非小细胞肺癌凋亡相关基因bcl-2和bax表达及其与端粒酶的关系

目的 探讨凋亡相关基因bcl-2与bax及端粒酶在非小细胞肺癌中的表达及其意义。方法 分别检测50例非小细胞肺癌细胞中bcl-2与bax表达情况及端粒酶活性，并测定人端粒酶逆转录酶(hTERT)含量。结果 凋亡相关基因bcl-2与bax的表达与细胞分化程度相关，分化程度愈高，bcl-2表达率愈低，而bax表达率愈高。bax的表达与TNM分期及N分期均相关，分期愈低，bax表达愈高，而bcl-2的表达则与分期无关。两者与细胞类型，端粒酶的表达均无相关性。结论 bcl-2/bax与端粒酶分别在不同途径上控制肿瘤细胞的凋亡，bax的表达多发生在早期非小细胞肺癌，对肺癌的早期诊断可能有一定的帮助。     

下调PRR11表达抑制胃癌HGC-27细胞增殖和侵袭

目的研究富含脯氨酸蛋白11(Proline-rich protein 11,PRR11)表达对胃癌HGC-27细胞增殖和侵袭能力的影响,并初步探讨相关分子机制。方法利用载有PRR11 shRNA的慢病毒及空载体慢病毒转染胃癌HGC-27细胞系,建立PRR11蛋白低表达细胞系及阴性对照细胞系。Western blotting方法检测转染后HGC-27细胞中PRR11蛋白表达;采用CCK-8和Transwell小室分别研究两组细胞增殖与侵袭能力的差异。采用Western blotting检测胃癌细胞中cyclin D1和MMP-2蛋白的表达。结果 PRR11蛋白低表达HGC-27细胞较对照组HGC-27细胞的增殖和侵袭能力减弱,PRR11蛋白低表达细胞中cyclin D1和MMP-2蛋白表达较少。结论 PRR11低表达介导的胃癌细胞增殖和侵袭能力减弱,可能与cyclin D1和MMP-2表达减少密切相关。     

下调gankyrin通过调节β-catenin/cyclin D1信号通路抑制胃癌细胞增殖

背景:Gankyrin是一个含锚蛋白重复序列的原癌蛋白,其高表达参与了多种恶性肿瘤的发生、发展进程。目的:探讨下调gankyrin表达对胃癌细胞增殖能力的影响及其可能机制。方法:以携带gankyrin siRNA的慢病毒载体转染人胃癌细胞株MKN28,分别采用MTT实验、流式细胞术和蛋白质印迹法检测下调gankyrin表达对MKN28细胞增殖、细胞周期分布及其β-catenin/cyclin D1信号通路的影响。结果:慢病毒载体的转染效率在90%以上,转染gankyrin siRNA后,MKN28细胞gankyrin蛋白表达显著受抑(P0.01)。与未转染慢病毒和转染对照病毒的细胞相比,转染gankyrin siRNA的MKN28细胞体外生长于第3天起显著受抑,细胞周期G1期细胞比率增高,S期细胞比率降低,细胞中的β-catenin和cyclin D1表达水平降低,差异均有统计学意义(P0.01)。结论:下调胃癌细胞中的gankyrin表达可通过抑制β-catenin/cyclin D1信号通路引起细胞周期G1期阻滞和细胞增殖抑制,gankyrin有望成为胃癌靶向治疗的新靶点。     

