MBP序列特异性shRNA质粒载体的构建及鉴定

目的:以pGenesil-1质粒为介导,构建针对MBP基因的shRNA表达载体。方法:设计特异性表达MBP shRNA结构的互补DNA序列3个及1个阴性对照序列,退火并克隆至质粒载体pGenesil-1中,转化DH5α菌株,进行重组质粒的酶切鉴定及测序分析。结果:经酶切鉴定及测序分析,筛选出的重组体测序结果和靶序列相同,成功构建了shRNA重组质粒载体pGenesil-1-MBP-1、pGenesil-1-MBP-2、pGenesil-1-MBP-3及pGenesil-1-MBP-neg。结论:成功构建MBP shRNA表达载体,为进一步研究MBP基因在细胞中的作用机制奠定基础。     

脑胶质瘤组织中cyclin D1的表达及其意义

背景与目的:细胞周期素D1(cyclinD1)在许多肿瘤中有过度表达,过量的cyclinD1是许多肿瘤发生的重要原因。但在脑胶质瘤中是否存在cyclinD1的过度表达尚有争论,本研究旨在检测人脑胶质瘤组织中cyclinD1的表达水平,探讨cyclinD1与脑胶质瘤恶性程度及预后的关系。方法:采用免疫组化SP法检测84例脑胶质瘤组织中cyclinD1的表达水平;分析脑胶质瘤组织中的cyclinD1表达强度、阳性细胞率与肿瘤恶性程度、预后之间的关系。结果:(1)32例低级别胶质瘤中cyclinD1平均阳性细胞率为(9.82±9.75)%,52例高级别胶质瘤中平均阳性细胞率为(27.45±21.03)%,两组均数比较差异有显著性(P<0.01);(2)低级别胶质瘤cyclinD1阳性率为31.25%(10/32),高级别胶质瘤阳性率为61.53%(32/52),差异有显著性(P<0.01);(3)复发组cyclinD1阳性率为76.19%(16/21),未复发组的cyclinD1阳性率为24.00%(6/25),差异有显著性(P<0.01);(4)死亡组cyclinD1阳性率为66.67%(14/21),未死亡组的cyclinD1阳性率为32.00%(8/25),差异有显著性(P<0.05)。死亡组21例中,高级别胶质瘤cyclinD1阳性率为86.66%(13/15),低级别胶质瘤阳性率为16.66%(1/6),差异有显著性(P<0.01)。结论:CyclinD1表达水平随胶质瘤病理分级的增高而增高;cyclinD1表达水平越高,患者预后越差。Cyc     

靶向活化T细胞核因子1基因的shRNA质粒表达载体的构建

 目的　利用pRNAT-U6.1／Neo质粒构建靶向NFAT1基因的3个shRNA,为下一步探索肺癌基因治疗的RNA干扰途径奠定基础。方法　设计3对shRNA（NFAT1-sh-1、NFAT1-sh-2、NFAT1-sh-3）,以人类基因同源性差的无关序列（NFAT1-sh-NC）作为阴性对照。筛选与人类其他基因同源性小、特异性针对目的基因的siRNA。合成并克隆至带有U6启动子的pRNAT -U6.1／Neo质粒中,构建质粒表达载体,通过凝胶电泳鉴定后转化至大肠杆菌中进行培养,并提取质粒进行DNA测序鉴定。结果　成功构建和筛选出靶向NFAT1基因的3对shRNA质粒表达载体,GC比例为40 ％～55 ％。结论　构建的3个靶向NFAT1基因shRNA质粒表达载体有助于肺癌靶向基因治疗RNA干扰的研究。为筛选有效的基因干扰载体,以及对荷瘤肺癌裸鼠靶向RNAi抗肿瘤细胞生长的研究鉴定了基础。     

cyclin D1、pRb在大肠癌中的表达及其临床意义

目的研究大肠癌中cyclin D1和pRb的表达与临床病理参数之间的关系以及二者之间的关系.方法采用免疫组化方法S-P法检测67例大肠癌、40例癌旁粘膜和40例正常粘膜中cyclin D1和pRb的表达.结果大肠癌中cyclin D1和pRb的阳性率分别为44.8%和100.0%,明显高于癌旁粘膜和正常粘膜(P＜0.05),cyclin D1在低分化癌的过表达率高于高分化癌(P＜0.05),C+D期的过表达率明显高于A+B期(P＜0.01);pRb在C+D期的过表达率高于A+B期(P＜0.05),pRb的过表达率与肿瘤的分化程度无关(P＜0.05).相关分析表明,cyclinD1和pRb无明显相关(P＞0.05).结论cyclin D1参与了大肠癌的发生并与肿瘤的分化程度和Duke's分期有关,而pRb与肿瘤的分化程度无关.     

