Biomedical subjects

Li-yun Zhang

Publications and source records attributed to Li-yun Zhang.

10 recordsLinked to original sources

PubMed2007

[Construction of the expression vector pET32a/His MBL-CLR and its expression in E.coli].

AIM: To construct pET32a/His MBL-CLR recombinant prokaryotic expression plasmid and to express mannan-binding lectin-CLR (MBL-CLR) protein in E.coli METHODS: The human MBL-CLR gene was amplified by PCR from pGEM-MBL plasmid, and was inserted into prokaryotic expression vector pET32a. After identified by restriction mapping and sequencing, the recombinant plasmid pET32a/His MBL-CLR was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by Immobilized Metal Affinity Chromatography (IMAC) and identified by SDS-PAGE, Western blot and indirect enzyme-linked immunosorbent assay (ELISA) using the antibody from BALB/c mice immunized with the recombinant human MBL protein. RESULTS: The cDNA fragment of 180 bp was amplified from pGEM-MBL plasmid and the recombinant expression vector pET32/His MBL-CLR was constructed. The recombinant plasmid was consistent with those expected by restriction maps and sequence. Three components of relative molecular mass 30,000, 60,000 and 120,000 in the purified recombinant product were detected by SDS-PAGE and all the components could be recognized by anti-6His antibody in Western blot assay. The three components were correspondingly with the band of the monomer and oligomer of the fusion protein. The purified recombinant product could react with the antibody against the recombinant human MBL protein in the indirect ELISA. CONCLUSION: The prokaryotic expression strains that efficiently express recombinant human MBL-CLR and the recombinant human MBL-CLR-Trx fusion protein were obtained successfully, which will help the further structure-function research of MBL molecule.

Animals↗

PubMed2006

[Effects of infliximab and etanercept, two types of anti-tumor necrosis factor-alpha inhibitor on serum level of matrix metalloproteinase 3 expression in patients with ankylosing spondylitis].

OBJECTIVE: To evaluate the effect of Infliximab and Etanercept two types of anti-tumor necrosis factor-alpha (TNF-alpha) inhibitors, on the serum level of matrix metalloproteinase 3 (MMP-3) in the pathogenesis of ankylosing spondylitis (AS). METHOD: 47 patients with AS, 40 males and 7 females, aged 17 - 51, were treated with Infliximab (5 mg/kg i.v at weeks 0, 2, and 6); and 26 patients with AS were treated with Etanercept (25 mg, twice a week for 12 weeks). Clinical data including Bath AS indices (BASDAI and BASFI) and sera were collected at baseline and other different times. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were measured. Serum levels of MMP-3 were measured with MMP-3 ELISA kits. RESULTS: Two, six, and ten weeks after Infliximab treatment the levels of ESR, CRP, and serum MMP-3 of the AS patients were all significantly lower than the baseline level (all P < 0.01), and the serum MMP-3 levels were all significantly correlated with ESR (all P < 0.05). In the Etanercept group 1, 2, 4, 8, and 12 weeks after treatment the levels of serum MMP-3, ESR, CRP, BASDAI, and BASFI were all significantly lower than the baseline levels (all P < 0.01); before the treatment ESR was significantly correlated with CRP (r = 0.80, P < 0.01), BASDAI was significantly correlated with BASFI (r = 0.48, P < 0.05), and MMP-3 was significantly correlated with ESR (r = 0.74, P < 0.01) and with CRP (r = 0.72, P < 0.01); and 12 weeks after the treatment significant correlation still existed between ESR and CRP (r = 0.40, P < 0.05), BASDAI and BASFI (r = 0.89, P < 0.01), and MMP-3 and ESR (r = 0.43, P = 0.029), however, there was no significant correlation between CRP and serum level of MMP-3 (r = 0.37, P = 0.061). CONCLUSION: Infliximab and Etanercept, 2 anti-TNF-alpha inhibitors, not only significantly decrease the ESR and CRP, but also decrease the serum level of MMP-3 of patients with AS. MMP-3 is involved in the pathogenesis and disease activity of AS. MMP-3 is also a potentially useful marker of AS disease activity and useful parameter to assess the effectiveness of anti-TNF-alpha inhibitor in treatment of AS.

Adolescent↗

PubMed2006

[A short-term efficacy and safety study of infliximab in active ankylosing spondylitis].

