Difference of Gene Expression Profiles between Barrett＇s Esophagus and Cardia Intestinal Metaplasia by Gene Chip

The difference of gene expression profile changes in Barrett＇s esophagus （BE） and cardia intestinal metaplasia （CIM） epithelium was studied and the novel associated genes were screened in the early stage by cDNA microarray. The cDNA retro-transcribed from equal quantity mRNA from BE and CIM epithelial tissues were labeled with Cy3 and Cy5 fluorescence as probes. The mixed probe was hybridized with three pieces BiostarH-40s double dot human whole gene chip. The chips were scanned with a ScanArray 4000. The acquired images were analyzed using GenePix Pro 3.0 software. It was found a total of 141 genes were screened out that exhibited differentially expression more than 2 times in all three chips. It was identified that in gene expression profiles of BE, 74 genes were up-regulated and 67 down-regulated as compared with CIM. The comparison between the difference of gene expression profile changes in BE and CIM epithelia revealed that there existed the difference between BE and CIM at gene level. 141 genes with the expression more than two time were probably related to the occurrence and development of BE and the promotion or progress in adenocarcinoma.     

Association of gene FNI with pulmonary metastasis of human fibrosarcoma

Objective To investigate the genes involved in pulmonary metastasis of human fibrosareoma HT1080 cells in nude mice. Methods HT1080 cells were injected into the tail vein of BALB/C nude mice. RNA samples were extracted from pulmonary metastatic tissues and normal control lung tissues, purified using Atlas Pure Total RNA Labeling System （Clonetech Laboratories）. cDNA probes labeled with ^32p were prepared and hybridized to a cDNA membrane constructed with spots of 1 176 human cancer related genes and radioactivities on the membrane were measeured by BAS 5 000.     

Development of gene microarray in screening differently expressed genes in keloid and normal-control skin

Background Keloid is an intricate lesion that is probably regulated by many genes. In this study, the authors used the technique of complementary DNA (cDNA) microarray to analyse abnormal gene expression in keloids and normal control skins.Methods The polymerase chain reaction (PCR) products of 8400 genes were spotted in an array on chemical-material-coated-glass plates. The DNAs were fixed on the glass plates. The total RNAs were isolated from freshly excised human keloid and normal control skins, and the mRNAs were then purified. The mRNA from both keloid and normal control skins were reversely transcribed to cDNAs,with the incorporation of fluorescent dUTP, for preparing the hybridisation probes. The mixed probes were then hybridised to the cDNA microarray. After thorough washing, the cDNA microarray was scanned for differing fluorescent signals from two types of tissues. Gene expression of tissue growth factor-β1 (TGF-β1) and of c-myc was detected with both RT-PCR and Northern blot hybridisation to confirm the effectiveness of cDNA microarray.Results Among the 8400 human genes, 402 were detected with different expression levels betweenkeloid and normal control skins. Two hundred and fifty genes, including TGF-β1 and c-myc, were upregulated and 152 genes were down-regulated. Higher expressions of TGF-β1 and c-myc in keloidwere also revealed using RT-PCR and Northern blot methods.Conclusion cDNA microarray analysis provides a powerful tool for investigating differential gene expression in keloid and normal control skins. Keloid is a complicated lesion with many genesin volved.     

Electromagnetic Field Change the Expression of Osteogenesis Genes in Murine Bone Marrow Mesenchymal Stem Cells

