RNA干扰程序性细胞死亡因子4表达对缺氧/复氧H9C2心肌细胞凋亡的影响及其机制

目的 探讨RNA干扰程序性细胞死亡因子4（PDCD4）表达对缺氧/复氧（H/R）H9C2心肌细胞凋亡的影响及其机制。方法 将H9C2心肌细胞分为对照组、H/R组、H/R+NC组和H/R+siPDCD4组。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法（Western blot）分别检测4组H9C2心肌细胞中PDCD4 mRNA和蛋白水平,流式细胞仪检测其凋亡率,酶联免疫吸附试验（ELISA）检测其上清液中丙二醛（MDA）、超氧化物歧化酶（SOD）、乳酸脱氢酶（LDH）水平,Western blot检测磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（AKT）信号通路相关蛋白水平。结果H/R组H9C2心肌细胞PDCD4 mRNA和蛋白、活化的Caspase-3、Bcl-2相关X(Bax)蛋白、上清液中LDH、MDA水平及凋亡率均高于对照组,而上清液中SOD水平、H9C2心肌细胞Bcl-2和p-AKT蛋白水平均低于对照组（P<0.05）。H/R+siPDCD4组H9C2心肌细胞PDCD4 mRNA和蛋白、活化的Caspase-3、Bax蛋白、上清液中LDH、MDA水平及凋亡率均低于H/R组,而上清液中SOD、H9C2心肌细胞Bcl-2和p-AKT蛋白水平均高于H/R组（P<0.05）。结论 RNA干扰PDCD4表达可抑制H/R引起的H9C2心肌细胞凋亡,其作用机制可能与激活PI3K/AKT信号通路进而降低氧化应激反应有关。     

ENST00000418539.1调控miR-24对H2O2诱导心肌细胞氧化应激损伤的影响

目的探讨长链非编码RNA ENST00000418539.1(LncRNA ENST00000418539.1)是否通过调控miR-24的表达影响过氧化氢(H₂O₂)诱导心肌细胞氧化应激损伤。方法体外培养大鼠心肌细胞H9c2,200μmol/L H₂O₂处理心肌细胞48 h建立模型,分别将乱序无意义阴性序列(si-NC)、ENST00000418539.1小分子干扰RNA(si-ENST00000418539.1)、si-ENST00000418539.1与阴性对照(anti-miR-NC)、si-ENST00000418539.1与miR-24特异性寡核苷酸抑制剂(anti-miR-24)转染至心肌细胞,使用H₂O₂处理心肌细胞。实时荧光定量聚合酶链反应检测ENST00000418539.1、miR-24的表达量;流式细胞术检测细胞凋亡率;检测丙二醛(MDA)、乳酸脱氢酶(LDH)水平;双荧光素酶报告实验验证ENST00000418539.1、miR-24的靶向关系;蛋白免疫印迹法检测半胱氨酰天冬氨酸特异性蛋白酶3(Caspase-3)、半胱氨酰天冬氨酸特异性蛋白酶9(Caspase-9)的表达。结果与对照组比较,模型组细胞凋亡率明显升高(P<0.05),Caspase-3、Caspase-9蛋白水平明显升高(P<0.05),MDA、LDH水平明显升高(P<0.05);与模型组、si-NC组比较,si-ENST00000418539.1组细胞凋亡率明显降低(P<0.05),Caspase-3、Caspase-9蛋白水平明显降低(P<0.05),MDA、LDH水平明显降低(P<0.05);双荧光素酶报告实验证实ENST00000418539.1可靶向结合miR-24。与si-ENST00000418539.1+anti-miR-NC组比较,si-ENST00000418539.1+anti-miR-24组细胞凋亡率明显升高(P<0.05),Caspase-3、Caspase-9蛋白水平明显升高(P<0.05),MDA、LDH水平明显升高(P<0.05)。结论干扰ENST00000418539.1表达可负向调控miR-24的表达,从而抑制H₂O₂诱导心肌细胞凋亡及减轻氧化应激损伤。     

