丹皮酚联合5-FU对食管癌EC9706细胞增殖及凋亡的影响

目的:探讨丹皮酚(paeonol,Pae)单独及联合5-Fu对人食管癌EC9706细胞的增殖抑制及凋亡诱导作用.方法:采用6种浓度的Pae(7.81、15.63、31.25、62.50、125.00、250.00 mg/L)、3种浓度的5-FU(12.50、25.00、50.00 mg/L)及Pae(31.25 mg/L)和5-FU(12.50 mg/L)联合分别处理EC9706细胞24、48、72 h.同时设对照组(细胞不做处理),采用MTT法检测各个时间段细胞的增殖情况:采用流式细胞术检测4种浓度的Pae(31-25、62.50、125.00、250.00 mg/L)处理EC9706细胞72 h后细胞周期的变化:倒置显微镜下观察各Pae组细胞各时间段形态学变化,HE染色光镜下观察凋亡细胞:采用免疫细胞化学法检测经Pae(31.25 mg/L)、5-FU(12.50mg/L)单独和联合作用48 h后细胞中凋亡相关蛋白Bcl-2及Bax的表达.结果:Pae、5-FU可明显抑制EC9706细胞增殖,并随着浓度的增加和作用时间的延长而增强(P<0.05),Pae与5-FU联合用药比单用Pae或5.FU抑制效果更明显(P<0.05);Pae作用后EC9706细胞中G0/G1期和G2/M期细胞比例下降、S期细胞比例上升(Pae 125.00 mg/L组:G0/G1期21.18%±2.28% vs 62.17%±5.23%、G2/M期0.76%±0.54% vs 9.92%±3.10%、S期78.06%±2.82% vs 27.91%±2.13%,均P<0.05):HE染色光镜下可见典型的肿瘤细胞凋亡改变:Pae、5-FU可下调EC9706细胞中Bcl-2蛋白表达,同时增强EC9706细胞中Bax蛋白的表达,联合用药组较单药组作用更为明显(2.21±0.14 vs 5.67±0.30,4.22±0.34;8.55±0.33 vs 3.90±0.27,6.28±0.26,均P<0.05).结论:Pae可明显抑制人食管癌EC9706细胞的增殖.促进其凋亡,Pae联合5-FU作用更为明显.     

siRNA阻断NF-κB信号通路联合5-FU对食管鳞癌细胞凋亡的促进作用

目的: 利用RNAi技术特异性的抑制NF-κB亚单位p65的表达,观察其对p65表达的抑制作用及联合5-FU对食管鳞癌细胞Eca109和EC9706的影响.方法:将终浓度为50 nmol/L的p65 siRNA转染到食管鳞癌细胞EC9706和Eca109中,通过RTPCR检测0、24、48和72 h时段p65 mRNA的表达水平.Western blotting法检测p65和Bcl-2蛋白表达,Annexin V/PI复染结合流式细胞仪检测细胞凋亡,显微镜下观察p65 siRNA与5-FU单独或联合应用对食管鳞癌细胞形态学特性的影响.结果:EC9706和Eca109细胞转染p65 siRNA 24、48和72 h后,p65 mRNA的表达水平随时间的延长逐渐下调,在72 h的阻断效率最为明显,与0 h相比,差异有显著性(0.12±0.01 vs 0.28±0.05,0.1±0.01 vs 0.38±0.04,均P<0.05),转染72 h后,p65和Bcl-2蛋白表达水平下调.EC9706和Eca109转染p65siRNA后,细胞凋亡指数明显升高(6.65%±0.27% vs 2.03%±0.08%,8.03%±0.06% vs 2.66%±0.25%,均P<0.05);p65 siRNA转染72 h后,EC9706和Eca109细胞增殖较慢;当p65 siRNA与5-FU联合作用,细胞增殖明显受到抑制.结论:p65 siRNA可阻断NF-κB信号通路,下调NF-κB下游基因中抗凋亡蛋白Bcl-2的表达,表明活化的NF-κB信号通路可成为食管鳞癌基因治疗中一个重要的分子靶点.     

