目的 构建APC膜结合蛋白1(APC membrane recruitment protein 1,AMER1)基因不同区域片段重组质粒,并检测其在人肺腺癌A549细胞中的表达.方法 以人基因组DNA为模板,PCR扩增AMER1基因不同功能区域片段2-1135AA、2-327AA、2-785AA、327-785AA、785-1135AA及327-1135AA,扩增片段分别经T4 DNA酶与EGFP-N1载体连接,构建AMER1不同功能片段重组质粒,产物分别转化至感受态E.coli DH5α中,提取阳性克隆进行双酶切及测序鉴定;用脂质体2000介导各重组质粒转染肺腺癌A549细胞,同时设转染空载体EGFP-N1为对照组,q-PCR法检测AMER1不同功能区域片段mRNA转录水平.结果 经双酶切及测序鉴定,AMER1不同功能区域片段重组质粒构建正确;与对照组比较,A549细胞中6个重组质粒AMER1基因mRNA转录水平均显著升高(P均＜0.01).结论 成功构建了AMER1基因不同功能片段截短子重组质粒,并在A549细胞中高表达,为进一步研究AMER1不同功能区域在肺腺癌中的作用及相关分子机制提供了生物学工具.

microRNA(miRNA)是一类广泛存在于真核生物体内的内源性非编码小分子RNA,成熟的miRNA由20～25个核苷酸序列组成.近些年随着研究者对miRNA研究的不断深入,发现其在胆囊癌的发生、发展、预后、化疗耐药以及早期诊断中具有重要作用.对miRNA在胆囊癌中作用的研究进展进行了综述.

目的 筛选非小细胞肺癌(non-small cell lung cancer,NSCLC)预后的关键基因,并对其进行生物信息学分析及鉴定.方法 从公共数据库基因表达数据库(Gene Expression Omnibus,GEO)中下载NSCLC cDNA芯片集GSE19188、GSE101929、GSE40275、GSE 18842,利用在线工具GE02R对差异表达基因(differentially expressed genes,DEGs)进行筛选,并用Venny取交集.基于DAVID数据库对DEGs进行GO(Gene Ontology)分析及KEGG(Kyoto Encyclopedia of Gene and Genome)通路分析,运用STRING数据库及Cytoscape软件构建蛋白互作网络.使用CytoHubba插件鉴定核心关键基因,并对其生存分析和表达检测.结果 4个cDNA芯片集中共筛选得到130个差异表达基因,包括53个上调表达基因和77个下调表达基因.这些差异表达的基因主要集中在胞浆和细胞外区域,所参与的主要是细胞周期调控、有丝分裂、微管运动相关信号.筛选出的处于核心地位的20个基因中,9个基因与肺癌患者预后显著相关,包括UBE2C、TTK、CEP55、ASPM、PRC1、CCNB2、CCNA2、CCNB1和CDC6.结论 通过生物信息学分析,筛选出与正常组织相比,在NSCLC中异常表达的基因130个,并鉴定出其中与预后显著相关的9个核心基因,对进一步了解NSCLC发生发展的分子机制,筛选鉴定新的预后标记物及潜在性分子靶点具有积极意义.

目的:探讨三叶青根多糖(RTP)对肝癌细胞HepG2增殖、凋亡、迁移和侵袭的影响及其机制.方法:不同质量浓度(0.65、1.25、2.5、5、7.5、10 mg/mL)的RTP作用于肝细胞L-02不同时间(24、48和72 h),MTT法检测细胞增殖,筛选出对肝细胞L-02无毒性的RTP质量浓度.对肝细胞L-02无毒性质量浓度的RTP作用于HepG2细胞24、48和72 h后,MTT法检测细胞增殖,筛选出RTP对HepG2细胞的最佳作用质量浓度和时间.最佳质量浓度的RTP干预HepG2细胞一定时间(最佳作用时间)后,流式细胞仪检测细胞凋亡,Transwell检测细胞迁移和侵袭,qRT-PCR法和Western Blot法分别检测细胞中P21、Cyclin D1、Bcl-2、Bax、E-cadherin和MMP-2的mRNA和蛋白表达水平,qRT-PCR检测细胞中miR-151表达水平.转染miR-151抑制剂抑制HepG2细胞中miR-151表达,检测抑制miR-151表达后HepG2细胞的增殖、凋亡、迁移和侵袭及细胞中P21、Cyclin D1、Bcl-2、Bax、E-cadherin和MMP-2的mRNA和蛋白表达情况.结果:低质量浓度(0.65、1.25、2.5 mg/mL)RTP对肝细胞L-02无毒性.RTP作用HepG2细胞的最佳作用质量浓度和时间分别为1.25 mg/mL、48 h,1.25 mg/mL的RTP及抑制miR-151表达均可降低HepG2细胞活性、迁移和侵袭细胞数及细胞中Cyclin D1、Bcl-2和MMP-2的mRNA和蛋白表达水平(P<0.05),提高HepG2细胞凋亡率及细胞中P21、Bax和E-cadherin的mRNA和蛋白表达水平(P<0.05).并且RTP可抑制HepG2细胞中miR-151表达.过表达miR-151且同时用1.25 mg/mL的RTP作用HepG2细胞时,细胞活性、迁移和侵袭数及细胞中Cyclin D1、Bcl-2和MMP-2的mRNA和蛋白表达水平升高(P<0.05),细胞凋亡率及细胞中P21、Bax和E-cadherin的mRNA和蛋白表达水平降低(P<0.05).结论:RTP可抑制肝癌HepG2细胞增殖、迁移和侵袭,并诱导其凋亡,其作用机制可能与下调miR-151表达有关.

