Screening of the differentially expressed genes in lymphocytes of patients with unstable angina with suppression subtractive hybridization

AIM: To screen and identify the differentially expressed genes in lymphocytes of patients with unstable angina in order to find the molecular mechanism of unstable angina . METHODS: Suppression subtractive hybridizations (SSH) and dot blot hybridizations were performed to screen the relatively differentially expressed genes in lymphocyte RNA between the patients with unstable angina pectoris and stable angina pectoris. The obtained expressed sequence tags (ESTs) were used as probes to perform Reverse Northern blot with forward and reverse suppression products. And the positive ESTs were performed RNA slot hybridization with unstable and stable angina group. The obtained ESTs were sequenced and analyzed using BLAST (nr) at NCBI. RESULTS: Three up-regulated ESTs in the unstable angina group, and one down-regulated EST in the stable angina group were obtained. All of them are sequences of known genes. CONCLUSION: All these ESTs may be associated with the unstablization of plaque of coronary artery in patients with unstable angina.     

PCR-select differential screening of a subtracted cDNA library of heat-adapted rat liver

AIM: To reveal the molecular mechanisms of heat adaptation by study the change of gene expression in rat liver when the rats exposed to hyperthermia stress and heat adaptation. METHODS: Differentially expressed genes in rat liver in association with heat adaptation were identified using the suppression subtractive hybridization (SSH) technique to prepare subtracted cDNA libraries in heat adaptation. The PCR-amplified cDNA fragments generated by SSH were then ligated into the plasmid pGEM-T (Promega). PCR-select differential screening technique was used to filtrate differentially expressed genes. Each of these differentially expressed genes was sequenced and was compared to known sequences in the GenBank database. RESULTS: eight up-regulating expressed genes segments were isolated from the up-regulating differentially expressed library, three of which were known gene such as p53 and five genes were new expressed sequence tags. CONCLUSION: Up-regulating expressed genes such as p53 maybe participate in the signal transduction pathway of heat adaptation.     

辣椒细胞质雄性不育系和保持系SSH-cDNA文库的构建

以辣椒细胞质雄性不育系A1、A5为驱动方(driver),以其相应的保持系B1、B5为测验方(tester),利用抑制差减杂交技术构建了1个辣椒细胞质雄性不育保持系表达基因的正向差减cDNA文库。从差减文库中筛选到189个阳性单克隆,经高密度点阵膜杂交筛选和测序分析,共获得了42条表达序列标签(ESTs)。BLAST比对分析后,得到了35条有比对结果的序列,其中包括26条已知功能基因序列和9条未知功能基因序列,已知基因功能多与基础物质代谢、胁迫应答、信号转导等方面有关;有7条ESTs没有比对结果,可能代表一些新基因。     

Influence of ovariectomy and estrogen replacement on gene expression profile of myocardium in rats

AIM:To investigate the influence of ovariectomy and estrogen replacement treatment on profile of gene expression in myocardium by cDNA microarray,and to characterize the targeting genes of estrogen.METHODS:cDNA microarray containing 1 400 rat cDNAs was used to study the genes differentially expressed in myocardium between sham (Ⅰ),ovariectomy (Ⅱ,OVX) and estrogen replacement treatment (Ⅲ,OVX+E2) group.Then down-regulated genes in myocardium of OVX rats were further confirmed by RT-PCR.RESULTS:177 genes were differentially expressed in myocardium between sham and OVX rats,with 91 genes up-regulated and 86 genes down-regulated in OVX rats.164 genes were differentially expressed in myocardium between OVX and OVX+E2 rats,with 113 genes up-regulated and 54 genes down-regulated in OVX rats.There were 54 genes differentially expressed in OVX compared to sham and OVX+E2.They are involved in membrane channels and transporters (18),cell receptors (9),intracellular transducers/effectors/modulator (7) and metabolism (6).Most of the genes (45) were down-regulated in OVX rats and up-regulated in OVX+E2 rats.RT-PCR test confirmed the results of cDNA microarray.CONCLUSIONS:Long-term estrogen replacement may influence the expression of genes involved in membrane channels and transporters,cell receptors,intracellular transducers/effectors/ modulator and metabolism.Long-term estrogen replacement has some beneficial effects on ionic concentration and cardiac function which partially comes from the results of influence of expression on Na+,K+-ATPase and Na+/H+ exchanger.Estrogen has an inhibitory effect on the expression of dopamine receptor,which partially clarify the myocardial protection of estrogen.     

