蒿甲醚对人鼻咽癌细胞CNE-1放射敏感性的影响及其机制研究

Objective To evaluate the effect of artemether on the cell cycle and the radiosensitivity in human nasopharyngeal carcinoma cell line CNE-1.Methods Cell growth inhibition was assessed with MTT.The method of colony-forming was used to detect the radiation sensitivity.Cell cycle distribution was analyzed by using flow cytometry.The protein expressions of clyclin B1 and Weei were detected by using Western blot.Results The growth of CNE-1 cells was inhibited in a dose-dependent manner.The concentration of 20 μmol/L artemether had radiosensitive effect on CNE-1 cells at 24 h after administration,and SER was 1.481.When CNE-1 cell was irradiated,the G2/M cells increased (t =4.59,P ＜ 0.05).After exposure to combination of artemether and irradiation,the G2/M cells were decreased (t= 10.60,P ＜ 0.05).Western blot showed that artemether increased the level of cyclin B1 expression and inhibited the level of Weel expression.Conclusions The noncytotoxic concentration of artemether could enhance radiosensitization of CNE-1 cells.The radiosensitivity enhancement of artemether might depend on the exposure time.The effect is most obvious when radiation is delivered 24 h after expose to artemetherr.The radiosensitizing effect could be related to apoptosis.     

青蒿素对人鼻咽癌CNE细胞的放射增敏作用及机制的研究

Objective To investigate the radiosensitizing effects of artemisinin on CNE human nasopharyngeal carcinoma cells in vitro.Methods CNE human nasopharyngeal carcinoma cell line was used in this study.Cell growth kinetics was determined by MTT assay.Effect of the drug on radiosensitivity of CNE cells was analyzed by clonogenic assay.The change of cell cycle was measured by flow cytometry.Results The inhibition of CNE cells growth by artemisinin was increased with concentrations.Artemisinin (1 μmol/L)could enhance the radiosensitizing effects on CNE cell line,and the sensitizing enhancement ratio(SER)was 1.26.Artemisinin abrogated radiation-induced G2/M arrest of the tested CNE cells.Compared with the radiation alone group,the proportion of G2/M phase cells increased in radiation combined with drug group.Conclusions Artemisinin could reduce radiation-induced G2/M arrest and enhance the cytotoxicity of γ-irradiation on the CNE ceils.     

西妥昔单抗对人食管癌放射抗拒性细胞株KYSE-150R放射敏感性的研究

Objective To determine the effect of cetuximab(C225)on the radioresistant human esophageal squamous carcinoma eell line KYSE-150R.Methods A radioresistant human esophageal squamous carcinoma cell line KYSE-150R was established by fractionated irradiation.Morphological changes from KYSE-150 to KYSE-150R were observed by phase-contrast microscopy.Karyotype analysis was performed by G-banding.The radiosensitivities were analyzed by colony formation assays.Results The population doubling time of KYSE-150 and KYSE-150R were(23.6±0.2)h and(25.9±0.6)h (t=6.6,P<0.01),respectively.The chromosome number of KYSE-150R was increased and chromosome aberrations were observed from(69.3±1.9)h to(73.7±1.2)h(t=-8.83,P<0.01).The SF2,D0,Dq and N values of KYSE-150R were all higher than those of KYSE-150.After 5μg/ml of C225 added,the SF2,D0,Dq and N values were significantly decreased as compared to the control.After C225 treatment,the G0/G1 and G2/M phase cells were increased,while S-phase cells decreased(t=-4.478-4.308,P<0.05).Conclusion Cetuximab can enhance the radiosensitivity of radioresistant human esophageal squamous carcinoma cell line KYSE-150R.     

青蒿素对人鼻咽癌CNE细胞的放射增敏作用及机制的研究

Objective To investigate the radiosensitizing effects of artemisinin on CNE human nasopharyngeal carcinoma cells in vitro.Methods CNE human nasopharyngeal carcinoma cell line was used in this study.Cell growth kinetics was determined by MTT assay.Effect of the drug on radiosensitivity of CNE cells was analyzed by clonogenic assay.The change of cell cycle was measured by flow cytometry.Results The inhibition of CNE cells growth by artemisinin was increased with concentrations.Artemisinin (1 μmol/L)could enhance the radiosensitizing effects on CNE cell line,and the sensitizing enhancement ratio(SER)was 1.26.Artemisinin abrogated radiation-induced G2/M arrest of the tested CNE cells.Compared with the radiation alone group,the proportion of G2/M phase cells increased in radiation combined with drug group.Conclusions Artemisinin could reduce radiation-induced G2/M arrest and enhance the cytotoxicity of γ-irradiation on the CNE ceils.     

