沉默CDKN1A基因对鼻咽癌放射抗拒性CNE-2R细胞放射敏感性的影响

目的：探讨CDKN1A基因的表达与鼻咽癌放射敏感性的关系。方法构建慢病毒表达载体LV-CDKN1A-RNAi并转染鼻咽癌放射抗拒性CNE-2R细胞,设转染LV-CDKN1A-RNAi慢病毒的CNE-2R细胞为实验组,转染阴性对照慢病毒的CNE-2R细胞为阴性对照组,未转染的CNE-2R细胞为空白对照组。用CCK-8法、细胞克隆形成实验及流式细胞术分别检测各组细胞增殖、放射敏感性及细胞周期的变化。结果成功构建了CDKN1A基因沉默的CNE-2R细胞,CCK-8法检测显示实验组CNE-2R细胞在照射6 Gy后生长受到抑制,且随时间延长其抑制作用更为明显。细胞克隆形成实验显示实验组CNE-2R细胞放射敏感性增强（放射增敏比为SER=1.24）。流式细胞术检测显示实验组与对照组细胞相比, G0/G1期和G2/M期细胞分布在X射线照射6 Gy前后明显改变（P约0.05）。结论 CDKN1A基因沉默能增强鼻咽癌放射抗拒性CNE-2R细胞的放射敏感性,CDKN1A基因的表达可能与鼻咽癌放射敏感性相关,有望成为鼻咽癌治疗的新靶点。     

放射诱导的六株细胞系细胞周期和细胞凋亡变化的观察

目的:探讨放射诱导的6株细胞系细胞周期和细胞凋亡的变化特点.方法:正常肝细胞系HL-7702,肝癌细胞系HepG2和SMMC-7721,肺小细胞癌HCI-H460,肺腺癌A549和宫颈癌细胞系Hela常规培养48 h后接受4 Gy X射线照射;收获受照前(0h)和受照后6、12、24、36和48h的细胞,采用流式细胞术(FCM)检测各细胞系细胞凋亡和细胞周期.结果:在4 Gy X线照射前,SMMC-7721和HCI-H460细胞凋亡率明显高于其他4株细胞系,两者差异有统计学意义(t=20.98,P<0.005);在照射后12 h,与照射前相比6株细胞系细胞凋亡率均有显著增加(t=5.27,P<0.05),SMMC-7721、HCI-H460和A549同时伴有S期和G2～M期细胞比率的降低;在照射后36h HCI-H460出现第2个细胞凋亡峰,伴有极低比例的S期和G2～M期细胞;HepG2在照射后12h、HL-7702和Hela在照射后24h均有明显的G2/M期阻滞.结论:4 Gy X线诱导的细胞凋亡主要发生在射线照射后12～36h,6株细胞系可能均发生了"有丝分裂前凋亡";每株细胞还呈现了不同的细胞凋亡和细胞周期变化特点,具有组织细胞特异性.     

60Co分割照射对鼻咽癌细胞生长增殖影响的观察

目的:观察不同剂量射线分割照射对鼻咽癌细胞株CNE-2细胞生长和细胞周期素(Cyclin)D1及增殖细胞核抗原(PC-NA)的影响.方法:0、2.0、4.0、6.0 Gy60Coγ射线分割照射CNE-2细胞1次/d,共5 d.四唑盐(MTT)比色法测定细胞抑制率;流式细胞术检测细胞周期.PCR检测PCNA和CyclinD1表达水平.结果:2.0、4.0、6.0 Gy60Coγ射线分割照射5次/5 d后各组细胞生长抑制率分别为-17.05%、4.01%和1 5.52%;各照射组G1期细胞数量减少,S+G2/M期细胞数比例增多,X2=16.53,P<0.001.0、2.0、4.0、6.0 Gy60Coγ射线分割照射CNE-2细胞5次/5 d,Cyclin D1表达水平随照射分割剂量增加而下降直至不表达,P值分别为0.035、0.022和0.000.2.0 Gy照射CNE-2细胞5次/5 d,细胞生长增殖未受到抑制,PCNA表达无下调,P=0.471;4.0和6.0 Gy照射CN E-2细胞5次/5 d,细胞生长增殖受到抑制,PCNA表达下调,P值分别为0.028和0.016.结论:4.0、6.0 Gy60Coγ射线分割照射5次/5 d,细胞生长增殖受到抑制;2.0 Gy照射5次/5 d细胞生长增殖未受抑制;2.0、4.0、6.0 Gy60Coγ射线分割照射5次/5 d后,Cyclin D1和PCNA表达受抑制.     

