BACKGROUND: An ideal marked molecule has not been found to detarmine bone marrow mesenchymal stem cells (MSCs) so as to make sure the homogenicity.OBJECTIVE: To verify the in vitro differentiation from BrdU-treced MSCs into osteoblasts.DESIGN, TIME AND SETTING: A cytological observation/n vitro was performed in Shanxi Medical University from January to October 2008.MATERIALS: Wistar rats aging 4 weeks old were provided by Experimental Animal Center of Shanxi Medical University.METHODS: MSCs were isolated and cultured by using density gradient cantrifugation combined with attachment culture method.At about 80% confluence, trypsin was used for passage and amplification. MSCs at density of 5×1010/L were inoculated in a 25-mm culture dish with L-DMDM culture medium containing dexamethasone, β -phosphoglycarol, vitamin C, and 10% fetal bovine serum. The third-passaged MSCs were labeled in vitro with 10 μmol/L BrdU tracer. Thereafter, 10 visual fields were randomly selected to calculate numbers of positive and negative ceils so as to obtain BrdU tracing rate under a fluorescence microscope (×200).MAIN OUTCOME MEASURES: Inverted microscope was used to observe cell morphology; flow cytometry was used to detect cell surface antigen, differentiation into osteoblasts, and BrdU tracing rate in vitro.RESULTS: The purified MSCs which were like fibroblasts were adherent and fusiform. The third-passaged cells were changed equidirectionally and whirlpool-arranged, and the survival rate was more than 95%. The seventh-passaged cells still grew rapidly.CD44, CD71, and CD105 expressions were positive, but CD45 expression was negative. Black particles were visualized in MSCs after Von kossa staining. BrdU tracing rate was more than 90%.CONCLUSION: Density gradient centrifugation combined with attachment culture method can effectively isolate and purify rat MSCs which are cultured in vitro for a long period and differentiated into osteoblasts. BrdU tracer is safe, effective, and convenient to successfully label MSCs.

Objective To investigate the effects of immune system and immuno-related miRNAs expression in low-dose benzene and its homologue mixed-exposed workers in a short time. Methods A total of 40 workers were recruited from a petrochemical factory ,and their blood samples were collected before and after work to detect the levels of IgA,IgG,IgM,IFN-γ,IL-1β,IL-2,IL-8,TNF-αin serum and miRNA-146a,-155 expression in the peripheral blood mononuclear cell. In the meanwhile ,we measured the individual benzene and its homologue exposure level of recruited workers. Results According to the concentration of benzene ,the subjects were divided into benzene-exposed group(20 workers)and control group(20 workers). And toluene exposure level in benzene-exposed group was significantly higher than that in control group(P<0.05). Significant decrease of IgG was found in benzene-exposed workers after one work shift,compared with control group(P<0.05). Moreover,the interaction between benzene and toluene was significant correlated with the decrease of IL-1β and TNF-α(P < 0.05). Additionally,the interaction between miRNA-155 and miRNA-146a was significant correlated with the decrease of TNF-α(P<0.05). Conclusions:Occupational exposure to low-dose benzene and toluene in a short time could decrease immune function. And there may be an effect of miRNAs in the regulation of cytokine.

Objective: To investigate the value of DNA content in comet tail (TailDNA) in predicting the changes in peripheral blood cell counts in workers exposed to benzene. Methods: In 2011, cluster sampling was used to select 150 male workers exposed to benzene in a petrochemical factory. Cubital venous blood and urine samples were collected for routine blood rest, comet assay, and measurement of s-phenylmercapturic acid (SPMA) and urine creatinine. The population was divided into groups according to urinary SPMA or TailDNA, and routine blood test results within 3 years were collected to analyze the changes in blood cell counts. Results: The low-SPMA group had significantly higher white blood cell and neutrophilcounts in all years than the high-SPMA group (P<0.05) . The low-Tail DNA group had a significant increase in platelet count from 2012 to 2014 (P<0.05) , while the high-Tail DNA group had no significant change (P>0.05) . During the 4-year period, the high-TailDNA group had a significantly lower red blood cell count than the low-TailDNA group (P<0.05) . The high-TailDNA group showed a gradual reduction in white blood cell count over time (β=-0.113, P<0.05) , and the low-TailDNA group showed no trend of the reduction in white blood cell count (P>0.05) . Conclusion TailDNA can be used to predict the changes in blood cell counts in workers exposed to benzene.