紫外线照射对梁平柚果皮基因表达的影响

 采用SSH技术以紫外照射的梁平柚[Citrus grandis（L.）Osbeck]果皮作为实验方（tester）,以未被照射的正常果皮作为驱动方（driver）,构建了一个梁平柚果皮紫外诱导基因的正向差减文库。经菌液PCR检测后随机挑取200个阳性克隆测序,获得168条表达序列标签（ESTs）。比对这168条ESTs,发现有分属于57个基因的114条ESTs与已知基因高度同源,54条ESTs同源性较低或没有同源性。功能分析发现,这些ESTs主要参与抗逆防御、生长发育、细胞凋亡、转录与翻译、细胞分化、信号传导、能量代谢、糖类及氨基酸代谢以及次生代谢等。     

Screening differentially expressed genes involved in apoptosis of primary cultured rat cerebellar granule neurons by mRNA differential display RT-PCR

AIM: To Screen and identify differentially expressed genes that involved in apoptosis model in rat cerebellar granule neurons (CGNs).METHODS: The rat cerebellar granule neurons were isolated and primarily cultured.Fluorescent differential display RT-PCR (FDD RT-PCR) was performed to screen differentially expressed ESTs in the apoptosis model of primarily cultured rat CGNs.ESTs were subcloned into pGEM-T EasyTM vector and then sequenced.Alignment assay in non-redunant database was applied for encoding information.Reverse Northern blotting was used to appraise the results from DDRT-PCR.RESULTS: 164 pieces of differentially expressed ESTs were obtained by FDDRT-PCR.17 of them were subcloned and sequenced.5 ESTs of 17 were confirmed to be positive results by reverse Northern blotting.CONCLUSION: DD-PCR is a rapid,simple-operation and sensitive method for screening differentially expressed genes,which would contribute to the molecular mechanisms of apoptosis/survive of CGNs.     

Cortical gene expression pattern in rat focal cerebral ischemia

AIM: To investigate the genes differential expression in cortex during rat focal cerebral ischemia.METHODS: cDNA microarray chips containing numerous cDNAs were used to investigate the gene expression pattern between samples of focal cerebral ischemia and sham-control operation rats. RESULTS: Two hundred and eleven genes differentially expressed were screened out, among these genes, up-and down-regulated genes were 199 and 12, respectively. CONCLUSIONS: The analysis of gene expression pattern of focal cerebral ischemia based on cDNA microarray can realize high-throughput screening of the genes associated with the focal cerebral ischemia. The differential expression of genes may be related to the pathogenesis of focal cerebral ischemic diseases.     

Gene expression pattern of periphery blood mononuclear cells in patients with active lupus nephritis

AIM: To find new gene function associate with active lupus nephritis (LN) through study on the difference in gene expression of peripheral blood mononuclear cells between LN patients and healthy controls by gene chip. METHODS: The CSC-GE-80 chip containing 8 000 spots of cDNAs were used to investigate the difference of the expression. Both the total RNA from peripheral blood mononuclear cells of active LN patients and healthy donors were reversely transcribed to cDNA with the incorporation of fluorescent( cy3 and cy5) labeled dCTP to prepare the hybridization probes. After hybridization, the gene chip was scanned for the fluorescent intensity. The differentially expressed genes were screened. We repeated that in three groups of LN patients and healthy controls, respectively, and only the genes that have differential expression in all three chips were considered associated with LN. RESULTS: 75 genes were identified to be differently expressed in all three groups of LN patients as compared with healthy controls, including 42 up-regulated genes and 33 down-regulated ones. CONCLUSION: The present study represents a global view of gene expression of LN and provides important clues for further study of LN related genes. And it also suggests defensin α₁, S100A8, S100A9 may be involved in the pathogenesis of LN.     

