大鼠体内气管损伤修复过程及气管干细胞的定位研究

目的观察大鼠体内气管损伤修复过程,进行气管干细胞的定位.方法应用氟尿嘧啶(5-FU)诱发在体气管上皮损伤,动态观察修复过程;对损伤后气管上皮细胞行Hoechst33342荧光染色,并用RT-PCR法检测ABC转运蛋白ABCG2/bcrp1基因.结果1.5-FU作用30min后大鼠气管上皮细胞绝大部分脱落,可见少量间隔分布的类似裸核的细胞呈钉状位于基底膜上,免疫组化检测增殖细胞核抗原阴性,证明为G0期细胞.其中部分细胞Hoechst33342染色阴性,为侧群(side population,SP)细胞.2.将5-FU 去除3-6h后,上皮细胞形态变为扁平,9-12h 后细胞变为立方,细胞数目逐渐增多,24h上皮细胞数更多,连接成片,可见纤毛,48h 接近恢复假复层纤毛柱状上皮.3.RT-PCR检测ABCG2/Bcrp1阳性反应产物长度为272bp.结论5-FU打击后,残余的G0期气管上皮细胞中含有干细胞.     

Effects of low-intensity pulsed ultrasound on cell viability, proliferation and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells

Low-intensity pulsed ultrasound (LIPUS) acting on induced pluripotent stem cells–derived neural crest stem cells (iPSCs–NCSCs) is considered a promising therapy to improve the efficacy of injured peripheral nerve regeneration. Effects of LIPUS on cell viability, proliferation and neural differentiation of iPSCs–NCSCs were examined respectively in this study. LIPUS at 500 mW cm^?2 enhanced the viability and proliferation of iPSCs–NCSCs after 2 days and, after 4 days, up-regulated gene and protein expressions of NF-M, Tuj1, S100β and GFAP in iPSCs–NCSCs whereas after 7 days expression of only NF-M, S100β and GFAP were up-regulated. LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs–NCSCs in vitro for peripheral nerve tissue engineering.     

应用寡核苷酸芯片筛选维甲酸诱导神经母细胞瘤细胞系SH-SY5Y分化成神经元过程中的差异表达基因

目的:应用寡核苷酸芯片筛选维甲酸(RA)诱导神经母细胞瘤细胞系SH-SY5Y分化成神经元过程中的差异表达基因。方法:从人胎脑及不同类型神经系统肿瘤组织中获取目的基因,查询相应基因mRNA序列,设计并合成探针,制备了含218种基因的神经功能相关的寡核苷酸芯片。应用RA诱导SH-SY5Y8d分化成成熟神经元,提取对照组和实验组每天的总RNA,通过逆转录荧光标记cDNA探针并与芯片杂交,洗片后扫描获取图像,数据分析获得差异表达基因,并通过RT-PCR进行验证。结果:发现13种基因表达上调,没有得到下调基因。RT-PCR验证结果基本与芯片结果一致。结论:SH-SY5Y经RA诱导分化成神经元存在一些差异表达的基因,寡核苷酸芯片技术可为研究SH-SY5Y诱导分化成神经元的分子作用机理提供技术平台。     

沉默DNA-PKcs对细胞信号转导相关基因转录的影响

利用RNA干扰技术构建DNA-PKcs表达抑制细胞模型,探讨DNA-PKcs对HeLa细胞信号转导相关基因表达的调控作用.通过观察细胞对辐射及顺铂的敏感性,鉴定细胞表型变化.用寡核苷酸芯片检测细胞信号转导相关基因的转录谱,并用RT-PCR方法和SEAP检测系统进一步验证基因的表达变化.所筛选出的DNA-PKcs表达抑制细胞对辐射及顺铂的敏感性升高,15个与细胞信号转导相关的基因表达升高,其中7个是与干扰素信号转导反应相关的基因.8个表达下降,包括有细胞增殖分化相关基因,如NFAT.RT-PCR检测结果与芯片结果相一致,利用SEAT报告系统检测,进一步证实NFAT转录活性下调.实验结果表明,DNA-PKcs除了参与DNA修复外,还调控细胞信号转导相关基因的表达,而且大多与细胞增殖分化相关.     

利用GenMAPP筛查鼻咽癌差异表达基因

利用GenMAPP软件对鼻咽癌和正常鼻咽上皮基因微阵列表达谱结果进行分析,筛查鼻咽癌差异表达基因. 结果显示：在17 000个基因中,与正常鼻咽上皮相比,在鼻咽癌中发生2倍以上差异表达的基因共有339个,其中有160个基因在鼻咽癌中表达上调,179个表达下调. 这些基因分别与细胞增殖、基因转录、凋亡、信号转导、DNA损伤修复、肿瘤分化和浸润转移及细胞周期调节等相关. 鼻咽癌的发生发展存在多基因表达调控的改变,对其差异表达基因的研究有助于阐明鼻咽癌发生发展机制.     

