OBJECTIVETo 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.

METHODSThe 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.

RESULTSA 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.

CONCLUSIONThe PGRPd cDNA of mPGRP-L has been successfully cloned and expressed in E. coli, which provides the basis for further study of PGRP molecule.

Animals ; Base Sequence ; Carrier Proteins ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Female ; Liver ; chemistry ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Protein Structure, Tertiary ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo obtain highly purified tetanus toxin fragment C (TTC) with good immunogenicity.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONHighly purified and immunogenetic TTC protein has been successfully obtained, which provides a good model antigen for studying antigen presentation and immune responses in vivo.

Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Tetanus Toxin ; biosynthesis ; genetics ; immunology ; Tetanus Toxoid ; immunology

OBJECTIVETo establish a gastric cancer cell line with stable expression of metastasis-associated in colon cancer 1 (MACC1) and detect the changes in tumor-related gene expression profiles for investigating the possible regulation mechanisms between MACC1 and the differentially expressed genes.

METHODSThe full-length MACC1 cDNA was amplified from human embryonic kidney 293FT cells and cloned into the pBaBb-puro vector. The recombinant pBaBb-puro-MACC1 expression vector, after identification with restriction enzyme digestion, was transfected into 293FT cells, and the expression of fluorescent reporter gene was observed. pBaBb-puro-MACC1 vector was transfected into human gastric cancer BGC-823 cell line to establish BGC-823/pBaBb-puro-MACC1 cell line stably expressing MACC1. Quantitative RT-PCR and Western blotting were used to detect MACC1 expression in both BGC-823/pBaBb-puro-MACC1 and control BGC-823 cells. High-throughout cDNA microarray was used to screen the effects of MACC1 on the gene expression profiles of gastric cancer cells.

RESULTSThe recombinant pBaBb-puro-MACC1 plasmid was successfully constructed and verified by PCR and sequencing. BGC-823/pBaBb-puro-MACC1 cells showed significantly increased MACC1 mRNA expression as compared with the control cells. The results of cDNA microarray identified 33 up-regulated and 24 down-regulated genes in the cells after MACC1 transfection involved were in various cellular functions.

CONCLUSIONThe established BGC-823/pBaBb-puro-MACC1 gastric cancer cell line show some important molecular changes caused by MACC1.

Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Oligonucleotide Array Sequence Analysis ; Stomach Neoplasms ; metabolism ; pathology ; Transcription Factors ; genetics ; metabolism ; Transcriptome ; Transfection

OBJECTIVETo express the carbohydrate recognition domain (CRD) of Balb/C mouse mannan binding lectin A (MBL-A) in E.coli.

METHODSThe 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).

RESULTSA 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.

CONCLUSIONWe have successfully obtained mouse MBL-A CRD protein, which provides the base for further functional study of the MBL-A molecule.

Animals ; Carbohydrates ; chemistry ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Inclusion Bodies ; metabolism ; Mannose-Binding Lectin ; biosynthesis ; chemistry ; genetics ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics

OBJECTIVETo explore the incidence and location of Y chromosome microdeletions in Chinese azoospermia and severe oligozoospermia, as well as the relationship between the deletion region and testicular phenotype.

METHODSSemen samples or blood samples were collected from 664 Chinese patients (584 with azoospermia and 80 with severe oligozoospermia). DNA was extracted by incubating cells with a lysis buffer containing polymerase chain reaction (PCR) buffer and proteinase K, and was assayed for deletion of 15 sequence tagged sites (including 6 loci recommended by European Academy of Andrology and European Molecular Genetics Quality Network (EAA/EMQN) distributed in AZFa, AZFb and AZFc by 4 multiplex PCRs. The histological phenotypes of testes of some azoospermic patients harboring Y chromosome microdeletion were studied by fine needle aspiration.

RESULTSSixty-six (11.3%) cases of microdeletions were found in the 584 patients with azoospermia, and deletions of AZFc region are the leading group (72.7% of all deletions), followed by AZFbc (13.6%), AZFabc (6.1%), AZFb (4.5%) and AZFa (3.0%). In the 80 men with severe oligozoospermia, 10 (12.5%) cases of AZFc microdeletions were detected. While azoospermia (n=19) with AZFc region deletion showed variable testicular phenotype, deletions of AZFb+c and AZFa+b+c (n=7) resulted in severe impaired spermatogenesis characterized by Sertoli cell only syndrome and spermatogenic arrest at spermatogonia.