长链非编码RNA TUG1在胃癌患者中的表达及其对预后的影响

背景:近年胃癌的发生率呈逐步上升的趋势,长链非编码RNA牛磺酸上调基因1(TUG1)对胃癌的发生、发展可能起有一定的作用。目的:研究TUG1在胃癌组织中的表达及其对预后的影响,并进一步探讨TUG1与p27蛋白、细胞周期蛋白D1(cyclin D1)的相关性。方法:收集2013年6月—2013年12月青岛市市立医院48例手术切除的胃癌组织及其相应远端正常组织,采用qRT-PCR法检测TUG1 mRNA表达,并分析其与临床病理特征的关系。采用蛋白质印迹法检测p27、cyclin D1蛋白表达,并分析其与TUG1表达的相关性。采用Kaplan-Meier法分析TUG1表达与预后的关系。结果:胃癌组织中TUG1 mRNA表达显著高于相应正常组织(6.18±0.19对5.09±0.16,P0.05),且其表达与性别、年龄、肿瘤大小无关,与淋巴结转移、肿瘤分化程度和TNM分期有关(P0.01)。胃癌组织中p27蛋白表达显著低于正常组织(0.170 9±0.021 2对0.308 7±0.025 2,P0.01),cyclin D1蛋白表达显著升高(0.341 7±0.027 1对0.241 7±0.017 3,P0.01),且p27蛋白表达与cyclin D1蛋白表达呈负相关(r=-0.897,P0.01)。胃癌组织中TUG1表达与p27表达呈负相关(r=-0.730,P0.01),与cyclin D1表达呈正相关(r=0.809,P0.01)。TUG1高表达的胃癌患者生存时间明显低于TUG1低表达者(P0.05)。结论:长链非编码RNA TUG1可能通过p27/cyclin D1途径在胃癌的发生、发展中起癌基因的作用,通过检测TUG1表达对判断胃癌患者预后具有潜在的价值。     

反义细胞周期素D1基因对肝癌细胞株HepG2的作用

目的通过基因反义封闭技术体外抑制细胞周期素D1(cyclin D1)的表达,研究其对肝癌细胞cyclin D1蛋白表达和细胞增殖的影响。方法以肝癌细胞株HepG2为研究对象,通过转染可表达cyclin D1反义互补脱氧核苷酸(AScDNA)的质粒后,观察cyclin D1反义cDNA对HepG2细胞cyclin D1基因表达及体外增殖活性的影响。结果四甲基偶氮唑盐法检测细胞增殖活性显示转染表达反义cyclin D1的质粒后, HepG2细胞的增殖受到抑制,抑制作用在48 h左右最强;逆转录聚合酶链反应检测显示cyclin D1 mRNA基因的表达明显被抑制;免疫组织化学检测结果显示cyclin D1蛋白表达明显降低; 流式细胞仪检测结果显示G0/G1期的细胞比例增高,G2+M和S期的细胞比例下降,HepG2细胞周期在G1期被阻滞。结论cyclin D1反义cDNA可以特异性的抑制肝癌HepG2细胞株cyclin D1蛋白的表达,从而调控细胞周期,抑制肝癌细胞增殖。利用cyclin D1反义cDNA进行细胞周期调控对于肝细胞癌的生物治疗具有一定的应用前景。     

姜黄素抑制胃癌细胞cyclin D1表达的研究

[目的]研究姜黄素对胃癌细胞cyclin D1表达的影响,探讨其抗肿瘤作用机制。[方法]①胃腺癌SGC7901细胞在体外常规培养,用免疫荧光染色、流式细胞仪(FCM)检测转化生长因子α(TGF-α)及其受体即表皮生长因子受体(EGFR)的表达情况。②胃腺癌SGC7901细胞先用无血清培养48 h,以便于观察不同处理后细胞cyclin D1表达的变化。然后用2.5%低浓度血清培养,并加入TGF-α或大蒜油处理,或同时加入TGF-α和大蒜油处理,用免疫荧光染色、FCM检测细胞cyclin D1表达的变化。[结果]①胃癌细胞常规培养48 h后,TGF-α和EG-FR表达的阳性率分别为46.80%和57.78%。②当在细胞培养液中加入30μg/L TGF-α作用5 h,细胞cyclin D1表达的阳性率增加了8.43%(P<0.01);当加入20μmol/L姜黄素作用5 h,cyclin D1表达的阳性率下降了21.37%(P<0.01);同时加入TGF-α和姜黄素处理细胞,与单加TGF-α比较,cyclin D1表达下降了25.32%(P<0.01),下降程度大于单加姜黄素引起的下降程度。[结论]姜黄素可通过抑制TGF-α的自分泌、旁分泌促增殖环路,发挥其抑制胃癌细胞增殖作用。推测这是姜黄素抗肿瘤作用机制之一。     

热休克蛋白70 mRNA在老年人胃癌中表达的意义

目的探讨热休克蛋白70 mRNA(HSP70 mRNA)在胃癌发生中的意义.方法取胃癌44例,癌旁不典型增生20例,单纯不典型增生16例,浅表性胃炎20例.采用核酸原位杂交技术(ISH)检测HSP70 mRNA表达状况.结果在胃癌、癌旁不典型增生和单纯不典型增生中HSP70 mRNA的阳性率分别为61.36(27/44)、55.00%(11/20)和31.25%(5/16);在浅表性胃炎组阳性率为5.00%(1/20).HSP70 mRNA表达与癌细胞浸润深度和淋巴结转移无明显相关性.结论 HSP70 mRNA在胃癌形成早期有较高表达,检测HSP70 mRNA可以作为胃癌早期诊断的指标.     

