ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of human cytomegalovirus (HCMV) on the cell cycle of duct epithelial cell cultures of human salivary gland (HSG) in vitro and relative mechanism.</p><p><b>METHODS</b>HSG was cultured in vitro. Reverse transcriptase polymerase chain reaction (RT-PCR) and nest-RT-PCR were used respectively to investigate ie1/ie2 transcription in HSG infected by human cytomegalovirus(HCMV). The effects of HCMV on the cell cycle of HSG were studied by flow cytometry in vitro. The expression of cyclin D1 in HSG infected by HCMV was detected by Western blotting.</p><p><b>RESULTS</b>HCMV iel/ie2 transcription could be detected in HSG infected by HCMV. HCMV arrested productively infected cells in G1 stage. And cyclin D1 was down-regulated in HCMV infected HSG.</p><p><b>CONCLUSION</b>HCMV inhibits proliferation of HSG by affecting G1/S check point and down-regulating cyclin D1 in vitro.</p>
Subject(s)
Humans , Blotting, Western , Cell Culture Techniques , Cell Cycle , Physiology , Cyclin D1 , Genetics , Metabolism , Cytomegalovirus , Physiology , Epithelial Cells , Cell Biology , Virology , Flow Cytometry , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Salivary Glands , Cell Biology , MetabolismABSTRACT
The aim of this study was to explore the characteristics of Toll-like receptor expression in mesenchymal stem cells derived from bone marrow of healthy donor (BM-MSCs). BM-MSCs were isolated from bone marrow of healthy donor by Ficoll method. Expressions of CD34, CD45, HLA-DR, CD44 and CD71 in BM-MSCs were detected by flow cytometry. CD71 in BM-MSCs was assayed by immunocytochemistry. The adipocyte and osteoblast induction of BM-MSCs were detected by alizarin red stain and oil red stain respectively. TLR 1 - 10 mRNA levels in BM-MSCs were evaluated by semiquantitative RT-PCR. The results showed that expressions of CD34, CD45 and HLA-DR in BM-MSC were negative while the expressions of CD44 and CD71 were positive. CD71 in BM-MSCs was positive. After induced by osteoblast and adipocyte inductor, BM-MSCs were positive for alizarin red staining and oil red staining respectively. All of TLR 1 - 10 mRNA were found in BM-MSCs with high expression levels of TLR2, TLR3, TLR4, TLR7, TLR8, TLR9 and low expression levels of TLR1, TLR5, TLR6, TLR10. In conclusion, different levels of TLR 1 - 10 mRNA were expressed in BM-MSCs of healthy donor.
Subject(s)
Humans , Bone Marrow Cells , Metabolism , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , Metabolism , RNA, Messenger , Genetics , Toll-Like Receptors , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of HCMV infection on phenotypes of parotid duct epithelial cells and relative mechanisms.</p><p><b>METHODS</b>The expressions of immediate early antigen of HCMV, pan cytokeratin and cathepsin D etc. were detected by immunohistochemical staining in tissues of parotid cytomegalic inclusion disease.</p><p><b>RESULTS</b>Cytokeratin which acts as an epithelial marker became negative while staining of Cathepsin D was intensified in parotid duct epithelial cells after infected by HCMV.</p><p><b>CONCLUSION</b>It demonstrated that cytokeratin was lost through over-expression of Cathepsin D in parotid duct epithelial cells infected by HCMV.</p>
Subject(s)
Animals , Female , Humans , Infant , Male , Mice , Antigens, CD , Antigens, Differentiation, Myelomonocytic , Antigens, Viral , Cathepsin D , Cytomegalovirus , Allergy and Immunology , Physiology , Cytomegalovirus Infections , Metabolism , Pathology , Virology , Desmin , Epithelial Cells , Metabolism , Pathology , Virology , Glial Fibrillary Acidic Protein , Host-Pathogen Interactions , Immunohistochemistry , Keratins , Salivary Ducts , Metabolism , Pathology , Virology , VimentinABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of human cytomegalovirus (HCMV) on the proliferation of duct epithelial cells of human salivary gland (HSG).</p><p><b>METHODS</b>The expression of proliferating cell nuclear antigen (PCNA) and p53 were studied in 11 cases of parotid cytomegalic inclusive disease (PCID) using immunohistochemical staining method. The effects of human cytomegalovirus (HCMV) on the proliferation of HSG were investigated by MTT method in vitro. The expression of PCNA in HSG infected by HCMV was examined using immunocytochemical staining and Western blotting.</p><p><b>RESULTS</b>PCNA was expressed weakly in most of megalic inclusion cells which were positive for HCMV, while all the megalic inclusion cells were p53 negative in all 11 cases of PCID. HCMV inhibited proliferation of HSG in vitro in a time dependent and dose dependent manner. Down-regulation of PCNA was shown in infected cells.</p><p><b>CONCLUSION</b>HCMV inhibits proliferation of HSG and down-regulation of PCNA may be an expression of the inhibition.</p>
Subject(s)
Female , Humans , Male , Cell Division , Cells, Cultured , Cytomegalovirus , Genetics , Virulence , Physiology , Cytomegalovirus Infections , Genetics , Pathology , Down-Regulation , Epithelial Cells , Pathology , Parotid Gland , Pathology , Virology , Proliferating Cell Nuclear Antigen , Salivary Ducts , Pathology , Virology , Tumor Suppressor Protein p53ABSTRACT
<p><b>OBJECTIVE</b>To get the full length of human METH1 cDNA and express it steadily in mammalian cell stably.</p><p><b>METHODS</b>METH1 was amplified by RT-PCR, and cloned into pCDNA3.0 after confirmed by sequence analysis. HepG2 cells were transfected by Lipofectamine reagent and then selected in medium with G418. The expression level of METH1 was detected by RT-PCR and Western blot.</p><p><b>RESULTS</b>METH1 with expected length was effectively amplified, and completely matched the published sequence of encoding mature peptide [GI:5725505] as shown by sequence analysis. Eukaryotic vector expressing METH1 was obtained by gene cloning, cells expressing METH1 was got by selection with G418 at 3 weeks after transfection. RT-PCR and Western blot showed high level expression of METH1.</p><p><b>CONCLUSION</b>Full length of human METH1 gene is cloned successfully and expressed in HepG2 steadily, The results set up a basis for the study of effects of METH1 on hypertrophic scar angiogenesis.</p>
Subject(s)
Humans , ADAM Proteins , ADAMTS1 Protein , Angiogenesis Inhibitors , Genetics , Metabolism , Blotting, Western , Cell Line, Tumor , Cloning, Molecular , DNA, Complementary , Genetics , Metabolism , Disintegrins , Genetics , Metabolism , Metalloendopeptidases , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transfection , MethodsABSTRACT
<p><b>BACKGROUND</b>To study the composition and significance of the inclusion bodies of human cytomegalovirus (HCMV).</p><p><b>METHODS</b>Microdissection of inclusion bodies, PCR and Southern blot were adopted to detect DNA, and immunohistochemistry method and catalyzed signal amplification (CSA) were used to detect the different antigens of HCMV.</p><p><b>RESULTS</b>The inclusion bodies of HCMV were separated from the tissue section of human salivary gland. The fragments amplified by PCR from these dissected inclusion bodies were confirmed to be the DNA of HCMV. With the immunohistochemical method CSA, the immediately early and early antigens of HCMV were detected with monoclonal antibodies DDG9/CCH2, while matrix protein AAC10 was negative in the inclusion bodies.</p><p><b>CONCLUSION</b>The ingredient of inclusion bodies of HCMV included the DNA and the antigens expressed in specific stage of the virus.</p>