^60Co分割照射对鼻咽癌细胞生长增殖影响的观察

目的：观察不同剂量60Coγ射线分割照射对鼻咽癌细胞株CNE-2细胞生长和细胞周期素（Cyclin）D1及增殖细胞核抗原（PC-NA）的影响。方法：0、2.0、4.0、6.0Gy60Coγ射线分割照射CNE-2细胞1次/d,共5d。四唑盐（MTT）比色法测定细胞抑制率;流式细胞术检测细胞周期。PCR检测PCNA和Cyclin D1表达水平。结果：2.0、4.0、6.0Gy60Coγ射线分割照射5次/5d后各组细胞生长抑制率分别为-17.05%、4.01%和15.52%;各照射组G1期细胞数量减少,S＋G2/M期细胞数比例增多,χ2=16.53,P〈0.001。0、2.0、4.0、6.0Gy60Coγ射线分割照射CNE-2细胞5次/5d,CyclinD1表达水平随照射分割剂量增加而下降直至不表达,P值分别为0.035、0.022和0.000。2.0Gy照射CNE-2细胞5次/5d,细胞生长增殖未受到抑制,PCNA表达无下调,P=0.471;4.0和6.0Gy照射CNE-2细胞5次/5d,细胞生长增殖受到抑制,PCNA表达下调,P值分别为0.028和0.016。结论：4.0、6.0Gy60Coγ射线分割照射5次/5d,细胞生长增殖受到抑制;2.0Gy照射5次/5d细胞生长增殖未受抑制;2.0、4.0、6.0Gy60Coγ射线分割照射5次/5d后,CyclinD1和PCNA表达受抑制。     

低剂量X射线照射对K562细胞周期及凋亡影响的实验研究

目的 低剂量照射(low-dose radiation,LDR)可调节细胞信号传导,通过各种基因和蛋白的表达改变其分子生物学效应,由此产生了多样复杂且不同于大剂量照射的生物效应.用LDR及LDR联合大剂量照射(high-dose radiation,HDR)人红白血病细胞系K562(CML),探讨LDR对K562细胞周期进程及凋亡的影响.方法 按照K562细胞照射剂量不同分为4组,对照组(0 Gy)、LDR组(0.08、0.50 Gy)、HDR组(6 Gy)及LDR联合HDR组(0.08/6 Gy、0.50/6 Gy,LDR/HDR组).采用6 MVX射线照射,LDR与HDR间隔8 h;照射后按不同时间点收集细胞,流式细胞仪检测细胞周期变化与凋亡并分析细胞DNA倍体变化.结果 LDR后6h各组S期比例增加,对照组与LDR各组分别为55.38±2.22 vs 71.91±7.30、73.13±4.09(Z=-2.428,P=0.022;Z=-3.987,P＜0.001);12 h G2/M期细胞比例增加,出现G2/M阻滞,24 h达峰值,对照组与LDR各组分别为17.81±1.27 vs 26.61±7.82、29.02±2.76(Z=-3.684,P＜0.001;Z=-2.928,P＜0.001);48 h各组G0/G1细胞增多,72 h达峰值,对照组与LDR各组分别为27.07±1.19 vs 52.32±2.42、44.06±1.90(Z=-2.351,P=0.020;Z=-2.172,P=0.032).LDR/HDR后6hS期细胞比例增多,且G2/M期比例增加,即G2/M期阻滞,12 h达峰值并持续至24 h,12 h HDR组与LDR/HDR各组G2/M期分别为26.98±2.15 vs 56.27±1.57、69.31±2.51(Z=-2.564,P=0.021;Z=-7.759,P＜0.001).LDR后48 h凋亡率增加,96 h达峰值,对照组与LDR各组分别为1.82±0.12 vs3.21±0.20、6.28±0.30(Z=-3.959,P=0.003;Z=-9.705,P＜0.001);LDR/HDR照射后24 h凋亡率增加,120 h达峰值,HDR组与LDR/HDR各组分别为14.21±0.61 vs 17.38±0.92、27.91±1.07(Z=-2.986,P=0.027;Z=-6.973,P＜0.001).LDR后6h各组四倍体及八倍体细胞增加,且随照射剂量增大而增多,对照组与LDR各组八倍体细胞分别为2.41±0.15 vs 4.84±0.46、7.83±0.59,差异有统计学意义,H值分别为5.956和7.200,P值分别为0.025和0.001;LDR/HDR后6h各组四倍体及八倍体细胞增加,亦随LDR剂量增大而增多,HDR组与LDR/HDR各组八倍体细胞分别为6.49±1.05 vs 10.80±1.23、13.77±0.79,差异有统计学意义,H值分别为5.600和7.200,P值分别为0.05和0.001.结论 LDR能诱导K562凋亡,并能增强随后HDR对K562凋亡作用;LDR能诱导S/M期解偶联及G2/M期阻滞等细胞周期进程改变;LDR诱导K562凋亡可能与S/M期解偶联及G2/M期阻滞等改变细胞周期进程有关.     

