Construction and evaluation of recombinant plasmid containing human triggering receptor expressed on myeloid cells-1 gene / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a eukaryotic expression plasmid containing human triggering receptor expressed on myeloid cells-1(TREM-1) gene.</p><p><b>METHODS</b>The entire gene coding region of human TREM-1 was amplified from total RNA of human peripheral blood by means of RT-PCR. The fragment of TREM-1 was cloned to vector pUCm-T. After digestion by restriction endonuclease BamH I and Pst I, the fragment was subcloned into the eukaryotic expressing vector pEGFP-C3. This recombinant vector was transfected into 293 cells using liposome. The expression level of TREM-1 was determined by fluorescence microscope and Western blot assay. The recombinant TREM-1 vector was transfected into THP-1 cells. After stimulation with 100 ng/ml LPS for 24 h, the mRNA levels of TNF-α and IL-1β were measured using RT-PCR.</p><p><b>RESULT</b>The expression vector was constructed, and the result of the DNA sequencing showed that the constructed plasmid containing the TREM-1 gene. Fluorescence microscope and Western blot analysis showed that TREM-1 protein was expressed in 293 cells successfully. After transfection into THP-1 cells, recombinant TREM-1 could upregulate the mRNA levels of TNF-α and IL-1β.</p><p><b>CONCLUSION</b>Eukaryotic expression plasmid pEGFP-TREM-1 is successfully constructed and showed biological activity.</p>

IL-10 inhibits inducible expression pattern of beta-defensin-2 in human peripheral blood cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of IL-10 on the inducibility of human beta-defensin 2 (hBD-2) in human peripheral blood cells.</p><p><b>METHODS</b>Peripheral blood samples were collected from 22 healthy individuals and co-cultured with 0 ng/ml lipopolysaccharide (LPS), 100 ng/ml LPS, 10 ng/ml IL-10, or 100 ng/ml LPS plus 10 ng/ml IL-10 at 37 degree for 6 h. Total RNA was extracted from peripheral blood cells and the mRNA level of hBD-2 was detected by relative quantitative real-time PCR..</p><p><b>RESULT</b>No detectable level of hBD-2 mRNA was found in normal peripheral blood cells with stimulation of 0 ng/ml LPS or 10 ng/ml IL-10. The mRNA level of hBD-2 was increased to 166.9 +/- 35.14 after 100 ng/ml LPS challenge, while the mRNA level of hBD-2 was decreased to 30.40 +/- 9.18 after the co-stimulation of 100 ng/ml LPS plus 10 ng/ml IL-10; which was significantly lower than that with LPS alone (P<0.05).</p><p><b>CONCLUSION</b>The expression of hBD-2 gene can be induced by LPS.IL-10 inhibits the inducible expression of hBD-2.</p>

Interleukin 10.G microsatellite in the promoter region of the interleukin-10 gene in severe sepsis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The highly polymorphic interleukin 10.G (IL10.G) microsatellite located in the promoter region of the interleukin-10 (IL-10) gene exerts a positive transcriptional regulatory effect on IL-10 gene expression and correlates with the in vitro IL-10 secretion. This study was conducted to investigate whether IL10.G microsatellite is associated with the incidence and/or the outcome of severe sepsis.</p><p><b>METHODS</b>One hundred and fifteen patients with severe sepsis who had been treated at the intensive care unit of the university hospital were studied. One hundred and forty-one healthy individuals served as controls. IL10.G microsatellite genotyping was performed with the following two methods: fluorescent based polymerase chain reaction (PCR) techniques and silver staining of the amplified DNA fragment in polyacrylamide gel. Alleles were defined according to the size of the amplified DNA product.</p><p><b>RESULTS</b>Ten alleles and 36 genotypes were detected both in the patients with severe sepsis and in the healthy controls. Allele IL10.G9 and allele IL10.G13 were the commonest alleles with the frequencies of 32.6% and 21.3% respectively in the patients with severe sepsis, and 34% and 27% respectively in the healthy controls. The allele frequencies of IL10.G microsatellite were neither different between the patients with severe sepsis and the healthy controls (P > 0.05), nor between survivors and non-survivors (P > 0.05). However, the frequency of one common allele IL10.G13 was slightly lower in the patients with severe sepsis than in the healthy controls (21.3% vs 27%, P > 0.05), and the frequency of allele IL10.G9 was slightly higher in the non-survivors than in the survivors (37.1% vs 28.1%, P > 0.05).</p><p><b>CONCLUSION</b>IL10.G microsatellite may neither contribute to the susceptibility to severe sepsis nor to the fatal outcome of severe sepsis.</p>