人脑胶质瘤组织中cyclin D1和cyclin E表达的研究

目的 :研究人脑胶质瘤中细胞周期素 (cyclin)D1和cyclinE的表达与肿瘤病理等级的关系。方法 :采用免疫组织化学法检测 5 2例人脑胶质瘤和 8例正常脑组织标本中cyclinD1和cyclinE的表达。结果 :人脑胶质瘤组中有 2 9例cyclinD1的表达。随着胶质瘤病理分级增高 ,高、低恶性度胶质瘤的阳性率和平均标记指数均显著升高 ,两者差异有统计学意义 ,P <0 0 5。cyclinD1与cyclinE的平均标记指数之间呈明显正相关 ,Pearson相关系数r =0 64 4,P <0 0 1。结论 :cyclinD1与cyclinE在胶质瘤中被异常表达 ,是肿瘤发生和恶性转化的重要促进因子。     

smad4 shRNA 真核表达质粒的构建及鉴定

 目的 针对smad4基因的不同部位,构建不同smad4shRNA表达质粒载体,在转录后水平抑制smad4的表达,对其克隆进行鉴定并挑选出抑制效率最高的克隆。方法 用DNA重组技术将针对人smad4基因不同部位所设计的3对shRNA序列克隆到真核表达质粒pGenesil-1中,构建smad4shRNA表达载体pGenesil-smad4-shRNA1、2、3。脂质体介导转染人宫颈癌细胞株HeLa,经G418筛选抑制smad4表达的稳定细胞克隆。结果 3个smad4shRNA表达载体pGenesil-smad4-shRNA1、2、3经限制性酶切及序列分析证明基因插入正确。RT-PCR、Westernblotting均证实：3种shRNA重组质粒中pGenesil-smad4-shRNA2可明显降低细胞内smad4mRNA丰度及smad4蛋白表达。结论 成功构建了smad4shRNA表达载体pGenesil-smad4-shRNA1、2、3,并筛选出特异而高效地阻断smad4表达的克隆。此实验结果为进一步研究smad4蛋白分子的生物学功能及应用奠定了基础。     

CD147 shRNA真核表达质粒的构建及鉴定

目的针对CD147基因的不同部位,构建不同CD147 shRNA表达质粒载体,在转录后水平抑制CD147的表达,对其克隆进行鉴定并挑选出抑制效率最高的克隆.方法用DNA重组技术将针对人CD147基因的不同部位所设计的3对shRNA序列克隆到真核表达质粒pGE-1中,构建CD147 shRNA表达质粒载体pGE-1 CD147shRNA1、2、3.用阳离子脂质体介导转染人卵巢癌高转移细胞系HO-8910pm,经G418筛选后获得克隆进行鉴定.结果3个CD147 shRNA表达载体pGE-1CD147shRNA1、2、3经PCR、限制性酶切及部分序列分析证明基因插入正确.半定量RT-PCR、Western blotting均证实:转染细胞8910 pm/pGE-1 CD147shRNA,与亲本细胞相比,CD147的表达在mRNA和蛋白水平均明显下降.结论成功构建了CD147 shRNA表达载体pGE-1 CD147shRNA,并筛选出特异而高效地阻断CD147表达的克隆.此实验结果为进一步研究CD147蛋白分子的生物学功能及应用奠定了基础,并对shRNA设计靶序列的选择有一定指导意义.     

膀胱癌中cyclin D1和MMP-2的表达及其临床意义

目的:探讨cyclin D1和MMP-2在膀胱癌中的表达及其与临床病理特征之间的关系。方法:应用逆转录-聚合酶链反应（RT-PCR）方法检测127例膀胱癌和配对癌旁组织以及15例正常膀胱组织中cyclin D1和MMP-2基因的表达,免疫印迹法观测cyclin D1和MMP-2蛋白表达情况。分析两者与膀胱癌临床病理特征之间的关系及两者的相关性。结果:cyclin D1和MMP-2 mRNA在膀胱癌中的表达明显高于癌旁组织和正常膀胱组织。正常膀胱组织中cyclin D1和MMP-2 mRNA的表达率分别为13.3%、20.0%,癌旁组织为26.0%、33.1%,均显著低于癌组织81.1%、88.2%（P<0.05）。类似于基因表达,cyclin D1和MMP-2蛋白在膀胱癌中的表达相对较高。cyclin D1和MMP-2基因在膀胱癌中的表达与病理分级、临床分期和淋巴转移相关(P<0.05),cyclin D1表达还与肿瘤大小有关（P<0.05）,两者均与患者年龄和性别无关（P>0.05)。膀胱癌组织中cyclin D1和MMP-2 mRNA的表达呈正相关(r=0.771,P<0.01)。结论:cyclin D1和MMP-2联合检测可为膀胱癌诊断和治疗提供参考。     

