三氧化二砷对人宫颈癌Hela细胞系的放射增敏作用

目的 探讨三氧化二砷（As2O3）对人宫颈癌Hela细胞系的放射增敏作用。方法 MTT法确定As2O3对Hela细胞的半数致死浓度（LD30），采用集落形成法观察20％该浓度的As2O3对Hela细胞的放射增敏作用。结果（1）细胞生长抑制随As2O3浓度的增加而增强；（2）As2O3＋照射组的细胞存活率低于单纯照射组，耽值小于单纯照射组（1．58Gy、2．11Gy），Dq值也小于单纯照射组（0．27Gv、0．64Gv），存活曲线肩区（Dq）变小，放射增敏比（SER）1．34。结论 As2O3对宫颈癌细胞有明显的放射增敏作用，其作用机理有待进一步研究。     

MLN2238对宫颈癌HeLa细胞生长抑制和放射增敏及其机制探讨

目的：探讨蛋白酶体抑制剂MLN2238对宫颈癌HeLa细胞生长抑制和放射增敏作用及其机制。方法：采用CCK-8法检测MLN2238对HeLa细胞生长的抑制效应,克隆形成实验检测MLN2238辐射敏感性的变化,流式细胞术检测细胞凋亡率的变化,线粒体荧光探针显像检测线粒体活性变化,蛋白质印迹法测定细胞中蛋白表达情况。结果：MLN2238对HeLa细胞有明显的抑制作用,0．05μmol／L抑制率为3．95％,0．1μmol／L为14．89％,0．5μmol／L为29．37％,1μmol／L为38．95％,5μmol／L为54．44％,10μmol／L为70．52％,30／μmol／L为81．76％,其效应呈剂量依赖性,F=1172．02,P〈0．001。0．1μmol／L的MLN2238对HeLa细胞有放射增敏作用,其放射增敏比（sensitizingen—hancementratio,SER）为1．40。MLN2238联合X射线对HeLa细胞存在交互作用,对照组细胞凋亡分数为（2．64±0．07）％,单药组为（2．76±0．38）％,单照4Gy组为（9．50±0．14）％,单照8Gy组为（21．04±0．04）％,联合4Gy组为（11．12土0．19）％,联合8Gy组为（26．18±0．35）％,细胞凋亡率逐渐增加,差异有统计学意义,F兰20．23,P=0．0022。0．1μmol／L的MLN2238可影响细胞线粒体活性,导致线粒体膜损伤加重,增加细胞凋亡。MLN2238与X射线联合作用能增加凋亡相关蛋白Bax／Bcl-2的表达。结论：MLN2238对宫颈癌HeLa细胞有生长抑制和放射增敏作用,MLN2238对宫颈癌HeLa细胞可能通过线粒体途径介导的凋亡而增强其辐射敏感性。     

青蒿素对人宫颈癌细胞放射增敏作用及其机制的研究

目的:研究青蒿素(artemisinin)对人宫颈癌HeLa细胞放射敏感性增强的作用机制.方法:MTT法检测青蒿素对正常人皮肤成纤维细胞GM0639、宫颈鳞癌SiHa细胞和宫颈腺癌HeLa细胞的生长抑制作用.克隆形成实验检测青蒿素对GM0639、SiHa和HeLa细胞的放射增敏作用.Western 印迹法检测CyclinB1和Wee1蛋白在HeLa细胞中表达的变化.结果:MTT法检测结果显示,不同浓度的青嵩素(27.67～442.75 nmol/L)对GM0639、SiHa和HeLa细胞作用24和48 h时均有细胞生长抑制作用;而青蒿素(110.69 nmol/mL)分别作用GM0639、SiHa和HeLa细胞6 h后,对3株细胞的生长抑制率分别为0.93±0.25、0.84±0.10和0.77±0.03,对照组(未加药组)3株细胞的生长抑制率分别为0.92±0.13、0.83±0.09和0.75±0.21,加药组和对照组之间差异无统计学意义(P>0.05).青蒿素对GM0639和SiHa细胞无放射增敏作用,对HeLa细胞有放射增敏作用,SER=1.12;青嵩素和照射联合作用HeLa细胞后,细胞中CyclinB1蛋白表达比照射组增加,Wee1蛋白表达比照射组减少.结论:青蒿素能增加HeLa细胞的放射敏感性,其作用机制可能与上调CylinB1及下调Wee1的蛋白表达有关.     