Background Lung cancer has been a common malignant tumor with a leading cause of morbidity and mortality, current molecular targets are woefully lacking comparing to the highly progressive cancer. The study is designed to identify new prognostic predictors and potential gene targets based on bioinformatic analysis of Gene Expression Omnibus (GEO) database. Methods Four cDNA expression profiles GSE19188, GSE101929, GSE18842 and GSE33532 were chosen from GEO database to analyze the differently expressed genes (DEGs) between non-small cell lung cancer (NSCLC) and normal lung tissues. After the DEGs functions were analyzed, the protein-protein interaction network (PPI) of DEGs were constructed, and the core gene in the network which has high connectivity degree with other genes was identified. We analyzed the association of the gene with the development of NSCLC as well as its prognosis. Lastly we explored the conceivable signaling mechanism of the gene regulation during the development of NSCLC. Results A total of 92 up regulated and 214 down regulated DEGs were shared in four cDNA expression profiles. Based on their PPI network, TOP2A was connected with most of other genes and was selected for further analysis. Kaplan-Meier overall survival analysis (OS) revealed that TOP2A was associated with worse NSCLC patients survival. And both GEPIA analysis and immunohistochemistry experiment (IHC) confirmed that TOP2A was aberrant gain of expression in cancer comparing to normal tissues. The clinical significance of TOP2A and probable signaling pathways it involved in were further explored, and a positive correlation between TOP2A and TPX2 expression was found in lung cancer tissues. Conclusion Using bioinformatic analysis, we revealed that TOP2A could be adopted as a prognostic indicator of NSCLC and it potentially regulate cancer development through co-work with TPX2. However, more detailed experiments are needed to clarify its drug target role in clinical medical use.

目的 体外培养条件下,比较肝肿瘤细胞系C3A和HepG2的代谢功能与蛋白合成能力,以评估其作为生物型人工肝种子细胞的可行性,为生物型人工肝的研究打下基础,为临床医学及组织工程的发展做出贡献.方法 2种肝细胞在同样处理条件下,体外培养1周,观察2种细胞生物学特性,包括细胞形态、大小等,检测细胞对安定代谢和白蛋白的合成情况.结果 在体外培养条件下,2种肝细胞都生长良好,C3A的白蛋白合成功能和安定代谢功能均较HepG2细胞强.结论 2种肝细胞的增殖能力均较好,C3A细胞系的安定代谢功能和白蛋白合功能较HepG2细胞好,但是总体功能仍偏弱,HepG2细胞功能则相对较差,仍需进一步改进.

全球肝癌的发病率居于恶性肿瘤的第五位,其发病率逐年递增.在我国,肝癌合并肝硬化发生率约为70%-85%,多数患者就诊时已处于中晚期,治疗难度大、预后不佳、生存期仅在6个月左右[1]. 我国中晚期肝癌患者多数位于Child-Pugh (CP)分级的B或C级,即不适宜接受手术治疗[2],因此,经导管肝动脉化疗栓塞术 (transcatheter arterial chemoem boliza, TACE)是目前治疗不宜手术的中晚期肝癌的主流技术[3]. 经TACE后肿瘤组织虽然坏死明显, 但同时造成癌旁组织的严重缺血缺氧, 局部促血管生成因子 [以血管内皮生长因子(VEGF)为代表]表达增加. 而VEGF过表达及其信号通路的激活在肿瘤进展和血管生成中发挥重要作用. 因此,如何降低TACE治疗中的促血管生成因子、从而提高TACE的临床效果成为我们需要解决的问题.

