This study was purposed to investigate the angiogenesis-promoting activities of human mesenchymal stem cells (hMSCs) modified by hepatocyte growth factor (HGF) and the underlying mechanisms. The hMSCs were transfected by recombinant adenoviral vector carrying human HGF gene and seeded onto the chicken chorioallantoic membrane. Three days later, the number of blood vessels was counted and their angiogenic response was compared with those of hMSCs of same generation, recombinant basic fibroblast growth factor (bFGF) and alpha-MEM as control. The expression levels of bFGF, VEGF, angiopoietin-1 and angiopoietin-2 were evaluated by RT-PCR assay. The results showed that gene-modified hMSCs exhibited greatest activity to promote angiogenesis while the angiogenic response was nearly same between groups treated by hMSCs and bFGF, all of which were significantly higher than that observed in control (p < 0.01). RT-PCR analysis revealed that hMSCs constitutively expressed multiple angiogenesis-associated growth factors and their levels seemed up-regulated by HGF gene transfer. It is concluded that HGF gene-modified hMSCs show a potent angiogenesis-promoting function and may be useful in the treatment of ischemic disorders.
Animals ; Cells, Cultured ; Chick Embryo ; Chickens ; Hepatocyte Growth Factor ; genetics ; Humans ; Mesenchymal Stromal Cells ; Neovascularization, Physiologic ; genetics ; Transfection

The volatile components of roots and stems of Zanthoxylum nitidum were investigated by supercritical fluid carbon dioxide extraction (SFE-CO2) and gas chromatography-mass spectrometry(GC-MS). Thirty-one and fifty-one compounds were identified in the supercritical extracts from roots and stems of Z. nitidum, respectively, and total twenty-seven compounds were the common constituents. Among them, the major constituents in root and stem supercritical extracts were spathulenol (18.49 and 26.18%), n-hexadecanoic acid (14.24% and 12.79%), ar-tumerone (6.95% and 8.88%), oleic acid (8.39% and 5.71%) and hexanoic acid (4.39% and 7.78%). The in-vitro MTT assay showed that the volatile components of roots and stems of Z. nitidum did not exhibited any cytotoxic activity against human cancer Huh-7 and normal IEC-6 cells. These results indicated the same nature of the volatile constituents in the root and stem of Z. nitidum. This investigation may provide further evidence for expansion of medicinal parts of Z. nitidum.
Animals ; Cell Line, Tumor ; Cell Survival ; drug effects ; Chromatography, Supercritical Fluid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; toxicity ; Gas Chromatography-Mass Spectrometry ; methods ; Humans ; Mice ; Plant Roots ; chemistry ; Plant Stems ; chemistry ; Zanthoxylum ; chemistry

A 2,4 -dichlorophenol degrading Pseudomonas strain GI241-1 was isolated from a soil sample. The dienelactone hydrolase gene, designated as dcpD which encodes dienelactone hydrolase involved in transforming cis-2-chloro-dienelactone into 2-chloromaleylacetic acid, was cloned from this bacterium strain. The gene cloning strategy was to construct genomic library after location of its neighbouring gene by Southem blot and to screen the aim transformant by dot blotting. Sequencing results showed that length of dcpD is 702bp. The sequence of dcpD and the deduced amino acid are different from the relative sequences registered in the GenBank.