应用抑制性消减杂交技术从枳中筛选根特异表达基因

为筛选分离植物根组织特异表达基因,以枳根RNA为试验组,叶RNA为驱动组,构建了枳根消减文库。从该消减文库中共获得有效序列1 177个,其片段长度主要分布在200 ~ 500 bp;序列拼接后得到455个非重复序列,其中245个与已知基因匹配,具有结合功能、催化活性和转运活性等生物学功能,可参与植物的代谢、细胞生长、个体发育、应激反应等生物学过程。实时定量PCR检测结果显示这些基因中的主要乳液蛋白、早期结瘤素样蛋白和贝壳杉烯酸氧化酶等基因在根中高表达。     

Effects of lovastatin on differentially expressed genes in HepG2 cells

AIM: To analyze the lovastatin-induced differential gene expression in HepG2 cells using a cDNA microarray assay. METHODS: Total RNA was extracted from the lovastatin-treated HepG2 cells and control group. cDNA was synthesized from RNA with Cy3/Cy5-labelled dCTP. Then the hybridization was conducted. The result was analyzed using Imagene and Genespring software. RT-PCR was carried to confirm the hybridization results. RESULTS: 30 genes were up-regulated while 11 genes were down-regulated in lovastatin-treated HepG2 cells, involved in some major functional areas including signal transduction, cell cycle regulation, tumor immunity, and so on. CONCLUSION: The analysis of differentially expressed genes in lovastatin-treated HepG2 cells is helpful to explore the mechanism of the anti-tumor activity of statins.     

黄龙病诱导下椪柑SSH文库的构建与分析

以实生苗和平椪柑(Citrus reticulate Blanco)为材料,采用抑制性差减杂交技术,分别以感染黄龙病与未感染黄龙病的椪柑叶片为检测方(tester)和驱动方(driver),成功构建了黄龙病诱导的差减cDNA文库。挑选了100个阳性克隆并成功测序得到71条EST,经NCBI基因库同源性比对,有41条非冗余高质量EST序列找到了同源序列,另有10条非冗余未搜索到同源序列。同源序列的基因涉及抗逆防御、运输、能量代谢、光合作用、蛋白代谢、信号转导、抗氧化等代谢途径和生理生化过程。值得注意的是文库中有由病原引起的韧皮部相关的凝集素蛋白的前体积累。挑选了2条进行Q-PCR定量分析,结果表明感病1周表达量增强不大,2周后tester的表达量明显高于driver,说明材料感病早期这些基因表达增强。     

The value of intrapericardial injection with a trans-diaphragmatic approach in the gene therapy of chronic heart failure rats

AIM: To study the feasibility, security and validity of the intrapericardial injection with a trans-diaphragmatic approach in chronic heart failure rats and its value in the study of gene therapy for heart diseases, and further investigate whether adeno-associated virual gene transfer of sarcoplasmic reticulum Ca2+-ATPase （SERCA2a） gene can improve ventricular function in chronic heart failure (HF) rats. METHODS: An animal model of heart failure was obtained by creating descending aortic constriction in rats. Recombinant adeno-associated virus, carrying enhanced green fluorescent protein (EGFP) gene and recombinant adeno-associated virus carrying SERCA2a gene, were respectively injected into pericardium of heart failure rats in different groups (group HF+EGFP and group HF+SERCA2a) by intrapericardial injection with a trans-diaphragmatic approach. After 30 days, hemodynamic parameters were measured and analyzed. Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein, and Western blotting was performed to detect the expression of SERCA2a. RESULTS: Green fluorescence was detected in cryosection of the hearts in all rats in group HF+EGFP and the expression of green fluorescent protein was ubiquitously. The expression of SERCA2a in all rats in group HF+SERCA2a was more than those in group HF and group HF+EGFP. And overexpression of SERCA2a improved the systolic and diastolic function of heart failure rats significantly and the hemodynamic parameters were similar with those of the controls. CONCLUSION: The intrapericardial injection with a trans-diaphragmatic approach suggests a simple, safe, efficient and cheap technique for the gene therapy of chronic heart failure. Gene thransfer of SERCA2a may be a new approach for the treatment of chronic heart failure.     

