[Purification of human MMP-1 fusion protein and clinical detection of MMP-1 by its polyclonal antibody].

OBJECTIVES: To obtain the fusion protein of MMP-1 and the rabbit-anti-human matrix metalloproteinase-1(MMP-1) polyclonal antibody, and to detect the serum MMP-1 in patients by this polyclonal antibody. METHODS: The fusion protein was purified by affinity chromatography. Two rabbits were immunized with the purified fusion protein, and the immune sera of rabbits were collected. Antibodies (IgG) obtained from the immune sera were purified by ammonium sulfate fractionation, and the indirect sandwich enzyme immunoassay was used to detect the serum MMP-1 in 24 samples of patients with chronic liver disease or cirrhosis and in 12 normal sera as controls. RESULTS: The purified MMP-1 fusion protein was successfully obtained; the purified specific polyclonal antibodies of rabbit-anti-human MMP-1 were also obtained from the immune sera of the rabbits, and could respond to human MMP-1. Compared with the normal controls, the OD value of serum MMP-1 significantly increased in patients with chronic hepatitis or liver cirrhosis (P < 0.01). CONCLUSION: The rabbit-anti-human MMP-1 polyclonal antibody may be used in the diagnosis of initial stage of hepatic fibrosis.

[Gene expression profile of peripheral blood monocyte in patients with fulminant hepatitis B by cDNA microarray].

OBJECTIVE: To investigate peripheral blood monocyte (PBMC) gene expression profile in patients with fulminant hepatic failure (FHF) by cDNA microarray. METHODS: Microarrays consisting of 8,192 human cDNAs and labelled cDNAs prepared from PBMC in both 10 FHF patients and 10 asymtomatic surface antigen carriers (ASC) were applied to analyze gene expression. Relative ratios of gene expression in individuals were obtained by comparing the hybridization results, by GenePix 4000B scanning and by ImaGene3.0 software analysis, of Cy5-labelled cDNA from FHF patients with those of Cy3-labelled cDNA from ASC. RESULTS: 249 genes out of 8,192 were identified differently, at least two times. Most of the genes (79%) involved in cell signaling transduction, cell cycles, metabolism, inflammatory response and apoptosis, whose mRNAs were differently regulated. CONCLUSIONS: These results suggest that HBV infection alters a broad range of cellular genes expression during developing into FHF and provide a framework for future functional study on the genes expressed differently.

[Study of the inhibiting effect of anti-NKG2D polyclonal antibody on cytotoxicities of NK and LAK cells].

AIM: To investigate the effect of anti NKG2D polyclonal antibody(pAb) on cytotoxicities of NK and LAK cells. METHODS: Peripheral blood mononuclear cells(PBMCs) were separated by centrifugation on Ficoll-Hypaque gradients. LAK cells were induced from PBMCs by PHA (10 mg/L) and rhIL-2 (1x10(6)U/L). Then NK cells were sorted by flow cytometry(FCM). The cytotoxicities of NK and LAK cells were analyzed by MTT colorimetry after NKG2D molecule on NK and LAK cells were blocked with anti-NKG2D pAb. RESULTS: FCM analysis proved that both purity and activity of obtained NK cells were high.The anti-NKG2D pAb could inhibit significantly cytotoxicities of NK and LAK cells to K562 and HepG2 cells, for NK cells,having decreased 82.9% and 75.6%, for LAK cells,having decreased 52.8% and 50.2%, respectively.The anti-NKG2D pAb had no effect on cytotoxicities of NK and LAK cells to CNE cells. CONCLUSION: The anti-NKG2D pAb can inhibit cytotoxocities on tumor cells by NK and LAK cells through NKG2D molecule on two effector cells.

[Construction of recombinant eukaryotic expression vector of NKG2D gene and its expression in COS-7 cells].

OBJECTIVE: To construct recombinant eukaryotic expression vector of NKG2D, and to examine its expression in COS-7 cells. METHODS: Human NKG2D cDNA was obtained from peripheral blood mononuclear cells using RT-PCR, and then the target gene was cloned into pMD18-T vector. A eukaryotic expression plasmid containing target gene was constructed by DNA recombinant technique, and was confirmed by double restriction enzymes digestion and DNA sequence analysis. The recombinant plasmid with encapsuled lipofectamine was transfected into COS-7 cells, and the transfected COS- The 7 cells containing expressive target gene were confirmed by RT-PCR and flow cytometry. RESULTS: The sequence of NKG2D cDNA obtained from the recombinant eukaryotic expression vector was identical with that published on GeneBank. The NKG2D gene was expressed successfully in COS-7 cells. CONCLUSION: The recombinant expression plasmid containing NKG2D gene is constructed successfully. After being transfected to COS-7 cells, the NKG2D protein is expressed by the engineering COS-7 cells line, which lays a foundation for further studying biological activity of NKG2D.

[Distribution of hepatitis B virus genotype in Hunan Province and its clinical significance].

OBJECTIVE: To study the distribution of hepatitis B virus genotype in Hunan Provine and its clinical significance. METHODS: HBV genotype was determined by the restriction fragment length polymorphism analysis in 185 PCR positive patients, including 42 asymptomatic HBV carriers (ASC), 38 chronic mild or moderate hepatitis (CH), 80 fulminant hepatic failure (FHF), and 25 hepatocellular carcinoma (HCC) patients in Hunan Province. RESULTS: Of the 185 patients, 136 (73.5%) were genotype B, and 49 (26.5%) were genotype C. There was a statistical significance in the distribution of genotype B between FHF and ASC, and between HCC and ASC (83.7% vs. 57.1%, 76% vs. 57.1%, P < 0.01, respectively). Vertical transmission and HBeAg positivity were higher in genotype C than in genotype B (38.8% vs. 13.2%, 57.1% vs. 30.9%, respectively, P < 0.001). The ALT value was significantly higher in genotype B than in genotype C (P < 0.001). CONCLUSION: Genotypes B and C exist in Hunan. Genotypes B is the major genotype in this area and associated with the development of severe liver diseases. Genotype C is associated with vertical transmission.

[Structure and regulative function of the 5' end flanking sequence of gene CCR5].

OBJECTIVE: To construct the pCAT reporter genes containing the 5'-end flanking of CC chemokine receptor 5(CCR5) gene in different sequence lengths and identify the sequence, which regulates the gene expression of CCR5 by the CAT analysis system. METHODS: The target sequences were amplified by pyrobest DNA polymerase, and were inserted into the upstream of CAT gene located in the pCAT enhancer vector by the directional clone technique respectively; the regulative sequence was identified by analyzing the CAT activities of reconstructed plasmid in Hela cells. RESULTS: The region, containing 486 bp upstreaming from exon 1, stimulated the reporter gene activity which was about 3 times that of the pCAT enhancer vector in transfected cells. CONCLUSION: There is an up-regulative element of gene transcription in the region of -1(-)-486 bp in CCR5 gene upstream.