In order to identify the differentially expressing gene of bone marrow mesenchymal stem cells （MSCs） stimulated by electromagnetic field （EMF） with osteogenesis microarray analysis, the bone marrow MSCs of SD rats were isolated and cultured in vitro. The third-passage cells were stimulated by EMFs and total RNA was extracted, purified and then used for the synthesis of cDNA and cRNA. The cRNA of stimulated group and the control group was hybridized with the rat oligo osteogenesis microarray respectively. The hybridization signals were acquired by using X-ray film after chemiluminescent detection and the data obtained were analyzed by employing the web-based completely integrated GEArray Expression Analysis Suite. RT-PCR was used to identify the target genes： Bmp1, Bmp7, Egf and Egfr. The results showed that 19 differentially expressing genes were found between the stimulated group and the control group. There were 6 up-regulated genes and 13 down-regulated genes in the stimulated group. Semi-quantitative RT-PCR confirmed that the expressions of Bmpl, Bmp7 mRNA of the stimulated group were up-regulated （P〈0.05） and those of Egf, Egfr were down-regulated （P〈0.05）. It was suggested that the gene expression profiles of osteogenesis of the bone marrow MSCs were changed after EMF treatment. It is concluded that the genes are involved in skeletal development, bone mineral metabolism, cell growth and differentiation, cell adhesion etc.     

Effect of Jinguo Weikang Capsule （金果胃康胶囊） on Proto-oncogene Expression of Gastric Mucosa in Rats with Gastric Precancerous Lesions

Objective： To study the effect of Jinguo Weikang Capsule （金果暖康胶囊, JWC） on the gene expression of H-ras, epidermal growth factor receptor （EGFR）, P53 and C-myc of the gastric mucosa in rats with gastric precancerous lesions, and to investigate the action mechanism of JWC on gastric precancerous lesions. Methods： A rat model with paratypical proliferation of the gastric epithelium mucosa was established by using ^60Co irradiation. Rats were divided into the normal group, model group, high-, medium-, low-dose JWC treatment groups, and the vitacoenzyme control group, and were treated for 30 days. The expression of H-ras, EGFR, P53 and C-myc genes of the gastric mucosa was detected by using immunohistochemical methods. Results： The expression and over-expression rates of H-ras, EGFR, P53 and C-myc gene in the high- and medium-dose JWC treatment groups were significantly lower （P〈0.05） as compared with those of the model group. Conclusion： JWC can inhibit the expression of the H-ras, EGFR, P53 and C-myc genes expression of the gastric mucosa in rats, which may be one of mechanisms involved in suppressing or reversing gastric carcinogenesis.     

Detection of cytokine expression patterns in the peripheral blood of patients with acute leukemia by antibody microarray analysis

The cytokines of acute leukemia （AL） patients have certain expression patterns, forming a complex network involved in diagnosis, progression, and prognosis. We collected the serum of different AL patients before and after complete remission （CR） for detection of cytokines by using an antibody chip. The expression patterns of cytokines were determined by using bioinformatics computational analysis. The results showed that there were significant differences in the cytokine expression patterns between AL patients and normal controls, as well as between acute myeloid leukemia （AML） and acute lymphoblastic leukemia （ALL）. In confirmatory test, ELISA revealed the expression of uPAR in AL. Moreover, the bioinformatic analysis showed that the differentially expressed cytokines among the AL groups were involved in different biological behaviors and were closely related with the development of the disease. It was concluded that the cytokine expression pattern of AL patients is significantly different from that of healthy volunteers. Also, differences of cytokine expression patterns exist between AML and ALL, and between before and after CR in the same subtype of AL, which holds important clinical significance for revealing disease progression.     

Microarray analysis of genes differentially expressed in placentas of pregnancy—induced hypertension patients

Objective:To uncover new clue for the research of the etiology of pregnancy-induced hypertension(PIH)by testing the gene expression difference between preeclamptic placentas and normal ones.Methods:mRNA level of 4 PIH placentas were examined using 4000 feature cDNA microarray in comparison with the pooled control consisting of total RNA from 4 cases of PIH placentas after the control cDNA and experimental cDNA were labeled by cy3 and cy5 respectively.Results:Fifty-eight to 131 genes were found down or up-regulated in 4 runs of hybridization.Among the differentially expressed genes,22 genes,including genes encoding secreted protein ADRP,CYR61,EPI AND HIF2,had the concordance in at least 2 cases were up-regulated or down-regulated.Conclusion:cDNA microarray is a high throughput and time-saving method to monitor the altered gene expression and the result could provide interesting clue and strategy for the etiological research of PIH.     

