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Differentiation of bone marrow mesenchymal stem cells induced by alveolar epithelial cells of rats exposed to silica dust

Ling YAN ; Ming HUANG ; Zong-Jun ZHANG ; Dan-Yan CAO ; Na ZHAO ; Ying-Xun ZHANG ; Xiao-Jing ZENG ; Wei-Hui LIANG ; Han-Lin HUANG ; Yong-Shun HUANG

China Occupational Medicine.2016;43(01):1-7. doi:10.11763/j.issn.2095-2619.2016.01.001

OBJECTIVE: To observe whether bone marrow mesenchymal stem cell( BMMSC) could be induced by alveolar epithelial cell( AEC) of rats exposed to silica dust or not. METHODS: BMMSCs were isolated and cultivated from 6specific pathogen free healthy male SD rats through bone marrow adherent method. The AECs from other 6 rats randomly selected from the same batch were cultivated by immune adherent purification method. Three rats were treated with 1. 0 m L( 40 g/L mass concentration) of silicosis dust suspension by one time intratracheal injection as silicosis dust exposure model,and the other 3 rats were given 0. 9% sodium chloride solution as normal. Experimental group was the co-culture of BMMSCs and AECs from silicosis dust exposure rats. Control group A was the co-culture of BMMSCs and AECs from normal rats. Control group B was the culture of BMMSCs alone. The morphology changes were observed by the inverted phase contrast microscope at the time points of the 4th and the 8th day. Double immunofluorescence staining using aquaporin 5( AQP5) and surfactant protein C( SP-C) was performed on the treated BMMSCs. The fluorescence staining was observed using the inverted fluorescence microscope( IFM) and laser scanning confocal microscope( LSCM). Integral optical density( IOD) analysis was conducted on fluorescence of 2 kinds of proteins by Image-pro plus 6. 0 graphic analysis software. RESULTS: After the co-culture,the BMMSCs in experimental group and control group A changed from long spindle shape to cubic and polygonal shape,the variation of morphology on day 8 was more obvious than that on day 4,and the change in control group A was less obvious than that of experimental group. There was no obvious morphology change in BMMSCs of control group B. By IFM and LSCM,on day 4 and day 8,the expression of green fluorescence AQP5 and red fluorescence SP-C were all observed in BMMSCs of experimental group and control group A. The BMMSCs of control group B only showed a little green fluorescence expression of AQP5,no expression of red SP-C fluorescence was seen. Both by IFM and LSCM,on day 4 and day 8,the 2 kinds of IOD of BMMSCs in experiment group were higher than those of control group A and B at the same time points( P < 0. 01); the IOD of control group A was higher than that of control group B at the same time point( P < 0. 01). The IOD of experiment group and control group A on day 8 were higher than those on day4 in the same group( P < 0. 01). CONCLUSION: AEC of rats exposed to silica dust can effectively induce BMMSC to be differentiated into AEC.

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Effects of different light on the ethology and melatonin secretion in depressive rats

Shu-Zhe ZHOU ; Wei-Min DANG ; Guo-Yi ZHANG ; Tian-Hang ZHOU ; Jian LIN ; Tian-Mei SI ; Ji-Tao LI ; Zhong-Kai HE ; Can-Tao ZHONG ; Sheng WANG ; Li ZHAO ; Yong-Zhi WANG ; Wei WEI ; Zhen-Lie HUANG ; Kuo ZHANG ; Zhi-Zhong CHEN ; Yi LIU ; Yang LIU ; Rong-Sheng ZHAO ; Hai-Ming SUN ; Si-Heng LI ; Rong-Feng NIU ; Yu-Zhen TONG ; Yan-Tao MA ; Xin YU

China Occupational Medicine.2016;43(01):8-14. doi:10.11763/j.issn.2095-2619.2016.01.002