沙库巴曲缬沙坦对缺氧心肌细胞线粒体动力系统和细胞凋亡的影响

目的 验证沙库巴曲缬沙坦(S/V)能否通过调节线粒体动力系统改善缺氧大鼠胚胎心肌细胞(H9c2)凋亡水平,发挥心脏保护作用。方法 培养H9c2心肌细胞,建立糖氧剥夺模型(OGD),将细胞分为对照组、造模组、药物组。对照组正常培养心肌细胞,造模组采用OGD建模,药物组采用OGD建模后加用S/V 20μmol/L干预处理,每组重复5遍。采用流式细胞术检测细胞凋亡及活性氧(ROS),JC-1检测线粒体膜电位,蛋白质免疫印迹法(WB)检测线粒体融合蛋白1(Mfn1)、线粒体融合蛋白2(Mfn2)、动力相关蛋白1(Drp1)、线粒体分裂蛋白1(Fis1)、细胞色素C(CytC)、B细胞淋巴瘤2(Bcl-2)、Bcl-2关联X蛋白(Bax)及含半胱氨酸天冬氨酸蛋白水解酶3(Caspase-3)表达情况。采用GraphPad Prism 8统计软件进行数据分析,多组间比较采用单因素方差分析,两两比较采用LSD-t检验。结果 H9c2心肌细胞建立OGD模型,经S/V处理后,光镜下心肌细胞形态学明显改善;流式细胞技术分析结果显示S/V明显降低细胞内ROS水平,抑制心肌细胞凋亡(P＜0.05);荧光显微镜分析结果显示S/V明显改善线粒体膜电位水平(P＜0.05);WB结果显示S/V可明显提升Mfn2、Mfn1、Bcl2蛋白表达水平,降低Drp1、Fis1、CytC、Bax及Caspase-3蛋白表达水平(P＜0.05)。结论 S/V可能通过促进线粒体融合、抑制线粒体分裂调节线粒体稳态,减少ROS生成,减轻心肌细胞凋亡。     

右美托咪定联合Tol样受体4抑制剂对缺氧复氧心肌细胞凋亡和炎症反应的影响及其机制

目的探讨右美托咪定(DEX)联合Toll样受体4(TLR4)抑制剂TAK-242对缺氧复氧(H/R)心肌细胞凋亡和炎症反应的影响及其机制。方法心肌细胞H9C2分为对照(Con)组、H/R组(H/R损伤)、DEX组(1.0μmol/L DEX,再行H/R损伤)、TAK-242组(30μmol/L TAK-242,再行H/R损伤)和DEX+TAK-242组(30μmol/L TAK-242及1.0μmol/L DEX,再行H/R损伤处理)。各组细胞复氧培养6 h后,采用MTT法、流式细胞仪、试剂盒、酶联免疫吸附法检测细胞增殖、凋亡、乳酸脱氢酶(LDH)释放率、白介素1β(IL-1β)和肿瘤坏死因子-α(TNF-α)含量,Western blot检测B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解的caspase-3(cleaved caspase-3)、TLR4和核因子B p65(NF-κB p65)蛋白表达。结果五组细胞凋亡率、LDH释放率、Bax、Bcl-2、cleaved caspase-3、TLR4、NF-κB p65蛋白表达水平、IL-1β、TNF-α含量比较差异均有统计学意义(F=316.938、330.004、839.933、169.750、378.365、476.535、298.527、99.219、293.498,P<0.05)。与Con组相比,H/R组细胞的凋亡率、LDH释放率、Bax、cleaved caspase-3、TLR4和NF-κB p65蛋白表达水平、细胞上清液中IL-1β和TNF-α含量均明显升高(均P<0.05),Bcl-2蛋白表达水平明显降低(均P<0.05)。与H/R组相比,DEX组、TAK-242组和DEX+TAK-242组的细胞凋亡率、LDH释放率、Bax蛋白、cleaved caspase-3、TLR4和NF-κB p65蛋白、IL-1β、TNF-α的表达水平均明显降低,Bcl-2蛋白表达水平明显升高(均P<0.05)。与DEX组、TAK-242组相比,DEX+TAK-242组的细胞凋亡率、LDH释放率、Bax、cleaved caspase-3、TLR4和NF-κB p65蛋白表达、IL-1β、TNF-α表达水平更低,Bcl-2蛋白的表达更高(均P<0.05)。结论DEX和TAK-242联合可协同抑制H/R引起的心肌细胞凋亡和炎症反应,其作用机制可能与协同抑制TLR4/NF-κB通路有关。     