人脑胶质瘤组织中cyclin D1和cyclin E表达的研究

目的：研究人脑胶质瘤中细胞周期素(cyclin) D1和cyclin E的表达与肿瘤病理等级的关系。方法：采用免疫组织化学法检测52例人脑胶质瘤和8例正常脑组织标本中cyclin D1和cyclin E的表达。结果：人脑胶质瘤组中有29例cyclin Dl的表达。随着胶质瘤病理分级增高，高、低恶性度胶质瘤的“阳”性率和平均标记指数均正著升高，两者差异有统计学意义，P<0．05。cyclin Dl与cyclin E的平均标记指数之同呈明显正相关，Pearson相关系数r-0．644，P<0．0l。结论：cyclin Dl与cyclin E在胶质瘤中被异常表达，是肿瘤发生和恶性转化的重要促进因子。     

凋亡抑制基因 Survivin在肝癌细胞中的表达及其与PTEN、cyclin D1蛋白表达的关系

目的:探讨Survivin基因的表达在肝癌发生、发展中的作用及其与PTEN、cyclin D1蛋白表达的相互关系.方法:采用流式细胞术对肝细胞癌(48例)、癌旁肝硬化(32例)、肝硬化(6例)、正常肝组织(6例)Survivin、PTEN、cyclin D1基因蛋白的表达进行定量检测.结果:Survivin和cyclin D1蛋白表达的FI值明显高于癌旁组织、肝硬化和正常肝组织,而肝细胞癌的PTEN蛋白的FI值明显低于癌旁组织、肝硬化和正常肝组织.Survivin和PTEN蛋白的表达量与分化程度、预后有关,与肿瘤大小、门静脉癌栓、肝内转移无关.随着肿瘤恶性程度加重,Survivin与PTEN蛋白表达呈显著负相关;Survivin蛋白与cyclin D1蛋白表达之间呈显著正相关.结论:Survivin、PTEN及cyclin D1基因在肝细胞癌的发生、发展中起着不同的作用,联合检测Survivin和PTEN对判定肝细胞癌预后可提供一定的依据.     

人细胞周期蛋白cyclin D1基因RNAi表达载体的构建与鉴定

[目的]构建针对人细胞周期蛋白cyclinD1基因的RNAi真核表达载体,检测其对人卵巢癌HO-8910细胞cyclinD1基因表达的干扰效率。[方法]以cyclinD1为靶向设计,合成的4条互补寡核苷酸链克隆至pSUPER载体中,得到重组质粒pSUPER-C1￣4,行双酶切分析后测序鉴定;运用脂质体介导转染HO-8910细胞,G418筛选;采用RT-PCR检测对cyclinD1基因mRNA表达的干扰,并筛选有效的siRNA干扰序列。[结果]经酶切测序鉴定证实,重组质粒中已插入目的序列;RT-PCR表明有4个重组载体均不同程度抑制目的基因的转录,但以pSUPER-C2的干扰效率最强。[结论]在卵巢癌细胞系筛选出有效干扰cyclinD1基因表达siRNA序列,成功构建针对cyclinD1基因的RNAi真核表达载体有效抑制目的基因的表达。     

人体胰腺癌细胞株PANC-1中四环素可诱导的周期素D1表达抑制系统的建立

[目的]在人体胰腺癌细胞株PANC-1中建立一个四环素可调控周期素D1表达系统。研究抑制周期素D1对胰腺癌细胞的影响。[方法]反义周期素D1质粒通过两次稳定转染进入胰腺癌细胞株PANC-1细胞中，其表达调控系统采用Tet-Off系统（四环素调控系统）。通过抑制周期素D1表达对PANC-1细胞生长、集落形成能力以及周期素蛋白表达的影响，评价此系统的可调控性和有效性。[结果]通过第一次转染pTet—Off质粒，选择两个最佳表达克隆行第二次转染DTRE-反义周期素D1质粒，并通过免疫印记测定挑选出能在Tet—Off系统中最有效地表达反义周期素D1的克隆。通过Tet—Off系统对反义周期素D1的调控．发现周期素D1的表达抑制可明显地抑制胰腺癌细胞生长和集落能力，并可导致胰腺癌细胞形态学改变。其抑制作用与四环素调控浓度和时间有关。[结论]此研究在PANC-1胰腺癌细胞株中建立了一个高效、可诱导的反义周期素D1的表达系统。通过这个系统的建立，可进一步在体内和体外研究周期素D1的抑制对胰腺癌细胞的影响．并可结合其它治疗手段如化疗来探讨联合治疗在临床的潜在应用价值。     