OBJECTIVE: To observe the impact of energy saving light,incandescent light and circadian light on the ethology of depressive rats and explore its possible mechanism on affecting the secretion of melatonin. METHODS: Thirty rats aged 6weeks were randomly selected from 40 specific pathogen free health female SD rats after they adapted to the living environment,depressive rat models were established in the rats by bilateral ovariectomy combined with isolated living and chronic unpredictable mild stress stimulation at the age of 11-14 weeks. Then these 30 ovariectomized rats were randomly divided into 3 intervention groups,including an energy saving light group,an incandescent light group and a circadian light group,with 10 rats in each group. The rats in these 3 groups were given specific experimental light intervention for 3 weeks respectively at the age of 17 weeks. The other 10 rats were raised in conventional environment as the control group. Their body weights were measured at the age of 17,19,20 and 21 weeks. The ethology tests were carried out by sucrose preference test and the open-field test at the age of 7,14 and 20 weeks respectively. The melatonin levels in peripheral blood of 7 time points from 19: 30 to 8: 30 were measured in the rats at age of 21 weeks. One rat in each group at every time point was randomly selected for examination. RESULTS: At the age of 17 weeks before light-intervention,the body weights of rats in 4 groups showed no significant difference( P > 0. 05). After light-intervention,at the age of 17-20 weeks,the body weights of rats in 3 intervention groups were gradually increased with the increase of age( P < 0. 05).There was no significant difference between body weights of rats at the age of 21 weeks and those at the age of 20 weeks in each group( P > 0. 05). At age of 7 weeks,no significant differences were found in sucrose consumption and standing scores among these 4 groups( P > 0. 05). After the depressive models were established,at the age of 14 weeks before light-intervention,in rats of these 3 intervention groups,the sucrose consumption and standing scores were lower than those of the control group( P < 0. 05),and there was no significant difference found in the above 2 indexes among these 3intervention groups( P > 0. 05). At the age of 20 weeks after light-intervention,the sucrose consumption and standing scores were not significantly different from each other among the 4 groups( P > 0. 05). The peak levels of melatonin in the peripheral blood of rats in these 3 intervention groups were higher than that in the control group. The peak levels onsets of melatonin in peripheral blood of rats in the circadian light group and the energy saving light group were earlier or 2 hours delayed compared to that of control group,while it was similar between the incandescent light group and control group.CONCLUSION: The circadian light,the energy saving light and the incandescent light are similarly effective in improving the behaviors of depressive rats. The circadian light can delay the onset of peak level of melatonin in peripheral blood.

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Cognitive and memory function evaluation on young and middle-aged workers occupationally exposed to aluminum

Pei REN ; Rui LI ; Jin FAN ; Yu-Zhou YUAN ; Qiao NIU

China Occupational Medicine.2016;43(01):15-25. doi:10.11763/j.issn.2095-2619.2016.01.003

OBJECTIVE: To analyze the characteristic changes of cognitive and memory function in young and middle-aged workers occupationally exposed to aluminum. METHODS: By cluster sampling method,358 workers aged 19. 0-55. 0 years and engaged in aluminum electrolytic work for more than 1. 0 year were selected as research objects. The cognitive and memory function were tested and evaluated by Mini-Mental State Examination( MMSE),Clock-drawing Test( CDT),Digit-span Test [DST,including Digit-span Forward Test( DSFT) and Digit-span Backward Test( DSBT) ],Verbal Fluency Test( VFT),Fuld Object Memory Evaluation( FOME) and Simple Reaction Time( SRT). Plasma aluminum was measured using graphite furnace atomic absorption spectrometry and used as internal exposure indicator. The research objects were divided into low-,medium- and high-dose aluminum exposure groups based on the median( M) and the 25 th and the 75 th percentile( P25,P75) of plasma aluminum level. RESULTS: The levels of plasma aluminum M( P25,P75) was135. 47( 87. 42,202. 24) μg / L. The DST,DSFT and VFT scores in the high-dose exposure group were lower than those of low- and medium-dose exposure aluminum groups [DST: 16( 13,19) vs 18( 14,21) scores,16( 13,19) vs 18( 15,20) scores,P < 0. 05; DSFT: 10( 8,12) vs 11( 8,12) scores,10( 8,12) vs 11( 9,12) scores,P < 0. 05; VFT: 36( 26,46) vs 40( 30,50) scores,36( 26,46) vs 40( 30,50) scores,P < 0. 05) ]. Comparison of MMSE,CDT,DSBT,FOME scores and SRT showed no significant difference among all groups( P > 0. 05). The multiple stepwise linear regression analysis results showed that plasma aluminum level was negatively correlated with VFT score( P < 0. 05). The VFT scores dropped with the increase of plasma aluminum level. The scores of MMSE,CDT,FOME and SRT showed no correlation with plasma aluminum level( P > 0. 05). CONCLUSION: Long-term occupational aluminum exposure could induce the damage of cognitive and memory function in young and middle-aged workers. The damage includes auditory attention,auditory memory span and verbal executive function. The mainly damage had a dose-effect relationship.