盐酸羟考酮通过JNK/p38MAPK信号通路调控异丙肾上腺素诱导的心肌细胞凋亡的机制

目的探究盐酸羟考酮对异丙肾上腺素(ISO)诱导的心肌细胞凋亡的影响及其作用机制。方法体外培养心肌细胞H9c2,设置对照组、ISO组、盐酸羟考酮1μmol/L组、盐酸羟考酮5μmol/L组、盐酸羟考酮10μmol/L组、ISO+SB203580〔c-Jun氨基末端激酶/p38丝裂原活化蛋白激酶(JNK/p38MAPK)信号通路阻断剂〕组。噻唑蓝(MTT)法检测心肌细胞存活率;流式细胞学法检测各组心肌细胞的凋亡率;Western印迹检测心肌细胞的B淋巴细胞瘤(Bcl)-2、B淋巴细胞瘤-2相关蛋白(Bax)及JNK/p38MAPK信号通路相关蛋白的表达;测定培养细胞上清液乳脱氢酶(LDH)、肌酸激酶(CK)含量。结果与对照组比较,ISO组心肌细胞存活率降低,各给药组心肌细胞存活率明显升高(P<0.05);与对照组比较,ISO组心肌细胞的凋亡率明显升高(P<0.05),而盐酸羟考酮可显著降低细胞凋亡率(P<0.05);与ISO组比较,盐酸羟考酮10μmol/L组LDH、CK水平及Bax、磷酸化(p)-JNK、p-p38MAPK表达水平显著降低(P<0.05),Bcl-2表达水平显著升高(P<0.05);阻断JNK/p38MAPK信号通路可显著增加细胞存活率(P<0.05),降低细胞凋亡率(P<0.05),促进Bcl-2表达(P<0.05),而抑制Bax表达(P<0.05)。结论盐酸羟考酮对ISO诱导的心肌细胞损伤具有抗凋亡作用,其作用机制可能与抑制JNK/p38MAPK信号通路的活化有关。     

Effect of WFDC 2 silencing on the proliferation,motility and invasion of human serous ovarian cancer cells in vitro

ObjectiveTo investigate effect and possible mechanisms of silencing human WFDC2 (HE4) gene on biological behavior changes as cell proliferation, apoptosis, movement and invasion of human serous ovarian cancer cell line SKOV3.MethodsLentiviral WFDC2 gene sequence of small interfering siRNA was stablely transfected into SKOV3 identified by Q-PCR and western-blot. Obtained SKOV3 stable strains with silenced HE4 were measured by proliferation, apoptosis, migration, and invasion.ResultsGene sequencing showed that the oligonucleotides were successfully inserted into the expected site. After silencing HE4 in the SKOV3, proliferation was significantly inhibited (P<0.05). G₀/G₁ phase was arrested by the cell cycle (P<0.01) and capacity of the migration and invasion decreased significantly (P<0.01). Slight early apoptosis ratio and no change of late apoptosis were found without change of Caspase-3 or Bcl-2 protein. Proteins involed in ERK pathway as phosphorylated protein as p-EGFR, p-ERK decreased and protease protein involved in tissue remoding as matrix metalloproteinases MMP-9, MMP-2 and cathepsin B decreased compared with control group.ConclusionsHE4 gene plays an important role in regulating proliferation, apoptosis, migration, invasion of serous ovarian cancer cells by ERK pathway and protease system. Its role in apoptosis needs to be further explored, and it may be a potential target for serous ovarian cancer.     

间歇性低氧大鼠模型心肌氧化应激损伤变化及依达拉奉的干预作用

目的:通过建立间歇性低氧(IH)大鼠模型,探讨IH对大鼠心肌氧化应激损伤的影响及其可能作用机制,并进一步了解依达拉奉的干预作用,为临床阻塞性睡眠呼吸暂停低通气综合征及其相关心血管并发症的研究及防治提供新思路。方法:选取健康雄性Wistar大鼠80只,随机分为正常对照(NC)组、IH组、IH+依达拉奉组、IH+生理盐水(NS)组,每组20只。采用气体控制装置向密闭模拟舱中充入氮气、氧气及压缩空气的方法建立IH大鼠模型。造模4周后,检测大鼠血清乳酸脱氢酶(LDH)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)及心肌组织丙二醛(MDA)、超氧化物歧化酶(SOD)、羟自由基的含量;测定心肌细胞线粒体腺嘌呤核苷三磷酸(ATP)水平;光镜、透射电镜观察心肌形态学及超微结构改变;反转录-聚合酶链反应技术检测心肌组织Bcl-2、Bax、半胱氨酸蛋白酶3(Caspase-3) mRNA的表达情况。结果:(1)与NC组相比,IH组及IH+NS组LDH、CK、CK-MB、MDA、羟自由基含量明显增加,Bax、Caspase-3 mRNA表达明显升高( P值均<0.05);而SOD活力显著降低,ATP含量显著减少,Bcl-2 mRNA表达明显下降( P值均<0.05)。(2)光镜和透射电镜下,NC组心肌组织未见明显损伤,而IH组及IH+NS组心肌组织的形态学及超微结构均受损。(3)经依达拉奉干预后,血清LDH、CK、CK-MB及心肌组织MDA、羟自由基含量明显降低,Bax、Caspase-3 mRNA表达下降( P值均<0.05);SOD活力明显升高,ATP含量增加,Bcl-2 mRNA表达水平升高( P值均<0.05);且光镜及电镜下心肌组织损伤程度得到一定缓解。(4)心肌细胞Caspase-3 mRNA表达水平与CK( r=0.575)、CK-MB( r=0.460)、MDA( r=0.643)、羟自由基( r=0.454)、Bax mRNA( r=0.741)呈正相关,与ATP( r=-0.525)、Bcl-2 mRNA( r=-0.578)呈负相关。 结论:(1)IH可通过增加氧化物、降低抗氧化物及激活Bcl-2、Bax、Caspase-3引起大鼠心肌氧化应激损伤;(2)IH所致的心肌氧化应激损伤可能通过线粒体介导的细胞凋亡实现;(3)依达拉奉对IH所致的大鼠心肌损伤具有干预作用。     