Detection of microvesicle miRNA expression in ALL subtypes and analysis of their functional roles

Microvesicles （MVs） are the heterogeneous mixtures of vesicles. MVs released by leukemia cells constitute an important part of the leukemia microenvironment. MVs might act as important reser- voirs of microRNAs （miRNAs）. It is worth evaluating whether MVs possess some unique miRNA con- tents that are valuable in understanding the pathogenesis. In this study, we investigated the miRNA ex- pression patterns of Nalm-6-derived MVs, Jurkat-derived MVs and normal cell-derived MVs using miRNA microarrays. The potential target genes regulated by differentially expressed miRNAs were also predicted and analyzed. Results demonstrated that 182 miRNAs and 166 miRNAs were differentially expressed in Nalm-6-MVs and Jurkat-MVs, respectively. Many oncogenes, tumor suppressors and sig- nal pathway genes were targeted by these aberrantly expressed miRNAs, which might contribute to the development of B-ALL or T-ALL. Our findings expanded the potential diagnostic markers of ALL and provided useful information for ALL pathogenesis.     

IDENTIFICATION OF DIFFERENTIAL GENES IN OVARIAN CANCER USING REPRESENTATIONAL DIFFERENCE ANALYSIS OF cDNA

Objoctive To identify differential genes between normal ovarian epithelium tissue and ovarian epithelial cancer using representational difference analysis of cDNA （cDNA-RDA）. Methods cDNA-RDA was performed to identify the differentially expressed sequences between cDNAs from cancer tissue and cDNAs from normal ovarian tissue in the same patient who was in the early stage of ovarian serous cystadenocarcinoma. These differentially expressed fragments were cloned and analyzed, then sequenced and compared with known genes. Results Three differentially cxpressed cDNA fragments were isolated using cDNA from normal ovarian tissue as tester and cDNA from cancer tissue as driver amplicon by cDNA-RDA. DP Ⅲ- 1 and DP Ⅲ-2 cDNA clone showed significant homology to the cDNA of alpha actin gene; DPⅢ-3 cDNA clone showed significant homology to the cDNA oftransgelin gene. Conclusion cDNA-RDA can bc used to sensitively identify the differentially expressed genes in ovarian serous cystadenocarcinoma. Ovarian serous cystadenocarcinoma involves alteration of multiple genes.     

Effect of oxymatrine on hepatic gene expression profile in experimental liver fibrosis of rats

Objective:To investigate the effects of oxymatrine on hepatic gene expression profile in a rat model of liver fibrosis.Methods:Forty healthy male SD rats were randomly divided into three groups,a normal group（n=8）,a model group（n=16）,and an oxymatrine treatment group（n=16）.Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride（CCI₄）.The rats in the treatment group received oxymatrine via celiac injection at a dosage of 40 mg/kg once a day at the same time.The rats in the model and normal groups received saline at the same dosage via celiac injection.Serum levels of aspartate aminotransferase （AST）,alanine transarninase（ALT）,alkaline phosphatase（AKP）,hyaluronic acid（HA）,and laminin（LN）were assayed.The deposition of collagen was observed with HE and Masson staining.Effect of oxymatrine on hepatic gene expression profile was detected by oligonucleotide microarray analysis with Affymetrix gene chip rat U230A. Quantitative real-time polymerase chain reaction（QRT-PCR）was carried out to confirm the expression changes of six genes.Results:Oxymatrine significantly improved liver function,lowered serum levels of HA and LN,and decreased the degree of liver fibrosis,compared with the model group（P<0.05）.A total of 754 differentially expressed genes were identified by gene chip between the model group and the normal group,among which 438 genes increased and 316 genes decreased over two folds.Compared with the model group,86 genes were downregulated markedly in the oxymatrine group（P<0.05）,including collagen I and other genes related to extracellular material（ECM）,integrin signal transduction genes,early growth response factor genes,and proinflammatory genes;28 genes were upregulated significantly（P<0.05）,including cytochrome P450（CYP450） superfamily genes,glycolipids metabolism and biological transformation related genes.Six genes were confirmed with QRT-PCR,consistent with the result from microarray.Conclusion:Oxymatrine could affect the expression of many functional genes and may be useful in the prevention and treatment of liver fibrosis.     