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Effects of DNA methylation of thymocyte differentiation antigen-1 in beryllium sulfate induced human fetal lung fibroblast fibrosis

Yao WANG ; Zhi-Hong LIU ; Cong-Hui WEI ; Ai-Hong GONG ; Qing YAN ; Qing-Feng ZHANG ; Kai WANG ; He-Jing WANG

China Occupational Medicine.2016;43(01):20-25. doi:10.11763/j.issn.2095-2619.2016.01.004

OBJECTIVE: The change of DNA methylation of thymocyte differentiation antigen-1( Thy-1) was observed in beryllium sulfate( Be SO4) stimulated human fetal lung fibroblast( MRC-5 cell) to explore the effects of Thy-1 in Be SO4 induced lung fibrosis. METHODS: MRC-5 cell culture in vitro model was used. The final concentrations of Be SO4were1. 0,10. 0 and 100. 0 μmol / L( low-,medium- and high-dose groups). The control was untreated. Other 2 intervention groups were the 5-azacytidine( AZC) intervention group( 10. 0 μmol / L of AZC and 10. 0 μmol / L Be SO4) and the trichostatin A( TSA) intervention group( 0. 5 μmol / L of TSA and 10. 0 μmol / L Be SO4). The cells were collected 24,48 and 72 hours after exposure. Real-time quantitative polymerase chain reaction( PCR) was used to determine the relative expression of collagen typeⅠ( Col Ⅰ),collagen type Ⅲ( Col Ⅲ),α-smooth muscle actin( α-SMA) and Thy-1 mRNA.The nested landed methylation specific PCR was used to detect the Thy-1 DNA methylation level. RESULTS: At 24 hours,the relative expression level of Col Ⅲ mRNA in MRC-5 cells showed an increasing trend with increasing dose( P < 0. 05);at 48 and 72 hours,the relative expression levels of Col Ⅰ,Col Ⅲ and α-SMA mRNA in MRC-5 cells increased with the increasing dose( P < 0. 05). All these 3 indicators in MRC-5 cells of 3 dose groups increased with the increase of expose time( P < 0. 05). The relative expression level of Thy-1 mRNA in MRC-5 cells of all 3 dose groups were lower than that in control( P < 0. 05). The relative expression level of Thy-1 mRNA of the high-dose group was lower than that of the lowdose group( P < 0. 05). The Thy-1 DNA methylation levels in the medium- and high-dose groups were both higher than that of the control( P < 0. 05). The Thy-1 DNA methylation levels of the 3 dose groups increased with the increasing dose( P < 0. 05). The Thy-1 DNA methylation levels of MRC-5 cells in the 2 intervention groups were higher than that of the control( P < 0. 05),but there was no significant difference when compared with the medium-dose group( P > 0. 05).CONCLUSION: Be SO4 stimulation can induce the fibrosis of MRC-5 cells. In this process,the Thy-1 DNA methylation level increases,while the Thy-1 mRNA expression level decrease. Thy-1 DNA methylation might be one of the important mechanisms of lung fibrosis induced by Be SO4.