西妥昔单抗对人食管癌放射抗拒性细胞株KYSE-150R放射敏感性的研究

Objective To determine the effect of cetuximab(C225)on the radioresistant human esophageal squamous carcinoma eell line KYSE-150R.Methods A radioresistant human esophageal squamous carcinoma cell line KYSE-150R was established by fractionated irradiation.Morphological changes from KYSE-150 to KYSE-150R were observed by phase-contrast microscopy.Karyotype analysis was performed by G-banding.The radiosensitivities were analyzed by colony formation assays.Results The population doubling time of KYSE-150 and KYSE-150R were(23.6±0.2)h and(25.9±0.6)h (t=6.6,P<0.01),respectively.The chromosome number of KYSE-150R was increased and chromosome aberrations were observed from(69.3±1.9)h to(73.7±1.2)h(t=-8.83,P<0.01).The SF2,D0,Dq and N values of KYSE-150R were all higher than those of KYSE-150.After 5μg/ml of C225 added,the SF2,D0,Dq and N values were significantly decreased as compared to the control.After C225 treatment,the G0/G1 and G2/M phase cells were increased,while S-phase cells decreased(t=-4.478-4.308,P<0.05).Conclusion Cetuximab can enhance the radiosensitivity of radioresistant human esophageal squamous carcinoma cell line KYSE-150R.     

沉默STAT1基因增强放射抗拒鼻咽癌CNE-2R细胞放射敏感性

目的 研究基因STAT1沉默对人放射抗拒鼻咽癌细胞放射敏感性的影响.方法 慢病毒介导的STAT1基因转染放射抗拒鼻咽癌细胞CNE-2R,荧光定量RT-PCR技术检测沉默效果.MTT法检测转染前后细胞增殖活性,流式细胞技术检测细胞周期及凋亡,克隆形成实验检测转染前后细胞放射敏感性变化.结果 慢病毒转染后放射抗拒鼻咽癌细胞STAT1表达降低(F=429.87,P<0.05)、细胞生长抑制(F3=.88~4.63,P<0.05)、凋亡率增加(F=38.13,P<0.05)、放射敏感性增加(F=252.80,P<0.05)、细胞周期中G₀/G₁、S和G₂/M期未见明显差异(P>0.05).结论 沉默STAT1基因能增加放射抗拒鼻咽癌细胞 CNE-2R的放射敏感性.     

沉默GRAMD1A及抑制STAT5信号通路对肝癌细胞Huh7放射敏感性的影响

目的 探讨沉默GRAMD1A及抑制STAT5信号对肝癌细胞Huh7放射敏感性的影响,旨在为肝癌临床联合治疗提供新思路。方法 慢病素感染构建沉默GRAMD1A的Huh7细胞株,采用qPCR和Western blot进行验证,qPCR和荧光素酶报告实验检测沉默GRAMD1A后对Huh7细胞中STAT5及其下游基因表达的影响;以克隆形成率和细胞凋亡为指标检测沉默GRA株。结果 构建后的Huh7细胞经2 Gy照射后,沉默GRAMD1A联合照射组细胞克隆形成能力较阴性对照联合照射组显著降低,差异有统计学意义（t=8.494,P<0.05）;沉默GRAMD1A联合照射组细胞凋亡较阴性对照联合照射组显著增加,差异有统计学意义（t=3.560,P<0.05）。沉默GRAMD1A后Huh7细胞放射敏感性明显增加,且细胞中STAT5及其下游基因表达显著降低。SH-4-54抑制剂联合照射组较二甲基亚砜联合照射组细胞克隆存活能力显著降低,差异有统计学意义（t=8.660,P<0.05）,SH-4-54抑制STAT5通路后,Huh7细胞放射敏感性显著增加。结论 沉默GRAMD1A可能通过STAT5信号通路增强肝癌细胞Huh7的放射敏感性,表明GRAMD1A在肝癌发生发展中起重要作用,将可能为肝癌靶向治疗及联合治疗提供新靶点。     

