Construction of lymphocyte subtracted cDNA library of unstable angina

AIM:To construct a subtracted cDNA library of differentially expressed genes in human unstable angina lymphocytes.METHODS:Suppression subtractive hybridizations (SSH) were performed between the patients with unstable angina pectoris and stable angina pectoris. Lymphocyte RNA, the obtained forward and reverse cDNA fragments were directly inserted into T/A cloning vector and transformed into E.coli JM 109 to construct a subtractive cDNA library. The inserting fragments were screened by blue and while blot screening and bacterium liqulid PCR.RESULTS:Each subtractive cDNA library contained more than 2000 positive bacteria clones. Most of them distributed between 200-600 bp inserts.CONCLUSION:The library is efficient and lays solid foundation for screening and cloning new and specific expressed genes in unstable angina lymphocyte RNA.     

A novel cDNA clone related with rat liver regeneration

AIM:To study a novel gene that probably related with liver regeneration, which was found by representational difference analysis(RDA). METHODS:cDNA sequence, tissue distribution and functions of the novel gene were studied by slot blot, Northern blot, RT-PCR, cDNA library screening and sequence analyzing. RESULTS:Two full-length clones were isolated from cDNA library of rat fetal livers and the sequence analysis identified that the positive cDNA encoded 76 amino acids only; Using the cDNA as a probe, the novel gene showed a specific liver distribution, a moment increasing expression in one hour after partial hepatectomy (PH) and high expression in fetal liver or liver tumor by Northern blot; EGF quickly induced its high expression in primary culture rat hepatocytes(FCS free).CONCLUSION:These results show that the novel gene is an early phase response gene that is closely related to a liver regeneration adjustment. It may encode peptide or has longer sequence at N tip.     

草原龙胆盐胁迫差减文库的构建及分析1

为了研究草原龙胆抗盐性机理,初步获得其抗盐性相关基因,分别以400 mmol·L-1的NaCl及清水处理的草原龙胆叶片mRNA为tester和driver方,利用抑制差减杂交技术构建了cDNA文库。经过测序和与GO数据库序列比对,获得了658条单一序列,其中61条序列分子功能未知,535条序列无同源性,62条序列有显著同源性。此62条序列注释按照功能分为21类。获得了活性氧清除系统基因DHAR、GST、TRX、CAT,转录因子SMT3,调控基因丝氨酸/苏氨酸激酶基因等。结果说明草原龙胆的盐抗性相关基因与数据库中的模式植物基因既有一定的共性,同时绝大部分的序列又无法用已知的基因进行解释。     

Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

Background  

We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector.     

Whole cell screening of phage-display peptide library for mimicry peptides of glioma SWO-38

AIM and METHODS:The Ph.D.-7 phage display library was used to isolate peptides specific for glioma SWO-38 cell by whole cell screening.Moreover,binding efficiency analysis was carried out to test the binding specificity of the clones obtained.RESULTS:After three rounds of biopanning,a high concentration of phage clones was obtained and two of them were found to be highly specific to glioma SWO-38.CONCLUSION:Highly specific clones against neurtral glioma cel s can be obtained from a phage display library by simple procedures.     

华东葡萄抗白粉病杂种后代cDNA文库的构建及EST序列分析

以抗白粉病的华东葡萄杂种后代6-12-4株系为试材,利用SMARTTM技术构建了白粉病病原菌诱导的全长cDNA文库。经检测,原始文库滴度为2.50×106pfu·mL-1,重组率达到99%,扩增文库滴度为3.0×109pfu·mL-1,重组率达到95%,插入片段长度在500～2000bp。随机挑取300个克隆,测序获得260条质量较高的ESTs,聚类及拼接后,共得到183个一致序列(所获序列已经提交GenBank,登录号为FG579859-FG580039)。经蛋白功能预测分析,获得含有TPR结构域的小的富含谷氨酰胺的蛋白、MYB类转录因子、水通道蛋白、富含脯氨酸蛋白、亲环素蛋白、ACI14蛋白、转脂蛋白等抗病相关基因,涉及到植物的信号转导,胁迫诱导,防卫反应等方面。这些EST序列为利用中国野生华东葡萄进行抗白粉病育种研究和利用提供了有价值的抗病基因片段,为丰富世界葡萄属植物抗白粉病基因提供序列数据。     

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。     

Expression of undulin in experimental rat liver fibrosis

AIM: To observe the expression of undulin (Un) in liver tissue and to clarify the diagnostic significance of serum Un in experimental rat liver fibrosis. METHODS: Rat liver fibrosis was induced by carbon tetrachloride. The expression of Un in liver tissue was detected by immunohistochemical method. Serum Un levels was measured by enzyme immunoassay. RESULTS: Expression of Un increased in fibrotic liver than normal liver, and it was mainly distributed in portal tract stroma, central veins and fibrotic septa in fibrotic liver. Also, the level of serum Un was significantly higher in fibrotic liver than normal liver. CONCLUSION: These data suggest that Un should be a component of the hepatic extracellar matrix, and its expression could be increased greatly in fibrotic rat liver. Serum Un levels may be used as an indicator in liver fibrosis diagnosis.     