4'',5,7-三羟基异黄酮抗骨髓瘤细胞增殖机制的研究

目的:为了研究4',5,7-三羟基异黄酮(genistein,Gen)是否能抑制人多发性骨髓瘤(multiple myeloma,MM)细胞株XGI的增殖,研究Gen处理骨髓瘤细胞后B细胞淋巴瘤,白血病.2基因0,c1.2)、bcl-xl、细胞周期蛋白D1(cyclin D1)、细胞间黏附因子1(ICMA-1)基因表达变化及骨髓瘤细胞中核转录因子κB(NF-κB)表达的变化.方法:利用体外培养人MM细胞株XG1,依据剂量梯度分别给予Gen,用噻唑蓝染色法(MTT)观察不同药物浓度的Gen对MM细胞的增殖影响;5、10、15μ/mL Gen与溶剂对照组分别作用XG1细胞48 h后,半定量逆转录聚合酶链反应(RT-PCR)法检测XG1细胞bcl-2、bcl-xl、细胞周期蛋白D1、ICMA-1基因表达;应用5 μg/mL Gen处理XG1细胞,用免疫组织化学法观察Gen处理前后细胞内NF.KB的表达情况.结果:Gen作用XG1细胞48 h,随浓度增加,细胞增殖逐渐下降;5、10、15 μg/mL Gen处理组mRNA的表达均低于溶剂对照组(P<0.05),伴随Gen处理浓度逐渐增加,bcl-2、bcl-xl、细胞周期蛋白D1、ICMA-1基因mRNA的表达逐渐下降;Gen能够使NF-κB在XG1细胞内重新分布,胞浆内出现NF-κB表达,胞核内NF-κB表达下降.结论:Gen可能通过下调骨髓瘤细胞核中NF-κB的表达来抑制bcl-2、bcl-xl、细胞周期蛋白D1、ICMA-1基因mRNA的表达,进而抑制骨髓瘤细胞的增殖、黏附、转移.     

小干扰RNA沉默bcl-2基因抑制胃癌细胞生长的研究

目的利用RNA干扰技术,研究靶向bcl-2的小干扰RNA(siRNA)在体内外对胃癌细胞生长的影响,探讨其对胃癌治疗的可行性。方法化学合成bcl-2基因的siRNA,脂质体法将bcl-2 siRNA转染胃癌SGC 7901细胞,采用实时定量PCR和Western印迹观察bcl-2 siRNA转染前后胃癌细胞bcl-2基因的表达变化,四甲基偶氮唑盐(MTT)实验、流式细胞检测技术和端粒重复序列扩增法(TRAP)分别检测胃癌细胞增殖、凋亡和端粒酶活性变化。并将转染bcl-2 siRNA的胃癌SGC 7901细胞接种于裸鼠皮下,观察其在裸鼠体内成瘤及生长情况。结果数据经方差分析,bcl-2 siRNA转染胃癌SGC 7901细胞后,明显抑制bcl-2基因表达,并与其浓度和作用时间相关,以100 nmol/L bcl-2 siRNA对bcl-2基因表达抑制效果最佳。以该浓度的bcl-2 siRNA转染胃癌SGC 7901细胞后,bcl-2 mRNA和蛋白表达抑制分别为79．9%和85．3%；经bcl-2 siRNA作用的SGC 7901细胞生长明显缓于对照组(P＜0．05),细胞凋亡率高于对照组(P＜0．05),端粒酶活性明显低于对照组(P＜0．05)。转染100 nmol/L bcl-2 siRNA的胃癌SGC 7901细胞接种裸鼠皮下后,肿瘤出现时间晚,体积小．生长受到明显抑制(P＜0．05)。结论靶向bcl-2的siRNA可明显下调靶基因bcl-2的表达,在体内外抑制胃癌SGC 7901细胞的生长,为探索胃癌基因治疗提供新的策略。     