沉默EZH2诱导舌癌Tca-8113细胞凋亡及其对放射敏感性的影响

目的 探讨zeste基因增强子同源物2（enhancer of zeste homolog 2,EZH2）对舌鳞癌细胞凋亡及放射敏感性的影响。方法 以舌鳞癌Tca-8113细胞为研究对象,细胞转染EZH2小干扰RNA（EZH2 siRNA1、EZH2 siRNA2）及小干扰RNA阴性对照（siRNA-NC）,RT-PCR和Western blot检测EZH2表达水平,筛选干扰效果较好的EZH2 siRNA2继续研究。将转染EZH2 siRNA2后的Tca-8113记为EZH2 siRNA2组,同时以不做处理的细胞为对照组,用8 Gy剂量照射转染siRNA对照组和EZH2 siRNA2细胞,并依次命名为照射组和联合组,四甲基偶氮唑盐（MTT）检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测信号转导与转录因子3（STAT3）、磷酸化的STAT3（p-STAT3）、活性Caspase-3（Cleaved Caspase-3）表达。siRNA-NC组和EZH2 siRNA2组细胞用0、2、4、6、8 Gy剂量照射处理后,细胞克隆实验检测放射敏感性。结果 EZH2 siRNA1、EZH2 siRNA2均能够干扰舌鳞癌细胞中EZH2的表达,且EZH2 siRNA2干扰效果最好（t_mRNA=8.660,P_mRNA<0.01;t_蛋白=2.883,P_蛋白<0.05）。下调EZH2 siRNA2组细胞凋亡率为（29.90±1.64）%,联合组凋亡率为（38.17±1.59）%,二者比较,差异有统计学意义（t=4.742,P<0.05）,同时下调EZH2还可以降低p-STAT3水平,促进Cleaved Caspase-3蛋白表达。干扰EZH2表达后Tca-8113细胞辐射增敏比为1.668。结论 干扰EZH2表达能够协同放疗促进舌鳞癌细胞凋亡,抑制舌鳞癌细胞增殖,增加舌鳞癌细胞放射敏感性。     

HAVCR2基因沉默对辐射诱发基因组不稳定肝细胞凋亡及细胞周期的影响

目的 研究HAVCR2基因沉默对辐射诱发基因组不稳定肝细胞凋亡及细胞周期的影响。方法 利用慢病毒介导的RNA干扰（RNAi）技术沉默辐射诱发基因组不稳定肝细胞中HAVCR2基因的表达,通过流式细胞术检测HAVCR2基因沉默对细胞周期及凋亡的影响;利用实时荧光定量PCR方法检测HAVCR2基因沉默后p53基因的表达变化。结果 慢病毒介导的RNAi技术能有效沉默HAVCR2基因的表达（t=19.21,P<0.05）,与对照组相比,HAVCR2基因的沉默可导致基因组不稳定肝细胞G₂期阻滞（t=-3.41,P<0.05）,细胞凋亡率降低（t=3.65,P<0.05）;实时荧光定量PCR检测结果表明,HAVCR2基因的沉默诱发基因组不稳定肝细胞p53基因的表达下调（t=4.82,P<0.05）。结论 HAVCR2siRNA使辐射诱发基因组不稳定肝细胞凋亡率降低,使基因组不稳定肝细胞发生G₂期的阻滞。     

过表达miR-29c靶向抑制AKT2增强肝癌HepG2细胞放射敏感性的实验研究

目的 探讨miR-29c靶向AKT2对肝癌细胞HepG2放射敏感性的影响。方法 RT-PCR检测人正常肝THLE-3细胞和肝癌HepG2细胞中miR-29c表达。给予不同剂量（0、2、4、6和8 Gy）的X射线照射后,RT-PCR检测HepG2细胞中miR-29c表达变化。经生物信息学预测并采用双荧光素酶报告基因实验和Western blot检测miR-29c与AKT2的靶向关系。采用脂质体2000将miR-29c mimic/AKT2基因重组质粒和miR-29c inhibitor/慢病毒载体AKT2 shRNA转染至HepG2细胞中,并给予不同剂量X射线照射后,克隆形成实验和MTT实验检测miR-29/AKT2对HepG2细胞存活率和细胞活力的影响。结果 与THLE-3细胞相比,HepG2细胞中miR-29c明显降低,差异有统计学意义（t=17.816,P<0.05）;HepG2经2、4、6和8 Gy X射线照射后,细胞存活率较THLE-3细胞显著降低（t=4.541、6.823、7.218、9.363,P<0.05）,HepG2细胞中miR-29c表达显著下降（t=5.599、9.262、10.470、10.873,P<0.05）。miR-29c过表达可降低HepG2细胞存活率和细胞活力（t_存活率=4.307、7.668、7.668、6.894,P<0.05;t_细胞活力=3.443、8.116、13.434,P<0.05）;反之,抑制miR-29c表达则升高HepG2细胞存活率和细胞活力（t=4.003、6.713、7.141,P<0.05;t_细胞活力=4.282、5.113,P<0.05）。双荧光素酶报告基因实验表明,AKT2是miR-29c的靶基因,Western blot检测结果显示,miR-29c可负向调控AKT2蛋白表达。沉默AKT2后,HepG2细胞的存活分数及细胞存活率趋势与miR-29c过表达相一致;反之,AKT2过表达则与抑制miR-29c表达相一致。结论 miR-29c可通过靶向AKT2增加肝癌细胞HepG2放射敏感性。     

