Plasma mannan?binding lectin and MBL?associated serine protease 2 in patients with hepatocellular carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To detect the plasma levels of mannan?binding lectin (MBL) and MBL?associated serine protease?2 (MASP-2) in patients with hepatocellular carcinoma (HCC) and explore their role in the tumorigenesis and progression of HCC.</p><p><b>METHODS</b>The plasma levels of MBL and MASP?2 were detected by enzyme?linked immunosorbent assay in 64 HCC patients and 30 healthy control subjects. The correlation of MBL and MASP?2 with the clinical parameters of HCC patients were analyzed.</p><p><b>RESULTS</b>The plasma levels of MBL (P=0.014) and MASP?2 (P=0.002) were significantly higher in HCC patients than in the healthy controls, but the MBL?to?MASP?2 ratio did not differ significantly between the two groups. In HCC patients, plasma MBL level was positively correlated with vascular invasion (r=0.253, P=0.047) and total bilirubin level (r=0.283, P=0.024). The plasma level of MASP?2 was positively correlated with TNM stage (r=0.276, P=0.027) and negatively correlated with plasma albumin level (r=0.?0.317, P=0.015). ROC curve analysis revealed an area under curve of 0.665 for MBL (P=0.010) and 0.694 for MASP?2 (P=0.003). The sensitivities of MBL and MASP?2 were 50% and 89.1% in the diagnosis of HCC, respectively.</p><p><b>CONCLUSION</b>MBL and MASP?2 are associated with the inflammatory state and disease progression in patients with HCC.</p>

Mechanism of bone marrow mesenchymal stem cells in alleviating pulmonary alveolitis in mice exposed to silica dust / 中国职业医学

OBJECTIVE: To explore the mechanism of bone marrow mesenchymal stem cells( BMSCs) in alleviating pulmonary alveolitis in mice exposed to silica dust. METHODS: Five specific pathogen free healthy male C57 BL /6 mice were used to isolate BMSCs using bone marrow adherent method. The poly-potent differentiation ability of BMSCs were identified by 3 differentiation-inducing experiments. Forty-five mice of similar background were randomly divided into 3groups: control group,silica group and BMSCs transplantation group. The mice of the control group were given 20. 0 μL of0. 90% sodium chloride solution by one time intratracheal injection. The mice of silica group and BMSCs transplantation group were first received 20. 0 μL( 250 g / L mass concentration) of silica dust suspension by one time intratracheal injection; followed by 500. 0 μL of 0. 90% sodium chloride solution or 500. 0 μL of BMSCs suspension( cell density 1 ×109/ L) by tail vein infusion 6 hours later. Mice were euthanized on the 3rd day of the experiment. Lung functional coefficient and pathologic changes in the lung were examined. The level of cytokines in bronchoalveolar lavage fluid( BALF) was detected by enzyme linked immunosorbent assay. Wright-Giemsa staining was used for staining cells in BALF for counting. Flow cytometry( FCM) was used to measure the percentage of macrophages of BALF in the mice. RESULTS: BMSCs were successfully induced to differentiate into osteogenic,adipogenic and chondrogenic cells and developed into osteoblast,adipogenic cells and chondroblast. On the 3rd day of the experiment,the mice in silica group showed histopathological changes similar to pulmonary alveolitis; while there was no obvious inflammatory change observed in the BMSCs transplantation group,and the structure of lung tissue appeared normal. The lung coefficient of the silica group was higher than that of the control group( P < 0. 05); the lung coefficient of BMSCs transplantation group was lower than that of the silica group( P < 0. 05),but it showed no significant difference when compared to the control group( P > 0. 05). The interleukin( IL)-1β,IL-6 and chemokine ligand 3 levels in BALF in the silica group were higher than those of the control group( P < 0. 05),and the above 3 indices in the BMSCs transplantation group regaining the level of the control group( P > 0. 05) were lower than those of the silica group( P < 0. 05). The level of tumor necrosis factor-α in BALF in silica group and BMSCs transplantation group were higher than that of the control group( P < 0. 05),but there was no significant difference between silica group and BMSCs transplantation group( P > 0. 05). The level of IL-10 in BALF showed no significant difference in these 3 groups( P > 0. 05). Wright-Giemsa staining results showed that the number of total cells and macrophages in BALF in the silica group was higher than that of the control group( P < 0. 05),and the above cell number of BMSCs transplantation was lower than that of silica group( P < 0. 05),but it showed no significant difference when compared to the control group( P > 0. 05). The FCM result showed that the percentage of macrophages was in accordance with that of the Wright-Giemsa staining. CONCLUSION: The BMSCs can alleviate pulmonary alveolitis in the mice exposed to silica dust by inhibiting the amounts and activity of alveolar macrophages and down-regulating the expression of IL-1β and IL-6 in BALF.

