ABSTRACT
<p><b>OBJECTIVE</b>To screen and clone hepatocyte protein interacting with hepatitis C virus NS5ATP4A protein for studying its biological functions.</p><p><b>METHODS</b>Bait plasmids of hepatitis C virus NS5ATP4A were constructed. After verifying that hepatitis C virus NS5ATP4A protein could be steadily expressed in AH109 yeast strain, yeast-two hybrid assay was performed by mating AH109 with Y187 which pre-transformed with liver cDNA library plasmids pACT2, and the diploidy yeast cells were plated on quadruple dropout (QDO) medium and assayed for X-a-gal activity. Nineteen yeast colonies which grew on QDO and had a-gal activity were obtained, and then the library plasmids were extracted and sequenced.</p><p><b>RESULTS</b>Seven genes were screened out and one of them was a formerly unknown gene. They were associated with RNA synthesis, protein translation, cell cycling and tumor immunity.</p><p><b>CONCLUSION</b>NS5ATP4A binding proteins were successfully screened, which offers new clues for further studying the signal transduction pathway of NS5ATP4A and the pathogenic mechanism of HCV.</p>
Subject(s)
Humans , Base Sequence , Cloning, Molecular , Gene Library , Genome, Viral , Hepacivirus , Metabolism , Hepatocytes , Metabolism , Molecular Sequence Data , Protein Binding , Sequence Homology , Two-Hybrid System Techniques , Viral Nonstructural Proteins , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To construct TIMP-1 siRNA eukaryotic expression vectors and evaluate their effect on TIMP-1 mRNA expression in hepatic stellate cells.</p><p><b>METHODS</b>The combinant lone DNA with cutting sites of BamH I and Xho I enzyme according to the sequences of 447-465, 552-540 TIMP-1 of rats and nonspecific sequence were selected and cloned to pGEM-T vector and sub-cloned to pRNAT-U6.2. They were then identified by double enzyme digestion analysis and DNA sequencing. Three plasmids were transfected into T6 separately through an oligofectamine package. TIMP-1 mRNA expression was evaluated by RT-PCR.</p><p><b>RESULTS</b>Targeting sequences of TIMP-1 siRNA eukaryotic expression vectors were correct. TIMP-1 mRNA expression was significantly reduced by transfecting them into the T6.</p><p><b>CONCLUSION</b>We successfully constructed two TIMP-1 siRNA eukaryotic expression vectors and the transfected cells can significantly suppress the TIMP-1 expression.</p>
Subject(s)
Animals , Rats , Cell Line , Gene Silencing , Genetic Vectors , Hepatic Stellate Cells , Plasmids , RNA, Small Interfering , Tissue Inhibitor of Metalloproteinase-1 , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of Huchang Qingfei concentrated pellets on the expression of E-cadherin (E-cd) in the lung tissue from mice infected with Mycoplasma pneumoniae (MP).</p><p><b>METHOD</b>A mice model of Mycoplasmal pneumonia (MPP) was developed by repeatedly intranasal infectious route. Transmission electronic microscope (TEM) and immunohistochemistry stain were performed to observe the pathological changes and expression of E-cd in lung tissues.</p><p><b>RESULT</b>Under TEM it was found that the cellular membrane was ruptured, mitochondria was denatured, crista was broken in the pulmonary cells of the model group; the all above parameters in Huchang medicated group were improved obviously. The immunohistochemistry test showed that strong positive brown stain of E-cd expression was found in the pulmonary epithelial cell membrane and bronchial periphery in the model group, however, in the medicated group, the E-cd expression level in the cellular membrane was decreased and the expression ratio was dropped significantly as compared with the model controls.</p><p><b>CONCLUSION</b>Huchang Qingfei concentrated pellets can inhibit the overexpression of E-cd in the lung tissue of mice with MP-infection, which may be helpful for prevention and treatment of pulmonary injury caused by MPP.</p>
Subject(s)
Animals , Female , Male , Mice , Cadherins , Metabolism , Cell Membrane , Metabolism , Drug Combinations , Drugs, Chinese Herbal , Pharmacology , Epithelial Cells , Metabolism , Lung , Metabolism , Pathology , Mycoplasma pneumoniae , Plants, Medicinal , Chemistry , Pneumonia, Mycoplasma , Metabolism , Microbiology , Pathology , Random AllocationABSTRACT
<p><b>OBJECTIVE</b>To study the effects and mechanism of Huchang Qingfei pellets on immune function in rats infected with mycoplasma pneumoniae.</p><p><b>METHOD</b>A rat model of mycoplasmal pneumonia (MP) was developed in repeated intranasal infectious routes and then the humoral and cellular immunocompetences were detected by radioimmunoassay, immune-turbidimetry and flow cytometry.</p><p><b>RESULT</b>The levels of serum IgG,IgM and IL-2, IL-6 were enhinced obviously, the complement C3 and TNF-alpha were decreased and the ratio of CD4+ /CD8+ was improved significantly in the Huchang groups as compared with MP model group.</p><p><b>CONCLUSION</b>Huchang Qingfei pellets can reinforce immune function via preventing both cellular and humoral immunity from depression in the rats with MP.</p>