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Effect of automotive paint volatile on learning and memory ability and amino acid neurotransmitters in brain tissue of mice

Guo-Ying ZHENG ; Qing-Zhao LI ; Tai-Shan LI ; Mao-Jing LI ; Shui-Yuan CHENG

China Occupational Medicine.2016;43(01):26-29. doi:10.11763/j.issn.2095-2619.2016.01.005

OBJECTIVE: To explore the effect of automotive paint volatile on the learning and memory ability and its influence mechanism of the amino acid neurotransmitters in the brain tissue of mice. METHODS: Thirty specific pathogen free healthy male Kunming mice were randomly divided into control group,primer group and topcoat group,10 mice in each group. By static inhalation intoxication method,the primer group and topcoat group were exposed to corresponding paint volatile 600 and 580 mg / m3 respectively every day for 4 weeks,6 days per week,one time a day,2 hours each time.The mice of the control group were kept in the exposure cabinet without any paint volatile. After the exposure,the neuroethology examination was carried out by Morris water maze test. The amino acid levels of glutamic acid( GLU),aspartic acid( ASP),γ-aminobutyric acid( GABA) and glycine( GLY) in brain tissue of mice were detected by high performance liquid chromatography. RESULTS: Compared with the control group,the escape latency and the first crossing platform time were longer( P < 0. 05),target quadrant staying time and platform crossing times were decreased( P <0. 05),and the levels of GLU,ASP,GABA and GLY in brain tissues were decreased in the primer group and the topcoat group( P < 0. 05). There was no significant difference between the primer group and the topcoat group in the above indexes( P > 0. 05). CONCLUSION: Automotive paint volatiles exposure may lower the learning and memory abilities of mice,and could reduce the secretion of amino acid neurotransmitters in the brain tissue of mice.

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Toxicity of 1,2-dichloroethane and its metabolites on human astrocytes

Guang-Chao LAI ; Guo-Zhong LUO ; Li-Hai ZENG ; Bi-Zhu ZHANG ; Hong-Ling LI ; Yi-Chen GE ; Xiao-Yong LIU ; Zhen-Lie HUANG

China Occupational Medicine.2016;43(01):30-36. doi:10.11763/j.issn.2095-2619.2016.01.006

OBJECTIVE: To explore the toxicity of 1,2-dichloroethane( 1,2-DCE) and its metabolites on human astrocytes( HAs). METHODS: Different doses of 1,2-DCE( 5. 00,10. 00,25. 00,50. 00 and 100. 00 mmol/L),2-chlorohydrins( 5. 00,25. 00,50. 00,100. 00 and 200. 00 mmol/L),2-chloroacetaldehyde( 1. 00,5. 00,10. 00,20. 00 and 50. 00 mmol / L) and chloroacetic acid( 0. 01,0. 05,0. 10,0. 50 and 1. 00 mmol / L) were used for treating HAs in vitro during their logarithmic phase. After 24 hours of culture,the morphology of HAs was observed by fluorescent inverted phase contrast microscope. The survival rate and the inhibition ratio of HAs were detected by CCK-8 colorimetry to estimate the50% inhibiting concentration in 24 hours( 24 h-IC50). The apoptosis of HAs was tested by double-labeling and flow cytometry using Annexin Ⅴ-fluorescein isothiocyanate and propidium iodide. RESULTS: The morphology of HAs changed in varying degrees after 24 hours exposure to 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid. The changes included smaller size of cells,pseudopodia tapering,increased intracellular particles and suspension of circular cells and decreased transparency of cells. With the increasing does of 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid exposure,the survival rates of HAs decreased( P < 0. 01),while its inhibition ratios increased( P <0. 01). They all showed dose-effect relationship. 24 h-IC50 of the above 4 chemicals were 56. 25,235. 00,26. 43 and1. 38 mmol / L,respectively. The 1,2-DCE,2-chlorohydrins and chloroacetic acid could induce the apoptosis of HAs and the apoptosis rate of HAs was positively correlated with the 3 kinds of chemicals( P < 0. 01). CONCLUSION: 1,2-DCE and its metabolites 2-chloroacetaldehyde,2-chlorohydrins and chloroacetic acid can lead to toxic damage and induce the apoptosis of HAs. Chloroacetic acid has the strongest toxicity among the metabolites.