Molecular mechanisms of liver regeneration following cold ischemia injury after liver transplantation in rat

AIM: To study the molecular mechanisms of liver regeneration following different cold ischemia (CI) times after liver transplantation in a rat model. METHODS: A model of rat orthotopic liver transplantation was established. The rats were divided into 3 groups: 1 h CI group, 8 h CI group and 16 h CI group. Survival rate in each group was recorded. Specimen were collected at predetermined intervals from 90 min, 1, 4 and 7 d post-reperfusion. The patterns of TNF-α, IL-6 and STAT3 activation were determined in liver grafts with 1 h, 8 h and 16 h CI times. Expression of cyclin D1 and hepatocyte replication with bromodeoxyuridine (BrdU) were confirmed by immunohistochemistry. The results of TNF-α and IL-6 expression in all groups were analyzed after whole liver transplantation. Statistical analysis was used to compare BrdU positively stained hepatocytes at 48 h post-reperfusion. RESULTS: Liver transplantation was successfully performed in all experimental groups. Survival rate in each group was 100% (>14 d). Compared with 1 h CI, TNF-α expressions in whole liver grafts with 8 h and 16 h CI were markedly increased at 90 min after reperfusion(P<0.05). Compared with 1 and 8 h CI, IL-6 expression in liver grafts preserved for 16 h were markedly increased at 90 min after transplantation (P<0.05). With 8 and 16 h CI, STAT3 activity was markedly increased. Cyclin D1 expression in 8 CI group was demonstrated with cytoplasmic and nuclear staining at 24 h in liver grafts. Cyclin D1 expression was mainly nuclear in 16 h CI group. Extensive hepatocyte replication was present. The numbers of hepatocytes with positively stained nuclei in 16 h CI group were more than those in 1 and 8 h CI group at 48 h after transplantation(P<0.05). CONCLUSION: Rat whole liver grafts with 16 h CI injury still initiate and complete liver regeneration and graft recovery after liver transplantation. Liver regeneration following transplantation may be through TNF-α/IL-6/STAT3/cyclin D1/DNA synthesis pathways.     

Expression of peroxisome proliferator-activated receptor α in rat fatty liver

AIM:To investigate the role of expression of peroxisome proliferator-activated receptor α(PPAR α) in pathogenesis of rat fatty liver.METHODS:The rats were treated with a low dose of carbon terachloride (CCl₄) and fed a high fat diet to produce fatty liver. We determined the concentrations of triglyceride (TG), total cholesterol (TC), free fatty acid (FFA) in liver and the alanine aminotransferase (ALT) activity, tumor necrosis factor-α (TNF-α), FFA in serum and the degree of hepatocytic steatosis. Total RNA of liver was extracted, and the expression of PPAR α were analyzed by semi-quantitative RT-PCR method.RESULTS:In model group, the hepatocytic PPAR α mRNA expression decreased to 0.41±0.28, compared to 1.41±0.29 in the control group (P＜0.01). The contents of TG, TC, FFA in model rat liver were (1.88±0.20) mmol·L^-1, (11.03±1.12) mmol·L^-1 and (1 260.38±151.27) μmol·L^-1, respectively, compared to (0.53±0.10) mmol·L^-1, (1.25±0.25) mmol·L^-1 and (334.30±27.09) μmol·L^-1 in the control group (P＜0.01). The activity of ALT, concentrations of TNF-α and FFA in serum were also increased remarkably in model group.CONCLUSION:Oxidation of fatty acid and utilization of lipids in liver are affected by reducing the expression of PPAR α, which result lipid accumulation in liver.     

Expression of calpain 2 and Bax in rat fibrotic liver tissues

AIM：To observe the expression of calpain 2 and Bcl-2-associated X protein (Bax) in rat fibrotic liver tissues and to explore their effects on hepatic fibrosis.
METHODS：Forty male Wistar rats were randomly divided into four groups (each n=10): 4-week control group, 8-week control group, 4-week liver fibrosis group and 8-week liver fibrosis group. Liver fibrosis model was induced by subcutaneous injection of 40% CCl4 (3 mL/kg) every 3 days for 4 or 8 weeks. The apoptosis of hepatocytes was detected by TUNEL. Additionally, the mRNA expression of calpain 2 and bax was determined by real-time PCR, and the protein expression of calpain 2 and Bax was determined by immunohistochemistry and Western blotting. RESULTS：Real-time PCR showed that the mRNA expression of calpain 2 and bax in liver tissues was elevated in 4-week and 8-week liver fibrosis groups. The results of immunohistochemistry and Western blotting revealed that there was no difference of calpain 2 protein expression in liver tissues between 4-week liver fibrosis group and control group, but that in 8-week liver fibrosis group was obviously increased. The protein expression of Bax in 4-week and 8-week liver fibrosis groups was higher than that in control groups. Additionally, the numbers of apoptotic hepatocytes in 4-week and 8-week liver fibrosis groups were obviously increased compared with control groups.CONCLUSION：Calpain 2 and Bax may play important roles in the process of liver fibrosis.     