siRNA阻断NF-κB信号通路联合5-FU对食管鳞癌细胞凋亡的促进作用

目的: 利用RNAi技术特异性的抑制NF-kappaB亚单位p65的表达, 观察其对p65表达的抑制作用及联合5-FU对食管鳞癌细胞Eca109和EC9706的影响. 方法: 将终浓度为50 nmol/L的p65 siRNA转染到食管鳞癌细胞EC9706和Eca109中, 通过RT-PCR检测0、24、48和72 h时段p65 mRNA的表达水平. Western blotting法检测p65和Bcl-2蛋白表达, Annexin V/PI复染结合流式细胞仪检测细胞凋亡, 显微镜下观察p65 siRNA与5-FU单独或联合应用对食管鳞癌细胞形态学特性的影响. 结果: EC9706和Eca109细胞转染p65siRNA 24、48和72 h后, p65 mRNA的表达水平随时间的延长逐渐下调, 在72 h的阻断效率最为明显, 与0 h相比, 差异有显著性(0.12±0.01 vs 0.28±0.05, 0.1±0.01 vs 0.38±0.04, 均P<0.05), 转染72 h后, p65和Bcl-2蛋白表达水平下调. EC9706和Eca109转染p65siRNA后, 细胞凋亡指数明显升高(6.65%±0.27% vs 2.03%±0.08%, 8.03%±0.06% vs 2.66%±0.25%, 均P<0.05); p65 siRNA转染72 h后, EC9706和Eca109细胞增殖较慢; 当p65 siRNA与5-FU联合作用, 细胞增殖明显受到抑制. 结论: p65 siRNA可阻断NF-kappaB信号通路, 下调NF-kappaB下游基因中抗凋亡蛋白Bcl-2的表达, 表明活化的NF-kappaB信号通路可成为食管鳞癌基因治疗中一个重要的分子靶点.     

丹参酮ⅡA联合5-FU对低氧下人胃癌SGC7901细胞增殖、凋亡的影响及与HIF-1α和突变型P53表达的关系

目的:观察低氧环境下不同浓度的丹参酮ⅡA(Tan Ⅱ A)联合5-氟尿嘧啶(5-FU)对人胃癌SGC7901细胞增殖、凋亡的影响及与HIF-1α和突变型P53的表达.方法:用氯化钴(CoCl<,2>)创建低氧模型,采用0、0.5、1.0、2.0、5.0、10.0 mg/L的TanⅡA联合10.0 mg/L的5-FU分别作用于低氧SGC7901细胞24、48和72 h,MTT法检测细胞活力:用上述同样方式作用于低氧SGC7901细胞24、48和72 h后,Hoechst染色法检测细胞凋亡:TanⅡA(0、0.5、2.0、10.0 mg/L)联合10.0 mg/L的5-FU作用于低氧SGC7901细胞48h后,免疫细胞化学二步法检测IHIF-1α及突变型P53的表达.结果:低氧环境下,不同浓度的Tan Ⅱ A联合10.0 mg/L 5-FU呈时间、剂量依赖性地抑制SGC7901细胞的增殖(均P<0.01),10.0 mg/LTan Ⅱ A联合5-FU作用细胞72 h后,其抑制率为67.46%.0.5-10.0 mg/L TanⅡA联合5-FU作用细胞24、48、72 h.TanⅡA呈时间、剂量依赖性地促进SGC7901细胞凋亡(均P<0.01).HIF-1α及突变型P53表达明显高于常氧组(t=22.786,13.914,均P<0.01),不同浓度的Tan Ⅱ A联合10.0 mg/L 5-FU作用细胞48 h后,HIF-1α、突变型P53蛋白表达明显降低(F=182.234,130.062,均P<0.01),且二者呈高度正相关(n=5,r=0.995,P<0.01).结论:在低氧环境下,TanⅡA可能通过抑制HIF-1α及突变型P53蛋白表达从而增强5-FU抑制SGC7901细胞增殖及诱导凋亡作用.     