目的 通过检测HBsAg阳性母亲分娩的新生儿外周血中T/B淋巴细胞亚群水平,分析HBV宫内传播对新生儿免疫功能的影响.方法 选择2011年1月至2014年12月太原市第三人民医院妇产科HBsAg阳性母亲分娩新生儿220例作为研究对象.采用酶联免疫吸附试验(ELISA)方法检测HBsAg阳性母亲及新生儿外周血HBV血清学标志物,实时荧光定量PCR检测HBsAg阳性母亲及新生儿外周血HBVDNA含量,流式细胞术(FCM)检测新生儿外周血T/B淋巴细胞亚群水平.新生儿出生24 h内静脉血HBsAg阳性或/和HBVDNA值＞103 copies/ml者判定为发生HBV宫内传播.结果 HBsAg阳性母亲分娩新生儿HBV宫内传播发生率为11.36％.宫内传播新生儿组(n=25)与非宫内传播新生儿组(n=195)外周血T淋巴细胞亚群(CD3+、CD4+、CD8+)相对计数[(60.71±13.64)％ vs (60.04±15.06)％,(43.37±12.69)％vs(43.77±13.39)％,(15.03±6.32)％vs(15.14±6.14)％],CD4+/CD8+[(3.42±1.66)％ vs(3.33±1.71)％]及B淋巴细胞中CD19+相对计数[(6.64±3.63)％vs(6.39 ±3.99)％]差异均无统计学意义(P均＞0.05).依据HBsAg阳性母亲分娩新生儿外周血HBVDNA不同载量,分为高载量组(HBVDNA≥107copies/ml)、低载量组(HBVDNA＜ 107 copies/ml)、阴性组(HBVDNA＜ 103 copies/ml).随着HBsAg阳性母亲分娩新生儿外周血HBVDNA载量增加,新生儿外周血CD3+、CD4+、CD8+相对计数及CD4 +/CD8+值逐渐增高,CD19+相对计数比例逐渐减低.高载量组新生儿CD3+、CD4+相对计数高于低载量组和阴性组,差异有统计学意义(P均＜0.05);3组新生儿CD8+、CD19+相对计数及CD4+/CD8+比较差异均无统计学意义(P均＞0.05).结论 随着HBVDNA载量的增加,HBsAg阳性母亲分娩的新生儿外周血中T淋巴细胞亚群(CD3+、CD4+)比例上升,新生儿细胞免疫功能处于活跃状态,易出现自身免疫反应,应采取相应措施防止自身免疫性疾病的发生.

Objective To investigate the expression of miRNA-10a and its function in gastric cancer(GC) and also its genomic promoter DNA methylation.Methods Detected the expression of miRNA-10a in 35 GC patients by real-time ?CR using Taqman probes.The association between miRNA-10a expression and clinicopathological factors was analyzed by SPSS 19.0.DNA methylation status was detected by methylation-specific PCR(MSP).The function of miRNA- 10a on cell proliferation was detected by CCK-8 kit.Results Out of 35 GC patients.the expression of miRNA-10a was down-regulated in 24 cases compared with the adjacent tissues.The low expression of miRNA-10a was significantly associated with gastric cancer TNM stage(P < 0.05).The methylation level in GC tissues was higher than in the matched adjacent non-neoplastic tissues(P < 0.05).0verexpression of miRNA-10a reduced the cell proliferation of GC cell lines.Conclusion miRNA-10a is down-regulated in majority of GC tissues possibly because of the high methylation level of its promoter in GC tissues.The inhibition of miRNA-10a on cell proliferation suggested that it acted as a tumor suppressor in gastric cancer.

Objective To investigate the impacts of miR-33b down-regulation by DNA Methylation in gastric cancer(GC) and the association between miR-33b level and various clinicopathological factors. Methods Expression of miR-33b was detected by Taqman real-time PCR in 42 paired GC samples. The association between miR-33b level and various clinicopathological factors was analyzed. Genomic DNA samples were amplificated by PCR after modification by sodium bisulfite using the EpiTect Bisulfite Kit then the methylation degree of CpG island upstream of miR-33b gene was detected by methylation specific PCR(MSP). Results The lower level expression of miR-33b was not associated with gender,age,venous invasion,position,borrmann typing,pT stage,pN stage,but significantly associated with gastric cancer metastasis(P <0. 05) and regulated by CpG island hypermethylation(P <0. 05). Conclusions miR-33b may be regulated by DNA methylation and act as a tumor suppressor in gastric cancer.

Objective To study the regulation of Erythroid Kruppel-like factor(ELKF) on the expression of miR-96 in Erythropoiesis.Methods K562 cells were induced to erythroid differentiation by Hemin.The expression of miR-96 during erythroid differentiation was detected by real-time PCR.Bioinformatic analysis was used to search for binding sites for EKLF within 2 kb upstream sequence of transcriptional start site(TSS) of miR-96.Biology function of these sites were detected by ChIP-PCR combined with Dual-Luciferase Reporter Assay.Results miR-96 expression was increased during erythroid differentiation of k562 cells induced by Hemin and peaked at 48 h(3.94156±0.63995,P0.05).Several binding sites within 2kb upstream sequence of TSS of miR-96,occupied with EKLF can recruit RNA polymerase Ⅱ(Pol Ⅱ) and promote the expression of downstream genes.ConclusionsEKLF directly activates the expression of miR-96 in Erythropoiesis.