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Objective To investigate gene expression profile in peripheral leucocytes of patients with severe preeclampsia (SPE) during 16-20 gestational weeks to see if there are different expression between normal pregnancy and SPE, and to provide the evidence for predicting the pathogenesis of preeclampsia in the future. Methods Eight hundred primipara who accepted pregnancy examination at the First Affiliated of Hospital SUN YAT-SEN University from August 2008 to December 2008 were selected into this study. The gestational age of all objects were confirmed as 16-20 weeks by ultrasonography. And they were followed up until delivered. Six patients developed severe preeclampsia (SPE group); and 40 pregnant women without any complications were chosen as the control. Human genome complementary DNA (cDNA) single-fluorescent chip were used to detect the different gene expression in peripheral leucocytes between normal pregnancy and SPE at 16-20 gestation weeks. Results There were different expressions in 983 genes between SPE group and control group, among which 719 genes were up-regulated and 264 genes were down-regulated in the SPE group. Up-regulating genes mainly involved in immunity, coagulation and fibrinolysis, signal transduction, cell adhesion, transcription and protein synthesis; and the expression of platelet and T cell activation antigen 1 (PTA1/CD226), bactericidal/permeability increasing protein (BPI), interleukin-8 (IL-8), protein kinase C (PKC), lymphocyte antigen 75 (LY-75), mucoprotein and EGFR pathway substrate 8 (EPS8) were significantly increased in SPE patients. Down-regulating genes mainly involved in apoptosis, calcium metabolism, lipid metabolism and cell transformation; and the expression of adrenomedullin (ADM), killer cell immunoglobulin-like receptors (KIR), vitamin D receptor (VDR), adipose differentiation-related protein (ADRP), parathyroid hormone (PTH) and mitogen-activated protein kinase kinase 4 (MKK4) were significantly decreased in SPE patients. Conclusions The gene expressions of peripheral leucocytes in pre-eclampsia patients were different from those of normal pregnant women during 16-20 gestational weeks. Gene CD226, BPI, IL-8, PKC, ADM, KIR and VDR might participate in the pathogenesis of SPE which should be further investigated.
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Objective To screen proteins from human pancreas cDNA library,which interact with hepatitis C virus(HCV)E1 protein.Methods The human pancreas cDNA library was amplified,purified and evaluated,and then the purified library plasmids were transformed into yeast strain Y187.The reconstructed plasmid pGBKT7-E1 was transformed into yeast strain AH109 and screened on the nutrient deficiency medium SD/-Trp.The transformed AH109 mated with Y187 that contained the library plasmids.The diploid yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade containing X-α-gal for selecting.The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5α.The plasmids in DH5α were extracted,sequenced and blasted.Result Sixteen proteins interacting with HCV E1 were found.Conclusion Some of the sixteen pancreatic proteins may be related with metabolisms of glucose and lipid.
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Objective: To clone a novel gene and explore its expression patterns in tissues and cells,so as to find its role in the process of encephalopathy in DS.Methods: On the base of our previous microarray's result together with the tissue type,we chose EST AI480014 to carry out RACE,then analyzed its expression profiles in liver,spleen,kidney,heart,brain by multi-tissues Northern blot,after that semi-quantitive RT-PCR was used to reexamine the expression profiles.Furthermore,we used ISH to find whether aim gene expressed in neuroglial cells cultured in vitro.Finally we performed semi-quantitive RT-PCR to explore whether it expressed differently between DS and normal.Results: We gained a 682 bp new cDNA fragment(DQ275636)which expressed in all the tissues examined and had no alternative splices in them.It expressed highly in brain especially in frontal lobe and hippocampus.According to the ISH result we convinced that it expressed in neuroglial cells.Using bioinformatics we mapped DQ275636 to chromosome 5q14.Conclusion: We have obtained a new gene fragment based on the(above) results.According to its expression character and tissue type,it can be suggested that this gene has a probable role in the process of encephalopathy in DS.