Inhibitory effect of bone marrow mesenchymal stem cells on early stage of inflammation in mice exposed to silica dust / 中国职业医学

OBJECTIVE: To study the effects of bone marrow mesenchymal stem cell( BMSC) transplantation on early stage of inflammation in mice exposed to silica dust. METHODS: Specific pathogen free healthy male C57 BL /6 mice were used.Five mice were used to isolate BMSCs using bone marrow adherent method. Thirty mice were randomly divided into 3groups: control group,silica group and BMSCs transplantation group. The mice of the control group were given 20. 0 μL of0. 90% sodium chloride solution by one time intratracheal injection. The mice of silica group and the BMSCs transplantation group first received 20. 0 μL( 250 g / L mass concentration) of silicosis dust suspension by one time intratracheal injection; followed by 500. 0 μL of 0. 90% sodium chloride solution in the silica group,and 500. 0 μL of BMSCs suspension( cell density 1 × 109/ L) by tail vein infusion in the BMSCs transplantation group 6 hours later. Mice were euthanized on the 7th day of the experiments. The histopathology changes in lung tissues were examined. The serum levels of interleukin( IL)-1β, IL-6 and IL-10 were detected by enzyme linked immunosorbent assay. Real-time quantitative polymerase chain reaction was used to detect the mRNA relative expression levels of above cytokines in the lung tissues. RESULTS: The positive rates of BMSCs surface molecules cluster differentiation( CD) 29,CD34,CD90,CD105 and CD106 were 67. 70%,0. 12%,39. 00%,37. 10% and 20. 10%,respectively. Histopathology examination showed the thickened alveolar walls,broadening alveolar septum and the damaged alveolar structure in silica group. In the BMSCs transplantation group,there was no obvious damage found in the lung tissue. There was no change in the alveolar cavity and alveolar structure was complete. The IL-1β and IL-6 levels in serum and mRNA relative expression of IL-1β and IL-6in lung tissue in the silica group were higher than those of the control group and BMSCs transplantation group( P < 0. 05).The IL-1β level in serum and mRNA relative expression of IL-1β in lung tissue in the BMSCs group were higher than those of the control group( P < 0. 05). The IL-10 level in serum and mRNA relative expression of IL-10 in lung tissue in all groups showed no statistical difference( P > 0. 05). CONCLUSION: The BMSCs can alleviate pulmonary inflammatory damage at early stage by down-regulating the expression of proinflammatory factors of IL-1β and IL-6.

Prokaryotic expression of Balb/C mouse MBL-A carbohydrate recognition domain / 南方医科大学学报