二氢姜黄素体外处理BRL-3A细胞对细胞凋亡通路的影响

目的 研究二氢姜黄素(DHC)对棕榈酸(PA)诱导的大鼠肝BRL-3A细胞线粒体凋亡通路的影响。方法 建立PA诱导的非酒精性脂肪性肝炎(NASH)细胞模型,将细胞分为对照组(正常培养基)、模型组(0.25 mM PA处理)和DHC处理组(16、32和64μM DHC处理),检测细胞培养上清乳酸脱氢酶(LDH)含量,采用流式细胞仪检测细胞凋亡,检测细胞线粒体膜电位、凋亡相关蛋白酶Caspase-3和Caspase-9活性,采用Western blotting 法检测线粒体凋亡相关蛋白Bcl-2和Bax蛋白表达量。结果 模型组细胞上清LDH、Caspase-3和Caspase-9活性分别为(100.1±4.2)%、(1.0±0.2)%和(1.0±0.1)%,均显著高于对照组,而线粒体膜电位为(0.5±0.1)%,显著低于对照组;32 μM DHC处理组LDH、Caspase-3和Caspase-9活性分别为(84.4±2.4)%、(0.8±0.1)%和(0.7±0.1)%,均显著低于模型组(P<0.05),而线粒体膜电位为[(0.6±0.1)%,显著高于模型组(P<0.05);与对照组比,模型组细胞凋亡率显著增加,而经DHC处理组,细胞凋亡率比模型组显著降低;模型组Bcl-2蛋白相对表达量为(1.0±0.1),显著低于对照组,而Bax蛋白相对表达量为(1.1±0.1),显著高于对照组;32 μM DHC处理组和64 μM DHC处理组Bcl-2蛋白相对表达量分别为(1.3±0.1)和(1.9±0.2),均显著高于模型组(P<0.05),而Bax蛋白相对表达量分别为(0.7±0.1)和(0.6±0.1),均显著低于模型组(P<0.05)。结论 DHC可能通过抑制线粒体介导的凋亡通路,缓解由PA诱导的BRL-3A细胞凋亡。     

Antiapoptosis and Mitochondrial Effect of Pioglitazone Preconditioning in the Ischemic/reperfused Heart of Rat

Purpose  Pioglitazone, used clinically in the treatment of type 2 diabetes mellitus, has been implicated as a regulator of cellular inflammatory and ischemic responses. The present study examined whether pioglitazone could inhibit cadiocyte apoptosis and reduce mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of the rat. Furthermore, we investigated whether the protective effect of pioglitazone was related to opening of the mitochondrial_ATP-sensitive potassium channels. Methods  Adult male Sprague–Dawley rats were subjected to 30 min of ischemia followed by 4 h of reperfusion. At 24 h before ischemia, rats were randomized to receive 0.9% saline, 5-hydroxydecanoate (5-HD, 10 mg kg⁻¹, i.v.) plus pioglitazone (3 mg kg⁻¹, i.v.) or pioglitazone (3 mg kg⁻¹, i.v.). One group served as sham control. We investigated mitochondrial structure, apoptosis rate and Bcl-2, Bax and Caspase-3 proteins by immunohistochemistry staining. RT-PCR was used to determine the expression of P38MAPKmRNA and JNKmRNA. Western blotting was used to measure the expression of P38MAPK, JNK and NFκB P65. A second group of rats were randomly divided into sham-operated, ischemia/reperfusion (I/R), pioglitazone treatment, 5-HD + pioglitazone and 5-HD groups and the size of myocardial infarction was determined. Primary cultured cardiomyocytes of neonatal Sprague–Dawley rats were divided into control, hypoxia reoxygenation, different concentrations of pioglitazone and 5-HD + pioglitazone groups. JC-1 staining flowcytometry was used to examine mitochondrial membrane potential (ΔΨm). Results  Pioglitazone decreased mitochondrial ultrastructural damage compared to I/R, and reduced infarct size from 34.93 ± 5.55% (I/R) to 20.24 ± 3.93% (P < 0.05). Compared with the I/R group, the apoptosis rate and positive cell index (PCI) of Bax and Caspase-3 proteins in the pioglitazone group were significantly decreased (P < 0.05), while the PCI of Bcl-2 protein was increased (P < 0.05). There was no significant difference between the I/R and 5-HD + pioglitazone groups. Compared with the sham-operated group, the expression of P38MAPK mRNA, JNK mRNA and protein of P38MAPK, JNK and NFκB P65 in I/R was increased (P < 0.05). Pioglitazone did inhibit the increase in expressions vs I/R (P < 0.05). The rate of loss ΔΨm cells in the pioglitazone group was significantly lower than in the hypoxia reoxygenation group, while the addition of 5-HD inhibited the effect of pioglitazone. Conclusion  Pioglitazone inhibited cadiocyte apoptosis and reduced mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of rat. These protective effects of pioglitazone may be related to opening mitochondrial_ATP-sensitive potassium channels.     