Effect of Tiantai No.1(天泰1号)on Gene Expression Profiles in Hippocampus of Alzheimer's Disease Rats by Bioinformatic Analysis

Objective:To study the effect of Tiantai No.1(天泰1号) on gene expression profile in hippocampus of Alzheimer's disease(AD) rat,molecular genetic target points of the effect of this drug were defined,its molecular genetic pharmacodynamic mechanism of anti-AD was further explored at molecular gene level,and a scientific basis was provided for its clinical availability and promotion.Methods:Thirty male SpragueDawley rats were divided into three groups with 10 rats per group:sham-operation group,model group and Tiantai No.1 group.Sterile surgical procedure was applied,the model group with bilateral hippocampal injection of Aβ_(1-40) was established,and normal saline was used instead of Aβ_(1-40)in the sham-operation group.One week after the models was made,rats were administered by gastric lavage once every day for three consecutive weeks.The rats of the sham-operation group and the model group were daily fed with purified water by lavage;the rats of the Tiantai No.1 group treated group were administered with Tiantai No.1 by lavage.Total RNAs of hippocampus tissues were extracted with Trizol,the changes of hippocampus gene expression profiles in the above three groups were analyzed by using Affymetrix rat whole genome expression profile microarray.Results:Microarray analysis showed that,compared with the sham-operation group,the hippocampus of the model group had 50 up-regulated genes with significant difference(fold change 2),and 21 down-regulated genes with significant difference(fold change 0.5);compared with the hippocampus of the model group,the hippocampus of the Tiantai No.1 group was found to have 5 up-regulated genes with significant difference(fold change 2) and 20down-regulated genes with significant difference(fold change 0.5).The functions of differentially expressed genes of the groups were involved in nervous system's development,neuronic differentiation and function-regulation,cellular growth and differentiation and apoptosis,synaptic occurrence and plasticity,inflammation and immune response,ion channels/transporters,cellular signal transduction,cellular material/energy metabolism and so on.Conclusion:Tiantai No.1 can regulate hippocampal function,and further regulate the brain function of animals in multiple gene target points by a number of ways.     

Study of brain-derived neurotrophic factor gene transgenic neural stem cells in the rat retina

Background Neural stem cells （NSCs） transplantation and gene therapy have been widely investigated for treating the cerebullar and myelonic injuries, however, studies on the ophthalmology are rare. The aim of this study was to investigate the migration and differentJatJon of brain-derived neurotrophic factor （BDNF） gene transgenic NSCs transplanted into the normal rat retinas. Methods NSCs were cultured and purified in vitro and infected with recombinant retrovirus pLXSN-BDNF and pLXSN respectively, to obtain the BDNF overexpressed NSCs （BDNF-NSCs） and control cells （p-NSCs）. The expression of BDNF genes in two transgenic NSCs and untreated NSCs were measured by fluorescent quantitative polymerase chain reaction （FQ-PCR） and enzyme-linked Jmmunosorbent assay （ELISA）. BDNF-NSCs and NSCs were infected with adeno-associated viruses-enhanced green fluorescent protein （AAV-EGFP） to track them in vivo and served as donor cells for transplantation into the subretinal space of normal rat retinas, phosphated buffer solution （PBS） served as pseudo transplantation for a negative control. Survival, migration, and differentiation of donor cells in host retinas were observed and analyzed with Heidelberg retina angiograph （HRA） and immunohistochemistry, respectively. Results NSCs were purified successfully by limiting dilution assay. The expression of BDNF gene in BDNF-NSCs was the highest among three groups both at mRNA level tested by FQ-PCR （P 〈0.05） and at protein level measured by ELISA （P 〈0.05）, which showed that BDNF was overexpressed in BDNF-NSCs. The results of HRA demonstrated that graft cells could survive well and migrate into the host retinas, while the immunohistochemical analysis revealed that transplanted BDNF-NSCs differentiated into neuron more efficiently compared with the control NSCs 2 months after transplantation. Conclusions The seed cells of NSCs highly secreting BDNF were established. BDNF can promote NSCs to migrate and differentiate into neural cells in the normal host retinas.     