Cyclin D1 Genotype and Expression in Sporadic Hemangioblastomas

Summary Central nervous system (CNS) hemangioblastomas are highly-vascularized tumors occurring in sporadic form or as a manifestation of von Hippel–Lindau disease (VHL). The VHL protein (pVHL) regulates various target genes, one of which is the CCND1 gene, encoding cyclin D1, a protein that plays a critical role in the control of the cell cycle. Overexpression of cyclin D1 is found in many cancers. The CCND1 gene contains a common G → A polymorphism (870G > A) that enhances alternative splicing of the gene. CCND1 genotype is associated with clinical outcome in a number of cancers although prognostic significance varies with tumor type. In VHL disease, CCND1 genotype has been suggested as a genetic modifier that influences susceptibility to hemangioblastomas. In order to analyze whether CCND1 genotype plays a role in sporadic CNS hemangioblastomas, we investigated CCND1 genotype in tumor tissue of 17 sporadic and also in five VHL-related CNS hemangioblastomas. In addition, in these tumors the extent and localization of cyclin D1 expression was investigated by immunohistochemistry. We found no deviation in CCND1 genotype distribution and allele frequencies from expected values. Also, there was no correlation between age at onset and CCND1 genotype. The expression of cyclin D1 as detected by immunohistochemistry was highly variable within and between tumors, without a clear correlation with CCND1 genotype. We conclude that, whereas variable but sometimes high cyclin D1 expression is a feature of sporadic hemangioblastomas, CCND1 genotype is unlikely to be an important genetic modifier in the oncogenesis of these tumors.     

颌骨骨肉瘤中cyclinD1和CDK4的表达及其意义

分析细胞周期素Ｄ１（ｃｙｃｌｉｎＤ１）和细胞周期素依赖激酶４（ＣＤＫ４）在颌骨骨肉瘤中的表达及意义。方法采用免疫组化ＡＢＣ法检测ｃｙｃｌｉｎＤ１和ＣＤＫ４在２０例颌骨骨肉瘤和８例骨软骨瘤中的表达。结果骨肉瘤中ｃｙｃｌｉｎＤ１和ＣＤＫ４的阳性率表达率分别为６５．００％（１３／２０）和６０．００％（１２／２０），两者的阳性表达存在正相关（γＳ＝０．４８，Ｐ〈０．０５）；而它们在骨软骨瘤中的阳性率均为１     

靶向mrp1基因shRNA表达载体构建

目的:构建靶向mrp1基因的shRNA表达载体.方法:根据mrp1基因序列设计并合成编码shRNA的DNA模板,将其定向克隆到psilencer2.1U6-neo质粒上,构建重组载体.经菌落PCR和DNA测序分析检测重组载体构建结果.结果:成功构建靶向mrp1基因shRNA表达载体.结论:为进一步研究mrp1基因在肺癌多药耐药中的作用以及探索肺癌的基因治疗奠定了基础.     

Cyclin D1 in Breast Cancer

Cyclin D1 protein plays an important part in regulating the progress of the cell during the G1 phase of the cell cycle. The cyclin D1 gene, CCND1, is amplified in approximately 20% of mammary carcinomas, and the protein is over- expressed in approximately 50% of cases. This has led to intensive study to ascertain whether cyclin D1 is a biological marker in breast cancer; however, the clinical work has produced unexpected results. Work in cell lines and in transgenic mice indicate that CCND1 is a weak oncogene and it was expected that, like c- erbB-2, over-expression of cyclin D1 protein would be associated with a poor prognosis. Early immunohistochemical prognostic studies produced equivocal results but we, and others, have recently shown that strong staining for cyclin D1 is more likely to be seen in well differentiated, estrogen receptor positive carcinomas. Furthermore, we have found that over-expression of cyclin D1 is actually associated with a good outcome, both in terms of prognosis and response to endocrine treatment. Cyclin D1 is frequently over- expressed in ductal carcinoma in situ but not in benign breast disease, including atypical ductal hyperplasia; hence its expression appears to be closely linked with carcinogenesis. In order to help explain the apparent beneficial effects of cyclin D1 over- expression, a number of closely associated cell cycle proteins have also been evaluated, including the cyclin dependent kinase inhibitor p27, which blocks the activating effects of cyclin D1. Initial reports show that high levels of p27 are associated with a good prognosis and we have shown a positive association between p27 and cyclin D1 expression. These clinical results of cyclin D1 are an example of how information obtained from basic cell biology studies needs to be complemented by clinical studies to ascertain the true worth of a prognostic marker.     