小鼠胚胎干细胞分化为肝细胞的基因芯片分析

弄清胚胎肝脏发育的分化调节机制,对指导干细胞在肝再生中的应用以及研究肝分化相关疾病分子机制具有重要意义．胚胎干细胞的全能性使得体外建立肝向分化模型成为可能,采用单层贴壁培养方式,分阶段加入成纤维细胞生长因子(FGF)、肝细胞生长因子(HGF)、制瘤素(OSM)等因子,诱导小鼠胚胎干细胞D3(mESC-D3)的肝向分化．分化细胞在光镜和电镜下呈现肝样细胞形态,RT-PCR、细胞免疫荧光检测以及PAS染色分析表明,这些细胞具有肝细胞特征性的基因表达和生化功能．采用干细胞分化相关基因芯片比较早期肝定向分化前后的基因表达差异,结果显示,48个差异表达基因中(大于2倍),20个上调、28个下调．进一步的生物信息学分析发现,它们集中体现在细胞外基质、细胞连接、FGF、BMP分子及Notch、Wnt信号通路上,提示这些改变可能与胚胎早期的肝向分化密切相关．     

雷公藤甲素对结肠癌细胞SW480 的基因表达谱的影响

目的:探讨雷公藤甲素对结肠癌SW480细胞的基因表达谱的影响。方法:雷公藤甲素处理结肠癌SW480细胞24h后,分别提取给药组和空白对照组SW480细胞总RNA,纯化并逆转录成用Cy3和Cy5标记的cDNA探针,经全基因芯片杂交,洗涤,通过生物信息学方法分析雷公藤甲素处理组和空白对照组SW480细胞基因表达谱的差异。结果:与空白对照组比较,共发现了902个差异基因,雷公藤甲素处理组有196个基因上调,706个基因下调。上调基因主要涉及细胞代谢。下调基因主要涉及wnt通路、细胞周期通路、Toll样受体通路以及MAPK等通路。结论:雷公藤甲素能导致结肠癌细胞基因表达谱的改变,这些基因改变可能参与了细胞增殖、分化、凋亡等过程。这些信息可能为探讨雷公藤抗结肠癌作用机制提供线索。     

Rv0646c,an esterase from <Emphasis Type="Italic">M. tuberculosis</Emphasis>, up-regulates the host immune response in THP-1 macrophages cells

5-Fluorouracil (5-FU) is a widely used chemotherapy agent for breast cancer, although drug resistance is a critical issue regarding the use of this agent in the disease. Calcium signaling is a well-known main cause of proliferation and apoptosis in breast cancer cells. Although previous studies have implicated TRPV1 inhibitor, anticancer, and apoptotic roles of Hypericum perforatum (HPer) in several cells, the synergistic inhibition effects of HPer and 5-FU in cancer and the stimulation of ongoing apoptosis have not yet been clarified in MCF-7 cells. Therefore, we investigated the apoptotic and antioxidant properties of 5-FU with/without HPer through activation of TRPV1 in MCF-7 cells. The MCF-7 cells were divided into four groups: the control group, the HPer-treated group (0.3 mM), the 5-FU-treated group (25 μM), and the 5-FU+HPer-treated group. The intracellular free calcium ion concentration ([Ca²⁺]_i) increased with 5-FU treatments, but they decreased with the HPer and HPer+5-FU treatments. The [Ca²⁺]_i is further decreased in the four groups by TRPV1 channel antagonist (capsazepine and 0.01 mM) treatments. However, mitochondrial membrane depolarization and apoptosis levels, and the PARP1, caspase 3, and caspase 9 expression levels were increased by 5-FU treatment, although the values were decreased by the HPer and 5-FU+HPer treatments. Cell viability level was also decreased by 5-FU treatment. In conclusion, antitumor and apoptosis effects of 5-FU are up-regulated by activation of TRPV1 channels, but its action was down-regulated by HPer treatment. It seems that HPer cannot be used for increasing the antitumor effect of 5-FU through modulation of the TRPV1.     

Identification of a potential ovarian cancer stem cell gene expression profile from advanced stage papillary serous ovarian cancer

Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.     

Differential gene expression in retinoic acid-induced differentiation of acute promyelocytic leukemia cells, NB4 and HL-60 cells