表皮生长因子受体抑制剂吉非替尼对食管癌EC9706细胞增殖、凋亡及细胞周期的影响

目的探讨表皮生长因子受体(EGFR)抑制剂吉非替尼对食管癌EC9706细胞增殖、凋亡和细胞周期的影响。方法将EC9706细胞培养并加入不同浓度的吉非替尼处理不同时间后,采用MTT法检测细胞增殖抑制率;Annexin V-FITC/PI双染法、流式细胞仪检测细胞凋亡率。PI染色、流式细胞仪检测细胞周期。结果吉非替尼对食管癌EC9706细胞增殖具有明显的抑制作用,且表现为剂量和时间依赖性;吉非替尼组细胞凋亡率明显高于正常对照组,且随着吉非替尼剂量的增加,凋亡率逐渐增加(P0.05);与对照组相比,10μg/ml吉非替尼处理24 h后的EC9706细胞,处于G0/G1期细胞比例明显增加,处于S期细胞比例明显减少(P0.05)。结论吉非替尼对食管癌EC9706细胞具有明显的增殖抑制作用,其机制与诱导凋亡及阻滞细胞周期有关。     

环氧合酶-2反义RNA在不同时间点对人食管癌细胞系生长的影响

背景:环氧合酶（COX）-2在人食管癌细胞系EC9706中高度表达,COX-2反义RNA能抑剂EC9706细胞的生长增殖。目的:探讨COX-2反义RNA在不同时间点对EC9706细胞生长的影响。方法:培养293细胞,扩增、纯化编码COX-2反义RNA的重组腺病毒Ad-AShcox-2,转染EC9706细胞,在不同时间点进行活细胞计数和3H-胸腺嘧啶核苷（TdR）掺入量测定。结果:扩增、纯化获得编码COX-2反义RNA的重组腺病毒Ad-AShcox-2,滴度为1.2×10~（12）PFU/ml。Ad-AShcox-2转染EC9706细胞后,在24h、48h、72h和96h时对细胞生长的抑制率分别为8.60％、24.33％、50.21％和75.26％。Ad-AShcox-2组EC9706细胞的3H-TdR掺入量从24h起开始减少,72～96h时达最低,与对照组相比有显著差异（P<0.001）。结论:COX-2反义RNA重组腺病毒感染人食管癌细胞后,从24h起开始对癌细胞产生抑制作用,72～96h时寸抑制作用最为明显。     

五味子多糖对裸鼠脑胶质瘤细胞增殖的影响

目的探讨不同浓度五味子多糖对体外培养裸鼠脑胶质瘤细胞生长的影响。方法体外培养原代裸鼠脑胶质瘤细胞,采用MTT方法,检测不同浓度五味子多糖作用24、48、72 h时对其增殖的影响。结果五味子多糖浓度在200μg/ml时,细胞生长无明显影响;在24、48 h时,药物浓度在400、800μg/ml时,对细胞生长的作用也不明显;但在72 h时,药物浓度在400和800μg/ml时,对细胞增殖的抑制作用很明显(P<0.01)。结论高浓度五味子多糖对体外培养裸鼠脑胶质瘤细胞的增殖具有抑制作用,提示五味子多糖可能在治疗脑胶质瘤方面发挥重要作用。     