Neuroprotective effect of pAkt and HIF-1α on ischemia rats

Objective:To explore the neuroprotective effect of pAkt and HIF-1α011 ischemia rats.Methods:The rat model of cerebral ischemia which induced by permanent middle cerebral artery occlusion was established.Silybin were given respectively.The behavior was measured by modified Longa method,brain water content were measured by the dry-wet melhod.Infarct volume was measured by image analysis method.Akt.HIF-1 a.Bcl-2,Bax.NF-κB protein expressions were delected by Western blotting.The Akt,HIF-1 a,Bcl-2,Bax,NF-κB mRNA expression were detected by RT-PCR.Results:The control group,low-dose silibinin group and high-dose silibinin group showed paralytic of the left body of rats in various degrees,the brain water content increased and different infarction size.There was no abnormal of the neurobehavioral assessment and no cerebral infarction in the blank group.Compared with the control group,there was no significant improvement of neurological function(t=1.341,P=0.188)or significant changes of the infarct volume(t=1.737.P=0.091)in the low-dose silibinin group,while there was significantly improvement of the neurological function in the high dose silibinin group(t=12.979,P0.001),and the infarct volume was significantly reduced(/-23.503.P0.001),the difference had statistically significant.The brain water content of lesion side of the control group increased(t=43.536,P0.001),while the brain water content of lesion side of the lowdose silibinin group and the high-dose silybin group were significantly reduced(t=25.571,P0.00l;t=42.426,P0.001).The differences were statistical significance.The p-Akt 473,p-Akt 308,HIF-1α,Bax,NF-κB protein and the Akt,Bax.NF-κB mRNA expression were increased of the control group,while the Bcl-2 protein and mRNA expression were decreased,the differences were statistically significant(P0.05).there was no significant change of the Akt protein expression and HIF-1 a mRNA in the control group(P0.05).In the high dose silybin group,the p-Akt 473,p-Akt 308,HIF-1 a,Bcl-2 protein and Akt.Bcl-2 mRNA expression were increased,while the Bax,NF-κB protein and Bax.NF-K B mRNA expression were decreased,the differences were statistically significant(P0.05),there was no significant change of the Akt protein expression and HIF-1 a mRNA in the high dose silybin group(P0.05).Conclusions:pAkt,HIF-1 a have neuroprotective effect on ischemia rats.     

过表达miR-29a对大鼠心肌细胞凋亡的影响

目的 探讨microRNA-29a (miR-29a)对大鼠心肌细胞凋亡的调控作用.方法 体外培养新生SD大鼠心肌细胞,合成人miR-29a的拟似物(mimic).用Lipofectamine RNAiMAX转染miR-29a的mimic进入心肌细胞,转染48 h后用荧光定量PCR方法检测心肌细胞miR-29a的表达变化,流式细胞仪检测细胞凋亡水平变化,Western blot法检测凋亡相关蛋白Caspase-3和Caspase-9前体的表达变化.结果 心肌细胞转染miR-29a的mimic 48 h后,心肌细胞中miR-29a的表达水平较对照组明显升高(P＜0.05);心肌细胞的凋亡水平也明显升高,凋亡相关蛋白Caspase-3和Caspase-9前体的含量则明显下降(P＜0.05).结论 在大鼠心肌细胞中过表达miR-29a能促进心肌细胞凋亡,其机制可能是通过Caspase-3和Caspase-9途径起作用.     