宫颈癌术前放化疗的疗效分析

[目的]探讨术前应用顺铂放化疗治疗Ⅱa~Ⅱb1期巨块型宫颈癌的临床疗效。[方法]对161例Ⅱa~Ⅱb1期宫颈癌的治疗进行回顾性分析,其中放化疗+手术(放化疗组)48例,放疗+手术组(放疗组)55例,单纯手术组(手术组)58例。对3组的近期疗效、组织学改变及预后情况进行比较。[结果]放化疗组近期疗效(93.75%)明显高于放疗组(80%),具有显著性差异(P<0.05)。放化疗组深肌层癌细胞损伤率为70%,明显高于放疗组(43.6%)及手术组(24.1%),具有显著性差异(P<0.01)。局部复发放化疗组为4.16%,放疗组为13.72%,手术组为20.68%,放化疗组与其他两组比较均具有显著性差异(P<0.05)。5年生存率放化疗组83.4%,放疗组74.86%,手术组55.82%,放化疗组与手术组比较具有显著性差异(P<0.05)。[结论]术前放化疗可提高巨块型宫颈癌疗效,增加手术机会,提高生存质量。     

甘氨双唑钠对宫颈癌放化疗增敏作用的研究

观察注射用甘氨双唑钠（CMNa）对中晚期宫颈癌的放化疗增敏作用及不良反应。方法：采用随机分组的方法,将60例经病理学确诊的中晚期宫颈癌患者分为增敏组（A组,放化疗加甘氨双唑钠）和对照组（B组,放化疗）,每组30例。两组放化疗方式相同。A组在放化疗同时,给予CMNa 800 mg/m2,给药后1h内接受放疗,每周3次,6~7周。B组行同步放化疗。结果：A组治疗结束CR率为83.3%（25/30）,明显高于B组的60%（18/30）,两组比较差异有显著性（P<0.05）,两组总有效率在放疗结束时均为100%,A组放疗结束3个月后CR率为86.7%（26/30）,总有效率为93.3%（28/30）,B组放疗结束后3个月CR率为80%（24/30）,总有效率为96.7%（29/30）,两组比较均无显著性差异（P>0.05）。两组患者发生不良反应的严重程度无明显差异。结论：甘氨双唑钠对提高中晚期宫颈癌同期放化疗临床近期完全缓解率有一定意义,并且未增加不良反应,但其远期疗效有待进一步扩大样本量并随访观察。     

动脉灌注顺铂放射增敏治疗晚期宫颈癌近期疗效观察

 本文对26例晚期宫颈癌放射治疗疗效进行近期临床观察。所有病例均经病理学证实。26例病人分为两组:放射增敏组和对照组,其中11例病人行动脉灌注顺铂放射增敏,15例行单纯放射治疗。对增敏组11例患者共行24次动脉灌注顺铂加栓塞治疗。所有病人皆接受根治性放疗。结果显示:放射增敏组的放疗半量、全量疗效皆优于对照组(P<0.05),放疗达PR、CR时的顺铂增敏比分别为134和118,同时也可看到较轻的副作用如恶心、呕吐等。     

顺铂与奈达铂同步放化疗治疗中晚期宫颈癌的临床对比研究

目的:探讨顺铂同步放化疗与奈达铂同步放化疗治疗中晚期宫颈癌的疗效和不良反应。方法:64例中晚期宫颈癌患者随机分为顺铂同步放化疗组(32例)和奈达铂同步放化疗组(32例),观察2组的近期和远期疗效以及不良反应。结果:顺铂同步放化疗组与奈达铂同步放化疗组的近期有效率分别为96.9%和100.0%(P>0.05)。顺铂同步放化疗组的3年生存率(71.88%)、局部复发率(12.50%)和远处转移率(6.25%)与奈达铂同步放化疗组(分别为70.97%、12.90%和6.45%)相比,差异均无统计学意义(P>0.05)。顺铂同步放化疗组的胃肠毒性和肾毒性发生率高于奈达铂同步放化疗组(P<0.05),而奈达铂同步放化疗组的血小板减少发生率高于顺铂同步放化疗组(P<0.05)。结论:顺铂单药同步放化疗与奈达铂单药同步放化疗均能明显提高中晚期宫颈癌患者的近期和远期疗效,并降低局部复发率和远处转移率。不良反应可以耐受。     