6MV-X射线对肝癌细胞凋亡及周期分布的影响

目的:研究人肝癌细胞接受不同剂量或时间的6MV-X线照射后,对细胞凋亡和周期进程的影响。方法:用流式细胞术检测6MV-X线照射4Gy后不同时间以及不同剂量6MV-X线照射后24h HepG2和PLC/PRF/5两种细胞的细胞凋亡和周期分布状况。结果:4Gy 6MV-X线照射后24h,HepG2细胞凋亡率高于PLC/PRF/5细胞,分别为(7.61±0.77)%和(5.63±0.87)%(P<0.05),且随时间和剂量增加,差异越显著;HepG2细胞受4Gy照射后12h,G0/G1期比例下降,为(40.56±1.59)%,后逐渐上升,G2/M期比例增高,为(13.28±1.02)%,随后逐渐下降;2-6Gy照射24h后,G0/G1期比例逐渐上升,G2/M期比例逐渐下降;PLC/PRF/5细胞G2/M期比例在照射后24h阻滞最明显(22.11±1.48)%,后逐渐降低,随照射剂量增加而增大。结论:2-6Gy 6MV-X线照射可诱导HepG2与PLC/PRF/5细胞凋亡,并改变周期进程,HepG2细胞发生G1和G2期阻滞,PLC/PRF/5发生G2期阻滞。     

健择对人鼻咽癌细胞系放射增敏机制的实验研究

目的：观察单纯放射及放射联合健择（gemcitabine）对人鼻咽癌细胞系（CNE-2）细胞周期改变的影响，探讨健择对CNE-2细胞的放射增敏机制。方法：用流式细胞仪技术分析^60Coγ射线单纯照射和照射联合健择对CNE-2细胞周期的影响。结果：单纯照射导致CNE-2细胞G1期阻滞，照射剂量〈6Gy时与照射剂量呈正相关，〉6Gy时G1期阻滞基本稳定在一定水平。5mg／L、10mg／L、15mg／L健择与CNE-2细胞共育24h后照射2Gy，以S期阻滞为主；随着单次照射剂量的提高，以G1阻滞明显。CNE-2细胞与健择15mg／L共育12h后照射2Gy，开始时以G1阻滞为主，但24h后以S期阻滞明显。且至少维持36h以上。结论：健择对CNE-2细胞有中度放射增敏作用；其机制可能与健择引起S期或G1期阻滞有关。     

