野生型P53、P16基因协同对肺腺癌细胞系生长抑制作用的研究

为了探讨野生型P53基因及P16基因在恶性肿瘤基因治疗中的作用,用腺病毒为载体将野生型P53基因转入高、低转移的肺腺癌细胞系Anip973、AGZY83-a和经野生型P16基因质粒转染的高、低转移肺腺癌细胞系Anip973（Anip973P16）、AGZY83-a（AGZY83-aP16）。对各组转染细胞进行生长曲线、MTT生长抑制率、原位末端标记、Western-blotting等技术检测分析。结果发现（1）野生型P53蛋白的过表达对上述肺腺癌细胞系均呈现出较强的生长抑制作用。（2）野生型P53蛋白的过表达对高转移肺癌细胞系Anip973的抑制作用明显高于低转移细胞系AGZY83-a。（3）野生型p53蛋白的过表达对经野生型P16基因转染的高、低转移的肺癌细胞Anip973、AGZY83-a抑制作用明显高于未经P16基因转染的细胞。野生型P53基因可以作为肺腺癌基因治疗的候选基因。肿瘤抑制基因P53、P16的联合转染可能是对肺腺癌进行基因治疗的有效手段。 Abstract：To investigate the suppression effect of tumor suppressor genes in lung adenocarcinoma cell lines,we transferred a pair of lung adenocarcinoma cell lines with different metastasis potential,Anip973(High-metastasis potential cell line) and AGZY83-a (Low-metastasis potential cell line)and this pair of cell lines transfected with P16 gene:AGZY83-a P16 and Anip973 P16 with wild type P53 gene with adenovirus vector.The suppression effects of P53 gene were evaluated by cell growth curve,MTT,western-blotting analysis and TUNEL technique.Overexpression of wild-type P53 gene in AGZY83-a,Anip973,Anip973 P16 and AGZY83-a P16 inhibited the growth of these four kinds of lung cancer cells and induced apoptosis of the cells.The suppression effect of P53 gene in Anip973 and Anip973 P16 was higher than AGZY83-a and AGZY83-a P16 while co-expression of P53 and　P16 in this pair of cell lines inhibited the cells more efficiently comparing with the expression of P53 alone.Wild-type P53 gene might act as a candidate gene in lung adenocarcinoma gene therapy while co-transfection of P53 and P16 genes was a more effective method.     

一个与非小细胞肺癌转移相关的基因――RAB5A基因

采用mRNA差异展示技术（mRNA DD）研究具有相同细胞来源,但转移能力高低不同的人肺腺癌细胞系AGZY83-a（低转移）和Anip973(高转移),分析在两个细胞系中基因差异表达的情况,发现在高转移细胞系中有RAB5A基因的表达。该基因为蛋白质入胞信号的调控者,为RAS超家族成员。为进一步证实其转录表达的调控改变情况,以及RAB5A高表达的临床意义,进一步采用RT-PCR和免疫组织化学的方法检测了50例临床非小细胞肺癌的手术标本,结果表明,RAB5A的表达有随转移发生而增强的趋势,而RAB5A的蛋白表达程度在有转移的病例中明显增强(P<0.05)。 Abstract：　Using mRNA differential display (mRNA DD)techniques, we analyzed the differences of gene expression between two human lung adenocarcinoma cell lines,AGZY83-a and Anip973. Anip973 was isolated from AGZY83-a, but manifested much higher metastatic potential than the parent line. The results showed that there were significant differences on gene expression between the two cell lines and that there was over-expression of RAB5Agene in the Anip973 cell line. The product of RAB5Agene was recognized as signal regulators of endocytotic pathway and protein trafficking at the cell surface, and belong to a member of the RAS superfamily. Furthemore, we compared to the expression of RAB5Agene and RAB5Aprotein in clinical samples of 50 cases non-small lung carcinoma and nearby lymph node by means of RT-PCR and immunohistochemistry method. The results indicated that the high expression of RAB5Ain metastatic tumor and the enhancement level of RAB5Ain metastatic tumor and the enhancement level of expression were corresponded with the increase of metastatic degree. And there were significance of difference on the expression degree of RAB5Aprotein between non-small lung carcinoma with metastasis and non- metastasis (P<0.05).     