Cyclin D1 overexpression associates with radiosensitivity in oral squamous cell carcinoma.

Overexpression of cyclin D1, a G1 cell cycle regulator, is often found in many different tumor types, including oral squamous cell carcinomas (SCC). Recent laboratory experiments have demonstrated that cyclin D1 levels can influence radiosensitivity in various cell lines. This study evaluated the relationship between cyclin D1 expression levels and radiosensitivity in nine oral SCC cell lines (HSC2, HSC3, HSC4, SCC15, SCC25, SCC66, SCC111, Ca9-22, and NAN2) and 41 clinical patients with oral SCC who underwent preoperative radiation therapy. Radiosensitivity of the nine oral SCC cell lines differed greatly in their response to radiation, assessed by a standard colony formation assay. Likewise, the expression of cyclin D1 varied, and the magnitude of the cyclin D1 expression correlated with increased tumor radiosensitivity. The similar significant association between the response to preoperative radiation therapy and cyclin D1 overexpression was observed in the oral SCC patients who were treated with preoperative radiation therapy. These results suggest that cyclin D1 expression levels correlate to radiosensitivity and could be used to predict the effectiveness of radiation therapy on oral SCC.     

胃癌eIF4E和cyclin D1表达及其临床病理意义

 目的研究真核细胞起始因子4E(eIF4E)及cyclin D1在胃癌中的表达及临床意义,探讨eIF4E与cyclin D1之间的相关性。方法应用免疫组化的方法检测91例胃癌及30例正常胃组织中eIF4E及cy-clinD1的表达。结果eIF4E、cyclin D1在正常胃组织中均为阴性表达,在胃癌组织中的阳性表达率分别为95.6%(87/91)和84.6%(77/91)。eIF4E及cyclin D1表达与胃癌浸润深度、淋巴结转移以及临床分期有关(P<0.05),但与肿瘤组织学分化程度、年龄、性别无关。胃癌组织中eIF4E与cyclin D1表达密切相关(P<0.05)。结论检测eIF4E和cyclin D1表达水平,对于判断胃癌的恶性程度有重要意义。     

ERK-1与Cyclin D1在肝细胞肝癌组织中的表达

 目的 探讨ERK-1与Cyclin D1在肝细胞肝癌组织中表达的临床意义、相关性及对预后的影响。 方法 应用免疫组织化学方法检测50例肝细胞肝癌组织、17例癌旁硬化肝组织及14例正常肝组织ERK-1与Cyclin D1的表达。 结果 1.ERK-1与Cyclin D1在肝细胞肝癌组织中的表达显著高于癌旁硬化肝组织和正常肝组织;2.ERK-1表达与临床分期、病理分级、年龄、肿瘤大小、癌栓等明显相关;3.Cyclin D1的表达与病理分级有关;4.ERK-1与Cyclin D1表达-~+者平均生存时间较短,生存率较低,预后较差;ERK-1与Cyclin D1表达++~+++者平均生存时间较长,生存率较高,预后较好;5.在肝细胞肝癌组织中ERK-1与Cyclin D1的表达呈正相关。 结论 ERK-1、Cyclin D1的表达水平的升高在肝癌的发生发展中发挥协同、促进的作用,监测它们的表达能为肝癌早期诊断及评价肝癌患者的预后提供帮助。     

HRCT Scans of Peripheral Non-Small Cell Lung Cancers and their Relationship with Cyclin D1 Expression: a Longitudinal Study

OBJECTIVE To investigate cyclin D1 expression in peripheral lung cancers and its relationship with CT signs and prognosis.METHODS Cyclin D1 expression in peripheral lung cancers and its relationship with the CT imaging and prognosis were analyzed retrospectively by means of SP immunohistochemistrv and spiral CT scanning in 92 patients with peripheral lung cancer verified by pathology.RESULTS Cyclin D1 expression was related to deep lobulafion,spiculate protuberance,short thin spinules sign and mediastina lymph node metastasis.Cyclin D1 expression was not related to tumor size,cavity,pleural indentation,histological type,djfferentiation,tumor TNM stage,age or sex.Cyclin D1 was a negative prognostic factor whose over-expression indicated a poor prognosis.CONCLUSION Cyclin D1 expression may play an important role in the occurrence,progress and CT scan results of lung cancers.Cyclin D1 was a negative factor whose over-expression implied a poor prognosis.Detection of the cyclin D1 and observation of the CT scan can be considered as indexes of clinical diagnosis and prognostic evaluation.