The aim of this study was to investigate if transfusion of mesenchymal stem cells (MSC) could exhibit beneficial effects on rheumatoid arthritis. Human bone marrow MSC were intraperitoneally injected into Wistar rats with collagen-induced arthritis at a dose of 10(7) on the next day (preventive group) or 2 weeks (treatment group) after collagen II induction, once a week for 2 weeks (preventive group) or 4 weeks (treatment group). The control group was given normal saline (NS) at corresponding time. The symptom scorings were documented weekly from the second week of the induction. On week 6, the hind joints of the rats were pathologically examined and the activation status of splenocytes was analyzed by flow cytometry. The results showed that all the rats developed arthritis and subsequent joint abnormality. On the sixth week, symptom scores of the rats that received MSC preventive (9.5 ± 0.5) or therapeutic (9.4 ± 0.6) infusions had no significant difference between each other, but were significantly greater than those of the NS controls (7.6 ± 0.6, P < 0.05). Consistently, pathological examination on the involved knees showed that the synovitis and arthritis scorings of MSC treated rats were greatly elevated compared with NS controls. Furthermore, the ratios of CD86(+) cells in the spleens of MSC prevention, MSC treatment and NS control groups were (4.16 ± 1.48), (4.06 ± 1.97) and (4.15 ± 2.04) respectively, while those of CD11b/c(+)CD86(+) cells were (1.04 ± 0.68), (0.95 ± 0.56) and (0.98 ± 0.44), all of which were significantly higher than those of healthy controls [(0.97 ± 0.18) and (0.30 ± 0.17), P < 0.05 for both parameters]. It is concluded that MSC infusion has little beneficial effects on collagen-induced arthritis in rats, conversely, MSC therapy aggravated the damage of the involved joints, its underlying mechanisms need to be further investigated.
Animals ; Arthritis, Experimental ; pathology ; prevention & control ; therapy ; Bone Marrow Cells ; Cells, Cultured ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Wistar ; Transplantation, Heterologous ; Treatment Outcome

Objective:To explore the protective effect of agmatine on mice with multiple organ failure (MODS) induced by yeast polysaccharide(ZYM) on the expression of inflammatory factors.Methods:ZYM induced inflammation model was established by intraperitoneal injection of ZYM in mice.All mice were divided into blank group,ZYM group and ZYM+AGM group.The mice feeding, white cell count,heart rate and so on were observed before and after the modeling to determine whether the model was successful.The liver function of mice,renal function,myocardial enzymes and other biochemical indicators were detected after the success of the model;and through the qPCR and ELISA method for detection of blood tumor necrosis factor alpha (TNF-alpha),interleukin 1 beta (IL-1 beta),interleukin 6 (IL-6),IL-10 gene and protein secretion level.Results: After the injection of ZYM,mice looked disorganized, activity and reduce consumption;the functional serological indexes of various organs of the mice were detected,which showed that the function of the viscera was serious.Compared with the blank control group,the serum parameters of ZYM group and ZYM+AGM group were significantly higher,and the inflammatory factors TNF-α,IL-1,IL-6 and IL-10 were significantly increased (P<0.05).Compared with ZYM group,ZYM+AGM serum markers of organ function decreased,inflammatory factor TNF-α,IL-1β,IL-6 decreased significantly (P<0.05),while there was no significant difference in IL-10 (P>0.05); and the mouse spirit,eating and activity had no significant change.Conclusion:Intraperitoneal injection of 500 mg/kg ZYM can successfully construct a model of MODS,AGM by reducing the release of inflammatory factors,play a protective role in the function of various organs of MODS mice.

OBJECTIVETo study the changes in serum cortisol levels in adolescents with type 1 diabetes (T1DM) and elevated depressive symptoms.

METHODSTwenty-eight adolescents with T1DM and 31 healthy peers were assessed for depressive symptoms using a depression self-rating scale developed by the Epidemiological Survey Center. Selected subjects were classified into four groups: T1DM with elevated depressive symptoms group (n=15), T1DM without elevated depressive symptoms group (n=13), elevated depressive symptoms without T1DM group (n=15), and normal control group (n=16). Fasting blood samples were collected in the morning, and the levels of serum cortisol were compared among the four groups. The correlations of serum levels of cortisol and glycosylated hemoglobin A1c (HbA1c) with the score of depression self-rating scale were evaluated by Pearson correlation analysis.

