相关基因14(ATG14)对肝癌细胞自噬的影响及其促进索拉菲尼诱导细胞凋亡的机制。方法 ATG14 siRNA和siRNA NC转染肝癌Huh7细胞,分别设为ATG14 siRNA组和siRNA NC组,正常培养肝癌细胞为空白对照组。实时荧光聚合酶链反应(PCR)检测3组细胞ATG14 mRNA表达情况;MTT法检测肝癌细胞增殖活力;流式细胞术测定细胞凋亡情况;蛋白质印迹法检测ATG14、LC3、p62、Caspase-3、Caspase-9和Bcl-2蛋白表达情况;免疫荧光检测LC3荧光点表达情况。结果 ATG14 siRNA组ATG14 mRNA相对表达量为0.47±0.19,低于空白对照组(1.07±0.09)和siRNA NC组(1.16±0.13),F=382.137,P<0.001。ATG14 siRNA组ATG14蛋白相对表达量为0.38±0.19,低于空白对照组(1.23±0.09)和siRNA NC组(1.19±0.11),F=32.621,P<0.001。空白对照组LC3Ⅱ/LC3Ⅰ蛋白相对表达量为0.94±0.12,低于ATG14 siRNA组的1.48±0.15,t=15.851,P<0.001;ATG14 siRNA组p62蛋白相对表达量为0.78±0.07,低于空白对照组的0.98±0.09,t=21.362,P<0.001。免疫荧光实验结果显示,ATG14 siRNA组LC3荧光强度为15.42±4.26,低于对照组的46.42±5.73,t=41.247,P<0.001。索拉菲尼干预Huh7细胞24 h后,空白对照组和ATG14 siRNA组细胞半抑制浓度分别为(7.87±0.38)和(6.15±0.22)μmol/mL,差异有统计学意义,t=31.747,P<0.001。对照组、索拉菲尼组和索拉菲尼+ATG14 siRNA组早期凋亡率分别为(1.61±0.33)%、(31.77±8.92)%和(58.14±3.12)%,F=37.843,P<0.001;晚期凋亡率分别为(5.56±1.28)%、(12.17±5.19)%和(18.33±3.42)%,F=59.872,P<0.001。空白对照组、索拉菲尼组和索拉菲尼+ATG14 siRNA组Caspase-3蛋白相对表达量分别为0.47±0.09、0.78±0.09和1.37±0.51,F=81.731,P<0.001;Caspase-9蛋白相对表达量分别为0.71±0.11、1.23±0.24和1.41±0.19,F=93.891,P<0.001;Bcl-2蛋白相对表达量分别为0.82±0.19、0.39±0.19和0.21±0.09,F=57.424,P<0.001。结论 ATG14 siRNA能够抑制肝癌细胞自噬,并通过抑制自噬提高索拉菲尼对肝癌细胞的诱导凋亡作用。

Expression of Serum hs-CRP and Lp-PLA2 in Arrhythmia Patients and its Significance

Objective:The purpose of this study is to examine the expression ofserum high-sensitivity C-reactive protein(hs-CRP)and lipoprotein-associated phospholipase A2(Lp-PLA2)in patients with arrhythmia and its significance.Methods:We selected 136 arrhythmia patients(observation group)who were treated at our hospital from February 2016 to March 2018.Among these patients,50 had premature ventricular contractions,42 had atrial fibrillation,24 had atrial flutter,and 20 had ventricular tachycardia.Simultaneously,we selected 120 healthy volunteers as the control group.We measured serum levels of hs-CRP and Lp-PLA2 in these subjects.Results:The levels of Lp-PLA2 and hs-CRP of the observation group were 210.06±30.46 pg/mL and 13.54±3.16,respectively,which were significantly higher than those of the control group(P<0.05).The serum levels of Lp-PLA2 and hs-CRP of patients with atrial flutter were 260.87±32.24 and 19.84±5.10 pg/mL,respectively,which were significantly higher than those of patients with premature ventricular contractions,atrial fibrillation,and ventricular tachycardia(P<0.05).Serum levels of Lp-PLA2 and hs-CRP were posi-tively correlated(r=0.413,P<0.05).Conclusion:Serum levels of hs-CRP and Lp-PLA2 in arrhythmia patients were significantly elevated and showed some association with the type of arrhythmia.