Effect of silencing Bcl-2 expression by small interfering RNA on radiosensitivity of gastric cancer BGC823 cells

ObjectiveTo explore the influence of silencing Bcl-2 expression by small interfering RNA (siRNA) on Bcl-2 protein expression, cell apoptosis rate and radiosensitivity of gastric cancer BGC823 cells.MethodssiRNA segment for Bcl-2 gene was designed and synthesized, then was induced into gastric cancer BGC 823 cells by liposome transfection. Bcl-2 protein expression was detected by Western Blotting. After X radiation, flow cytometry and clone forming assay were used to determine the effects of RNA interference on BGC823 cell apoptosis rate and radiosensitivity.ResultAfter the transfection of Bcl-2 siRNA, the positive expression rate of Bcl-2 protein in BGC823 cells was (35.45±2.35)%. Compared with the control group and negative siRNA transfection group, the rate was significantly decreased (P<0.01). The apoptosis rate of BGC823-RNAi cell was (10.81±0.91)%, which was significantly higher than the control group and negative siRNA transfection group (P<0.01). After 48h X radiation, the apoptosis rate of BGC823-RNAi was (28.91±1.40)%, which was significantly higher than the control group and the group without radiation (P<0.01). During clone forming assay D₀, D_q and SF₂ values in Bcl-2 siRNA1 transfection group were all lower than those in the control group. The radiosensitivity ratio was 1.28 (the ratio of D₀) and 1.60 (the ratio of D_q).ConclusionsSpecific siRNA of Bcl-2 gene can effectively inhibit the expression of Bcl-2 gene, enhance the radiosensitivity and apoptosis of gastric cancer BGC823 cells, having good clinical application perspective.     

Inhibition of fatty acid synthase (FASN) affects the proliferation and apoptosis of HepG2 hepatoma carcinoma cells via the β-catenin/C-myc signaling pathway

Introduction and objectivesResearch in the last few years has proven that inhibition of fatty acid synthase (FASN) suppresses the migration and invasion of hepatoma carcinoma cells. This study aims to explore the effect of fatty acid synthase knockdown on the apoptosis and proliferation of HepG2 cells.Materials and methodsThe human liver cancer cell line HepG2 was cultured and then transfected with FASN-specific siRNA and negative control RNAi. After 48 h, cells and protein lysates were used for western blotting, CCK-8 (cell counting kit-8) assays, flow cytometry and other tests. To assess cell apoptosis, Bax, Bcl-2 and caspase-3 were detected; to assess proliferation, CDK4 (cyclin-dependent kinases 4) and P21 were detected; and to determine the signaling pathway involved, β-catenin and C-myc were also detected.ResultsInhibition of FASN in HepG2 cells can decrease proliferation and promote apoptosis. Flow cytometry and CCK-8 assays demonstrated that the apoptosis rate of FASN-specific siRNA-transfected cells was significantly increased compared to that of the control cells (p < 0.01). In addition, the cell cycle analysis revealed that FASN-specific siRNA-transfected cells induced G1 phase arrest (p < 0.05), but an increasing trend in G2 (p < 0.05).Compared with expression in negative RNAi-transfected cells, the expression of Bcl-2 and CDK-4 was reduced and the expression of Bax, caspase-3 and P21 was increased in FASN-specific siRNA-transfected cells (p < 0.05). Regarding the signaling pathway, the expression of β-catenin and C-myc was significantly reduced when compared to that in negative control cells (p < 0.05).ConclusionsInhibition of FASN suppressed the cell survival of HepG2 cells by inhibiting the β-catenin/C-myc pathway. This result suggests the potential treatment value of FASN for hepatoma carcinoma (HCC).     

Comparative study on the antitumor effects of gemcitabine polybutylcyanoacrylate nanoparticles coupled with anti-human MUC1 and CA199 monoclonal antibodies on pancreatic cancer in vitro and in vivo

Background and Study AimsThis study was designed to compare the antitumor effects of anti-human MUC1 monoclonal antibody with those of anti-human CA199 monoclonal antibody coupled with drug-loaded polybutylcyanoacrylate nanoparticles on human pancreatic cancer cell lines and pancreatic cancer-bearing model animals and to screen more efficient targeting molecules.Patients and MethodsGemcitabine-loaded nanospheres were prepared by emulsion polymerization (GEM-PBCA-NP), and then, anti-MUC1 monoclonal antibody was coupled with GEM-PBCA-NP (MUC1-GEM-PBCA-NP), and anti-human CA199 monoclonal antibody was coupled with GEM-PBCA-NP (CA199-GEM-PBCA-NP), using the chemical crosslinking method. The cell-killing rates were detected using MTT assay. The changes in the tumor cell cycle and apoptosis after treatment were detected using flow cytometry. Then, the subcutaneous planting method was adopted to establish an animal model of pancreatic cancer: two nanometer microspheres were injected into the body of nude mice via the tail vein; the tumor suppression effect was detected after treatment; then, the groups were compared.ResultsIn vitro, the cell-killing rate of each experimental group was significantly different from that of the control group (P < 05). The MUC1-GEM-PBCA-NP group had a significantly higher cell-killing rate than the other groups (P < 05). The apoptosis rate of the MUC1-GEM-PBCA-NP treatment group was significantly higher than that of other groups (P < 05). In vivo, the tumor inhibition rate of the MUC1-GEM-PBCA-NP treatment group was 72.69% ± 4.29%, which was significantly higher than those of other groups (P < 0.05). The tumor inhibition rate of the CA199-GEM-PBCA-NP treatment group was 56.58% ± 5.11%, which was significantly higher than those of other control groups (P < 0.05). At the end of treatment, the average tumor mass of the MUC1-GEM-PBCA-NP treatment group was 433.55 ± 12.49 mg, which was significantly lower than those of other groups (P < 0.05).ConclusionCompared with CA199-GEM-PBCA-NP, MUC1-GEM-PBCA-NP is more effective in vitro and in vivo. MUC1 could be a target molecule in treating pancreatic cancer.     