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Effect of cadmium chloride on the expression of KIM-1 in in vitro HK-2 cell culture

Fang-Lin LI ; Ning-Yu ZHANG ; Li-Hua DENG ; Jie SI-TU ; Jin-Lin WANG ; Zhi-Min LI

China Occupational Medicine.2016;43(01):37-41. doi:10.11763/j.issn.2095-2619.2016.01.007

OBJECTIVE: To investigate the effect of cadmium chloride on the expression of kidney injury molecule-1( KIM-1)in human renal tubular epithelial cells( HK-2 cells). METHODS: HK-2 cells at logarithmic phase were divided into a control group and 5 treatment groups that were treated with 5. 0,10. 0,20. 0,50. 0 and 100. 0 μmol / L of cadmium chloride dissolved in phosphate buffer solution. Cell pathology observation was carried out after 24 hours of cultivation. The methyl thiazolyl tetrazolium assay was used to calculate the survival rate of HK-2 cells. The expression of KIM-1 mRNA and protein were detected by the reverse transcription-polymerase chain reaction and Western blotting analysis respectively.RESULTS: There were no cellular morphologic change in HK-2 cells in the control group,the 5. 0 and 10. 0 μmol / L groups;the HK-2 cells showed different degree of swellings or vacuoles in the 20. 0 and 50. 0 μmol / L groups; a large number of cells were found dead in the 100. 0 μmol / L group. The cell survival rates of HK-2 cells in the 20. 0,50. 0 and 100. 0μmol /L groups were lower than those of control group,the 5. 0 and 10. 0 μmol /L groups( P < 0. 05). The pairwise comparison among survival rates of the 20. 0,50. 0 and 100. 0 μmol / L groups showed significant difference( P < 0. 05).The expression levels of KIM-1 mRNA and protein in the 20. 0 and 50. 0 μmol / L groups were higher than those of control group,the 5. 0 and 10. 0 μmol / L groups( P < 0. 05). The levels of KIM-1 mRNA and protein in the 50. 0 μmol / L group were higher than those of the 20. 0 μmol / L group( P < 0. 05). CONCLUSION: Cadmium chloride at certain concentration can increase the expression of KIM-1 mRNA and protein in HK-2 cells. Therefore,the expression of KIM-1 could be used as one of the effect biomarkers for cadmium induced kidney tubule injury.

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Clinical analysis on four cases of occupational chronic toxic peripheral neuropathy caused by 1-bromopropane

Xiang GUO ; Hui LIN ; Jie SI-TU ; Jian HE ; Qian-Ling ZHENG ; Zhen-Lie HUANG ; Yong-Shun HUANG

China Occupational Medicine.2016;43(01):42-47. doi:10.11763/j.issn.2095-2619.2016.01.008

OBJECTIVE: To analyze the clinical features of occupational chronic toxic peripheral neuropathy caused by1-bromopropan( 1-BP). METHODS: Clinical data of 4 patients who suffered from occupational chronic toxic peripheral neuropathy caused by 1-BP were collected for retrospective analysis. RESULTS: The 4 male patients were ultrasonic cleaning operation workers in a hardware vacuum coating enterprise. They were exposed to high levels of 1-BP for 9-11 months. The main clinical manifestations were varying degrees of sensory disorder and dyskinesia. The main symptoms were progressive increase of numbness and fatigue in the lower extremities. These symptoms might be accompanied by unsteady gait.Physical examination showed muscle strength weakness in the double lower limbs. The hypalgesia,pselaphesia,topesthesia and pallesthesia decreased in the double lower limbs or 4 limbs. The bilateral achilles tendon reflex mainly showed reduced or disappeared. One case had sensory ataxia. Electroneuromyography examination showed different levels of peripheral nerve damage among the cases. The motor nerve conduction velocity and sensory nerve conduction velocity reduced commonly. The axon and myelin sheath damage were visible. On the basis of GBZ / T 247-2013 Diagnosis of Occupational Chronic Toxic Peripheral Neuropathy Caused by Chemicals,these cases were diagnosed as occupational chronic toxic peripheral neuropathy caused by 1-BP. CONCLUSION: Long-term exposure to high level 1-BP can lead to chronic poisoning with peripheral nervous system damage. The diagnosis can be made based on the 1-BP exposure history,clinical features and the neurogenic damage found in electroneuromyography examination.