不同转移能力的肺腺癌细胞系金属蛋白酶活性分析

为了探讨肺腺癌细胞系中金属蛋白酶的活性与其转移能力之间的相关性,选取9个转移能力不同的肺腺癌细胞系,分别对它们所分泌的金属蛋白酶MMP－2、MMP－9进行活性分析。结果发现,金属蛋白酶的活性与肺腺癌细胞系的转移能力有一定的相关性,其中MMP－9的活性与肺腺癌细胞系的转移相关性更显著。 Abstract:To investigate the relationship between the MMP activities and metastatic ability in human lung adenocarcinoma cell lines with differential metastatic potential.Nine lung adenocarcinoma cell lines were tested for their MMP activities by zymographic analysis methods.The MMP production capabilities of carcinoma cells rose following the increase of their metastatic potentials.The result suggested that there was some relationship between MMP－9 activties and metastatic potential in human lung adenocarcinomas.     

Characterization of mouse clonal mesenchymal stem cell lines established by subfractionation culturing method

AIM:To characterize single-cell-derived mouse clonal mesenchymal stem cells (mcMSCs) established with bone marrow samples from three different mouse strains. METHODS:We established mcMSC lines using subfractionation culturing method from bone marrow samples obtained from long bones.These lines were characterized by measuring cell growth, cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability. Nonclonal MSCs isolated by the conventional gradient centrifugation method were used as controls. RESULTS:All mcMSC lines showed typical nonclonal MSC-like spindle shape morphology. Lines differed inoptimal growth density requirement.Cell surface epitope prof iles of these mcMSC lines were similar to those of nonclonal MSCs. However, some lines exhibited different expression levels in a few epitopes, such as CD44 and CD105. Differentiation assays showed that 90% of the mcMSC lines were capable of differentiating into adipogenic and/or chondrogenic lineages, but only 20% showed osteogenic lineage differentiation. T-cell suppression analysis showed that 75% of the lines exhibited T-cell suppression capability. CONCLUSION:mcMSC lines have similar cell morphology and cell growth rate but exhibit variations in their cell surface epitopes, differentiation potential, lineage-specifi c gene expression and T-cell suppression capability.     

Choline Transporters in Human Lung Adenocarcinoma： Expression and Functional Implications

Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN 1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H 1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential ＂choline-starvation＂ strategy of cancer interference through targeting choline transporters, especially CTL1.     

N-Myc downstream-regulated gene 2 (NDRG2) is a negative regulator of cell growth and its expression is decreased in cancers

Using subtraction cloning, we identified the human N-Myc Downstream- Regulated Gene-2 (hNDRG2), located at 14q11.2, as a candidate tumor suppressor gene. Semi-quantitative RT-PCR showed that the expression of hNDRG2 in 15 of 27 (56%) human GBM tissues and all 6 human glioblastoma cell lines was significantly lower than that in the normal brain. The expression of hNDRG2 also was evaluated in 60 lung-carcinoma patients. 17 of 26 cases of squamous carcinoma and 4 of 11 cases of small cell lung cancer displayed     

Suppression of type 1 Insulin-like growth factor receptor expression by small interfering RNA inhibits A549 human lung cancer cell invasion in vitro and metastasis in xenograft nude mice

Cancer invasion and metastasis, involving a variety of pathological processes andcytophysiological changes,contribute to the high mortality of lung cancer.The type 1 insulin-like growthfactor receptor (IGF-1R),associated with cancer progression and invasion,is a potential anti-invasion andanti-metastasis target in lung cancer.To inhibit the invasive properties of lung cancer cells,we successfullydown-regulated IGF-1R gene expression in A549 human lung cancer cells by small interfering RNA (siRNA)technology,and evaluated its effects on invasion-related gene expression,tumor cell in vitro invasion,andmetastasis in xenograft nude mice.A549 cells transfected with a plasmid expressing hairpin siRNA forIGF-1R showed a significantly decreased IGF-1R expression at the mRNA level as well as the proteinlevel.In biological assays,transfected A549 cells showed a significant reduction of cell-matrix adhesion,migration and invasion.Consistent with these results,we found that down-regulation of IGR-1Rconcomitantly accompanied by a large reduction in invasion-related gene expressions,including MMP-2,MMP-9,u-PA,and IGF-1R specific downstream p-Akt.Direct tail vein injections of plasmid expressinghairpin siRNA for IGF- 1R significantly inhibited the formation of lung metastases in nude mice.Our resultsshowed the therapeutic potential of siRNA as a method for gene therapy in inhibiting lung cancer invasionand metastasis.     

Profiling of differentially expressed genes in LRRC4 overexpressed glioblastoma cells by cDNA array

Our previous study has shown that LRRC4 is a novel member of the leucine-rich repeat （LRR） superfamily and has the potential to suppress brain tumor growth. In order to further analyze the functions of LRRC4 on the maintenance of normal function and suppression of tumorigenesis in the central nervous system, we investigated alterations in gene expression related to neurobiology by the Atlas array in two inducible dual-stable LRRC4-overexpressing cell lines. Seventeen of 588 genes spotted on the Atlas membrane showed altered expression levels in LRRC4 transfected U251MG Tet-on cells, which are involved in cell proliferation and cell cycle progression, tumor invasion and metastasis, and neurotransmitter synthesis and release. In addition, cell invasion assay results showed that LRRC4 can inhibit the U251MG cell migration. These studies represent the first cDNA array analysis of the effects of LRRC4 on the involvement of different neurobiological genes in U251MG glioblastoma cells and provide new insights into the function of LRRC4 in glioma.     

MiRNA-218, a new regulator of HMGB1, suppresses cell migration and invasion in non-small cell lung cancer

MieroRNAs （miRNAs） function as negative regulators of gene expression involved in cancer metastasis. The aim of this study is to investigate the potential roles of miR-218 in non-small cell lung cancer and validate its regulation mech- anism. Functional studies showed that miR-218 overexpres- sion inhibited cell migration and invasion, but had no effect on cell viability. Enhanced green fluorescent protein reporter assay, real-time polymerase chain reaction and western blot analysis confirmed that miR-218 suppressed the expression of high mobility group box-1 （HMGB1） by directly targeting its 31-untranslated region. Accordingly, silencing of HMGBI accorded with the effects of miR-218 on cell migration and invasion, and overexpression of HMGB1 can restore cell migration and invasion which were reduced by miR-218. In conclusion, these findings demon- strate that miR-218 functions as a tumor suppressor in lung cancer. Furthermore, miR-218 may act as a potential thera- peutic biomarker for metastatic lung cancer patients.     

Analysis of pathway activity in primary tumors and NCI60 cell lines using gene expression profiling data

To determine cancer pathway activities in nine types of primary tumors and NCI60 cell lines, we applied an in silico approach by examining gene signatures reflective of consequent pathway activation using gene expression data. Supervised learning approaches predicted that the Ras pathway is active in ～70% of lung adenocarcinomas but inactive in most squamous cell carcinomas, pulmonary carcinoids, and small cell lung carcinomas. In contrast, the TGF-β, TNF-α, Src, Myc, E2F3, and β-catenin pathways are inactive in lung adenocarcinomas. We predicted an active Ras, Myc, Src, and/or E2F3 pathway in significant percentages of breast cancer, colorectal carcinoma, and gliomas. Our results also suggest that Ras may be the most prevailing oncogenic pathway. Additionally, many NCI60 cell lines exhibited a gene signature indicative of an active Ras, Myc, and/or Src, but not E2F3, β-catenin, TNF-α, or TGF-β pathway. To our knowledge, this is the first comprehensive survey of cancer pathway activities in nine major tumor types and the most widely used NCI60 cell lines. The "gene expression pathway signatures" we have defined could facilitate the understanding of molecular mechanisms in cancer development and provide guidance to the selection of appropriate cell lines for cancer research and pharmaceutical compound screening.     