DNA-PKcs、Ku80及ATM备选宫颈癌放疗增敏靶点的体外研究

背景与目的:DNA双链断裂(DNA double strand break,DSB)是细胞受辐射后最致命的损伤,而DNA依赖蛋白激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)、Ku80和ATM(ataxia-telangiectasia mutated)为DSB的主要修复蛋白.宫颈癌是以放疗为主要治疗手段的肿瘤.但其肿瘤细胞对放射线的敏感性不同.本实验拟研究3种DSB修复蛋白的表达与宫颈癌细胞放射敏感性的关系.并探讨DSB修复蛋白成为宫颈癌放疗增敏靶点的可能性.方法:免疫组化法检测41例宫颈癌患者组织中DNA-PKcs、Ku80和ATM蛋白的表达情况;Western blot检测8株肿瘤细胞(包括4株宫颈癌细胞)中3种蛋白的表达,克隆形成实验检测SF2 (suivival fraction at 2 Gv)、α值,分析蛋白表达水平和SF2、α值的关系;利用靶向抑制DNA-PKcs的shRNA表达质粒和小分子抑制剂LY294002,分别抑制HeLa细胞DNA-PKcs蛋白表达和活性后,克隆形成实验和流式细胞仪检测HeLa细胞受6 MVX线照射后的SF2、α值和凋亡率变化.结果:在41例宫颈癌组织中,Ku80、DNA-PKcs和ATM的阳性率分别为70.73%、68.29%和19.51%:8株肿瘤细胞中Ku80、DNA.PKcs和ATM蛋白的相对表达量与各细胞SF2、α值各不相同,作Pearson线性相关分析后得出DNA-PKcs的表达水平与SF2之间有明显的正相关关系(r=0.72,P=0.04);靶向抑制DNA-PKcs的shRNA可以促进HeLa细胞的放射敏感性,其SF2值为0.37,显著低于对照HeLa细胞的0.53(P＜0.05);单独接受50 μmol/L LY294002作用1 h HeIa细胞的凋亡率未见明显增加,但先经LY294002处理再照射6 Gy的HeLa细胞在48 h和72 h的凋亡率比单独照射6 Gy的HeLa细胞凋亡率显著增加(48 h点:t=3.25,P=0.03;72h点:t=3.01,P=0.04).结论:DNA-PKcs在宫颈癌组织中表达较高,且其表达水平可以预示肿瘤细胞的放射敏感性:抑制DNA-PKcs的表达或活性可以促进HeLa细胞的放射敏感性.     

曲古霉素A对宫颈癌HeLa细胞的毒性及放射增敏作用

目的探讨曲古霉素A（TSA）对宫颈癌HeLa细胞的毒性及放射增敏作用。方法MTT法检测TSA处理宫颈癌HeLa细胞12、24、48、72 h的细胞毒性及TSA作用24 h的10%细胞抑制浓度（10％ inhibition concentration,IC10）和半数抑制率IC50对HeLa细胞不同分割放疗方式的增敏效果;克隆形成法分析IC10 的TSA对HeLa细胞放射增敏作用的影响,单击多靶模型拟合细胞存活曲线,计算放射增敏比（sensitive enhencement ratio,SER）。结果0.05~0.8 μmol/L TSA对HeLa细胞具有增殖抑制作用,且呈一定的时间－剂量依赖性,TSA作用24 h的IC10和IC50分别为0.0312和0.5865。0.6 μmol/L以上浓度及药物作用时间超过48 h的TSA对HeLa细胞的增殖抑制作用并无明显增加。IC10曲古霉素A的SER为1.6858,IC10的TSA对每次2.0 Gy、1.0 Gy（×2）、0.5 Gy（×4）分割放射组的SER分别为1.4496、1.6410、1.9554。结论TSA对宫颈癌HeLa细胞具有良好的细胞毒性及放射增敏作用,且对低剂量多次分割放疗的增敏作用更强。     

