Search

Objective: To construct the standard recombinant plasmids for 7 common haplotypes of mannan-binding lectin (MBL) gene., Methods: The DNA samples with known haplotypes and genotypes of MBL gene were used as the templates for amplifying the fragments of MBL gene haplotypes including the promoter region and exon 1 with sequence-specific primer-polymerase chain reaction (SSP-PCR) method. The amplified fragments were cloned into T vector and the bases located at codon 52 and codon 57 of exon 1 in MBL gene were mutated respectively by site-directed mutagenesis. All the 7 recombinant plasmids were identified by PCR and direct sequence analysis., Results: From the DNA samples with known haplotypes and genotypes of MBL gene, the standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA and LYPB of MBL gene were constructed by SSP-PCR and molecular cloning technique. From the recombinant plasmids of HYPA and LYQA, the standard plasmids of haplotypes HYPD and LYQC of MBL gene were constructed by site-directed mutagenesis, respectively., Conclusion: The constructed standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA, LYPB, HYPD and LYQC of MBL gene provide standard controls for detecting the SNPs, haplotypes and genotypes of MBL gene with such genotyping methods us SSP-PCR and real-time PCR.

Objective: To explore the role of complement C3 in delayed-type hypersensitivity (DTH)., Methods: After inducing DTH reaction in the footpads of C3 knockout C3(-/-) and wild-type (C3(+/+) ) mice with ovalbumin (OVA), the thickness of the footpad was measured and HE and immunohistochemical staining preformed to identify the number and types of the infiltrating mononuclear cells in the footpad tissues. T lymphocytes were separated from the spleens of the mice and incubated in 96-well plates with serial dilutions of OVA or mitogens in the presence of mitomycin C-treated macrophages, and the proliferation of the T cells was assessed by (3)H-TdR incorporation assay., Results: The footpad thickness of DTH-C3(-/-) mice was significantly smaller than that of DTH-C3(+/+) mice. The number of the infiltrating mononuclear cells in the footpad tissue of C3(-/-) mice was obviously decreased in comparison with that of C3(+/+) mice, and the cells were characterized mainly as CD4(+) T lymphocytes. No significant difference in the proliferation of mitogen-stimulated splenic T cells was noted between C3(-/-) and C3(+/+) mice, but after stimulation with the specific antigen OVA, significant reduction in the proliferation of splenic T cells from C3(-/-) mice was observed as compared with the T cell proliferation in C3(+/+) mice., Conclusion: C3 defect results in impaired DTH responses in mice, which indicates the important role of C3 in DTH reaction.

Objective: To investigate the expression of endogenetic matrix metalloproteinases-9 (MMP-9) and transforming growth factor-beta(TGF-beta) and their role in the wound healing of blast injury., Methods: Rat models of blast injury under a humid and hot environment were established and the effusion from the wound surface was collected at 4, 24, 48 h and 5, 7, 14, 21 and 28 days after injury, respectively. The contents of MMP-9 and TGF-beta in the effusion of the wound were measured by zymography and enzyme-linked immunosorbent assay (ELISA), respectively., Results: During the wound healing of blast injury, MMP-9 and TGF-beta exhibited changes that followed a regular pattern, both reaching the peak value at 48 h after the injury. TGF-beta content reached the another peak on day 7. TGF-beta value and MMP-9 contents decreased in the second week after injury and their reduction was no longer parallel. Administration of tissue inhibitor of metalloproteinase (TIMP) in the early phase of injury showed no obvious effect, but during the 2 weeks after the injury, its administration caused decrease in MMP-9 content and increase in TGF-beta content in the effusion., Conclusions: In the early phase of wound healing, the elevation of MMP-9 and TGF-beta accelerated cell migration to promote the clearance of the inflammatory necrosis tissues, which might be one of the wound healing mechanisms. But overexpression of MMP-9 in the wound may hinder wound healing, and appropriate use of TIMP can accelerate the delayed wound healing.

Objective: To separate mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) from human plasma., Methods: A two-step affinity chromatography on underivatized sepharose 4B was employed for purification of MBL-MASP complex, followed by gel filtration on a Sephacryl S-300 column for separation of MBL and MASPs from the complex. The purification procedures were performed at 4 degrees Celsius with the addition of two proteolytic inhibitors, phenyl methylsulfonyl fluoride and 1,10-phenanthroline during affinity chromatography but not in the gel filtration buffer., Results: Preparations of highly purified MBL and proenzyme MASPs were obtained. The purified MBL was shown by SDS-PAGE and Western blotting to be a functional multimer composed of 28,000 and 32,000 peptide chains, with high bioactivity as demonstrated by ligand-binding assay and yeast agglutination experiment., Conclusion: A simple and convenient procedure is established successfully for the purification of MBL and the proenzyme MASPs.