RESULTSThe fasting serum cortisol levels in the 28 T1DM patients were significantly higher than in the 31 healthy peers (P<0.01). The fasting cortisol levels in the T1DM with elevated depressive symptoms group were significantly higher compared with those in the elevated depressive symptoms without T1DM group and normal control group (P<0.01). In adolescents with T1DM, serum HbA1c level was positively correlated with the score of depression self-rating scale (r=0.481, P=0.010).

CONCLUSIONSThe fasting serum cortisol levels in adolescents with T1DM and elevated depressive symptoms are significantly increased, suggesting that the patients with comorbidity of T1DM and depression develop dysfunction of the corticotropin-releasing hormone-adrenocorticotropic hormone-cortisol axis. The elevated depressive symptoms may be associated with a poor control of glucose metabolism.

Adolescent ; Adrenocorticotropic Hormone ; physiology ; Child ; Corticotropin-Releasing Hormone ; physiology ; Depression ; blood ; etiology ; Diabetes Mellitus, Type 1 ; blood ; Female ; Glucose ; metabolism ; Glycated Hemoglobin A ; analysis ; Humans ; Hydrocortisone ; blood ; Male

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibody Specificity ; CHO Cells ; Cricetulus ; Female ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Thrombomodulin ; immunology ; Transfection

2,4-Dichlorophenol is toxic and biorefratory organic pollutant. A 2,4-dichlorophenol degrading bacterial strain GT241-1, identified as Pseudomonas sp., was isolated from soil samples which was collected from drainage area of several 2,4-dichlorophenol producing factories. Strain GT241-1 had strong 2,4-dichlorophenol degrading ability, it could decompose 91% 2, 4-dichlorophenol of 90 mg/L within 48 hours at 25 - 30 degrees C, and could utilize 2,4-dichlorophenol, 2,4-dichlorophenoxyacetic acid (2,4-D), benzoate and catechol as sole carbon and energy source. Southern blot showed that 2,4-dichlorophenol hydroxylase gene (dcpA) of strain GT241-1 locates on the about 10kb EcoR I/Xba I fragment. This fragment was recovered, linked to the vecter pUC19 and transformed into the E. coli DH5alpha. A aim transformant, Z539, was obtained by dot blotting from about 1200 transformants. PCR and the sequencing results shew that the whole dcpA gene is contained within the 10kb EcoR I /Xba I fragment of pZ539. This fragment was shortened to about 2.4kb by HindmIII. The shorted fragment was subcloned to vecter pRSET-B to get a transformant BS1-12. The subcloned fragment was sequenced. Sequencing results showed that the whole length of the subcloned fragment containing dcpA is 2389bp and the nucleotide span of coding region is from number 276 to number 2072 (1797 bp), with ATG and TAA as start and stop codon respectively. The sequence analysis of dcpA and the deduced amino acid encoded by dcpA showed that they are different from the relative sequences registered in the GenBank. The subcloned fragment carry the promoter of dcpA, this can deduce from the fact that the upflow length of dcpA coding region is 275bp, and further confirmed by the 2,4-dichlorophenol hydroxylase activity measurement results. The 2,4-dichlorophenol hydroxylase activity of transformant Z539 and BS1-12 were detected, the results showed these transformants have 2,4-dichlorophenol hydroxylase activity. By comparison, the activity of these transformants were lower than that of the strain GT241-1.
Amino Acid Sequence ; Bacterial Proteins ; genetics ; metabolism ; Biodegradation, Environmental ; Chlorophenols ; metabolism ; Cloning, Molecular ; Environmental Pollutants ; metabolism ; Mixed Function Oxygenases ; genetics ; metabolism ; Molecular Sequence Data ; Pseudomonas ; enzymology ; genetics ; isolation & purification ; Soil Microbiology

BACKGROUNDConnective tissue growth factor (CTGF) is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. CTGF plays an important role in extracellular matrix production by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. We herein evaluated whether CTGF was required for extracellular matrix synthesis of meniscal fibrochondrocytes and/or angiogenesis during the repair of meniscal tears.