紫杉醇脂质体联合铂类同步放化疗治疗宫颈癌的随机对照研究

目的 评价紫杉醇脂质体联合铂类同步放化疗治疗宫颈癌的疗效和安全性.方法 对162例 Ⅱa～Ⅳ期宫颈癌患者进行随机对照研究.紫杉醇联合铂类组(对照组)71例,紫杉醇135 mg/m2,第1天;顺铂80 mg/m2或卡铂[曲线下面积(AUC)取值4～6],第2天.紫杉醇脂质体联合铂类组(试验组)91例,紫杉醇脂质体135 mg/m2,第1天;顺铂80 mg/m2或卡铂(AUC取值4～6),第2天.两组均3周为1个疗程,共治疗2～3个疗程.两组均同期予以根治性放疗.同步放化疗结束后6个月,根据肿瘤退缩情况判定疗效,并持续进行随访.结果 试验组和对照组的有效率分别为89.0%和90.1%,差异无统计学意义(P＞0.05).试验组和对照组的1年累积生存率分别为89.2%和91.4%,差异亦无统计学意义(P＞0.05).试验组的胃肠道反应、骨髓抑制、肌肉和关节酸痛、皮疹发生率分别为49.6%、78.0%、26.3%和4.4%,显著低于对照组(63.3%、81.6%、53.5%和12.6%,均P＜0.05),而脱发、肝功能损害、外周神经炎等方面差异并不明显(P＞0.05).结论 紫杉醇脂质体联合铂类同步放化疗治疗Ⅱa～Ⅳ期宫颈癌安全、有效,远期疗效有待进一步观察.

Abstract：

Objective To compare the efficacy, side effects and influence of two chemotherapy regimens, paclitaxel liposome combined with platinum and paclitaxel combined with platinum, on the survival rate in patients with cervical carcinoma receiving concurrent chemoradiotherapy. Methods One hundred and sixty two cases with primary cervical carcinoma diagnosed and treated in the Jiangxi Maternal and Children Hospital between January 2008 and November 2009 were enrolled in this randomized controlled trial. Seventy one cases were included in the paclitaxel group and 91 in the paclitaxel liposome group. The chemotherapy doses were as followings: paclitaxel liposome and paclitaxel 135 mg/m2;cisplatin 80 mg/m2 or carboplatin AUC 4-6, repeated every 21 days for two or three times. Radical radiotherapy was given to both groups at the same time. The efficacy was evaluated by the tumor regression and the patients were followed-up for six months. Results The overall response rates of paclitaxel group and paclitaxel liposome group were 90.1% and 89.0%, respectively (P>0.05). The 1-year cumulative survival rate was 91.4% for the paclitaxel group and 89.2% for the paclitaxel liposom group (P>0.05). The incidence rate of adverse effects such as rash, gastrointestinal toxicity, bone marrow suppression and muscle/joint pain in the paclitaxel liposome group was significantly lower than that in the paclitaxel group (P<0.05), while there was no significant difference regarding the hair loss, liver damage, and peripheral neuritis (P>0.05). Conclusions Paclitaxel liposome plus platinum is a safe and effective therapeutic regimen for stage Ⅱa-Ⅳ cervical carcinoma. However, the long-term efficacy of this regimen should be further observed.     

组蛋白去乙酰化酶抑制剂联合紫杉醇抑制宫颈癌细胞增殖的体外实验

目的：研究组蛋白去乙酰化酶抑制剂SAHA联合紫杉醇对宫颈癌HeLa细胞增殖的抑制效果及其机制。方法：设置空白对照组、紫杉醇（10 nmol/L）、SAHA（10 μmol/L）、紫杉醇（10 nmol/L）+SAHA（10 μmol/L）联合组,采用四甲基噻唑蓝（MTT）法检测各组HeLa细胞的生长抑制率,计算紫杉醇对HeLa细胞的IC₅₀。RT-PCR法检测各组HeLa细胞中抑癌基因p27 mRNA的相对表达,Western blot检测HeLa细胞乙酰化组蛋白H4（Ac-H4）的表达。结果：紫杉醇、SAHA、紫杉醇+SAHA联合组处理HeLa细胞24 h的相对抑制率分别为25.93%±5.32%、46.38%±3.66%、54.27%±4.02%,联合组抑制率与紫杉醇组相比明显升高,差异具有统计学意义（P < 0.01）;48 h的抑制率分别为29.12%±3.09%、65.26%±3.03%、77.02%±3.86%,联合组抑制率最强,显著高于SAHA组和紫杉醇组（P < 0.01）。经SAHA预处理后紫杉醇对HeLa细胞的IC₅₀较单独紫杉醇组均显著下降（P < 0.05或P < 0.01）。紫杉醇组、SAHA组、紫杉醇+SAHA联合组细胞中p27 mRNA的相对表达量分别为5.845±0.548、0.978±0.117和10.601±0.673,乙酰化组蛋白H4（Ac-H4）的相对表达分别为0.878±0.068、1.148±0.018、1.282±0.033,联合组中表达量均显著高于SAHA组和紫杉醇组（P均 < 0.01）。结论：SAHA联合紫杉醇在体外能显著抑制宫颈癌细胞HeLa的增殖,增强组蛋白乙酰化水平,诱导抑癌基因的表达。     

Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis

In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize HPV-positive cervical cancer cell lines to apoptotic stimuli such as rhTRAIL (recombinant human TNF-related apoptosis inducing ligand). In a panel of cervical cancer cell lines, CaSki was highly, HeLa intermediate and SiHa not sensitive to rhTRAIL-induced apoptosis. MG132 strongly sensitized HeLa and SiHa to rhTRAIL-induced apoptosis in a caspase-dependent and time-dependent manner. MG132 massively induced TRAIL receptor DR4 and DR5 membrane expression in HeLa, whereas in SiHa only DR5 membrane expression was upregulated from almost undetectable to high levels. Antagonistic DR4 antibody partially inhibited apoptosis induction by rhTRAIL and MG132 in HeLa but had no effect on apoptosis in SiHa. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in elevated levels of active p53 as demonstrated by p53 small interfering RNA (siRNA) sensitive p21 upregulation. Although p53 siRNA partially inhibited MG132-induced DR5 upregulation in HeLa and SiHa, no effect on rhTRAIL-induced apoptosis was observed. MG132 plus rhTRAIL enhanced caspase 8 and caspase 3 activation and concomitant cleavage of X-linked inhibitor of apoptosis (XIAP), particularly in HeLa. In addition, caspase 9 activation was only observed in HeLa. Downregulation of XIAP using siRNA in combination with rhTRAIL induced high levels of apoptosis in HeLa, whereas MG132 had to be added to the combination of XIAP siRNA plus rhTRAIL to induce apoptosis in SiHa. In conclusion, proteasome inhibition sensitized HPV-positive cervical cancer cell lines to rhTRAIL independent of p53. Our results indicate that not only DR4 and DR5 upregulation but also XIAP inactivation contribute to rhTRAIL sensitization by MG132 in cervical cancer cell lines. Combining proteasome inhibitors with rhTRAIL may be therapeutically useful in cervical cancer treatment.     

Anti-proliferative and pro-apoptotic effects of 3,3'-diindolylmethane in human cervical cancer cells

The antitumor effects of Indo-3-carbinol (I3C) have been proven in many human carcinoma cells. However, the roles of 3,3-diindolylmethane (DIM), an important polymer converted from I3C under pH 5.0-7.0, on the growth and proliferation of cervical cancer HeLa and SiHa cells still remain unrevealed. In the present study, we investigated the potential anti-proliferative and pro-apoptotic effects of DIM on HeLa and SiHa cells. Cell proliferation was detected by Cell Counting kit-8 and apoptosis was analyzed by flow cytometry. In addition, morphological changes accompanying cell apoptosis were observed using an inverted microscope after Hoechst 33258 staining. In addition, expression changes of proteins involved in the MAPK and PI3K pathways were determined by western blotting. DIM treatment inhibited the proliferation and induced apoptosis of HeLa and SiHa cells significantly in a time- and dose-dependent manner. Moreover, SiHa cells were more sensitive to DIM treatment than HeLa cells (P<0.05). In addition, the expression of ERK, p38 and p-p38, which are involved in the MAPK pathway, was downregulated by DIM treatment. Another protein involved in the MAPK pathway, JNK, was upregulated. Furthermore, DIM treatment significantly suppressed the expression of Akt, p-Akt, PI3K p110α, PI3K p110β, PI3K class III, GSK3-β, p-PDK1 and p-c-Raf which are involved in the PI3K pathway. These results demonstrate that DIM exerts antitumor effects on HeLa and SiHa cells through its anti-proliferative and pro-apoptotic roles, especially for SiHa cells. The molecular mechanism for these effects may be related to its regulatory effects on MAPK and PI3K pathway and apoptosis proteins. DIM may be a preventive and therapeutic agent against cervical cancer.     