Objective: To explore the methods for rapid in vitro culture of the dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) under serum-free conditions and ascertain whether intracellular signal transduction pathway differs between calcium ionophore (CI) and tumor necrosis factor (TNF)- alpha during their induction of dendritic cell differentiation., Methods: PBMCs isolated from healthy donors were plated in serum-free medium supplemented with 50 ng/ml rhGM-CSF. Cells cultured overnight were induced to differentiate with 100 ng/ml A23187 or 50 ng/ml TNF-alpha, given before or 30 min after pre-treatment with 0.5 mug/ml cyclosporine A (CsA). After culture for 40 h, the cell morphology was observed under phase-contrast microscope, and the surface markers on treated PBMCs were analyzed by flow cytometry. MTT colorimetry was employed to assess the proliferation of the allogeneic T cells., Results: PBMCs of healthy donors treated with 50 ng/ml rhGM-CSF in combination with 100 ng/ml CI or 50 ng/ml TNF-alpha for 40 h exhibited typical morphology of DCs with rapidly decreased CD14 expression and increased expressions of CD83 and co-stimulatory molecules (CD80 and CD86), showing also enhanced ability of stimulating allogeneic T cell proliferation. Calcineurin antagonist CsA inhibited the differentiation induced by CI, but not that induced by TNF-alpha., Conclusions: Under serum-free conditions, both CI and TNF-alpha are capable of inducing rapid DC differentiation from human PBMCs, but the intracellular signal transduction of CI-induced differentiation is different from that induced by TNF-alpha.

Objective: To construct pcDNA4/His C-MBL recombinant eukaryotic expression plasmid and examine its expression of mannan-binding lectin (MBL) in mammary cells., Methods: The target sequence was amplified by PCR from pGEM-MBL plasmid that contains wild-type human MBL cDNA, and inserted into eukaryotic expression vector PcDNA4/His C followed by restriction mapping and sequencing. The recombinant plasmid PcDNA4/His C-MBL was transformed into Chinese-hamster ovary (CHO) cells by electroporation, and the Zeocin-resistant clones were selected for analysis of mRNA expression by reverse transcription (RT)-PCR. The expressed product was purified by immobilized metal affinity chromatography (IMAC) and identified by SDS-PAGE and Western-blot analysis, and its immunoreactivity was detected by an indirect enzyme-linked immunosorbent assay ELISA using the anti-serum from Balb/C mice immunized with the recombinant protein., Results: The cDNA fragment of 750 bp was amplified from pGEM-MBL plasmid, which was shown by restriction enzyme digestion and DNA sequencing. The mRNA expression of Zeocin-resistant CHO cell clones was detected by RT-PCR. Three components of 29, 58 and 87 kD in the purified recombinant product were found by SDS-PAGE and the 29 kD component could be recognized by anti-6His antibody in Western blot analysis. The titers of the anti-serum from immunized mice were 1: 819 200 against the recombinant protein and 1:25 600 against both the natural human MBL and the recombinant trimeric carbohydrate-recognition domain (CRD) of human MBL, as determined by the indirect ELISA., Conclusion: The cell strains that express recombinant human MBL (rhMBL) and rhMBL protein have been obtained successfully, which provides the basis for further research of MBL molecule.

Objective: To investigate the single nucleotide polymorphisms (SNP) in the promoter region of mannan-binding lectin (MBL) gene in a Han population in Guangdong Province., Methods: A total of 167 blood samples were obtained from this Han population to isolate the genomic DNA from the leucocytes. The polymorphism alleles -550(G/C, named H/L alleles), -220(G/C, X/Y alleles) and +4(C/Tr, P/Q alleles) in the promoter region of MBL gene were detected by PCR with sequence-specific primers and molecular beacon real-time fluorescent PCR, and the frequencies of haplotypes and genotypes were analyzed., Results: The frequencies of several genotypes in the 167 samples were: LYP/LYP, 10(5.9%); HYP/LYQ, 7(4.2%); LYP/LYQ, 94(56.3%); LXP/LXP, 6(3.6%); LYQ/LYQ, 4(2.4%); LXP/LYQ, 29(17.4%); HYP/LYP, 3(1.8%); HYP/LXP, 2(1.2%); HYP/HYP, 12(7.2%)., Conclusion: The polymorphism genotypes in the promoter region of MBL gene in this chosen population are mostly LYP/LYQ and LXP/LYQ.

Objective: To obtain full-length cDNA encoding mouse mannan-binding lectin C(MBL-C) polypeptide., Methods: The cDNA encoding mouse MBL-C was isolated from Balb/c mouse liver cells by reverse transcriptase (RT)-PCR and inserted into pUC-T vector, and the encoding region structure of the DNA was analyzed to understand its evolutionary relationship with single human MBL homologues., Results: The amplified mouse MBL-C cDNA, which was 735 bp in length, encoded 245 amino acid residues, and its structural analysis showed 100% homology with published mouse MBL-C gene sequence and 71.4% homology with MBL gene of Chinese human., Conclusion: The gene encoding Balb/c mouse MBL-C has been successfully isolated, which may facilitate further study of the innate immune functions of MBL molecule in vivo.