Influence of Ginkgo biloba extract on the proliferation,apoptosis of ACC-2 cell and Survivin gene expression in adenoid cystic carcinoma of lacrimal gland

ObjectiveTo explore the influence of extract of Ginkgo biloba (EGB) on the proliferation, apoptosis of ACC-2 cell and Survivin gene expression in adenoid cystic carcinoma (ACC) of lacrimal gland.MethodsACC-2 cell in human with ACC of lacrimal gland was in vitro cultured. MTT method was used for cell proliferation detection. Annexin V/PI double-staining flow cytometer was used to detect cell apoptosis and cell cycle. Survivin gene expression was analyzed by RT-PCR and Western blotting.ResultsEGB had inhibitory effect on the proliferation of ACC-2 cell with significant dose-effect relationship, and there was statistical difference when compared with the control group (P<0.01). The inhibitory concentration 50 % (IC₅₀) is 88 mg/L. The flow cytometer test indicated that EGB can gradually increase ACC-2 cell in G₀-G₁ stage and decrease it in G₂-M and S stage. With the increase of dose, the apoptosis rate of ACC-2 cell was obviously increased (P<0.05 or P<0.01). EGB had certain inhibitory effect on Survivin gene expression of ACC-2 cell, and Survivin gene expression was decreased with the increasing of the EGB concentration (P<0.01).ConclusionsEGB can effectively inhibit Survivin gene expression of ACC-2 cell in human with ACC of lacrimal gland, induce the apoptosis of ACC-2 cell and inhibit tumor cell proliferation.     

脂联素通过减轻内质网应激介导的凋亡抑制心肌细胞钙化

目的通过培养SD大鼠乳鼠心室肌细胞并建立细胞钙化模型,观察脂联素对心肌细胞钙化的影响及其机制。方法采用Ⅱ型胶原酶消化法原代培养SD大鼠乳鼠心室肌细胞,α肌动蛋白免疫荧光法进行鉴定。选用培养72 h的单层心肌细胞,实验分为3组:对照组、钙化组、脂联素+钙化组。β-甘油磷酸钠和丙酮酸钠联合诱导制备心肌细胞钙化模型,茜素红和Von Kossa染色法对钙化心肌细胞进行鉴定,通过细胞钙含量、碱性磷酸酶活性和骨钙素的测定判断钙化程度。通过乳酸脱氢酶活性测定和流式细胞术检测心肌细胞凋亡。用定量实时聚合酶链反应测定骨保护素及内质网应激指标葡萄糖调节蛋白78、半胱氨酸天冬氨酸蛋白酶12的表达。结果与对照组相比,单纯心肌细胞钙化后,钙含量、碱性磷酸酶活性和骨钙素分泌量显著增加(P<0.01),骨保护素及内质网应激指标葡萄糖调节蛋白78和半胱氨酸天冬氨酸蛋白酶12在mRNA水平表达明显增高,乳酸脱氢酶活性和细胞凋亡率显著增加(P<0.01)。用脂联素进行干预后,可较大程度地逆转上述指标变化,与心肌细胞钙化组相比均具有显著差异(P<0.05),并且呈现一定的浓度依赖性。结论钙化心肌细胞可以诱导心肌细胞内质网应激介导的凋亡;脂联素可促进血管保护因子骨保护素的表达,并通过减轻内质网应激来减轻心肌细胞钙化及凋亡,对心肌细胞有保护作用。     