METHODSMeniscal fibrochondrocytes were isolated from the inner-1/2 of rabbit meniscus by trypsin collagenase treatment and further treated with 100 ng/ml CTGF in vitro. Characterization of fibrochondrocytes was identified by flow cytometry analyzing CD31, CD44, CD45 and CD105, and was further tested by type II collagen immunocytochemistry. Changes in gene expression of meniscal fibrochondrocytes were monitored by quantitative real-time polymerase chain reaction. Histological sections prepared from a 3-mm portion of a longitudinal tearing defect in the middle of the rabbit meniscus were subjected to fluorescence-immunohistochemistry analysis at 1, 4 and 10 weeks following surgical treatment with 1.5 µg of CTGF/fibrin-glue composites.

RESULTSQuantitative RT-PCR assay showed that types I and II collagen and vascular endothelial growth factor mRNA expression in the 100 ng/ml CTGF group were remarkably enhanced as compared to levels in the no-dose group at 14 days ((2.38 ± 0.63) fold, (2.96 ± 0.87) fold, (2.14 ± 0.56) fold, respectively). Likewise, fluorescence-immunohistochemical analysis revealed that in the group implanted with CTGF-fibrin glue, types I and II collagen, as well as the capillaries, completely filled the defect by 10 weeks, postoperatively. In contrast, only soft tissue repair occurred when PBS-fibrin glue was implanted.

CONCLUSIONSThese findings suggest that CTGF can significantly promote extracellular matrix deposition (types I and II collagen) within the meniscal avascular zone; CTGF can greatly heighten the expression of vascular endothelial growth factor activity simultaneously in vivo, further enhancing the repair of meniscal tears in the avascular zone.

Animals ; Cells, Cultured ; Chondrocytes ; cytology ; Collagen Type I ; metabolism ; Collagen Type II ; metabolism ; Connective Tissue Growth Factor ; pharmacology ; Flow Cytometry ; Immunohistochemistry ; Male ; Menisci, Tibial ; cytology ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Tibial Meniscus Injuries ; Vascular Endothelial Growth Factor A ; metabolism ; Wound Healing ; drug effects

OBJECTIVETo establish a model of human monocytic leukemia with CNS infiltration in BALB/c nude mice.

METHODSBALB/c nu/nu mice pre-treated by splenectomy, cytoxan intraperitoneal injection, and sublethal irradiation (SCI), were transplanted intravenously with 1 x 10(7) of human monocytic leukemic SHI-1 cells. The leukemic cells engrafted in the mice were detected by RT-PCR, histopathological examination, immunohistochemistry and FCM.

RESULTSThe survival time of SCI-nu/nu mice was 33-46 d. Paraplegia occurred in some of the mice. 5 weeks after transplantation, SHI-1 cells engrafted in SCI-nu/nu mice, multi-organs were involved and green solid neoplasms were formed in some organs. Histopathological examination found that SHI-1 cells infiltrated in liver, lung, kidney and testis of the mice and vertebral and skull bone marrow was replaced by leukemic cells. Leukemic cell penetrated through the surface of vertebrae, formed neoplasm, and entered the subdural space, but seldom involved the spinal parenchyma. In brain leukemia cells were filled in the subdural space and pia-arachnoid, covered the surface of cerebrum, cerebellum, spread along the virchow-robin space on the surface of pia mater, and eventually invaded the brain parenchyma.

CONCLUSIONSHI-1 cells could engrafted in the SCI-nu/nu mice, form an efficient and reproducible experimental model of CNSL and systematic leukemia. This model may be useful for studying the pathogenesis of CNSL.

Adult ; Animals ; Cell Line, Tumor ; Central Nervous System Neoplasms ; Humans ; Leukemia, Experimental ; pathology ; Leukemia, Monocytic, Acute ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Rats ; Xenograft Model Antitumor Assays ; methods