125I-UdR壳聚糖纳米微粒对肝癌细胞的内照射生物学效应

目的 评估¹²⁵I-UdR壳聚糖载药纳米微粒(¹²⁵I-UdR-CS-DLN)对肝癌细胞的内照射生物学效应.方法 采用激光共聚焦显微镜观察¹²⁵I-UdR-CS-DLN在肝癌细胞HepG2和人正常肝组织细胞HL-7702内的聚积和分布;通过MTT实验、流式细胞仪和单细胞凝胶电泳技术,评价内照射细胞生物学效应;采用TUNEL染色法观察兔肝原位肿瘤细胞经¹²⁵I-UdR-CS-DLN靶向治疗后的细胞凋亡.结果 纳米微粒作用30 min后,其在HepG2细胞质内的聚积大于HL-7702;当¹²⁵I-UdR-CS-DLN浓度大于37 kBq/ml时,HepG2细胞在纳米微粒作用后24、48 h的存活率显著低于HL-7702细胞(t=-4.46~6.31,P<0.05),且细胞周期G₁期阻滞明显, G₂/M期细胞明显受损;¹²⁵I-UdR-CS-DLN造成细胞DNA双链断裂的程度明显高于¹²⁵I-UdR,HepG2细胞的DNA损伤后修复能力显著低于HL-7702(Olive尾矩:t=2.94,P<0.05;彗尾DNA%:t=10.64,P<0.01);兔肝原位癌模型经介入被动靶向治疗后的TUNEL染色结果表明,¹²⁵I-UdR-CS-DLN可使兔肝原位肿瘤细胞产生明显的凋亡,而相同剂量¹²⁵I-UdR作用后肿瘤并未出现明显的凋亡.结论 ¹²⁵I-UdR-CS-DLN进入肝癌细胞的能力明显强于¹²⁵I-UdR,引起的DNA辐射损伤效应更强,可明显加剧肝癌细胞的凋亡,阻止DNA损伤修复.     

长链非编码RNA PRMT5-AS1调控电离辐射诱导肝癌细胞铁死亡机制的研究

目的 探讨长链非编码RNA PRMT5-AS1对电离辐射诱导的肝癌细胞铁死亡的影响。方法 在MHCC-97H细胞中构建PRMT5-AS1过表达模型,在HepG2细胞中构建PRMT5-AS1敲低模型。使用X射线照射,吸收剂量为10 Gy,剂量率为3 Gy/min。采用Western blot和qRT-PCR实验检测基因表达水平。采用台盼蓝染色流式细胞术检测PRMT5-AS1表达对受照肝癌细胞脂质过氧化以及铁死亡的影响。采用CCK-8实验检测PRMT5-AS1表达水平对电离辐射照射后肝癌细胞死亡的影响。双荧光素酶报告实验检测let-7c-5p与PRMT5-AS1和SLC7A11之间结合作用。结果 MHCC-97H细胞中过表达PRMT5-AS1能够显著降低电离辐射引起的细胞死亡(对照组vs. PRMT5-AS1过表达组:27.57% vs.18.30%,t=14.94,P<0.05)。HepG2细胞中敲低PRMT5-AS1可显著增加电离辐射引起的细胞死亡(对照组vs. PRMT5-AS1敲低组:17.26% vs.28.26%,t=13.63,P<0.05)。过表达PRMT5-AS1能够明显抑制由电离辐射诱导的细胞内脂质活性氧(ROS)水平增加(对照组vs. PRMT5-AS1过表达组:17.01% vs.12.52%,t=12.80,P<0.05),敲低PRMT5-AS1可显著增加电离辐射诱导的脂质ROS水平增加(对照组vs. PRMT5-AS1敲低组:14.54% vs.17.72%,t=5.93,P<0.05)。CCK-8实验结果表明,过表达PRMT5-AS1能够显著抑制Erastin诱导的细胞活性降低(对照组vs. PRMT5-AS1过表达组:87.92% vs.109.06%,t=2.87,P<0.05),敲低PRMT5-AS1则促进Erastin抑制细胞活性(对照组vs. PRMT5-AS1敲低组:82.56%vs.60.58%,t=38.35,P<0.05)。Western blot和荧光定量PCR结果表明,过表达PRMT5-AS1能够明显提高SLC7A11的蛋白和mRNA水平(t=26.24,P<0.05),敲低PRMT5-AS1后SLC7A11的蛋白和mRNA水平均显著降低(t=5.60,P<0.05)。荧光素酶报告基因实验表明PRMT5-AS1与let-7c-5p之间存在相互作用(t=9.74,P<0.05)。PRMT5-AS1可以与let-7c-5p形成ceRNA网络,靶向调节SLC7A11。let-7c-5p能够逆转由过表达PRMT5-AS1引起的SLC7A11表达水平增加、脂质ROS水平和细胞死亡减少(t=3.01、4.11,P<0.05),而敲低SLC7A11能够逆转PRMT5-AS1引起的脂质ROS抑制和细胞死亡减少(t=21.35、7.15,P<0.05)。结论 长链非编码RNA PRMT5-AS1通过PRMT5-AS1/let-7c-5p/SLC7A11轴抑制电离辐射诱导肝癌细胞铁死亡的发生。     