参蛤散对H9c2心肌细胞增殖的影响

目的 观察参蛤散对H9c2心肌细胞增殖的影响,为参蛤散后续研究提供依据。方法 采用H9c2心肌细胞株,制备参蛤散冻干粉进行干预,在24 h、48 h、72 h各时间点,用细胞计数试剂盒-8(CCK-8)法检测细胞吸光度值(OD值),细胞划痕实验检测细胞迁移率。结果 24 h参蛤散0.03 mg/mL、0.10 mg/mL、0.30 mg/mL、1.00 mg/mL组OD值高于对照组(P<0.05);48 h参蛤散各浓度组OD值均高于对照组(P<0.05);72 h参蛤散0.01 mg/mL组OD值高于对照组(P<0.05),其余组与对照组比较差异均无统计学意义(P>0.05)。24 h参蛤散0.10 mg/mL、0.30 mg/mL、1.00 mg/mL组细胞迁移率大于对照组(P<0.05);48 h参蛤散0.01 mg/mL、0.10 mg/mL、0.30 mg/mL组细胞迁移率大于对照组(P<0.05);72 h参蛤散0.01 mg/mL、0.30 mg/mL组细胞迁移率大于对照组(P<0.05)。结论 参蛤散可以促进H9c2心肌细胞增殖。     

Nimesulide联合奥沙利铂对人肝癌细胞增殖与凋亡的影响

目的:探讨选择性环氧合酶-2(cyclooxygenase-2,COX-2)抑制剂Nimesulide联合奥沙利铂(Oxaliplatin,L-OHP)对人肝癌细胞株SMMC-7721增殖与凋亡的影响.为肝癌的药物治疗提供依据.方法:预实验筛选Nimesulide有效终浓度与不同浓度L-OH P(0.5、1.0、2.0、5.0 mg/L)联合处理肝癌细胞48 h;另外选取L-OHP有效终浓度与Nimesulide筛选的浓度单独或联合处理肝癌细胞24 h、48 h、72 h;采用倒置显微镜观察细胞形态学改变,四氮唑盐比色法(MTT法)观察细胞增殖活力的改变,流式细胞仪检测细胞凋亡率.结果:不同浓度Nime sulide及L-OHP对SMMC-7721表现出一定程度的生长抑制作用,并呈剂量依赖性.Nimesulide(50μmol)或L-oHP(1 mg/L)单用时可显著抑制SMMC-7721细胞的增殖,选用Nimesulide 50μmol/L与L-OHP(0.5、1.0、2.0、5.0 mg/L)联合使用时,联合抑制作用呈现协同作用(Q＞1.15).L-OHP 1 mg/L、Nimesulide 50 μmol/L及两药联合处理SMMC-7721细胞24h、48 h、72 h,各处理组各时间点对肝癌细胞的抑制作用随着时间的延长而增大,并且两药联合作用有协同作用(Q＞1.15).流式细胞仪检测分析发现两种药物均可有效诱导SMMC-7721细胞的凋亡,且在上述浓度联合应用时具协同效应.结论:Niruesulide与L-OHP对人肝癌细胞株SMMC-7721均有抑制增殖和促进凋亡作用,而且两者联合应用有协同作用.     

肿瘤坏死因子相关凋亡诱导配体及其与5-Fu联合用药对T-24细胞生长的抑制作用

目的研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)单独应用及其与传统化疗药物5-氟尿嘧啶(5-Fu)联合应用对体外培养人膀胱肿瘤T-24细胞生物学行为影响及可能机制。方法倒置显微镜下观察不同浓度TRAIL作用不同时间后T-24细胞形态变化;MTT法检测不同浓度TRAIL单独或10 ng/ml TRAIL联合不同浓度5-Fu不同时间后T-24细胞生存率;流式细胞仪检测T-24细胞凋亡率。结果 TRAIL单独应用时对T-24细胞生长抑制作用有限,显微镜下部分细胞出现形态学改变,作用72 h后1、300、1 000 ng/ml组细胞生存率分别(92.52±3.60)%,(84.63±5.93)%和(79.45±2.04)%;单用200、400、600、800及1 000μmol/ml 5-Fu时细胞生长抑制率分别为(10.76±1.78)%、(14.17±2.75)%、(28.01±1.66)%、(20.17±1.68)%和(32.53±3.65)%,联合10 ng/ml TRAIL处理后0、200、400、600、800及1 000μmol/ml 5-Fu组细胞抑制率分别为分别为(3.85±0.88)%、(8.41±0.74)%、(16.39±2.72)%、(26.27±1.72)%、(28.99±3.36)%和(30.18±1.34)%;10、100、1 000 ng/ml TRAIL处理48 h后的细胞平均凋亡率为(3.75±0.31)%、(7.82±1.07)%、(7.78±3.15)%。结论 TRAIL单用或联合其他化疗药物对T24细胞生长抑制作用有限,同5-Fu联合应用时不能明显增加T-24细胞对5-Fu的敏感性。     