Effect of Xuefu Zhuyu Capsule (血府逐瘀胶囊) on Angiogenesis in Hindlimb Ischemic Rats

Objective: To investigate the effect and mechanism of Xuefu Zhuyu Capsule(血府逐瘀胶囊, XZC) on pro-angiogenesis in the hindlimb ischemic model rats. Methods: A total of 100 Sprague Dawley rats were randomly divided into a model group, a regular-dose XZC group(0.48 g·kg~(-1)·d~(-1)) and a high-dose XZC group(0.96 g·kg~(-1)·d~(-1)) using random number table method. The model of hindlimb ischemic rats were made through femoral artery embolization with Bletilla microsphere agent. XZC were given on the first day after embolization surgery and lasted 5 days. Finally 72 models were obtained with 12 in each group for each time point. The lower ischemic limb was amputated on the third day after embolization surgery. Histopathological characters and the number of blood vessels of granulation tissues were observed at 36 and 48 h after amputation, respectively. The main genes were obtained from microarray analysis and were validated using real-time quantitative polymerase chain reaction. Results: The vascular number of granulation tissues at both 36 and 48 h were characterized by new and fresh vessels. The number of angiogenesis in the high-dose XZC group at 36 and 48 h was greater compared with that in the regular-dose XZC and model groups(P0.01), and high-dose XZC at 36 h increased more vessels than that at 48 h(P0.01). Consequently, granulation tissues from the high-dose XZC group at 36 h were chosen for microarray analysis. In all, 2,085 differentially expressed genes(DEGs) were detected and 25 DEGs were determined to be directly related to angiogenesis. Four biological process terms were found including angiogenesis, regulation of angiogenesis, positive regulation of angiogenesis, and positive regulation of vascular endothelial growth factor receptor signaling pathway(P0.05). Microarray analysis also showed 49 pathways including 11 pathways related to angiogenesis. Conclusion: XZC promoted angiogenesis moderately and the mechanism involved multiple DEGs and multiple pathways.     

Discovery of chrysoeriol,a PI3K-AKT-mTOR pathway inhibitor with potent antitumor activity against human multiple myeloma cells <Emphasis Type="Italic">in vitro</Emphasis>

This study was designed to determine the impact of chrysoeriol on proliferation and cell cycle progression in the human multiple myeloma cell lines RPMI 8226 and KM3,and its related molecular mechanisms.Chryseoriol was identified by using the phosphorylated AKT-specific cytoblot high throughput assay.CCK-8 assay was employed to examine the growth inhibition rate and IC 50 (48 h) in peripheral blood mononuclear cells (PBMNCs),RPMI 8226 and KM3 cells treated with chrysoeriol at various concentrations.Cells were labeled with 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE),and the proliferation dynamics was detected by flow cytometry and analyzed with ModFit software.The cell cycles of RPMI 8226 and KM3 cells were measured by flow cytometry when the IC 50 concentration of chrysoeriol was adopted.The alterations in cell-cycle related proteins (Cyclin B1,Cyclin D1,p21) and proteins in PI3K-AKT-mTOR pathway were determined by Western blot analysis.The results showed the proliferation of multiple myeloma cells was significantly inhibited by chrysoeriol,resulting in cell cycle arrest in G 2 /M phase.Chrysoeriol could significantly reduce the expression of p-AKT (s473) and p-4eBP1 (t37/46) protein,meanwhile enhanced Cyclin B1 and p21 protein expression.Similar effects were not observed in PBMNCs from normal donors.It was concluded that chrysoeriol was a selective PI3K-AKT-mTOR pathway inhibitor.It restrained the proliferation of human multiple myeloma cells,but didn’t affect proliferation of PBMNCs from normal donors.It might exhibit the cell cycle regulatory effect via the inhibition of PI3K-AKT-mTOR signal pathway.