二甲双胍对人胃癌细胞株MKN45增殖和迁徙的影响

背景:近年发现二甲双胍具有潜在抑制肿瘤的作用,但其在消化系肿瘤尤其是胃癌中的研究较少。目的:研究二甲双胍对人胃癌细胞株MKN45增殖和迁徙的影响,并初步探讨其可能机制。方法:体外培养人胃癌细胞株MKN45.予10mmol/L二甲双胍进行干预(联合或不联合5-氟尿嘧啶),以MTT法检测细胞存活率,流式细胞仪检测细胞凋亡、线粒体膜电位,细胞划痕实验检测迁徙速率,RT-PCR检测AMPKα1、cyclin D1、Bcl-2、Bax、MMP-2、MMP-9mRNA表达。结果:与对照组相比,二甲双胍作用后,MKN45细胞存活率显著降低,并呈浓度-时间依赖性,细胞凋亡率显著增加,线粒体膜电位显著下降,平均迁徙速率显著降低,cyclin D1、MMP-2、MMP-9mRNA表达均显著减少,Bcl-2、BaxmRNA表达显著增加但两者之比降低,AMPKα1 mRNA表达无明显差异。二甲双胍与5-氟尿嘧啶联用对MKN45细胞的存活率无明显协同作用。结论:二甲双胍能抑制人胃癌细胞株MKN45增殖、迁徙,可通过改变线粒体膜通透性促进细胞凋亡。     

Long-term cultured IL-2-dependent T cell lines demonstrate p16(INK4a) overexpression, normal pRb/p53, and upregulation of cyclins E or D2

Acquisition of an immortal phenotype by circumvention of the normal senescence program can be an important step in tumor development and progression. The regulation of life-span checkpoints is complex and abrogation of these processes can occur at different levels. To better understand these mechanisms in long-term cultured lymphocytes we have characterized two human long-term cultured IL-2-dependent T cell lines regarding telomere length, telomerase activity, and the expression of selected cell cycle regulators (pRb, p53, cyclin E, cyclin D1, cyclin D2, cyclin D3, cdk4, p16(INK4a), p21(WAF1), p27(KIP1), c-myc, bcl-2, and NPAT). We compared these cell lines with a primary T lymphoblast population with a limited life span from the same donor. Both T cell lines with extraordinary growth capacity showed telomere length stabilization, high telomerase activity and demonstrated wild-type pattern of pRb and p53 but strong p16(INK4a) protein expression. The growth inhibitory activity of p16(INK4a) seemed to be abrogated by enhanced expression of cyclin D2, cdk4, and c-myc in one T cell line and overexpression of cyclin E in the second T cell line.     

4′,5,7-三羟基异黄酮抗骨髓瘤细胞增殖机制的研究

目的：为了研究4′,5,7-三羟基异黄酮（genistein,Gen）是否能抑制人多发性骨髓瘤（multipule myeloma,MM）细胞株XG1的增殖,研究Gen处理骨髓瘤细胞后B细胞淋巴瘤/白血病-2基因（bcl-2）、bcl-xl、细胞周期蛋白D1（cyclin D1）、细胞间黏附因子1（ICMA-1）基因表达变化及骨髓瘤细胞中核转录因子κB（NF-κB）表达的变化。方法：利用体外培养人MM细胞株XG1,依据剂量梯度分别给予Gen,用噻唑蓝染色法（MTT）观察不同药物浓度的Gen对MM细胞的增殖影响;5、10、15μg/mLGen与溶剂对照组分别作用XG1细胞48h后,半定量逆转录聚合酶链反应（RT-PCR）法检测XG1细胞bcl-2、bcl-xl、细胞周期蛋白D1、ICMA-1基因表达;应用5μg/mLGen处理XG1细胞,用免疫组织化学法观察Gen处理前后细胞内NF-κB的表达情况。结果：Gen作用XG1细胞48h,随浓度增加,细胞增殖逐渐下降;5、10、15μg/mLGen处理组mRNA的表达均低于溶剂对照组（P〈0.05）,伴随Gen处理浓度逐渐增加,bcl-2、bcl-xl、细胞周期蛋白D1、ICMA-1基因mRNA的表达逐渐下降;Gen能够使NF-κB在XG1细胞内重新分布,胞浆内出现NF-κB表达,胞核内NF-κB表达下降。结论：Gen可能通过下调骨髓瘤细胞核中NF-κB的表达来抑制bcl-2、bcl-xl、细胞周期蛋白D1、ICMA-1基因mRNA的表达,进而抑制骨髓瘤细胞的增殖、黏附、转移。     