shRNA转染对食管癌细胞TE13中CHK1和CHK2表达以及照射后G2/M期阻滞的影响

背景与目的:肿瘤细胞照射后常表现为细胞周期的变化,本研究采用细胞周期监测点激酶CHK1和CHK2基因的短发卡状RNA(shRNA)转染食管癌细胞,观察对其蛋白表达以及60Co γ射线照射后细胞周期的影响.方法:设计合成并构建质粒连接的CHK1和CHK2 shRNA,分别提取质粒DNA并采用脂质体转染TE13细胞并传代;采用Western blot、RT-PCR和流式细胞仪分别检测CHK1和CHK2蛋白和muRNA表达、以及5 Gy 60Co γ射线照射后细胞周期变化,克隆形成实验检测5 Gy γ射线照射后细胞存活率.结果:采用CHK1和CHK2 shRNA转染TE13细胞后,其mRNA和蛋白表达均明显降低.单纯5 Gy γ射线照射后24 h,TE13细胞G2/M期比例由未照射组的32.17%增至6147%;shRNA转染的TE13细胞在5 Gy γ射线照射后24 h,C2/M期比例由单纯照射组的61 47%降至28.13%(CHK1)和42.80%(CHK2),P＜0.05.5 Gy γ射线、CHK1 shRNA力5 Gy γ射线、以及CHK2 shRNA力5 Gy γ射线照射后,细胞存活率分别为27.0%、13.0%和21.0%.shRNA转染的子一代TE13细胞在转染后120 h,对CHK1和CHK2蛋白表达的抑制作用已基本消失:转染120 h后以5 Gy γ射线照射24 h,CHK1或CHK2 shRNA转染组G2/M期比例高于单纯照射组,P＜0.05;至转染后144 h和5 Gy γ射线照射后48 h,转染组与单纯照射组比较G2/M期比例无明显差别,P＞0.05.结论:采用质粒连接的人CHK1和CHK2 shRNA转染TE13细胞后,可以明显抑制其mRNA和蛋白表达并消除照射后G2/M期阻滞,增加放射敏感性;提示TE13细胞γ射线照射后G2/M期检测点可能受CHK1和CHK2激酶双重调节,但以CHK1为主.     

塞来昔布对人鼻咽癌CNE-2细胞生长抑制及放射增敏研究

目的 研究塞来昔布对人鼻咽癌细胞系CNE-2细胞生长影响及有无放射增敏作用.方法 (1)细胞生长抑制研究:用MTT法检测细胞生长抑制,流式细胞术检测细胞周期分布及凋亡,透射电子显微镜观察细胞凋亡形态,SP法检测细胞COX-2表达.(2)放射增敏研究:随机设置照射对照、药物对照、单纯照射、药物+照射组,其中成克隆实验单次照射2、4、6、8、10 Gy,细胞周期分布和凋亡实验单次照射6 Gy.结果 塞来昔布显著抑制CNE-2细胞生长并呈浓度和时间依赖性,IC50为80 μmol/L.细胞周期分布显示G0+G1期细胞显著升高(47.03±2.76:56.17±1.95,t=4.68,P=0.010),而S、G2+M期细胞明显下降(33.07±1.86:24.87±1.76,t=5.54,P=0.010;19.30±0.53:17.73±0.83.t=2.75,P=0.050)并呈浓度依赖性.凋亡率显著增高(1.57±0.47:10.47±0.31,t=27.39,P=0.000)并呈浓度依赖性.电镜观察到细胞皱缩、核质浓缩、核碎裂等凋亡形态学改变.SP法检测塞来昔布显著下调CNE-2中COX-2表达[17.48±0.34、12.82±0.51(t=13.20,P=0.00)].塞来昔布的放射增敏比(D0值比为1.74:1.52)为1.15.4个组别细胞周期分布结果 显示单纯照射、药物+照射组的G2+M期细胞明显高于照射对照、药物对照组(68.00±1.65、54.27±5.74、17.60±0.80、14.86±1.23,t=47.70,P=0.000;t=11.63,P=0.000),且单纯照射与药物+照射组间也不同(t=3.99,P=0.020);单纯照射、药物+照射组的细胞凋亡率也明显高于照射对照、药物对照组(4.83±0.97、9.50±1.35、1.33±0.36、2.28±0.42,t=4.67,P=0.01;t=8.81,P=0.000),且单纯照射与药物+照射组也不同(t=4.85,P=0.010).结论 塞来昔布能抑制人鼻咽癌CNE-2细胞生长和诱导凋亡,其机制可能涉及COX-2依赖途径.塞来昔布还能增强CNE-2细胞放射敏感性,可能机制与抑制放射后亚致死损伤修复、直接抑制细胞增殖和增强细胞对放射诱导凋亡率有关.     