OBJECTIVE: To evaluate the efficacy and safety profile of a loading regimen of the anti-TNFalpha antibody infliximab in ankylosing spondylitis (AS). METHODS: This was an open-labeled trial. Subjects eligible for this study were adults with a diagnosis of definite AS. Active disease was a Bath AS disease activity index (BASDAI) > or = 4 and spinal pain VAS > or = 4. Concurrent stable treatment with nonsteroidal anti-inflammatory drugs was permitted during the study. Subjects were not permitted to be on methotrexate, systemic corticosteroids, cytotoxic drugs or disease-modifying anti-rheumatic drugs for various time periods before screening. Infliximab 5 mg/kg was infused at weeks 0, 2, 6. All patients were followed up to 10 weeks. The primary endpoint was proportion of ASAS 20 responders at week 10. The secondary endpoints were the proportion of subjects achieving an ASAS partial remission, the change from baseline in bath AS functional index and in the physical component summary score of the short form-36 in health survey questionnaire at week 10. Other secondary endpoints, related to reducing signs and symptoms of AS and improving range of motion and physical function, were evaluated. RESULTS: 63 patients (79% males, 90% HLA-B(27)(+), median age 32 yr, median disease duration 10 yr) completed the treatment. The proportion of ASAS 20 responders at 2, 6, 10 week was 75%, 84%, 84% respectively. The proportion of ASAS partial remission patients at 2, 6, 10 week was 10%, 21%, 30% respectively. Results for other secondary efficacy endpoints showed that infliximab could provide substantial benefits to patients with AS by reducing clinical signs and symptoms and improving range of motion, physical function, and quality of life. Sixty-five percent of subjects reported treatment-related adverse events. The most frequently occurred were upper respiratory tract infection and skin and appendages. Secondary was elevation of liver enzymes. Most treatment-related adverse events were mild to moderate in severity. Two patients had serious dermatitis and one stopped treatment owing to an infusion reaction. Short-term follow up indicated that effect of Remicade lasted for about 2 to 8 months without any new side effect. CONCLUSION: A loading regimen of infliximab demonstrated consistent evidence of efficacy and was well tolerated in the treatment of active AS.

Adolescent↗

PubMed2005

[In vitro effect on the differentiation and maturation of monocyte-derived dendritic cells by mannan-binding lectin].

AIM: To explore the effect of mannan-binding lectin (MBL) on the differentiation and maturation of human peripheral blood monocyte-derived dendritic cells (MoDCs). METHODS: After MoDCs was stimulated with human natural MBL, MoDC's morphology was observed under inverted microscope and the expressions of CD1a, CD83, CD40, CD80, CD86 and HLA-DR on MoDCs were analyzed by FACS. The ability of MoDCs to stimulate the proliferation of allogenic T cells was detected by (3)H-TdR incorporation. The ability of MoDCs to up-take antigens was evaluated by zymosan granule phagocytosis test. The levels of IL-12 and TNF-alpha in the culture supernatant of MoDCs were determined by ELISA. RESULTS: The expressions of CD1a, CD83, CD40, CD80, CD86 and HLA-DR on the MoDCs were up-regulated by MBL. The ability of MoDCs to up-take zymosan granules decreased and the proliferation of native T cells induced by MoDCs was enhanced. MBL stimulated the production of IL-12 by MoDCs, but had no such effect on TNF-alpha secretion. CONCLUSION: MBL can induce differentiation and maturation of DCs in vitro, suggesting that MBL possibly participate in the adaptive immune response through modulation of functions of DCs.

Cell Differentiation↗

PubMed2005

[Study on enhanced leukocyte antigen-related tyrosine phosphatase in keratoconus].

OBJECTIVE: To study the enhanced expression of leukocyte antigen-related tyrosine phosphatase (LAR) in human keratoconus cornea. METHODS: Corneal buttons of patients received keratoplasty in Eye Center of Beijing Tongren from December 2001 to March 2002 were collected. Total RNA was isolated from the buttons from keratoconus (15 eyes), normal cornea (7 eyes), bullous keratopathy (6 eyes), non-neovascular cornea scar (3 eyes) and corneal dystrophy (2 eyes). Polymerase chain reaction (RT-PCR) was performed in this group. The results were displayed by agarose gels electrophoresis. In another group of patients, the corneal buttons of patients received keratoplasty in Eye Center of Beijing Tongren from July 2002 to December 2002 were collected. Total protein was extracted from the buttons of keratoconus (6 eyes), normal cornea (3 eyes), bullous keratopathy (3 eyes), non-neovascular cornea scar (3 eyes) and corneal dystrophy (2 eyes). Western blot was performed in this group. The results were displayed by enhanced chemical illumination. Bands of LAR RNA and LAR protein from these two tests were analyzed quantitatively. RESULTS: In RT-PCR study using the same amount of total RNA, high signal of LAR RNA was detected in RNA from the keratoconus, whereas low or no signal was detected in normal cornea and other diseases. Sequence analysis of the product of amplified LAR fragment from keratoconus showed deletion of exon 13. In Western blot study using the same amount of total protein, high signal of LAR was detected in keratoconus by the LAR antibodies, while low or no signal was detected in normal cornea and other diseases. CONCLUSIONS: LAR mRNA and protein are enhanced in keratoconus, whereas there are low or no expression in normal cornea, bullous keratopathy, non-neovascular cornea scar and corneal dystrophy. Elevated LAR in keratoconus indicates that LAR plays an essential role in the occurrence of keratoconus. Deletion of exon 13 in keratoconus may play a potential role in the abnormal LAR function.

Humans↗

PubMed2005

[Expressions and role of endogenetic matrix metalloproteinases-9 and transforming growth factor-beta during wound healing of blast injury].

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.

Animals↗

PubMed2005

[Construction of standard recombinant plasmids for 7 common mannan-binding lectin gene haplotypes].

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.

Base Sequence↗

PubMed2005

[Role of complement C3 in delayed-type hypersensitivity].

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.

Animals↗

PubMed2004

[Construction of the expression vector PcDNA4/His C-MBL and its expression in Chinese-hamster ovary cells].

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.

Animals↗

PubMed2004

[Purification and separation of mannan-binding lectin (MBL) and MBL-associated serine proteases complex from human plasma].

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.

Chromatography, Affinity↗