Telomerase activity correlates with cell cycle regulators in human hepatocellular carcinoma

AIMS/BACKGROUND: Mutation in cell cycle genes is the most common genetic change in malignant tumor cells. Telomerase activation, considered as essential in the immortality of cancer cells, is found in most cancers, where there may be an association with an active cell cycle. METHODS: In this study study we used the TRAP assay to determine telomerase activity in liver tumor specimens from 25 cases of hepatocellular carcinoma (HCCs) as well as in corresponding non-cancerous liver tissue in each patient. The expression of cyclin D1, cdk2, and cdk4 protein was also examined by Western blot. RESULTS: Twenty-one of the 25 cases of HCC were found to have increased telomerase activity, whereas only five out of the 25 non-cancerous liver samples were found to have weak telomerase activity. Telomerase activity was not found to be related to tumor size, HBsAg, HBeAg, anti-HCV, transaminase, or alpha-fetoprotein serum titer. Furthermore, three out of the 25 cases of HCC showed cyclin D1 overexpression, whereas 15 of the 23 cases of HCC showed decreased cyclin D1 expression. Down regulation of cyclin D1, cdk2, cdk4 protein correlated with telomerase activity (p<0.004, p<0.013, and p<0.001 respectively). CONCLUSION: The results indicate that genetic defects in HCC facilitate the reactivation of telomerase activity, a process which may be dependent on cyclin D1 with its cyclin dependent kinase (cdk) partner defect.     

Expression analysis of cyclins in pituitary adenomas and the normal pituitary gland

OBJECTIVES: The molecular events involved in pituitary tumour development are still poorly understood. The cyclins play an important role in the control of the cell cycle during cell proliferation and over-expression of the cyclins has been shown in many different tumour types. The aim of this study was to investigate whether, in comparison to the normal gland, ectopic expression of cyclins occurs in pituitary tumours, and whether differences in cyclin expression are seen with different pituitary tumour types or in association with different tumour behaviour. In contrast to work on cyclin D there are no published data on cyclin B, A and E in human pituitary tumours. METHODS: Sixty-seven surgically removed pituitary tumours and 10 specimens of normal human anterior gland were studied using immunohistochemistry to detect the nuclear expression of cyclin A, B, D and E. The microvascular density (as a measure of angiogenesis), Ki-67 labelling index (to assess cell proliferation) and bcl-2 expression had previously been investigated in this cohort. RESULTS: All tumours studied contained cells that immunostained positively for cyclin A, B, D and E. However the proportion of positive cells in each tumour type was different. In contrast, there were no cyclin D positive cells in the normal anterior pituitary gland studied, and labelling indices (LI) for cyclins A, B and E were significantly lower in the normal gland than in pituitary adenomas. The cyclin LIs for A, B, D and E were significantly higher in macroadenomas when compared to microadenomas. Non-functioning pituitary tumours (NFA) generally showed the highest cyclin LI. In particular, both recurrent and nonrecurrent NFA showed significantly higher cyclin D LI than other tumours. The ratio of cells expressing cyclin B compared to those expressing cyclin A was significantly higher in functionless tumours that regrew when compared to NFAs that did not (P<0.05). Cyclin D LI and the overall Ki-67 LI as a measure of cell proliferation were related (R2 = 11.4, P = 0.0033) and bcl-2 positive tumours had significantly higher cyclin D LI compared with bcl-2 negative tumours. There was a weak relationship between angiogenesis and the relative proportion of cells expressing D when compared to those in S phase (D/A ratio) (r2 = 10.5, P = 0.02). CONCLUSIONS: We have demonstrated that ectopic expression of cyclin D and over-expression of cyclins A, B and E, regulating different stages of the cell cycle is common in pituitary adenomas. In addition, cyclin expression was related to size and to pituitary tumour regrowth. The differences between functionless tumours that regrow and those that do not, may be due to reduced bcl-2 expression, increased cell proliferation, more cells at the G2/M stage (B/A ratio) and reduced cell differentiation with more aggressive subsequent tumour behaviour. Cyclin D expression and cell proliferation were related indicating that the cells entering the cycle become 'committed' to cell cycle progression. There was no relative over-expression of individual cyclins, and therefore no evidence of relative increase in cell cycle phase, indicating that the increased cyclin expression is more likely to be due to constant mitogenic stimulation rather than cell cycle regulatory failure. Although nuclear cyclin expression is a good marker of tumour growth and aggressive behaviour, the growth signal that leads to cyclin expression remains to be identified.     