靶向生存素基因的RNAi对肺腺癌A549细胞放射敏感性的影响

目的 构建靶向生存素(survivin)基因的RNA干扰(RNAi)载体,观察对肺腺癌A549细胞放射敏感性的影响,并探讨其机制。方法 根据survivin的cDNA序列设计干扰序列,构建干扰survivin的重组干扰质粒pGenesil2-survivin。酶切、测序鉴定正确后,经脂质体介导转染肺腺癌A549细胞;应用反转录-聚合酶链式反应(RT-PCR)和Western blot法检测survivin的表达;流式细胞术检测细胞凋亡的变化;克隆形成实验检测细胞的放射敏感性。结果 酶切和测序结果显示,载体构建正确;将质粒pGenesil2-survivin转染肺腺癌A549细胞48 h,survivin 蛋白水平及mRNA水平在正常组与pGenesil2组中无明显变化,5 Gy照射组增强,而转染pGenesil2-survivin后明显抑制;pGenesil2-survivin与5 Gy X射线照射都能诱导细胞凋亡增加(t₁=10.63,P<0.001;t₂=3.75 ,P<0.05),两者共同作用,凋亡增加更明显(t=4.83,P<0.05);克隆形成实验显示,pGenesil2与正常组D₀、D_q无明显变化,而pGenesil2-survivin则明显降低,说明其可以提高A549细胞的放射敏感性。结论 靶向生存素基因的RNAi能够明显抑制survivin mRNA与蛋白的表达,促进凋亡,增强A549细胞的放射敏感性。     

Lentivirus-mediated RNA interference of Ku70 to enhance radiosensitivity of human mammary epithelial cells

Purpose:?To investigate the radiosensitising effect of Ku autoantigen 70 (Ku70) and Ku autoantigen 80 (Ku80) knockdown by lentivirus-mediated RNA interference (RNAi) in the MCF10A immortalised human mammary epithelial cell line.

Materials and methods:?MCF10A cells were infected with lentiviral vectors for RNAi of Ku70. The Ku70-knockdown cell line (Ku70i) and a mock-infected control cell line (LVTHM) were used to perform radiation experiments. For the in?vitro Micronucleus (MN) assay, both cell lines were irradiated with doses of 2 and 4 Gy ⁶⁰Co γ-rays. For cell survival experiments, doses ranging between 0 and 8 Gy were used.

Results:?Western blot analysis showed that the Ku70 lentiviral vector was effective in silencing the expression of both Ku70 and Ku80. A significantly higher radiation-induced MN yield was obtained in the Ku70i cell line compared to the control LVTHM cell line. RNAi of Ku70 also resulted in a lower survival yield after irradiation compared to the control cell line. Analysis of cell death mechanisms showed that MCF10A cells (Ku70i and LVTHM) do not undergo apoptosis, but undergo post-irradiation cellular senescence.

Conclusion:?RNAi of Ku70 resulted in increased chromosomal and cellular radiosensitivity in the MCF10A human mammary cell line after irradiation with ⁶⁰Co γ-rays. These results further strengthen the role of the Ku protein in correct DNA double strand break (DSB) repair.