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation t(15;17), which results in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor alpha gene (RARalpha). APL can be effectively treated with the cell differentiation inducer all-trans retinoic acid (ATRA). NB4 cells, an acute promyelocytic leukemia cell line, have the t(15;17) translocation and differentiate in response to ATRA, whereas HL-60 cells lack this chromosomal translocation, even after differentiation by ATRA. To identify changes in the gene expression patterns of promyelocytic leukemia cells during differentiation, we compared the gene expression profiles in NB4 and HL-60 cells with and without ATRA treatment using a cDNA microarray containing 10,000 human genes. NB4 and HL-60 cells were treated with ATRA (10(-6)M) and total RNA was extracted at various time points (3, 8, 12, 24, and 48h). Cell differentiation was evaluated for cell morphology changes and CD11b expression. PML/RARalpha degradation was studied by indirect immunofluoresence with polyclonal PML antibodies. Typical morphologic and immunophenotypic changes after ATRA treatment were observed both in NB4 and HL-60 cells. The cDNA microarray identified 119 genes that were up-regulated and 17 genes that were down-regulated in NB4 cells, while 35 genes were up-regulated and 36 genes were down-regulated in HL60 cells. Interestingly, we did not find any common gene expression profiles regulated by ATRA in NB4 and HL-60 cells, even though the granulocytic differentiation induced by ATRA was observed in both cell lines. These findings suggest that the molecular mechanisms and genes involved in ATRA-induced differentiation of APL cells may be different and cell type specific. Further studies will be needed to define the important molecular pathways involved in granulocytic differentiation by ATRA in APL cells.     

Genetic networks in nonpermissive temperature-induced cell differentiation of Sertoli TTE3 cells harboring temperature-sensitive SV40 large T-antigen

To identify the molecular basis by which nonpermissive temperature (NPT) induces cell differentiation in Sertoli TTE3 cells harboring temperature-sensitive SV40 large T-antigen, we performed global scale microarray and computational gene network analyses. In TTE3 cells, inactivation of the large T-antigen by a NPT at 39 degrees C led to cell differentiation accompanying elevation of transferrin, a marker for differentiation of Sertoli cells, and CDKN1A, a cyclin-dependent kinase inhibitor. Of the 22,690 probe sets analyzed, NPT down-regulated 498 probe sets and up-regulated 432 probe sets by >2.0-fold. Hierarchical clustering analysis showed six gene clusters. In the down-regulated cluster I, a significant genetic network including fibronectin 1 was associated with cellular growth and proliferation. In up-regulated cluster IV, a significant genetic network including CDKN1A was associated with cellular differentiation. The present results provide additional novel insights into the molecular basis of cell differentiation induced by NPT in cells.     

Differential Gene Expression of Human CD34+ Hematopoietic Stem and Progenitor Cells Before and After Culture

Ex vivo expanded CD34⁺ hematopoietic stem and progenitor cells (HSPCs) have compromised homing and engraftment capacities. To investigate underlying mechanisms for functional changes of expanded HSPCs, we compared gene expression profiling of cultured and fresh CD34⁺ cells derived from cord blood using SMART-PCR and cDNA array: 20 genes were up-regulated while 25 genes were down-regulated in cultured CD34⁺ HSPCs. These differentially expressed genes are involved primarily in proliferation, differentiation, apoptosis, and homing. Revisions requested 27 September 2005; Revisions received 14 December 2005     

力生长因子E肽对成骨细胞增殖及基因表达的影响

为了探索力生长因子羧基端E结构域的后24个氨基酸组成的短肽(MGF-Ct24E)对成骨细胞生物学活性的影响,通过组织块培养法获得大鼠原代成骨细胞,采用MTT法和流式细胞仪检测细胞的增殖及细胞周期分布情况,基因芯片技术检测细胞基因表达谱,并用定量PCR实验验证芯片检测结果。结果显示MGF-Ct24E组的细胞增殖活性明显高于对照组,且在培养第一天促增殖效果最为显著。细胞周期结果显示MGF-Ct24E显著提高了S期和G2/M期的细胞所占比例。基因芯片检测发现差异表达基因共1397个,其中上调922,下调475,且差异表达的基因主要是关于细胞的增殖分化调节,生长因子结合和活性调节等方面。MGF-Ct24E对成骨细胞的这种增殖分化调控提示MGF-Ct24E在促进骨修复方面有着潜在的应用价值。     

5-Fluorouracil treatment after irradiation impairs recovery of bone marrow functions

Summary In mice, persisting radiation-induced growth retardation of hematopoietic tissue suggested that at least part of the surviving stem cells are genetically injured. Additional mitotic stress some time after the radiation insult might remove injured stem cells, thus improving the overall recovery of the irradiated bone marrow.Mice were treated with 5 Gy whole-body gamma irradiation. Two weeks later half of the animals were injected i.v. with 150 mg/kg 5-fluorouracil (5-FU), the other half remained untreated (5 Gy-controls). 2 or 10 weeks later, femoral cellularity and CFU-S content, proliferation ability of transplanted bone marrow and the compartment ratio (CR; ratio of splenic IUdR incorporation at day 3 and number of CFU-S transfused) were determined.Four weeks after 5 Gy and 2 weeks after 5-FU treatment all parameters showed significant impairment of recovery. 12 weeks after 5 Gy and 10 weeks after 5-FU CFU-S and CR were still reduced compared to the 5 Gy-controls. 5-FU treatment of unirradiated mice did not produce permanent effects on the quality of stem cells or the hematopoietic microenvironment. It is concluded, therefore, that an increased proliferation stimulus does not aid in the removal of injured CFU-S and may even impair recovery of bone marrow functions by increasing the proportion of genetically injured stem cells which continue proliferation.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday