一种基于基因表达模型识别酵母细胞周期条件特异调控子网的方法(英文)

由高通量微阵列技术产生的数据集可以用于解释生物系统基因调控的未知机制.生物过程是动态的,所以很有必要关注某些条件下特异的基因调控子网络.细胞周期是一个基本的细胞过程,识别酵母的细胞周期特异调控子网是理解细胞周期过程的基础,并且有助于揭示其他细胞条件的基因调控机理.使用一个基因表达微分方程模型(GEDEM),从静态网络中识别了动态的细胞周期相关调控关系.与已经报道的细胞周期相关调控相互作用相比,该方法识别了更多的真实存在的条件特异调控关系,取得了比当前的方法更好的性能.在大数据集上,GEDEM 识别了具有高敏感性和特异性的调控子网.组合调控的深入分析显示,条件特异调控子网的转录因子之间的相关性呈现出比静态网络中转录因子相关性更强,这说明条件特异网络比静态网络更加接近真实情况.另外,GEDEM 方法还识别更多潜在的共调控转录因子.

具有时滞的基因表达模型的稳定性与分支分析

应用频域法研究了一类具有三个时滞的基因表达模型的Hopf分支问题.基于Nyquist稳定性准则和Hopf分支定理,选取三个时滞的和τ作为分支参数,发现当τ超过某个临界值时,系统产生了Hopf分支.最后,对系统进行了数值仿真,数值仿真的结果验证了理论分析的正确性.

一类具有状态依赖时滞的基因表达模型的分支分析

研究了一类具有状态依赖时滞的基因表达模型的Hopf分支问题．基于Rouche定理和Hopf分支定理，选取状态依赖时滞丁作为分支参数，发现当τ通过一系列临界值时，系统出现了Hopf分支．最后，对系统进行了数值仿真，数值仿真的结果验证了理论分析的正确性．

完整基因表达模型中的精确概率分布

基因表达是细胞内部过程的中心环节,其建模与分析一直是人们关注的焦点.转录水平上的两类代表性基因表达模型(即构成式表达模型和爆发式表达模型)已经被广泛研究,特别是m RNA的分布已经解析地给出,但对于同时考虑转录和翻译的完整基因表达模型,如何解析地导出m RNA和蛋白质的分布以及它们的联合分布的问题至今未得到解决.本文应用作者自己发展起来的二项矩方法解决了此数学问题,尤其是给出了基因产物分布及有关统计量的解析表示.此外,本文还讨论了基因表达噪声的可调性.

人内皮高表达脂多糖相关因子1强制表达模型的建立及对ECV304细胞增殖的影响

目的设计和构建新基因内皮高表达脂多糖相关因子1(EOLA1)诱导表达载体,建立EOLA1强制表达模型,观察其对细胞增殖的影响。方法逆转录聚合酶链反应扩增EOLA1开放阅读框片段,引入NoⅠt和XhoⅠ酶切位点,定向亚克隆入可调控真核表达的载体pOPRSVⅠ,测序验证后共转染pOPRSVⅠ-EOLA1和pCMVLacⅠ质粒于ECV304细胞,经潮霉素和G418筛选,获得稳定转染细胞株。对异丙硫半乳糖苷(IPTG)诱导和非诱导的稳定转染细胞株进行计数,绘制细胞生长曲线。结果成功构建了EOLA1可调控真核表达载体pOPRSVⅠ-EOLA1。诱导后第4天,EOLA1稳定表达的ECV304细胞数量为(44±17)×104个,显著多于未诱导细胞的数量(27±11)×104个(P<0.01)。结论强制高表达EOLA1蛋白具有促进ECV304细胞增殖的作用。

Related Study on the Nm23-H1 Gene Expression in the Model of Ovarian Carcinoma Cell Lines with High Frequent Metastasis

Objective: To select the ovarian carcinoma cell lines with high frequent metastasis and study the association between nm23-H1 gene expression and metastasis of ovarian carcinoma. Methods: Each ovarian cancer cell line was transplanted subcutaneously into the flank of nude mice, and the metastatic behavior was evaluated by counting lung tumor foci at different time points. The metastatic tumors were cultured in vitro, then substrain was established and transplanted subcutaneously three times. The RNA level of nm23 in 8 human ovarian cancer cell lines were examined by northern-blot. Results: Of the 8 human ovarian cancer cell lines, 4 had high requent metastatic potentiality. The expression of nm23 RNA in human ovarian cancer cells was inversely related to metastatic behavior in the experimental animals (r=0.96, P=0.0001). Conclusion: The difference of the tendency of metastasis which was determined by genetic and molecular levels was significant among different type of cell lines and subtypes. The expression of nm23 mRNA in human ovarian carcinomas was correlated closely with the reduced metastatic behavior in experimental animals and may serve as a sensitive prognostic indicator for ovarian cancer.

Effects of Wei Chang An on expression of multiple genes in human gastric cancer grafted onto nude mice

AIM： To investigate the expression of multiple genes in Chinese jianpi herbal recipe Wei Chang An （WCA） in human gastric cancer cell line SGC-7901. METHODS： A human gastric adenocarcinoma cell line SGC-7902 grafted onto nude mice was used as the animal model. The mice were randomly divided into 3 groups, one control and the two representing experimental conditions. Animals in the two experimental groups received either WCA over a 34-d period or 5-fluorouracil （5-FU） over 6-d period starting at 8th d after grafting. Control animals received saline on an identical schedule. Animals were killed 41 d after being grafted. The expression profiles in paired WCA treated gastric cancer samples and the N.S. control samples were studied by using a cDNA array representing 14181 cDNA clusters. The alterations in gene expression levels were confirmed by Real-time Quantitative polymerase chain reaction （qPCR）. RESULTS： When compared with controls, the average tumor inhibitory rate in WCA group was 44.32% ± 5.67% and 5-FU 47.04% ± 22.33% （P 〈 0.01, respectively）. The average labeling index （LI） for PCNA in WCA group and 5-FU group was significantly decreased compared with the control group. Apoptotic index （AI） was significantly increased to 9.72% ± 4.52% using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling （TUNEL） method in WCA group compared with the controls 2.45% ± 2.37%. 5-FU group was also found to have a significantly increased AI compared with the controls. The expression of cleaved Caspase-3 in WCA group and 5-FU group was significantly increased compared with the control group respectively. There were 45 different expressed sequence tags （ESTs） among the control sample pool and WCA sample pool. There were 24 ESTs up-regulated in WCA samples and 21 ESTs down-regulated. By using qPCR, the expression level of Stat3, rap2 interacting protein x （RIPX）, regulator of differentiation 1 （ROD1） and Bcl-2 was lower in WCA group than that in control group respectively. By using SP immunohistochemical method the expression of Phospho-Stat3 （Tyr705） and Bcl-2 in WCA group and 5-FU group was significantly decreased compared with the control group respectively. CONCLUSION： WCA could inhibit gastric cancer cell SGC-7901 growth in vivo. WCA could induce gastric cancer cell apoptosis and suppress proliferation. Its mechanisms might be involved in the down-regulation of Star3, RIPX, ROD1 and Bcl-2 gene.

Establishment and primary application of a mouse model with hepatitis B virus replication

AIM： To establish a rapid and convenient animal model with hepatitis B virus （HBV） replication.METHODS： A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen （HBcAg） and hepatitis B surface antigen （HBsAg） in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen （HBeAg） was detected by enzyme- linked immunosorbent assay （ELISA）. Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid （polyIC） or phosphate-buffered saline （PBS）.RESULTS： After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS.CONCLUSION： A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.

Gene expression and MR diffusion-weighted imaging after chemoembolization in rabbit liver VX-2 tumor model

AIM: To investigate the dynamic characteristics and the correlation between PCNA, Bax, nm23, E-cadherin expression and apparent diffusion coefficient (ADC) on MR diffusion-weighted imaging (DWI) after chemoembolization in rabbit liver VX-2 tumor model. METHODS: Forty New Zealand rabbit liver VX-2 tumor models were included in the study. DWI was carried out periodically after chemoembolization. All VX-2 tumor samples in each group were examined by histopathology and Strept Avidin-Biotin Complex (SABC) immunohistochemical staining. RESULTS: The PCNA expression index in VX-2 tumors was higher than in the normal parenchyma around the tumor (P < 0.001). Nm23, Bax or E-caderin expression index in VX-2 tumors were lower than in the normal parenchyma around the tumor (all P < 0.001). PCNAand nm23 expression in the VX-2 tumor periphery first increased and then decreased (P < 0.001 and P = 0.03, respectively), while the expression of Bax and E-cadherin before and after chemoembolization was insignificant. When b-value was 100 s/mm2, there was a linear correlation between PCNA expression and ADC in the area of VX-2 tumor periphery (P < 0.001), and PCNA expression in VX-2 tumor periphery influenced the ADC. CONCLUSION: The potential of VX-2 tumor infiltrating and metastasizing decreases, while its ability to proliferate increases for a short time after chemoembolization. To some degree, the ADC value indirectly reflects the proliferation of VX-2 tumor cells.

Effect of ALA-PDT on the expressions of MMP-9, MMP-13 and TIMP-1 of hypertrophic scar model in rabbit ears

Objective: To investigate the effect of 5-aminolevulinic(ALA)-photodynamic therapy(PDT) on the expressions of MMP-9, MMP-13 and TIMP-1 of hypertrophic scar model in rabbit ears, and analyze the possible therapeutic mechanisms of ALA-PDT treatment to hypertrophic scars of rabbit ears. Methods: The experimental animals were randomly divided into normal control, negative control, high concentration of ALA-PDT, low concentration of ALA-PDT and PDT groups. The latter three groups received ALA-PDT treatment or PDT treatment once a week for 3 weeks. The specimens of the rabbits were collected respectively 1, 2 and 3 months after treatment to be used for RT-PCR and Western-blot test. Results: 1, 2 and 3 months after PDT treatment, the expressions of MMP-9 and MMP-13(including mRNA and protein) in hypertrophic scar tissues of three treatment groups were significantly higher than those of the negative control group(P<0.01), and the expression of TIMP-1 mRNA and protein of three treatment groups were significantly lower than that of the negative control group(P<0.01). There were also significant differences between high-concentration ALA-PDT treatment group and the low one(P<0.05). Conclusion: ALA-PDT is effective in treating hypertrophic scars of rabbit ears, and its possible therapeutic mechanisms are that ALA-PDT treatment generates oxidation activation effect to activate the activity of MMPs and induces the photoaging of fibroblasts of hypertrophic scar tissues of rabbit ears to inhibit the activity of TIMPs, which causes the up-regulation of MMPs and the down-regulation of TIMPs. Because of this, the degradation of collagen and ECM is accelerated and the formation of scars is suppressed.

Toll-like receptor 4 plays an anti-HBV role in a murine model of acute hepatitis B virus expression

AIM： Toll-like receptor 4 （TLR4） has been shown to be important for bacterial infection, especially to lipopolysaccharide signaling. Its possible role in HBV infection is studied in the present study. MATERIALS AND METHODS： pHBV3.6 plasmid, containing full-length HBV genome was used in the murine model of acute HBV expression by hydrodynamics in vivo transfection. TLR4 normal or mutant mouse strain was compared to investigate the possible role of TLR4 in acute HBV expression. RESULTS： After pHBV3.6 injection, the infiltrating leukocytes expressed TLR4 were observed nearby the HBsAg-expressing hepatocytes. The HBV antigenemia as well as the replication and transcription were higher in TLR4-mutant C3H/HeJ mice than in normal C3H/ HeN mice. The HBV-specific immune responses were impaired in the liver or spleen of the C3H/HeJ mice. Their inducible nitric oxide synthase （iNOS） expression on the hepatic infiltrating cells was also impaired. When adoptively transferring splenocytes from C3H/HeN mice to C3H/HeJ mice, the HBV replication was inhibited to the level as that of C3H/HeN. CONCLUSION： These results suggest that TLR4 plays an anti-HBV role in vivo through the induction of iNOSexpression and HBV-specific immune responses after HBV expression.

Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone

The thyroid hormones L-thyroxine and triiodo-L-thyronine have profound effects on postembryonic development of most vertebrates. Analysis of their action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH)or that of other hormones and factors that modulate its action. Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process(morphogenesis, cell death, re-structuring, etc.) in free-living embryos and larvae of most anurans. This article will first describe the salient features of metamorphosis and its control by TH and other hormones. Emphasis will be laid on the key role played by TH receptor (TR), in particular the phenomenon of TR gene autoinduction, in initiating the developmental action of TH. Finally, it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in other vertebrate species.

REGULAR EXPRESSION OF DISCOIDIN DOMAIN RECEPTOR 2 IN THE IMPROVED ADJUVANT-INDUCED ANIMAL MODEL FOR RHEUMATOID ARTHRITIS

Objective To investigate the expression of discoidin domain receptor 2 (DDR2) of fibroblast-like synovial cells in im- proved adjuvant-induced animal (AIA) model for rheumatoid arthritis (RA) and to provide evidence for DDR2’s antagonist use clinically. Methods AIA was modified by administrating 0.1 mL of complete Freund’s adjuvant (CFA, mixed with 5 mg Bacillus Calmette-Guerin vaccine/mL) into rats’ right hind paws and 0.125 mL tumor necrosis factor-α (2 U/mL) into right ankles and subpatellar fatty tissue. The expression of DDR2 in fibroblast-like synovial cells was assessed using immunohistochemistry, immunofluorescence histochemistry, and in situ hybridization methods. Levels of anti-collagen II antibody were measured using enzyme-linked immunosorbent assay. Results Given the terms mentioned above, we found a more practical rat model, apparently decreasing immunization time (average 3-5 days). DDR2 can be detected upon the 15th day of immunization; expression gradually increased with time going on, and reaching a peak 35 days after immunization before gradually decreasing. Serum anti-collagen II antibody showed similar expression patterns as DDR2, but reached peak later than DDR2, about 40 days after immunization. Conclusion Regular expression of DDR2 in animal models infers its important role in the pathological process of RA.

Meta-Analysis of the Association between Mir-196a-2 Polymorphism and Cancer Susceptibility

Objective MicroRNA plays a vital role in gene expression, and microRNA dysregulation is involved in carcinogenesis. The miR- 196a-2 polymorphism rs11614913 is reportedly associated with cancer susceptibility. This meta-analysis was performed to assess the overall association of miR-196a-2 with cancer risk. Methods A total of 27 independent case-control studies involving 10,435 cases and 12,075 controls were analyzed for the rs11614913 polymorphism. Results A significant association was found between rs11614913 polymorphism and cancer risk in four genetic models （CT vs. TT, OR-1.15, 95%CI=1.05-1.27; CC vs. TT, OR=1.23, 95%CI=1.08-1.39; Dominant model, OR=1.17, 95%CI=1.06-1.30; Additive model, OR-1.08, 95%CI=1.01-1.14）. In the subgroup analysis of different tumor types, the C allele was associated with increased risk of lung, breast, and colorectat cancer, but not with liver, gastric, or esophageal cancer. In the subgroup analysis by ethnicity, a significantly increased risk of cancer was found among Asians in all genetic models, but no associations were found in the Caucasian subgroup. Conclusions The meta-analysis demonstrated that the miR-196a-2 polymorphism is associated with cancer susceptibility, especially lung cancer, colorectal cancer, and breast cancer among Asian populations.

Screening difFerentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis

AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis.METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed.RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes.CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.

EFFECT OF KANGLEMYCIN C ON LYMPHOKIN A PRODUCTION AND GENE EXPRESSION OF MOUSE SPLENOCYTE AND MACROPHAGE

Aim. To study the effect of kanglemycin C (KC) on production and gene transcription of lymphokins. Methods.Cell proliferation and lymphokin activities were quantified with MTT colorimetry and ELISA, and gene transcriptions of lymphokins semi quantified with RT PCR. Results.Suppression of KC on proliferation of enriched T and B cell respectively mediated by Con A and LPS was declined by addition of exogenous IL 1, IL 2, and IL 6. KC 80 nmol/L markedly inhibited IL 2 and IL 6 production and mRNA transcription of incubated mouse splenocytes induced by Con A. Additionally, KC had some suppression on IL 1β and IL 6 productions of peritoneal macrophage stimulated by LPS (5μg/mL), whereas cyclosporine (CS) had not. [WT5”BX]Conclusion. [WT5”BZ]Immunosuppression of KC came true partially through the decrease of IL 1β, 2 and 6 productions, especially of IL 2. However, CS′s immunosuppression was mainly through the decrease of IL 2 procduction.

A robust statistical procedure to discover expression biomarkers using microarray genomic expression data

Microarray has become increasingly popular biotechnology in biological and medical researches, and has been widely applied in classification of treatment subtypes using expression patterns of biomarkers. We developed a statistical procedure to identify expression biomarkers for treatment subtype classification by constructing an F-statistic based on Henderson method Ⅲ. Monte Carlo simulations were conducted to examine the robustness and efficiency of the proposed method. Simulation results showed that our method could provide satisfying power of identifying differentially expressed genes （DEGs） with false discovery rate （FDR） lower than the given type I error rate. In addition, we analyzed a leukemia dataset collected from 38 leukemia patients with 27 samples diagnosed as acute lymphoblastic leukemia （ALL） and 11 samples as acute myeloid leukemia （AML）. We compared our results with those from the methods of significance analysis of microarray （SAM） and microarray analysis of variance （MAANOVA）. Among these three methods, only expression biomarkers identified by our method can precisely identify the three human acute leukemia subtypes.

ANDROGEN REPRESSION OF CYTOKERATIN GENE EXPRESSION DURING RAT PROSTATE DIFFERENTIATION:EVIDENCE FOR AN EPITHELIAL STEM CELL-ASSOCIATED MARKER

Cytokeratin (CK) 8 mRNA expression in developing and degenerating rat prostate was studied using in situ hybridization with an antisense RNA-probe. It was found that : 1) the CK 8 antisense probe was accumulated only within prostatic epithelial cells ; 2) after castration, CK 8 mRNA signals in ventral prostute(VP) sections were significantly increased, and elevated CK 8 mRNA expression persisted even long after prostate involution was complete; and 3) during prostate development,the strongest CK 8 mRNA staining was found in the early neonatal prostatic epithelia which were composed mainly of prostatic stem cells. Thereafter, a shift of CK 8 mRNA staining to peripheral regions and decreased overall CK 8 mRNA levels were noted. These data indicate that excessive expression of CK 8 mRNA is a characteristic of prostatic stem cells, and CK molecules are excellent markers for determing the hierarchical pathway of cell differentiation in prostate epithelium.

A data structure and function classification based method to evaluate clustering models for gene expression data

Objective:To establish a systematic framework for selecting the best clustering algorithm and provide an evaluation method for clustering analyses of gene expression data. Methods: Based on data structure (internal information) and function classification (external information), the evaluation of gene expression data analyses were carried out by using 2 approaches. Firstly, to assess the predictive power of clusteringalgorithms, Entropy was introduced to measure the consistency between the clustering results from different algorithms and the known and validated functional classifications. Secondly, a modified method of figure of merit (adjust-FOM) was used as internal assessment method. In this method, one clustering algorithm was used to analyze all data but one experimental condition, the remaining condition was used to assess the predictive power of the resulting clusters. This method was applied on 3 gene expression data sets (2 from the Lyer's Serum Data Sets, and 1 from the Ferea's Saccharomyces Cerevisiae Data Set). Results: A method based on entropy and figure of merit (FOM) was proposed to explore the results of the 3 data sets obtained by 6 different algorithms, SOM and Fuzzy clustering methods were confirmed to possess the highest ability to cluster. Conclusion: A method based on entropy is firstly brought forward to evaluate clustering analyses.Different results are attained in evaluating same data set due to different function classification. According to the curves of adjust_FOM and Entropy_FOM, SOM and Fuzzy clustering methods show the highest ability to cluster on the 3 data sets.

Gene expression analysis of pancreatic cystic neoplasm in SV40Tag transgenic mice model

AIM： To study the gene expression changes in pancreatic cystic neoplasm in SV40Tag transgenic mice model and to provide information about the prevention, clinical diagnosis and therapy of pancreatic cancer. METHODS： Using the pBC-SV40Tag transgenic mice model of pancreatic cystic neoplasm, we studied the gene expression changes by applying high-density microarrays. Validation of part gene expression profiling data was performed using real-time PCR.RESULTS： By using high-density oligonucleotide microarray, of 14113 genes, 453 were increased and 760 decreased in pancreatic cystic neoplasm, including oncogenes, cell-cycle-related genes, signal transduction-related genes, skeleton-related genes and metabolism-related genes. Among these, we confirmed the changes in Igf, Shh and Wnt signal pathways with real-time PCR. The results of real-time PCR showed similar expression changes in gene chip.CONCLUSION： all the altered expression genes are associated with cell cycle, DNA damage and repair, signal pathway, and metabolism. SV40Tag may cooperate with several proteins in promoting tumorigenesis.