Growth inhibitory effects and radiosensitization induced by fatty aromatic acids on human cervical cancer cells.

Evidences have been reported that phenylacetic (PA) and phenylbutyric (PB) fatty aromatic acids can exert tumor growth inhibition in vitro and in vivo. Moreover, clinical trials also showed some activity for these drugs to modulate the expression of genes implicated in tumor growth, metastasis, immunogenicity, and to potentiate the efficacy of cytotoxic agents. The aim of the study was to examine the effects of PA and PB on the growth as well as sensitization to cisplatin and radiation in human cervical cancer cells. The effects of PA and PB on the proliferative activity and apoptosis induction in cervical tumor tissue was investigated. Both PA and PB exhibited a time- and dose-dependent antiproliferative activity in SW756 and ME180 cell lines: after 72-h treatment, the IC50 (concentration able to inhibit 50% of cell growth) of PB was 1.9 +/- 0.2 mM and 1.5 +/- 0.2 mM in SW756 and ME180 cells, respectively, while the IC50 of PA was 13.0 +/- 1.7 mM and 10.0 +/- 1.2 mM in SW756 and ME180 cells, respectively. In tumor tissue biopsies obtained from patients affected by squamous cervical cancer, both drugs resulted in a marked reduction of the percentage of bromodeoxyuridine-labeled cells compared with untreated samples [19.0 +/- 1.63% in untreated tissues with respect to 1.30 +/- 0.54% and 4.20 +/- 2.50% of stained cells after treatment with PA (30 mM) (P < 0.0001) and PB (5 mM) (P < 0.0001), respectively]. Moreover, analysis of the staining with M30 monoclonal antibody revealed that PA (30 mM) and PB (5 mM) were able to produce a marked increase in the number of stained apoptotic nuclei with respect to untreated samples. Finally, PB and PA were shown to enhance the sensitivity of SW756 to radiation and to exert an additive effect when combined with cisplatin. A significant reduction of the processed form of p21ras and rhoB proteins in the membrane fraction of cells exposed to PA and PB was observed. When farnesol, which is able to circumvent the enzymatic step inhibited by PA and PB, was added to the medium only a partial reversal of the growth inhibition and potentiation of sensitivity to radiation induced by PA and PB were found. In conclusion, the growth inhibitory properties of fatty aromatic acids suggest that these molecules could represent the prototype of a new class of compounds with some therapeutic potential in cervical cancer.     

黑种草子总皂苷对人宫颈癌SiHa细胞生物学行为的影响

目的：研究黑种草子总皂苷提取物（TSNG）对人宫颈癌SiHa细胞的增殖、迁移、凋亡和自噬等生物学行为的影响。方法：将SiHa细胞分为空白对照组和不同浓度（0.8、0.9、1.0、1.1、1.2、1.4、1.6、1.8 mg/mL）的TSNG作用组,以及10 μmol/L羟氯喹（HCQ）干预组;四甲基噻唑蓝（MTT）法检测各组细胞的生长抑制率;划痕实验检测细胞的相对迁移率;流式细胞术Annexin V-FITC/PI染色法检测细胞凋亡率;Hoechst染色观察细胞核凋亡现象;单丹磺酰戊二胺（MDC）染色观察细胞内自噬体数量;Western blot实验检测LC3-Ⅱ和p62蛋白表达。结果：与空白对照组相比,TSNG对细胞的生长有抑制作用且呈剂量效应关系（P<0.05或0.01）;0.9、1.0、1.1 mg/mL TSNG处理组细胞相对迁移率分别为（20.40±2.49）%、（15.14±0.39）%、（5.05±1.04）%,均低于空白对照组的（36.63±2.52）%（P<0.01）;1.0、1.2、1.4 mg/mL TSNG处理组细胞的凋亡率分别为（39.10±0.22）%,（68.37±0.58）%和（80.93±0.12）%,均高于空白对照组的（10.73±0.82）%（P<0.05或0.01）;与空白对照组比较,TSNG处理组细胞核表现出凋亡现象细胞数量增多,且细胞内自噬体数量增加,LC3-Ⅱ和p62蛋白表达上升（P<0.05或0.01）;相较于TSNG作用组,HCQ预处理组LC3-Ⅱ蛋白表达无显著变化（P>0.05）。结论：TSNG对SiHa细胞体外增殖、迁移表现出明显的抑制作用,并在诱导细胞凋亡的同时阻断细胞自噬流。     