OBJECTIVE: To study the effect of aquaporin 4(AQP4) in regulating the permeability of blood-brain barrier(BBB) induced by subacute 1,2-dichloroethane(1,2-DCE) inhalation. METHODS: Specific pathogen free healthy CD-1 male Aqp4 genetically engineered mice(Aqp4~(+/+)and Aqp4~(-/-)) were randomly divided into control and low-, medium-and high-dose groups. The mice were exposed to 1,2-DCE at the dosages of 0.00, 100.00, 350.00 and 700.00 mg/m~3 for 6 hours per day for consecutive 28 days by systemic dynamic inhalation. After the end of 1,2-DCE exposure, the BBB permeability was evaluated by Evans blue staining. Real-time fluorescence quantitative polymerase chain reaction method was used to detect the mRNA expression of genes related to BBB tight junction protein(Tjp)1, Tjp2, Tjp3, claudin(Cldn)3, Cldn5, Cldn11, occludin(Ocln), matrix metalloproteinase(Mmp)2, Mmp9 and Na-K-Cl cotransporter-1(Nkcc1). RESULTS: The BBB permeability in mice showed significant change with 1,2-DCE dose and Aqp4 genotype(P<0.01). The BBB permeability of Aqp4~(+/+) genotype mice was higher in low-, medium-and high-dose groups than that of control group(all P values were <0.05). The permeability of BBB was lower in Aqp4~(+/+) genotype mice in the control group than that of Aqp4~(-/-) genotype mice in the same group(P<0.05), but BBB permeability was higher in Aqp4~(+/+) genotype mice in the three dose groups than that of Aqp4~(-/-) genotype mice in the same group(all P values were <0.05). The Cldn3 and Olcn mRNA relative expression in the brain cortex had statistical difference in mice with different genotype(all P values were <0.01). The mRNA relative expressions of Cldn3 and Olcn in the brain cortex were higher in Aqp4~(-/-) genotype mice than that of Aqp4~(+/+) genotype mice(all P values were <0.01). The relative mRNA expression levels of Tjp1, Tjp2, Tjp3, Cldn5, Cldn11, Mmp2, Mmp9 and Nkcc1 in the cerebral cortex of mice were not statistically significant in aspect of 1,2-DCE exposure dose and genotype(all P values were >0.05). CONCLUSION: Exposure to 1,2-DCE can increase BBB permeability in mice, and the mechanism may be associated with 1,2-DCE-induced down-regulation of Aqp4 and up-regulation of mRNA expression of the cerebral cortex TJP-related molecules Cldn3 and Ocln.

OBJECTIVE: To explore the molecular mechanism underlying 1,2-dichloroethane(1,2-DCE) induced apoptosis by screening differentially expressed proteins in human astrocytes( HAs). METHODS: HAs were cultured in complete medium with 1,2-DCE at various concentrations of 0-80 or 0-40 mmol/L. After 24 hours,apoptosis of HAs was evaluated using flow cytometry and staining with annexin Ⅴ-fluoresce in isothiocyanate and propidium iodide. An AAH-APO-1-2 protein chip was used to screen differentially expressed proteins and quantitative real-time polymease chain reaction(qRT-PCR) was used to verify related differentially expressed genes(DEGs). RESULTS: At 1,2-DCE concentrations of0-80 mmol/L,the total apoptosis rate of HAs increased with 1,2-DCE concentrations in a dose-dependent manner( P <0. 01). Seven different kinds of proteins were screened out by apoptotic protein chip. Among them,the expression of insulin-like growth factor-binding protein( IGFBP)-1,IGFBP-4 and cytochrome C( Cyto C) were up-regulated,while the expression of P27,cysteine aspartic acid specific protease-3( Caspase-3),B-cell lymphoma-2 interacting mediator of cell death( BIM) and BH3 interacting domain death agonist( BID) were down-regulated compared with the control group. The result of DEGs verified by qRT-PCR showed that the expression of mRNA of IGFBP-1,IGFBP-4 and Cyto C at 1,2-DCE concentrations of 40 mmol/L was up-regulated. This result was in consistent with the trend of target expression in the protein chip. The mRNA expression of Caspase-3,BIM and BID was also up-regulated. CONCLUSION: 1,2-DCE induces apoptosis of HAs through mitochondrial pathway.