Survey of molecular profiling during human colon cancer development and progression by immunohistochemical staining on tissue microarray

AIM： To explore the molecular events taking place during human colon cancer development and progression through high-throughput tissue microarray analysis. METHODS： We constructed two separate tissue microarrays containing 1.0 mm or 1.5 mm cylindrical samples acquired from 112 formalin-fixed and paraffinembedded blocks, including carcinomas （n = 85）, adenomatous polyps （n = 18）, as well as normal paracancerous colon tissues （n = 9）. Immunohistochemical staining was applied to the analysis of the consecutive tissue microarray sections with antibodies for 11 different proteins, including p53, p21, bcl-2, bax, cyclin D1, PTEN, p-Aktl, β-catenin, c-myc, nm23-h1 and Cox-2. RESULTS： The protein expressions of p53, bcl-2, bax, cyclin D1, β-catenin, c-myc, Cox-2 and nm23-h1 varied significantly among tissues from cancer, adenomatous polyps and normal colon mucosa （P = 0.003, P = 0.001, P = 0.000, P = 0.000, P = 0.034, P = 0.003, P = 0.002, and P = 0.007, respectively）. Chi-square analysis showed that the statistically significant variables were p53, p21, bax, β-catenin, c-myc, PTEN, p-Aktl, Cox-2 and nm23-h1 for histological grade （P = 0.005, P = 0.013, P = 0.044, P = 0.000, P = 0.000, P = 0.029, P = 0.000, P = 0.008, and P = 0.000, respectively）, β-catenin, comyc and p-Akt1 for lymph node metastasis （P = 0.011, P =0.005, and P = 0.032, respectively）, β-catenin, c-myc, Cox-2 and nm23-h1 for distance metastasis （P = 0.020, P = 0.000, P = 0.026, and P = 0.008, respectively）, and cyclin D1, β-catenin, c-myc, Cox-2 and nm23h1 for clinical stages （P = 0.038, P = 0.008, P = 0.000, P = 0.016, and P = 0.014, respectively）. CONCLUSION： Tissue microarray immunohistochemical staining enables high-throughput analysis of genetic alterations contributing to human colon cancer development and progression. Our results implicate the potential roles of p53, cyclin D1, bcl-2, bax, Cox-2, β-catenin and c-myc in development of human colon cancer and that of bcl-2, nm23-h1, PTEN and p-Akt1 in pro     

应用基因转染方法调节HSP70在人胃癌细胞株SGC7901中的表达

目的　为探讨 ＨＳＰ７０ 在人胃癌中过度表达的意义, 调节ＨＳＰ７０ 在人胃癌细胞系ＳＧＣ７９０１ 中的表达．方法　应用脂质体介导的基因转染方法,将表达针对ＨＳＰ７０的反义ＲＮＡ 表达载体及ＨＳＰ７０ 表达载体转入ＳＧＣ７９０１ 细胞,通过ｈｙｇｒｏ ｍ ｙｃｉｎｅ 抗性筛选, 挑选阳性克隆;对阳性克隆从ＲＮＡ 水平（ 点杂交及ＲＮａｓｅ 保护试验） 及蛋白质水平（ Ｗｅｓｔｅｒｎｂｌｏｔ 及免疫组化） 进行研究．结果　ＨＳＰ７０ 在转染细胞中的表达得到了调节． 为进一步研究ＨＳＰ７０ 对胃癌形成与发展的作用创造了条件．结论　经过ＨＳＰ 反义或正义ＲＮＡ 转染的ＳＧＣ７９０１ 细胞具有不同的ＨＳＰ７０ 表达水平可用于胃癌发生机制的研究．