Origins, lineage-specific expansions, and multiple losses of tyrosine kinases in eukaryotes

Tyrosine kinases are important components of metazoan signaling pathways, and their mutant forms are implicated in various malignancies. Searching the sequences from the genomes of 28 eukaryotes and the GenBank, we found tyrosine kinases not only in metazoans but also in the green algae Chlamydomonas reinhardtii, the potato late blight pathogen Phytophthora infestans, and the protozoan pathogen Entamoeba histolytica, contrary to the current view that tyrosine kinases are animal-specific. Based on a phylogenetic analysis, we divided this gene family into 43 subfamilies and found that at least 19 tyrosine kinases were likely present in the common ancestor of chordates, arthropods, and nematodes. Interestingly, most of the subfamilies have conserved domain organizations among subfamily members but have undergone different degrees of expansion during the evolution of metazoans. In particular, a large number of duplications occurred in the lineage leading to the common ancestor of Tagifugu and mammals after its split from the Ciona lineage about 450 to 550 MYA. The timing of expansion coincides with proposed large-scale duplication event in the chordate lineage. Furthermore, gene losses have occurred in most subfamilies. Interestingly, different subfamilies have similar net gain rates in the chordates studied. However, the tyrosine kinases in mouse and human or in fruit fly and mosquito mostly have a one-to-one relationship between species, indicating that static periods of 90 Myr or longer in tyrosine kinase evolution have followed large expansion events.     

A roadmap of tandemly arrayed genes in the genomes of human, mouse, and rat

Tandemly arrayed genes (TAGs) play an important functional and physiological role in the genome. Most previous studies have focused on individual TAG families in a few species, yet a broad characterization of TAGs is not available. Here we identified all TAGs in the genomes of humans, mouse, and rat and performed a comprehensive analysis of TAG distribution, TAG sizes, TAG orientations and intergenic distances, and TAG functions. TAGs account for about 14-17% of all genes in the genome and nearly one-third of all duplicated genes, highlighting the predominant role that tandem duplication plays in gene duplication. For all species, TAG distribution is highly heterogeneous along chromosomes and some chromosomes are enriched with TAG forests, whereas others are enriched with TAG deserts. The majority of TAGs are of size 2 for all genomes, similar to the previous findings in Caenorhabditis elegans, Arabidopsis thaliana, and Oryza sativa, suggesting that it is a rather general phenomenon in eukaryotes. The comparison with the genome patterns shows that TAG members have a significantly higher proportion of parallel gene orientation in all species, corroborating Graham's claim that parallel orientation is the preferred form of orientation in TAGs. Moreover, TAG members with parallel orientation tend to be closer to each other than all neighboring genes in the genome with parallel orientation. The analyses of Gene Ontology function indicate that genes with receptor or binding activities are significantly overrepresented by TAGs. Computer simulation reveals that random gene rearrangements have little effect on the statistics of TAGs for all genomes. Finally, the average proportion of TAGs shows a trend of increase with the increase of family sizes, although the correlation between TAG proportions in individual families and family sizes is not significant.     