电离辐射对食管癌细胞周期及MDC1、53BP1蛋白表达的影响

目的 观察60Co γ射线照射后食管癌细胞周期、细胞凋亡及其相关蛋白表达的变化,为食管癌放射治疗、靶向治疗提供理论依据。方法 食管癌细胞株TE 13进行不同剂量（0、1、2、5、10、15Gy）照射后,应用流式细胞术分别检测照射后1、2、12、24和48h细胞周期和凋亡指数的变化;同时采用Western blot方法检测MDC1和53BP1蛋白表达情况。结果 TE 13细胞照射后12、24、48h,TE 13细胞的G0/G1期、G2/M期和S期的变化呈现明显剂量依赖性,1Gy和2Gy照射后12h,细胞G2/M期阻滞开始出现;5、10、15Gy照射后24h,细胞G2/M期阻滞最为明显,与对照组（0Gy组）相比,差异具有统计学意义（P<0.05）;15Gy照射后12h、24、48h,TE 13细胞的凋亡增加非常显著（P<0.01）;不同剂量照射后1、2、24h,TE 13细胞MDC1和53BP1蛋白表达未见明显变化（P>0.05）。结论 TE 13细胞经不同剂量放射线照射后,细胞周期出现明显的G2/M期阻滞,细胞凋亡指数明显增加,但对MDC1和53BP1蛋白表达未见明显影响。     

Biomarkers in clear cell renal cell carcinoma

The treatment of advanced renal cell carcinoma (RCC) has evolved significantly following the identification of the von Hippel–Lindau (VHL) gene and the function of its protein, and subsequent development of antiangiogenic therapies. A series of clinical trials resulted in the approval of three new agents with significant activity in this disease. Additional studies are now underway to identify subsets of patients most likely to benefit. This article reviews the current therapy for advanced RCC and the development of biomarkers in RCC. This requires the identification of disease characteristics at a clinical, genetic and molecular level associated with response and/or surrogate measures of clinical benefit. Currently, a variety of prognostic factors (lactate dehydrogenase, performance status, disease-free interval, hemoglobin and calcium levels) are utilized to predict the survival of RCC patients. The use of validated biomarkers in either serum/plasma, urine or tissue could enhance this process, as well as define at the molecular and genetic levels, factors associated with response to therapy and/or the development of resistance. Examples include plasma VEGF levels, VHL gene mutation status and carbonic anhydrase IX levels in tumor tissue, among others. Validation of such biomarkers is crucial in order for them to be clinically useful.     

Biomarkers in clear cell renal cell carcinoma

The treatment of advanced renal cell carcinoma (RCC) has evolved significantly following the identification of the von Hippel-Lindau (VHL) gene and the function of its protein, and subsequent development of antiangiogenic therapies. A series of clinical trials resulted in the approval of three new agents with significant activity in this disease. Additional studies are now underway to identify subsets of patients most likely to benefit. This article reviews the current therapy for advanced RCC and the development of biomarkers in RCC. This requires the identification of disease characteristics at a clinical, genetic and molecular level associated with response and/or surrogate measures of clinical benefit. Currently, a variety of prognostic factors (lactate dehydrogenase, performance status, disease-free interval, hemoglobin and calcium levels) are utilized to predict the survival of RCC patients. The use of validated biomarkers in either serum/plasma, urine or tissue could enhance this process, as well as define at the molecular and genetic levels, factors associated with response to therapy and/or the development of resistance. Examples include plasma VEGF levels, VHL gene mutation status and carbonic anhydrase IX levels in tumor tissue, among others. Validation of such biomarkers is crucial in order for them to be clinically useful.     

Non-small and small cell lung carcinoma cell lines exhibit cell type-specific sensitivity to edelfosine-induced cell death and different cell line-specific responses to edelfosine treatment

The unique signal transduction pathways that distinguish non-small cell lung carcinoma (NSCLC) from small cell lung carcinoma (SCLC) are poorly understood. We investigated the ability of edelfosine, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) to inhibit cell viability among four NSCLC cell lines and four SCLC cell lines. The differential sensitivity of cells to edelfosine's cytostatic and cytotoxic effects has been attributed to edelfosine-induced changes in the activities of many enzymes, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), p38 kinase, and poly(ADP-ribose) polymerase (PARP). To investigate the role of these enzymes in edelfosine-induced cytotoxicity, we correlated edelfosine-induced changes in enzyme activity and cell viability among the different NSCLC and SCLC cell lines. We found that NSCLC cells are much more susceptible to the cytotoxic effects of this drug than are SCLC cells. Three out of the four edelfosine-sensitive NSCLC cell lines (NCI-H157, NCI-H520, NCI-H522) exhibit G2/M arrest, significant apoptosis and some degree of JNK activation in response to drug treatment. In contrast, none of the SCLC cell lines exhibit edelfosine-induced G2/M arrest or significant apoptosis. A comparison of the edelfosine-induced effects among the sensitive and resistant lung cancer lines indicates that there is little correlation between edelfosine-induced cytotoxicity and altered activities of JNK, ERK, p38, or cleavage of PARP. These results demonstrate that edelfosine-induced changes in JNK, ERK, p38, or PARP are not good predictors of cell susceptibility to edelfosine-induced cytotoxicity. Thus, edelfosine-induced inactivation of PLC may disrupt signaling cascades downstream of PLC that are unique to individual cellular environments. These findings also identify edelfosine as one of the few potential chemotherapeutic agents that has a greater cytotoxic effect against NSCLC cells than SCLC cells.     

桥接整合因子1通过AKT-mTOR通路诱导非小细胞肺癌H1975细胞周期阻滞

目的：研究桥接整合因子1（bridging intergrator 1,Bin1）基因过表达后对非小细胞肺癌细胞株H1975细胞周期的影响及其作用机制。方法：构建携带Bin1基因的CMV-MCS-GFP-SV40-Neomycin-Bin1质粒,并转染H1975细胞（Bin1+组）,另设置空白质粒转染组（Bin1-组）及空白对照组（Ctrl组）,利用RT-PCR和Western blotting分别检测3组细胞中Bin1在mRNA和蛋白质水平的表达情况。流式细胞术检测不同处理组H1975细胞周期的变化,Western boltting分别检测各组中AKT、mTOR磷酸化水平及细胞周期相关蛋白（周期蛋白D1、CDK4、Rb）的表达情况。结果：与Bin1-组、Ctrl组比较,Bin1+组H1975细胞中Bin1在mRNA、蛋白水平表达明显上调（均P<0.05）; H1975细胞阻滞在G1期\[（60.53±1.89）% vs（46.14±1.56）%、（47.33±2.07）%,均P<0.05\]; Bin1+组H1975细胞内p-AKT、p-mTOR表达下调（均P<0.05）,AKT、mTOR表达变化无统计学差异（P>0.05）;周期蛋白D1、CDK4的表达量均明显下调(P<0.05),Rb表达量明显增加（P<0.05）。结论：Bin1基因在H1975细胞株过表达后明显诱导细胞周期阻滞,其机制可能是通过抑制AKT-mTOR通路及其细胞周期相关蛋白实现的。     

A human squamous cell carcinoma cell line.

An epithelial cell line COLO 16 has been established from a human squamous carcinoma, characterized and maintained for over two years. The cells produce a parathyroid-like hormone and carcinoembryonic antigen. The line is definitely not a "HeLa contaminant." The cell line is available to other investigators.