Apoptosis of rabbit retinal cell after eyeball rupture

ObjectiveTo establish rabbit eyeball rupture model by air gun in order to observe and analyze the early injury condition and reasons of retinal cell after eyeball rupture.MethodsForty eight healthy rabbits were randomly divided into control group and 1, 3, 6, 12 and 24 h after injury groups. After anesthesia, the rabbit eyeball rupture model was established by air gun. Then the early pathological changes of rabbit retina were observed, and apoptotic index (AI), oncosis index (OI), the relationship between the expression amounts of apoptosis-related genes and AI were analyzed.ResultsObvious pathological lesion appeared in retina 6 h after injury. Irreversible damage occurred 12-24 h after injury. The results of AI and OI indicated that the OI peak appeared 6 h after injury and then gradually declined, while the AI increased with the prolongation of time, and the AI was higher than OI in 12 h after injury. Immunohistochemical results indicated that there was no obvious bcl-2 protein expression change. Compared with the control group and the 3, 6, 12 and 24 h after the injury groups, the expressions of p53 and Caspase-3 were significantly improved and peaked at 12 h (P<0.01). Positive correlation existed among p53, Caspase-3 expression amount and cell apoptosis amount.ConclusionsApoptosis and oncosis of visual cells are the main reasons of retinal cell injury. p53 and Caspase-3 are the important factors in promoting the retinal cell apoptosis after eyeball rupture.     

Mitochondrial pathway of the lysine demethylase 5C inhibitor CPI-455 in the Eca-109 esophageal squamous cell carcinoma cell line

BACKGROUNDEsophageal cancer is a malignant tumor of the digestive tract that is difficult to diagnose early. CPI-455 has been reported to inhibit various cancers, but its role in esophageal squamous cell carcinoma (ESCC) is unknown.AIMTo investigate the effects and mechanism of the lysine demethylase 5C inhibitor, CPI-455, on ESCC cells.METHODSA methyl tetrazolium assay was used to detect the inhibitory effect of CPI-455 on the proliferation of Eca-109 cells. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential were assessed by flow cytometry. Laser confocal scanning and transmission electron microscopy were used to observe changes in Eca-109 cell morphology. The protein expression of P53, Bax, lysine-specific demethylase 5C (KDM5C), cleaved Caspase-9, and cleaved Caspase-3 were assayed by western blotting.RESULTSCompared with the control group, CPI-455 significantly inhibited Eca-109 cell proliferation. Gemcitabine inhibited Eca-109 cell proliferation in a concentration- and time-dependent manner. CPI-455 caused extensive alteration of the mitochondria, which appeared to have become atrophied. The cell membrane was weakly stained and the cytoplasmic structures were indistinct and disorganized, with serious cavitation when viewed by transmission electron microscopy. The flow cytometry and western blot results showed that, compared with the control group, the mitochondrial membrane potential was decreased and depolarized in Eca-109 cells treated with CPI-455. CPI-455 significantly upregulated the ROS content, P53, Bax, Caspase-9, and Caspase-3 protein expression in Eca-109 cells, whereas KDM5C expression was downregulated.CONCLUSIONCPI-455 inhibited Eca-109 cell proliferation via mitochondrial apoptosis by regulating the expression of related genes.     

白皮杉醇对过氧化氢诱导的血管平滑肌细胞凋亡的影响与机制

目的:研究白皮杉醇(PCT)对过氧化氢(H2O2)诱导的血管平滑肌细胞(VSMCs)凋亡的影响以及所涉及的相关分子机制。方法:组织贴块法培养SD大鼠VSMCs并进行不同分组与处理;MTT法检测各组VSMCs的存活率;LDH、超氧化物歧化酶(SOD)试剂盒测定各组VSMCs中LDH与SOD活力;Annexin V-PI染色检测各组VSMCs凋亡情况,Hoechst 33258染色观察各组VSMCs细胞核形态变化;Western blot检测各组VSMCs中凋亡相关蛋白(Bcl-2、Bax、caspase-3)及PI3K/AKT/eNOS通路蛋白表达水平。结果:不同浓度与时间过氧化氢(H2O2)处理VSMCs后使其存活率降低,而不同浓度PCT预处理可提高细胞存活率,差异有统计学意义(P<0.05、P<0.01或P<0.001)。与对照组相比,H2O2组VSMCs中LDH活力升高而SOD活力下降,细胞凋亡率增加,细胞中Bax、caspase-3蛋白表达升高而Bcl-2蛋白表达下降,p-PI3K、p-Akt、p-eNOS蛋白表达下降,且均呈剂量依赖关系;与H2O2组相比,PCT预处理组VSMCs LDH活力下降而SOD活力升高,细胞凋亡率降低,细胞中Bax、caspase-3下降,Bcl-2蛋白表达升高,p-PI3K、p-Akt、p-eNOS的蛋白表达升高,且均呈剂量依赖关系,差异有统计学意义(P<0.05或P<0.01)。结论:PCT对H2O2引起的VSMCs损伤有保护作用,其机制可能是抑制LDH活力、增强SOD活力以及增强PI3K/AKT/eNOS信号通路进而阻碍VSMCs凋亡的发生。