辣椒Me3基因介导抗根结线虫相关基因的SSH分析

以辣椒（Capsicum annuum L.）双单倍体HDA149为试验材料,接种南方根结线虫（Meloidogyne incognita）12、24、36 h的根尖材料作为测验方(Tester),相应的未接种根尖材料作为驱动方(Driver),构建一个南方根结线虫诱导Me3 基因表达早期的抑制消减杂交cDNA文库,通过高密度点阵膜杂交差异筛选,获得了309条表达序列标签(EST)。通过测序,除去重复序列共得到259条EST序列,在GenBank上进行BLAST分析,30个EST片段未发现同源性基因,229个为具有同源性的已知基因,其中71个为功能未知的基因。在显微观察和抗性相关EST表达谱分析的基础上,推测在Me3基因介导的不亲和互作中,编码NBS-LRR结构的抗病基因参与了寄主对根结线虫的识别,激发了过敏性坏死反应的信号基因, 同时多种抗性相关的转录因子基因表达, 使以代谢为基础的防御反应和保护机制发挥抗线虫作用。     

Effects of myocardial extracts on transdifferentiation of bone marrow-derived mesenchymal stem cells of rats

AIM: To investigate the effects of myocardial extracts of rats on transdifferentiation of bone marrow-derived mesenchymal stem cells (MSCs). METHODS: After establishing stable culture methods for rat MSCs, they were induced by high level of myocardial extracts on day 3 of primary culture for 1 week. Immunocytochemistry technique was used to detect MHC and troponin-T, and the expression of Nkx2.5, α-MHC and ANP genes were identified by RT-PCR technique 3 weeks later. RESULTS: MSCs of rats expressed cardiac MHC and troponin-T proteins, Nkx2.5 and α-MHC genes after induction. However, they neither expressed ANP gene nor formed myotubes and intercalated disk. CONCLUSION: Myocardial extracts can induce MSCs to transdifferentiate into cardiomyocytes.     

Effect of basil polysaccharide on rat model bearing NuTu-19 cells

AIM：To study the effect of basil polysaccharide on the rat model bearing NuTu-19 cells.METHODS：The rat model bearing NuTu-19 cells was treated with basil polysaccharide.The changes of the tumor cell biology and histopathology were observed.Total RNA was isolated from the tumor tissue.Hybridization assay was performed using gene expression microarray.The results were analyzed by photo disposal software-GenePix Pro3.0.RESULTS：Compared with control,the amounts of ascites in the rats treated with basil polysaccharide were decreased (P<0.05) and the scores of abdominal carcinoma metastasis were changed significantly (P<0.01).268 differentially expressed genes were found in the tumor specimen from the rats exposed to basil polysaccharide.CONCLUSION：Basil polysaccharide may be a new anti-tumor drug and it has multi-targets.Gene expression microarray has huge advantage and value in the study of the drug target.     

利用抑制消减杂交技术分离辣椒细胞质雄性不育育性恢复相关EST

 以辣椒(Capsicum annuum L. ) 细胞质雄性不育系23A、121A, 和其相应的近等基因恢复系23C、121C为试验材料, 利用抑制消减杂交( SSH) 技术成功构建了CMS恢复基因诱导表达的消减cDNA文库。结合高密度点阵膜杂交差异筛选, 获得了282个阳性克隆。通过测序, 除去重复序列共得到175个Unique ESTs。在GenBank上进行BLAST分析, 55个EST片段未找到对应的同源序列, 可能代表了新基因;120个EST片段找到了对应的同源序列, 包括103个已知功能基因和17个未知功能基因。按照MIPS功能分类法, 将其分为14个功能组, 涉及代谢、胁迫应答、蛋白活性、转录因子、信号转导等多方面的功能。