Effect of K‐ras antisense oligodeoxynucleotides on human pancreatic cancer cell line PaTu 8988s

OBJECTIVE : To study the effect of K‐ras antisense oligodeoxynucleotides (ASODN) on human pancreatic cancer cell line PaTu 8988s at different times after treatment. METHODS : Human pancreatic cancer cells (PaTu 8988s) in exponential growth stage were used at a cell concentration of 1 × 10⁵/mL; 0.5 mL of the cell suspension was placed in each well of replicate 24‐well culture plates in the presence of different concentrations (50 and 100 μg/mL) of ASODN and sense oligodeoxynucleotides (SODN). Cell counts and 3‐[4,5‐dimethylthiazolzyl]‐2,5‐diphenyl tetrazolium bromide (MTT) assays were carried out 24, 48 and 72 h after treatment. RESULTS : At 12, 24, 48 and 72 h after ASODN treatment, the following rates of inhibition were observed: for 50 μg/mL, 42.3, 66.6, 69.6 and 74.6%, respectively; for 100 μg/mL, 66.2, 91.4, 98.2 and 98.3%, respectively. CONCLUSION : The inhibitory effect of ASODN began at 12 h post‐treatment and became more marked at 48–72 h. The higher the concentration of ASODN, the earlier the peak of inhibitory rate appears.     

伊班膦酸钠对食管鳞癌EC9706细胞增殖及VEGF表达的作用

目的了解双膦酸盐(BPs)对人食管癌EC9706细胞株增殖的影响,探讨伊班膦酸钠对VEGF表达的作用。方法倒置显微镜观察细胞形态变化,进行细胞形态学观察;采用MTT法检测伊班膦酸钠对EC9706细胞增殖的抑制作用;采用免疫细胞化学检测伊班膦酸钠对EC9706细胞VEGF表达的影响。结果随着伊班膦酸钠药物浓度的增加和作用时间的延长,实验组细胞密度下降,细胞碎片增多,核染色质和胞浆皱缩,胞膜和核膜增厚,折光性差,细胞贴壁能力下降。实验组不同浓度的伊班膦酸钠对食管癌EC9706细胞均有抑制作用,且与药物浓度和作用时间呈正相关,与对照组相比,差异均有统计学意义(P0.05)。对照组及10、20、40μg/ml浓度VEGF蛋白的表达率分别为43.81±3.61、30.92±3.30、21.57±3.17、9.05±1.38,各实验组与对照组比较、各实验组组间比较,差异均有统计学意义(P0.05)。结论伊班膦酸钠对食管癌EC9706细胞有增殖抑制作用,并且具有时间及剂量依赖性。伊班膦酸钠可以从蛋白水平抑制食管癌EC9706细胞VEGF的表达。     

Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent Chlorogenic acid in vitro

Objective:To examine the acanthamoebicidal effects of ethyl acetate,aqueous and butanol fractions of dried flower buds of Lonicera japonica(L.japonica) Thunb.(Flos Lonicerae) in vitro.Methods:Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR.They were exposed to ethyl acetate,water and butanol fractions of L.japonica Thunb.at concentrations ranging from 0.5 mg/m L to 1.5 mg/m L.The extracts were evaluated for growth inhibition at 24,48 and 72 h,respectively.Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment.Results:Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h,49.2% after 48 h and 33.7% after 72 h Chlorogenic acid,the major active constituent of L.japonica Thunb.at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h,84.0% after 48 h and 72.3% after 72 h.This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h.Conclusions:Results obtained from this study show that ethyl acetate fraction at 1.5 mg/m L is the most potent fraction of L.japonica Thunb.and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.     