<p><b>OBJECTIVE</b>To express the carbohydrate recognition domain (CRD) of Balb/C mouse mannan binding lectin A (MBL-A) in E.coli.</p><p><b>METHODS</b>The target gene fragment was obtained by PCR from the plasmid pmMBL-A harboring mouse MBL-A gene. The PCR product was recombined with the prokaryotic expression vector pET-41a(+) and the resulting recombinant plasmid was identified by PCR, restriction analysis and sequencing before transformation into E.coli BL21(DE3) cell for expression of the target protein. After washing and renaturation, the protein was purified on GST-Tag purification resins and analyzed by SDS-PAGE, Western blotting and enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>A DNA fragment of about 450 bp was amplified by PCR and the recombinant plasmid pET41a-mMBL-A-CRD was constructed by linking the fragment with pET41a(+) vector. The result of restriction enzyme analysis and sequencing of the selected clones were consistent with those by computer analysis. The recombinant vector was expressed in E.coli BL21(DE3), and the expressed protein existed mainly as inclusion bodies, whose relative molecular mass was about 47,000 by SDS-PAGE analysis. After washing, renaturation and purification, the purity of recombinant protein was about 90%. Western blotting suggested immunoreactivity of the purified protein with anti-GST antibody, and its sugar binding activity was verified by ELISA.</p><p><b>CONCLUSION</b>We have successfully obtained mouse MBL-A CRD protein, which provides the base for further functional study of the MBL-A molecule.</p>

Gene cloning, optimized expression and immunogenicity evaluation of tetanus toxin fragment C / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain highly purified tetanus toxin fragment C (TTC) with good immunogenicity.</p><p><b>METHODS</b>The gene fragment encoding TTC was amplified from Clostridium tetani plasmid DNA by PCR, inserted into the vector pET43.1a (+) and expressed in E. coli BL21(DE3)plysS. After purification using Ni2+-chelate affinity chromatography, the expressed fusion protein was digested by thrombin and the resultant TTC protein was purified with Ni2+-chelate affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purifed TTC protein was then used to immunize mice to test its immunogenecity.</p><p><b>RESULTS</b>The 1373-bp gene fragment encoding TTC was obtained, and the constructed recombinant expression vector pET43.1a (+)-TTC was successfully expressed in E. coli BL21(DE3)plysS. SDS-PAGE identified a recombinant fusion protein with relative molecular mass (Mr) of 117 000, which accounted for 22% of the total bacterial protein. The TTC protein with Mr of 50 000 was obtained after purification of the thrombin digestion products of the fusion protein, with a purity reaching 95.5%. Both the fusion protein and TTC protein could be recognized by anti-tetanus toxin antibody as shown by Western blotting. The titer of the anti-serum from mice immunized with the TTC protein was 1:25 600, and the anti-serum could specifically bind to tetanus toxin.</p><p><b>CONCLUSION</b>Highly purified and immunogenetic TTC protein has been successfully obtained, which provides a good model antigen for studying antigen presentation and immune responses in vivo.</p>

Cloning and prokaryotic expression of the gene encoding PGRP domain of mouse long peptidoglycan recognition protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the gene coding for the peptidoglycan recognition protein (PGRP) domain (PGRPd) of mouse long PGRP (mPGRP-L) and express the protein in E. coli.</p><p><b>METHODS</b>The cDNA fragment encoding PGRPd of mPGRP-L was obtained by RT-PCR from the total RNA of Balb/C mouse liver cells and cloned into pUCm-T vector. The recombinant plasmid were identified by PCR, restriction endonucleases and sequence analysis. The PGRPd gene fragment was amplified by PCR from the recombinant plasmid, inserted into pQE-30 vector and transformed into E. coli strain M15, and the expressed PGRPd protein was purified.</p><p><b>RESULTS</b>A cDNA fragment of about 500 bp was amplified by RT-PCR and the recombinant plasmid, pmPGRPd, was constructed by linking the fragment to pUCm-T vector. The results of restriction mapping of the recombinant vector were consistent with those of computer analyses. Sequence analysis showed that the cloned gene fragment (518 bp) had identical sequence with the gene encoding PGRPd of mPGRP-L gene in GenBank. The recombinant expression vector pQE-PGRPd was constructed and expressed in E. coli M15. SDS-PAGE showed that the expressed product existed mainly in the lysate supernatant as a soluble protein with relative molecular mass of 29 kD.</p><p><b>CONCLUSION</b>The PGRPd cDNA of mPGRP-L has been successfully cloned and expressed in E. coli, which provides the basis for further study of PGRP molecule.</p>