南京市正常人群NQO1、CYP1A1、mEH基因的多态性研究

应用PCR技术,对南京市正常人群中NQO1、CYP1A1、mEH-外显子3、mEH－外显子4基因型多态性进行了研究。88例样本中,相关基因野生型纯合子（wt/wt）、杂合子（wt/vt）、突变型纯合子（vt/vt）三种基因型的频率分布及基因频率分别是：NQO1 29.5%（0.304）,51.1%(0.495)和19.3%(0.202);CYP1A?135.2%(0.329)、44.3%(0.489)和20.5%(0.181);mEH－外显子3为26.1%(0.297),56.8%(0.496),17.0%(0.207);mEH－外显子4为83.0%(0.826),15.9%(0.165),1.1%(0.008)。以上结果与国外的有关报道存在一定差异,在不同地区中国人群的频率分布特征基本一致,种族差异可能是造成有关基因型分布差异的重要原因。 Abstract：The polymorphisms of NQO1, CYP1A1, mEH-Exon3 ,and mEH-Exon4 genes in normal Nanjing population (88 cases) were investigat ed by PCR approach. The results showed that the population frequency distributio ns of genotypes of wild-type,heterozygote, homozygous variant were respectively: NQO1? 29.5%,51.1%,19.3%;CYP 1A1 35.2%,44.3%,20.5%;mEH-exon3 26.1 %,56.8%,17.0%;mEH-exon4 83.0%,15.9%,1.1%. The frequency distributions o f genotypes in Nanjing population differ from those of other countries and do no t show marked differences compared with other different area in Chinese populati on. The ethnic difference might be an important reason which results in the diff erences of related genotypes.     

人vasostatin的克隆、表达、纯化及活性检测

从成人肝脏cDNA文库中,PCR扩增得到人vasostatin基因编码区序列,将此序列插入原核表达载体pQE30进行表达,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定表明产物以包涵体形式存在,表达量占菌体总蛋白量的50%以上.包涵体洗涤后溶于8 mol/L尿素溶液,在变性条件下通过镍-氨三乙酸(Ni-NTA)金属螯合亲和层析柱进行纯化后,再经透析进行复性.N端氨基酸序列、分子质量、等电点等理化指标的测定结果与理论值相符.用内皮细胞增殖试验、内皮细胞迁移试验以及鸡胚尿囊膜血管生成试验等方法进行活性检测,证实复性的表达产物具有抑制内皮细胞增殖和迁移、抑制鸡胚尿囊膜血管生成的功能.     

基因敲除与学习、记忆：现状、问题和展望

基因敲除技术的应用使学习、记忆分子机制的研究出现了新的突破.目前已报道了多种学习、记忆以及LTP、LTD有缺陷的基因敲除动物,发现多种基因在学习、记忆的形成过程中必不可少.然而,现有研究的一个较大问题是忽视了遗传背景基因在表型改变中的作用,被认为由突变靶基因造成的表型缺陷实际上可能是由背景基因而不是由突变基因造成的.要排除背景基因的作用,必须建立新的ES细胞,选择纯遗传背景的小鼠品系,并且在时间、范围和程度上对基因敲除进行精细的控制.     

水稻抗白叶枯病基因的鉴定、定位和克隆进展

由水稻黄单胞菌水稻变种Xoo引起的水稻白叶枯病是全球性的重要病害之一。已有31个水稻白叶枯抗性基因被鉴定并报道,其中18个被定位到染色体上,5个被克隆。简要综述了水稻白叶枯抗性基因的鉴定、定位和克隆的进展,并讨论了合理利用抗性基因防治白叶枯病的前景。     

家蚕chi、gluE和fruA基因与微生物相应基因的同源性及基因水平转移初探

通过对家蚕(Bombyxm mori)大规模EST的分析,发现家蚕的chi、gluE和fruA基因分别与微生物的相应基因存在高度的氨基酸序列同源性,且进化关系很近,但与线虫(Caenorhabditis elegans)、果蝇(Drosophila melanogaster)、按蚊(Anopheles gamble)以及家蚕近缘昆虫的类似基因之间的相似性却非常低。这表明它们可能分别与微生物的同源基因具有共同的祖先,即微生物的基因水平转移给了家蚕,进化途径不属于垂直遗传。