Construction of pETNF-P16 plasmid and its expression properties in EC9706 cell line induced by X-ray irradiation

AIM: Recombined plasmid pETNF-P16 was constructed to investigate its expression properties in esophageal squamous carcinoma cell line EC9706 induced by X-ray irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: Recombined plasmid pETNF-P16 was constructed and transfected into EC9706 cells with lipofectamine. ELISA,Western blot, and immunocytochemistry were performedto determine the expression properties of pETNF-P16 in EC9706 after transfection induced by X-ray irradiation. RESULTS: Eukaryotic expression vector pETNF-P16 was successfully constructed and transfected into EC9706 cells. TNFα expressions were significantly increased in the transfected cells after different doses of X-ray irradiation than in those after 0Gy irradiation (1 192.330-2 026.518 pg/mL,P&lt;0.05-0.01), and the TNFα expressions and P16 were significantly higher 6-48 h after 2 Gy X-ray irradiation (358.963-585.571 pg/mL, P&lt;0.05-0.001). No P16 expression was detected in normal EC9706 cells. However, there was strong expression in the transfected and irradiation groups. CONCLUSION: X-ray irradiation induction could significantly enhance TNFα and P16 expression in EC9706 cells transfected with pETNF-P16 plasmid. These results may provide important experimental data and therapeutic potential for gene-radiotherapy of esophageal carcinoma.     

己酮可可碱对人食管癌EC9706细胞增殖及NF—κB p65、COX-2、Ki-67蛋白表达的影响

目的探讨己酮可可碱（PTX）对肿瘤细胞的增殖抑制作用及机制。方法体外培养人食管癌EC9706细胞,用不同质量浓度的PTX进行干预,采用MTT法检测细胞增殖抑制率（IR）,免疫组化染色SP法检测核转录因子-κB（NF—κB p65）、环氧化酶-2（COX-2）、Ki-67蛋白表达。结果PTX处理后EC9706细胞IR明显升高,NF-κB P65、COX-2蛋白及Ki-67蛋白表达明显下降（P〈0．05）,且呈时间-浓度依赖性。结论PTX可抑制EC9706细胞增殖,可能机制为下调NF-κB、COX-2及Ki-67蛋白表达;此为PTX治疗食管癌提供了理论依据。     

Effect of blocking IGF-Ⅰ receptor on growth of human hepatocellular carcinoma cells

AIM: To study the expression level and localization of insulin-like growth factor -Ⅰ receptor (IGF-IR) in HepG2 cells and Chang liver cells, and to observe the effect of anti-IGF-IR monoclonal antibody (αIR3) on the growth of HepG2 cells.METHODS: The expression of IGF-IR in HepG2 cells and Chang liver cells was detected by immunohistochemistry.The influences of αIR3 on proliferation and apoptosis were examined by the 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and electron microscopy, respectively. Flow cytometry (FCM) was applied for the analysis of cell cycle and apoptosis was observed under electron microscope.RESULTS: IGF-IR was located in the membranes of both HepG2 and Chang liver cell lines, and the expression level of IGF-IR was higher in HepG2 cells than in Chang liver cells. Treated with 0.1 μg/mL αIR3 for 48 h in vitro, the cell growth index (GI) of HepG2 cells was significantly higher than that of control (103.41% vs 100%, P ＜ 0.01).However, the αIR3 for 24 h at final concentration of 4.0 μg/mL made the GI of HepG2 cells lower than that of control (93.37% vs 100%, P ＜ 0.01). Compared with control, treated with αIR3 for 48 h at final concentrations ranging from 1.0 μg/mL to 4.0 μg/mL markedly reduced the GIs of HepG2 cells (97.63%, 97.16%, 95.13%,92.53% vs 100%, P ＜ 0.05 or P ＜ 0.01), treated with αIR3 for 72 h at final concentrations ranging from 0.2 μg/mL to 4.0 μg/mL decreased the GIs of HepG2 cells obviously (95%, 91.63%, 90.77%, 89.84%, 88.51% vs 100%, P ＜ 0.01), and treated with αIR3 for 96 h at final concentrations ranging from 0.5 μg/mL to 4.0 μg/mL made GIs of HepG2 cells lower significantly (88.86%,83.97%, 79.81%, 77.24%, 70.51% vs 100%, P ＜ 0.05 or ,P ＜ 0.01). Moreover, treated with αIR3 from 24 h to 96 h at final concentrations ranging from 0.2 μg/mL to 4.0 μg/mL reduced the GI of HepG2 cells from 97.63% to 70.51% in a dose- and time-dependent manner. Also,αIR3 treatment for 72 h at final concentration from 0.5 μg/mL to 2.0 μg/mL increased the proportion of G0/G1 phase cells(61.73%, 67.1%, 83.7%,76.87% vs 44.47%,P ＜ 0.01) and significantly decreased that of S phase cells(28.63%, 25.13%, 15.63%, 23.13% vs 53.17%, P ＜ 0.01), in contrast to the proportion of G2/M phase cells.The apoptotic rates of HepG2 cells were increased more than that of control (7.83%, 16.13%, 21.1%, 37.73% vs 4.13%, P ＜ 0.01).CONCLUSION: The malignant cell phenotype of human hepatocarcinoma cell is related to overexpression of IGFIR. The blockage of IGF-IR with αIR3 may contribute to the inhibition of proliferation and induction of apoptosis in HepG2 cells.     