Identification of hepatic macrophages in pig serum induced rat liver injury

AIM: Our previous study identified two subsets of liver macrophages in normal rat liver, here we plan to identify and study the liver macrophages in pig serum induced rat liver injury.METHODS: Immunologically mediated liver injuries were chronically induced in rats by injection of pig sera (IPS). The histology and immunofluorescent characteristics of the livers were studied. The liver macrophages were isolated and identified by flow cytometry. The mRNA expression of the genes relating to inflammation, fibrogenesis and Ki67 in both subsets of liver macrophages was quantitatively analyzed by the method of quantitative real time PCR (qPCR). RESULTS: The IPS livers were characterized by intensive septum formation, positive α-SMA+ myofibroblast and proliferation of ED2+ cells. Immunofluorescent image and Indian ink staining showed that the liver macrophages were in different size, different autofluorescence and different distribution. GdCl3 only deleted the liver macrophages of big size/strong autofluorescence. Two subsets of macrophages were isolated and identified from the rat livers, namely ED2high/AFhigh and ED2dim/AFdim, both of which in IPS group increased in number as compared to control group. ED2high/AFhigh subset expressed cytokines mainly related with inflammation, whereas ED2dim/AFdim subset expressed cytokines mainly related with fibrosis. The mRNA level of Ki67 expression was significantly higher in ED2dim/AFdim subset than that in ED2high/AFhigh subset, and it was significantly higher in macrophages isolated from IPS group than that from control group. CONCLUSION: The liver macrophages can be divided into two subsets in pig serum induced rat liver injury model. The cells of two subsets play different roles in the pathogenesis of pig serum induced liver injury. The ED2dim/AFdim cells proliferate actively and express cytokines mainly related with fibrosis, indicating an important role in the formation of pig serum induced liver fibrosis.     

Searching and cloning of differentially expressed genes in liver tumor by restriction fragment differential display PCR

AIM:The aim of this study was to isolate and clone the liver tumor-associated genes by restriction fragment differential display PCR (RFDD-PCR) methods that developed recently. METHODS:Total RNA was extracted from primary carcinoma of the liver and adjacent noncancerous tissue separately. The method of RFDD-PCR was used to search for the differentially expressed genes. Each gene segment was sequenced after cloning. The sequence homogeneity were analyzed and compared with BLAST through Internet. The data were then transported into the Translated BLAST Searches to acquire the related information such as the corresponding protein function. RESULTS:Eighteen cDNA bands were found significantly different at their expression levels with 16 higher and 2 lower in primary carcinoma of the liver cells. Three of them were selected to reamplication by PCR, and then were cloned, identified and sequenced. One of them had no homology as compared with known genes and was likely to be a new one. One had relatively high homology with human ribosomal protein genes (86%). The other one has relatively high homology with selenoprotein P genes (98%). CONCLUSIONS:Compared with DDRT-PCR method, RFDD-PCR is better for rapid screening of the differentially expressed genes. It is simple, sensitive, reproducible and less false positiveness. Three differentially expressed genes are found, their functions and another 15 genes need further analysis.     

Functional analysis of differential microRNA expression profile in mouse fibrotic liver tissues induced by CCl4