Twist基因转染对人宫颈癌细胞生物学行为影响的观察

目的:构建人Twist基因真核表达载体,并建立稳定高表达Twist的人宫颈癌HCC94细胞系,研究转染前后HCC94细胞生物学行为。方法:用RT-PCR法扩增人低分化宫颈癌HCE1细胞中Twist开放阅读框架,将PCR产物克隆到pZsGreen-C1真核表达载体,构建重组质粒pZsGreen-C1/Twist并鉴定。采用脂质体法把重组载体转染入人高分化宫颈癌HCC94细胞,经G418筛选获得稳定高表达Twist的细胞克隆,并进行荧光,蛋白质印迹法和Realtime RT-PCR检测;MTT法和侵袭小室实验研究转染前后HCC94细胞的增殖、黏附和迁移能力。结果:成功构建pZsGreen-C1/Twist真核表达载体,筛选出稳定高表达人Twist的p-tw-1克隆,经荧光显微镜观察其转染效率>70%。Realtime RT-PCR扩增曲线和熔点曲线显示,Twist基因有效扩增,p-tw-1组、p-tw-v(转染空载体)组和HCC94(未转染)组mRNA相对表达量分别为4.14±2.26、2.03±1.10和1.90±0.78,p-tw-1组相对于其他两组水平明显上调,差异有统计学意义,F=10.24,P=0.001;而对照组之间差异无统计学意义,P=0.146。蛋白质印迹法检测结果显示,Twist基因稳定表达,p-tw-1组、p-tw-v组和HCC94组IOD与GAPDH比值分别为0.65、0.33和0.30。p-tw-1组细胞从第4天起生长趋势快于p-tw-v组和HCC94组,差异有统计学意义,F=24.113,P=0.000;而两对照组之间差异无统计学意义,P=0.229。P-tw-1、P-tw-v和HCC94组平均穿膜细胞数分别为(263.67±18.04)、(126.00±17.69)和(116.33±9.87)个,p-tw-1组细胞数明显增多,差异有统计学意义,F=83.093,P=0.000;p-tw-v与HCC94组之间差异无统计学意义,P=0.478。P-tw-1、P-tw-v和HCC94组黏附细胞平均A值分别为0.38±0.01、0.53±0.01和0.57±0.01,p-tw-1组A值明显降低,差异有统计学意义,F=65.086,P=0.000;p-tw-v组与HCC94组之间差异无统计学意义,P=0.244。结论:Twist基因高表达可增强宫颈癌细胞增殖和迁移能力,降低其黏附能力;Twist真核表达载体和稳定高表达Twist的HCC94细胞系构建为进一步研究Twist功能奠定了基础。     

Processing of clustered DNA damage in human breast cancer cells MCF-7 with partial DNA-PKcs deficiency

Complex DNA damage such as double strand breaks (DSBs) and non-DSB bistranded oxidative clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1-10bp on opposing DNA strands) are considered the hallmark of ionizing radiation. Clustered DNA lesions are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role during the processing of DSBs. To evaluate the role of DNA-PKcs in the processing of complex DNA damage in human MCF-7 breast cancer cells we used small interfering RNAs (siRNAs) to target the silencing of the gene Prkdc coding for DNA-PKcs. MCF-7 cells with knockdown DNA-PKcs expression showed a marked decrease in their efficiency to process DSBs and OCDL after exposure to radiotherapy-relevant gamma ray doses. For the detection and measurement of complex DSBs and OCDL, we used the gamma-H2AX assay and an adaptation of pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. An accumulation of all types of DNA damage was detected for the siRNA-treated MCF-7 cells compared to controls. These findings point to the important role of DNA-PKcs in the processing of complex DNA damage and its potential association with breast cancer development.