吡喹酮异构体对肝星状细胞系LX⁃2增殖及活化的作用

目的　观察左旋吡喹酮（L⁃PZQ）和右旋吡喹酮（D⁃PZQ）体外对人肝星状LX⁃2细胞系增殖及活化的作用差异。方法　利用转化生长因子⁃β（TGF⁃β）刺激激活LX⁃2细胞;采用CCK⁃8法检测以0 ~ 50 μg/mL梯度浓度吡喹酮刺激24 h后的LX⁃2细胞增殖情况;采用实时荧光定量PCR（qPCR）和免疫印迹试验检测15 mg/mL吡喹酮刺激LX⁃2细胞24 h 后的Ⅰ型胶原、Ⅲ型胶原和α⁃平滑肌肌动蛋白（α⁃SMA）基因表达水平和48 h后蛋白表达水平,以分析LX⁃2细胞活化情况。结果　L⁃PZQ和D⁃PZQ两药物各浓度组LX⁃2细胞存活率差异均有统计学意义（F = 6.119、79.180,P均< 0.05）;药物浓度< 30 μg/mL时,L⁃PZQ和D⁃PZQ对激活型LX⁃2增殖能力均无显著影响（P均> 0.05）;L⁃PZQ浓度> 50 μg/mL和D⁃PZQ浓度> 40 μg/mL时,均对激活型LX⁃2增殖能力产生抑制作用（P均< 0.05）;D⁃PZQ浓度为40 μg/mL和50 μg/mL时,对激活型LX⁃2增殖的抑制作用均强于L⁃PZQ（t = 3.419、8.776,P均< 0.05）。空白组、TGF⁃β诱导组、L⁃PZQ组和D⁃PZQ组LX⁃2细胞中Ⅰ型胶原、Ⅲ型胶原和α⁃SMA基因（F = 21.55、79.99、46.70,P 均< 0.05）和蛋白表达水平（F = 20.12、30.29、32.93,P 均< 0.05）差异均有统计学意义。L⁃PZQ对激活型LX⁃2细胞中Ⅲ型胶原和α⁃SMA基因表达水平具有显著抑制作用（P均< 0.05）,但对Ⅰ型胶原基因表达水平及Ⅰ型胶原、Ⅲ型胶原和α⁃SMA蛋白表达水平均无显著影响（P均> 0.05）;D⁃PZQ对激活型LX⁃2细胞中Ⅰ型胶原、Ⅲ型胶原和α⁃SMA基因和蛋白表达水平均有显著抑制作用（P均< 0.05）;D⁃PZQ对激活型LX⁃2细胞中Ⅰ型胶原、Ⅲ型胶原和α⁃SMA基因表达抑制作用强于L⁃PZQ（P均< 0.05）。结论　L⁃PZQ和D⁃PZQ对LX⁃2细胞增殖及活化均有一定抑制作用,且D⁃PZQ的抑制作用强于L⁃PZQ。     