AIM:To discover the expression profile of microRNAs (miRNAs) in mouse fibrotic liver tissues induced by carbon tetrachloride (CCl4), and to investigate the functions of these differential miRNAs based on the gene ontology (GO) analysis and KEGG Pathway analysis. METHODS:The mice were randomly divided into normal group and model group. Liver fibrosis was induced by subcutaneous injection of CCl4. miRNA expression profile of the liver tissues was assayed by a mouse miRNA microarray (Agilent 12.0). The differential expression of miRNAs between the normal and model mice was screened, and GO analysis and KEGG Pathway analysis were performed to determine the functions of these differential miRNAs. RESULTS:Thirty-nine miRNAs with differential expression were discovered in the model mice compared with the normal mice, among which 23 were up-regulated and 16 were down-regulated. GO analysis and KEGG Pathway analysis indicated that most pathological processes of liver fibrosis regulated by miRNAs included cell proliferation and activation, cell apoptosis, cell cycle, cell adhesion, inflammatory reaction, cell migration, transforming growth factor β (TGF-β) signaling pathway, Wnt signaling pathway and proteometabolism process. GO analysis revealed that the key up-regulated miRNAs were mmu-miR-322, mmu-miR-15b, mmu-miR-195, mmu-miR-200b and mmu-miR-214, and the key down-regulated miRNAs were mmu-miR-16, mmu-miR-130a, mmu-miR-101b, mmu-miR-30a and mmu-miR-30e. Analyzing the target genes screened out by GO analysis and Pathway analysis simultaneously, we found that the key up-regulated miRNAs included mmu-miR-200b, mmu-miR-322, mmu-miR-106b, mmu-miR-23a and mmu-miR-15b, and the key down-regulated miRNAs included mmu-miR-16, mmu-miR-30e, mmu-miR-30c, mmu-miR-30a and mmu-miR-130a. CONCLUSION: Differential expression of miRNAs is discovered in mouse fibrotic liver tissues induced by CCl4 compared with the normal liver tissues. Most of the pathological processes involved in liver fibrosis may be regulated by miRNA, such as cell proliferation and activation, cell adhesion and apoptosis, cell migration and differentiation, metabolism, TGF-β receptor signaling pathway and so on.     

金钗石斛花芽cDNA表达文库的构建及鉴定

 cDNA文库是分离、筛选基因的重要平台。利用SMARTTM cDNA library construction技术, 成功构建了金钗石斛花芽cDNA表达文库。结果表明该文库的克隆数为3.02 ×10⁵ pfu, 插入片段的大小在0.5～2.5 kb范围, 插入片段的重组率接近100% , 说明所构建的cDNA文库质量较好。该文库的构建为克隆与金钗石斛花芽分化相关功能基因奠定了基础。     

Protective effects of aFGF compound solution on rat liver in hypothermic preservation

AIM: To investigate the protective effects of human acidic fibroblast growth factor (aFGF) on hypothermic preservation of rat liver. METHODS: Twenty-four Wistar rats were randomly divided into 2 groups (n=6): hypertonic citrate adenine (HCA) solution control group and experimental group (aFGF compound solution, containing 40 μg/L aFG_14-154 in HCA solution). The isolated livers were preserved in corresponding solution for 12 h or 24 h, and the content of malondialdehyde (MDA), the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the changes of liver weight in the 2 groups were analyzed. The histopathological changes of the livers were also examined. RESULTS: Compared with HCA control group, the levels of ALT, AST and MDA and the changes of liver weight were significantly decreased in experimental group (24 h), and the changes of liver histopathology were obviously alleviated in experiment group. CONCLUSION: aFGF can reduce rat hepatic injury. The protective effects of aFGF may be related to attenuating cell swelling and improving the capacity of anti-oxidative damage in the liver.     

番茄果实酸性转化酶基因cDNA片段的克隆

根据酸性转化酶基因的保守序列设计引物 ,采用RT PCR方法 ,从番茄果实总RNA中扩增出目标cDNA片段 ,克隆到pMD18 T载体中。在所测的 10 38个核苷酸中 ,与GenBank中登记号为M810 81的番茄果实酸性转化酶基因高度同源 ,同源性为 99%。该基因片段在GenBank中已登记 ,登记号为AF4 90 5 30。     

Morphology of endoplasmic reticulum and expression of GRP78 in rat fibrotic liver

AIM: To observe the changes of endoplasmic reticulum and the biomarker of endoplasmic retidum stress (ERS), glucose-regulated protein 78(GRP78),in the process of liver fibrosis induced by carbon tetrachloride (CCl₄). METHODS: Male Wistar rats weighing 180 g to 220 g were divided into control group and liver fibrosis group. The rats in liver fibrosis group were induced by hypodermic injection of CCl₄ for 4 weeks or 8 weeks. The liver index and the serumactivity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed. The liver fibrosis and the morphological changes of endoplasmic reticulum were observed under light and electronic microscopes, respectively. Additionally, the expression of GRP78 at mRNA and protein levels was determined by real-time PCR and the method of immunohistochemistry, respectively. RESULTS: The liver index, serum ALT and AST activity in liver fibrosis group were obviously higher than those in control group. Swelling and reduced number of endoplasmic reticulum were observed in the hepatocytes of fibrotic rats compare to the controls. The levels of GRP78 protein and GRP78 mRNA in the liver of hepatic fibrotic rats were obviously higher than those in the control rats. CONCLUSION: In the process of liver fibrosis induced by CCl₄, the obvious morphological changes of endoplasmic reticulum and increased expression of ERS protein indicate that ERS plays an important role in the liver fibrosis.