人甲状腺髓样癌TT细胞胃动素基因表达的调控(英文)

目的:研究人甲状腺髓样癌TT细胞系中胃动素基因表达调控。方法:通过人甲状腺髓样癌TT细胞体外培养,观察经cAMP,生长激素或甲状腺雌激素诱导后,胃动素表达的改变以及胃动素对TT细胞生长、侵袭和转移的影响。结果:甲状腺髓样癌TT细胞表达胃动素mRNA,胃动素可抑制TT细胞的生长。当胃动素基因沉默后,TT细胞转移和侵袭能力增加。当TT细胞分别经cAMP、胃动素、生长激素或甲状腺刺激素孵育48小时后,胃动素基因转录增加,降钙素基因相关肽与胃动素mRNA比值持续下降。环磷酸腺苷可降低TT细胞增殖和c-myc基因的表达。结论:人甲状腺髓样癌细胞生长活性可能与甲状腺C细胞(低的降钙素基因相关肽与胃动素比率)分化的表型有关。     

人甲状腺髓样癌TT细胞胃动素基因表达的调控

目的:研究人甲状腺髓样癌TT细胞系中胃动素基因表达调控.方法:通过人甲状腺髓样癌TT细胞体外培养,观察经cAMP,生长激素或甲状腺雌激素诱导后,胃动素表达的改变以及胃动素对TT细胞生长、侵袭和转移的影响.结果:甲状腺髓样癌TT细胞表达胃动素mRNA,胃动素可抑制TT细胞的生长.当胃动素基因沉默后,TT细胞转移和侵袭能力增加.当TT细胞分别经cAMP、胃动素、生长激素或甲状腺刺激素孵育48小时后,胃动素基因转录增加,降钙素基因相关肽与胃动素mRNA比值持续下降.环磷酸腺苷可降低TT细胞增殖和c-myc基因的表达.结论:人甲状腺髓样癌细胞生长活性可能与甲状腺C细胞(低的降钙素基因相关肽与胃动素比率)分化的表型有关.     

Gene Transfer and Genome-Wide Insertional Mutagenesis by Retroviral Transduction in Fish Stem Cells

Retrovirus (RV) is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM) to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP) and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT) via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES) cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.     

人工转录因子促进外源基因在CHO细胞中的高效表达

以酵母转录因子GAL4中1-147位氨基酸序列为构建人工转录因子的DNA结合结构域,单纯疱疹病毒转录激活子VP16中12肽(DALDDFDLDMLG)的4个串联重复作为人工转录因子的功能结构域,用SV40的核定位序列(NLS)将两部分连接起来,构建了人工转录因子GVP4并将其克隆进入表达载体pcDNA3·1/Hygro( )中。将不同长度的人工转录因子结合序列构建在外源基因表达载体pcDNA3·1( )启动子CMV的上游,分别连接外源基因EGFP和tPA。用表达人工转录因子和外源基因的载体共转染CHO细胞,EGFP和tPA在含不同数量人工转录因子结合位点表达载体pcDNA3·1( )转染的CHO细胞中的表达水平呈现不同程度的提高。其中,以引入10个人工转录因子结合位点的表达载体的效果最明显,EGFP和t-PA的表达效率均提高2~3倍。结果表明,人工转录因子能有效地促进外源基因在哺乳动物细胞中的表达。     

胚胎干细胞的基因转录调控

胚胎干细胞作为一种具有多潜能性和自我更新能力的细胞,在人类等高等生物发育中占有重要地位;基于这一特性,胚胎干细胞在临床上具有极其广阔的应用前景。转录因子OCT4、SOX2和NANOG通过调节胚胎干细胞的基因转录,对其多潜能性和自我更新能力具有关键性的调控作用。对这一作用机制的研究,将对人类早期发育的了解和胚胎干细胞的临床应用具有积极意义。     

A Method to Evaluate Genome-Wide Methylation in Archival Formalin-Fixed,Paraffin-Embedded Ovarian Epithelial Cells

Background

The use of DNA from archival formalin and paraffin embedded (FFPE) tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue.

Methodology/Principal Findings

Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E).Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR) amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample.

Conclusions/Significance

We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples.     

Diversity of Gene Expression in Hepatocellular Carcinoma Cells

Understanding tumor diversity has been a long-lasting and challenging question for researchers in the field of cancer heterogeneity or tumor evolution. Studies have reported that compared to normal cells, there is a higher genetic diversity in tumor cells, while higher genetic diversity is associated with higher progression risks of tumor. We thus hypothesized that tumor diversity also holds true at the gene expression level. To test this hypothesis, we used t-test to compare the means of Simpson's diversity index for gene expression(SDIG) between tumor and non-tumor samples.We found that the mean SDIG in tumor tissues is significantly higher than that in the non-tumor or normal tissues(P 0.05) for most datasets. We also combined microarrays and next-generation sequencing data for validation. This cross-platform and cross-experimental validation greatly increased the reliability of our results.     

Nanog 基因在卵巢癌和卵巢肿瘤干细胞中的表达及意义

目的:探讨胚胎干细胞关键因子Nanog基因mRNA及其蛋白在卵巢癌和卵巢癌肿瘤干细胞中的表达及意义。方法:选取10例正常卵巢上皮组织、10例卵巢良性肿瘤及60例卵巢癌组织,采用逆转录酶-聚合酶链反应(RT-PCR)方法和免疫组织化学PV-6000两步法检测Nanog mRNA和蛋白表达水平;采用无血清悬浮培养法从SKOV-3卵巢癌细胞株中分离培养肿瘤干细胞,流式细胞术鉴定肿瘤干细胞CD117表达,采用RT-PCR和Western Blot方法检测SKOV-3卵巢癌细胞及肿瘤干细胞中NanogmRNA及其蛋白的表达水平。结果:Nanog mRNA在卵巢癌组织中的表达水平均高于正常卵巢组织和卵巢良性肿瘤组织(P<0.05);Nanog mRNA在不同分化程度及临床分期的卵巢癌组织中表达水平不同,低分化组高于高分化组(P<0.05);III-IV期高于I-II期(P<0.05);免疫组化结果同RT-PCR。从SKOV-3卵巢癌细胞株中成功分离出肿瘤干细胞,SKOV-3卵巢癌细胞和肿瘤干细胞Nanog mRNA相对含量分别为0.6044±0.0368,0.8736±0.0537,差异具有统计学意义(P<0.05),两种细胞Nanog蛋白相对含量分别为0.6364±0.0169 1.2788±0.0314,差别具有统计学意义(P<0.05)。结论:Nanog基因在卵巢癌组织和SKOV-3细胞系中均高表达,其在组织中的表达强度与临床分期及病理分级关系密切,且在肿瘤干细胞中表达高于一般卵巢癌细胞,其与卵巢癌的发生发展关系密切,可能是卵巢癌干细胞的表面标志物,有望成为新的标志物。     

Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.