纤维溶解酶原激活物抑制剂-1对大鼠胚肺成纤维细胞的影响

目的 研究纤维溶解酶原激活物抑制剂-1(PAI-1)对大鼠胚肺成纤维细胞增殖、转化及胶原合成的影响,探讨PAI-1在肺纤维化发生中的作用.方法 体外分离培养Wister 待产孕大鼠胚肺成纤维细胞,应用四甲基偶氮唑盐试验观察不同浓度(5、10、20、40、80 μg/L)的PAI-1刺激12、24和48 h后成纤维细胞的增殖率;选用PAI-1最适浓度20 μg/L刺激48和72 h后,细胞免疫化学法检测增殖细胞核抗原(PCNA)的表达;实时PCR法检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原24和48 h时mRNA表达的变化.结果各浓度PAI-1刺激成纤维细胞后,以20 μg/L 12 h增殖率及吸光度值最高,分别为62.6%和0.573±0.039.在12 h中,不同浓度组较对照组差异有统计学意义(F=111.112,P=0.000),选择20 μg/L为最适浓度.免疫细胞化学法检测20 μg/L PAI-1刺激48、72 h后,PCNA表达的积分吸光度值分别为3685±686和2530±477,较对照组差异有统计学意义(F=7.85,P=0.020).PAI-1刺激成纤维细胞24和48 h后,α-SMA表达上调(230±11)%和(159±9)%,较对照组差异有统计学意义(F=39.92,P=0.000).Ⅰ型胶原表达上调(92±8)%和(65±12)%,差异有统计学意义(F=32.61,P=0.001).结论 PAI-1可以促进成纤维细胞增殖、转化及胶原合成,可能促进肺间质纤维化的发生和发展.

Abstract：

Objective To explore the effect of plasminogen activator inhibitor-1 (PAI-1) on the proliferation and conversion of rat embryonic lung fibroblasts and the synthesis of collagen, and therefore to explore the function of PAI-1 in pulmonary fibrosis.Methods The embryonic lung fibroblasts from pregnant Wistar rats were isolated and cultured in vitro. The reproduction rate of fibroblasts at 12 h, 24 h, and 48 h after being stimulated by PAI-1 with different concentrations (5, 10, 20, 40, 80, and 100 μg/L) was measured by MTT assay. After being stimulated by PAI-1 with the most suitable concentration (20 μg/L) for 48 h and 72 h, the expression of proliferating cell nuclear antigen (PCNA) was measured by immunocytochemical technique, and the mRNA expression of α-SMA and type-1 collagen at 24 h and 48 h was measured by real-time PCR. Results PAI-1 with different concentrations stimulated the proliferation of fibroblasts. The highest proliferation rate and absorbance in concentration of 20 μg/L and at 12 h were 62.6% and 0.573±0.039. The comparison of different concentrations showed that the difference was significant(F=111.112,P=0.000). Therefore, 20 μg/L was selected as the most suitable concentration. Using immunocytochemical method, the optical density of PCNA at 48 h and 72 h were 3685±686 and 2530±477 after being stimulated with 20 μg/L PAI-1.The comparison showed significant difference(F=7.85,P=0.02). The expression of α-SMA increased (230±11)% and(159±9)% at 24 h and 48 h after being stimulated with 20 μg/L PAI-1, and the difference was significant(F=39.92, P=0.0003). The expression of type-1 collagen increased(92±8)% and (65±12)%, the difference being significant(F=32.61,P=0.0006). Conclusion PAI-1 can promote the proliferation and conversion of fibroblasts and the synthesis of collagen, which may be involved in the pathogenesis of pulmonary interstitial fibrosis.