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Correlation analysis between serum and bronchoalveolar lavage fluid TNF-α and the lung function of coal workers' pneumoconiosis

Wei GUO ; Ping WANG ; Hua-Min YANG ; Feng-Ling ZHAO ; Yu-Min L ; Han-Lin HUANG ; Jing WANG

China Occupational Medicine.2016;43(01):48-51. doi:10.11763/j.issn.2095-2619.2016.01.009

OBJECTIVE: To explore the effect and significance of tumor necrosis factor-α( TNF-α) in serum and bronchoalveolar lavage fluid( BALF) of coal workers' pneumoconiosis( CWP). METHODS: Twenty-eight cases of CWP were selected by purposive sampling method and divided into 3 groups based on different stages of pneumoconiosis. There were 7 cases in stage Ⅰ,10 cases in stage Ⅱ,and 11 cases in stage Ⅲ. The venous blood and BALF were collected.TNF-α in the serum and BALF were detected by chemiluminescence assay. The lung function was determined. RESULTS: In the serum,the TNF-α level of CWP patients was positively correlated with the CWP stage [Spearman correlation coefficient( rS) = 0. 843,P < 0. 01],and negatively correlated with the forced expiratory volume in one second/forced vital capacity( FEV1/ FVC)( rS=- 0. 503,P < 0. 01). In BALF,the TNF-α level of CWP patients was negatively correlated with the CWP stage( rS=- 0. 654,P < 0. 01),and positively correlated with the FEV1/ FVC( rS= 0. 432,P <0. 05). The TNF-α level in the serum was negatively correlated with the TNF-α level in BALF( rS=- 0. 561,P < 0. 01).CONCLUSION: With the development of pneumoconiosis,the levels of TNF-α in the serum and BALF showed different variation,and were correlated with the changes of FEV1/ FVC. The level of TNF-α could be used as a reference index for evaluating the severity of CWP.

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Clinical analysis on hard metal lung disease

Zhi-Hui LI ; Huan-Qiang WANG ; Tao LI

China Occupational Medicine.2016;43(01):52-56. doi:10.11763/j.issn.2095-2619.2016.01.010

OBJECTIVE: To analyze the clinical features,and diagnostic and therapeutic method of hard metal lung disease( HMLD). METHODS: By using literature metrology method,the open published case reports associated with HMLD from January 1980 to October 2015 were searched using the China Hospital Knowledge Database,Wanfang Database and Pub Med Database. The data of patients with hard metal dust exposure history were collected and analyzed based on the inclusive and exclusive criteria. RESULTS: Thirty-six cases of HMLD were collected. The onset age of patients was 21. 0-63. 0( 37. 2 ± 11. 7) years old. The median exposure time was 6. 0( 0. 6-43. 0) years,and the major working type was hard metal grinder. The main clinical type of HMLD maily was giant cell interstitial pneumonia( GIP),hypersensitivity pneumonitis and occupational asthma were also seen. HMLD was lack of characteristic clinical manifestation. The clinical symptoms of HMLD mainly included dry cough,dyspnea on exertion,restrictive pulmonary ventilation,and diffuse pulmonary dysfunction. The imaging study showed ground-glass opacity,diffuse small nodule shadow and reticular opacity shadow,which were mainly seen in the lower lobes of both lungs. There were 24 cases( 66. 7%) showed GIP in the lung tissue in pathological examination. The tungsten and cobalt elements were detected in lung tissue and bronchoalveolar lavage fluid in some cases. Among 22 patients treated with glucocorticoid after keeping away from hard metal dust exposure,the treatment was effective in 19 patients. The clinical symptoms of 6 patients were improved by avoiding hard metal dust exposure. CONCLUSION: HMLD belongs to the interstitial lung disease and there is no specific clinical manifestation. Glucocorticoid therapy is effective in most of the patients. The history of exposure to hard metal dust has important significance in making the diagnosis.

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