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Objective To detect the genes differentially expressed in sepsis-injured lungs and discover new genetic targets for management of sepsis-induced acute lung injury (ALI) and evaluate the role of cDNA microarray in the study of molecular pathogenesis of sepsis. Methods In a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), the gene expression patterns of the lungs of the animals in sepsis group and control group (Sham-operation ) were screened at 6 h and 12 h after CLP by using a commercially available cDNA microarray chips containing 2 201 cDNA clones. The cDNA of differentially expressed unique genes were sorted and analyzed. Results Of the 2 201 cDNA clones on the chip, 80 known unique genes had significant differential expression at 6 and/or 12 h after CLP as compared with those of mice in control group. 40 of the 80 genes were up-regulated and 40 down-regulated and they were related with a range of genetic functions, such as cell defence or immune/inflammatory reaction, acute-phase reaction or heat-shock reaction, redox regulation, cytoskeleton, cell apoptosis, cell signaling and cell metabolism etc. By functional analysis of these differentially expressed genes, some unique genes or expression patterns were interpreted in the context of septic ALI process and warrant further investigation. Conclusion cDNA microarray technique provides a powerful new tool for detecting differentially expressed genes and analyzing gene expression patterns in sepsis-injured tissues. Further study using this technique may yield great insight into the molecular pathologic mechanism of sepsis and discern new targets for therapeutic interventions.
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Objective To clone, express and purificate human MRPS17 cDNA in Escherichia coli (E. coli). Methods MRPS17 cDNA was obtained from total RNA isolated from primary cultured human hair papillary cells by RT-PCR and sequencing method, and then it was inserted into prokaryotic expression vector pET28a for the IPTG-induced expression in E. coli BL21 (DE3). The expression product fused with 6?His at C-terminal was analyzed by Western blotting, and purified by using Ni 2+-NTA ion exchange resin. The purity of MRPS17 protein was analyzed by SDS-PAGE. Results Human MRPS17 cDNA was obtained, and the expression plasmid pET28a-MRPS17 was constructed successfully. Western blot analysis showed that human MRPS17 with 13kD molecular weight was expressed in E. coli at 20℃. The purity of the recombinant MRPS17 was more than 90% after purification using Ni 2+-2NTA ion exchange resin. Conclusion The sequence of MRPS17 cDNA was consistent with the known sequence from the Genbank. MRPS17 is successfully induced and expressed in E. coli.
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Objective To clone the full-length cDNA of rat B cell lymphoma-2(bcl-2) gene,then construct and identify the cytomegavirus-mediated lentiviral expression vector of bcl-2 gene,and assess the gene expression in 293T cell,which is a human embryonic kidney cell line.Methods The full-length bcl-2-cDNA fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR) from the kidney tissue of a Wistar rat.The double-stranded oligonucleotides(dsOligoe) were then cloned into the pMD18-T plasmid.After confirmation of a correct construction by sequencing,the positive clone was subcloned into pGC-FU vector with enhanced green fluorescent protein(EGFP),and then transformed into DH5a competent cells.The restricted endonuclease and T4 DNA ligase were used to construct the lentiviral expression vector plasmid pGC-FU-bcl-2 which,combined with the lentiviral packing materials(pHelper 1.0,pHelper 2.0),was then transfected into 293T cell line to form the recombinant lentivirus pGC-FU-bcl-2,and it was used to transfect the 293T cells.The expression of pGC-FU-bcl-2 was further verified by detecting EGFP and bcl-2.Results 1) It was verified by DNA sequencing that the sequence of rat bcl-2 gene was consistent with reported sequence in GenBank.2) The bcl-2 gene was successfully combined in pGC-FU-bcl-2 recombinant plasmid which could be transfected into human embryonic kidney cells.3) The recombinant virus pGC-FU-bcl-2 could be obtained from the 293T cells by co-transfection of pGC-FU-bcl-2 and packing plasmids.4) Targeting gene could be cloned into 293T cells by the recombinant lentivirus with steady expression.The fluorescent protein could be observed under microscope and the expression of bcl-2 protein was detected by Western blotting.Conclusions The lentiviral expression vector containing EGFP and bcl-2 gene has been successfully constructed,with which the transfected 293 T cells can lead to a steady expression of bcl-2 protein.The present study provides a basis for the further study of the function of bcl-2 gene and a potential therapy for the diseases relating to apoptosis.