CONCLUSIONIn the Chinese men with azoospermia and severe oligozoospermia, the incidence of Y chromosome microdeletions and the frequency of the deletions of the three AZF regions are similar to those described previously in other populations. Massive deletions of AZFb+c and AZFa+b+c impair spermatogenesis severely.

Azoospermia ; genetics ; Chromosome Deletion ; Chromosomes, Human, Y ; genetics ; Humans ; Male ; Models, Genetic ; Oligospermia ; genetics ; Polymerase Chain Reaction

OBJECTIVETo evaluate the effectiveness of free graft transplantation two-stage urethroplasty for hypospadias repair.

METHODSFifty-eight cases with different types of hypospadias including 10 subcoronal, 36 penile shaft, 9 scrotal, and 3 perineal were treated with free full-thickness skin graft or (and) buccal mucosal graft transplantation two-stage urethroplasty. Of 58 cases, 45 were new cases, 13 had history of previous failed surgeries. Operative procedure included two stages: the first stage is to correct penile curvature (chordee), prepare transplanting bed, harvest and prepare full-thickness skin graft, buccal mucosal graft, and perform graft transplantation. The second stage is to complete urethroplasty and glanuloplasty.

RESULTSAfter the first stage operation, 56 of 58 cases (96.6%) were successful with grafts healing well, another 2 foreskin grafts got gangrened. After the second stage operation on 56 cases, 5 cases failed with newly formed urethras opened due to infection, 8 cases had fistulas, 43 (76.8%) cases healed well.

CONCLUSIONSFree graft transplantation two-stage urethroplasty for hypospadias repair is a kind of effective treatment with broad indication, comparatively high success rate, less complications and good cosmatic results, indicative of various types of hypospadias repair.

Adolescent ; Adult ; Child ; Child, Preschool ; Humans ; Hypospadias ; surgery ; Male ; Mouth Mucosa ; transplantation ; Penis ; abnormalities ; surgery ; Retrospective Studies ; Scrotum ; abnormalities ; surgery ; Skin Transplantation ; Treatment Outcome ; Urethra ; surgery ; Urologic Surgical Procedures, Male ; methods

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.

OBJECTIVETo 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.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONMBL and MASP?2 are associated with the inflammatory state and disease progression in patients with HCC.

BACKGROUND:Stem cells have been widely used in the treatment of spinal cord injury,but the clinical application is limited by immune rejection,difficulty in cell harvesting and purification.However,human nasal mucosa mesenchymal stem cells (hNM-MSCs) have no such problems,and its clinical application in the treatment of spinal cord injury has been not reported yet.OBJECTIVE:To observe the biological characteristics of autologous hNM-MSCs and its clinical efficacy in the treatment of advanced incomplete spinal cord injury.METHODS:NM-MSCs were isolated from the human nasal mucosa,cultured and identified in vitro.The ultrastructure of NM-MSCs was observed by transmission electron microscope and scanning electron microscope.Then the NM-MSCs were induced to differentiate into osteocytes,adipocytes,stem cell spheres,or neurons in vitro.Totally eight patients with incomplete spinal cord injury were enrolled and subjected to hNM-MSCs transplantation via lumbar puncture for 1-3 sessions of about 5× 107 cells each,with an interval of 5-7 days,after the approval of the medical ethics committee.All the patients were followed up for 6 months.Preoperative and postoperative therapeutic effect evaluations were performed on the basis of American Spinal Injury Association (ASIA) scores.RESULTS AND CONCLUSION:Under light microscope,the NM-MSCs were mainly spindle-shaped,positive for STRO-1 and exhibited a radial arrangement.NM-MSCs highly expressed CD73,CD90 and CD105,but did not express CD34 and CD45,with the purity of over 97％.And lots of podgy microvilli were seen on the surface of NM-MSCs under the scanning electron microscope,and two kinds of cell morphologies were seen under the transmission electron microscope.Moreover,hNM-MSCs had the ability to differentiate into osteocytes,adipocytes,stem cell spheres and neurons.During the 6-month follow-up,seven patients achieved neurological function recovery to different extents except for one patient,and no side effects were found.It is concluded that hNM-MSCs can become the ideal seed cells for tissue-engineered cell repair.Autologous NM-MSCs transplantation for the treatment of spinal cord injury can achieve the ideal effect,with the value of clinical application.

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.