OBJECTIVE To investigate the damage effect and mechanisms of cyclophosphamide(CTX)and its active metabolite derivative 4-hydroperoxycyclophosphamide(4-HC)to human neuroblas⁃toma SH-SY5Y cells.METHODS SH-SY5Y cells wer...OBJECTIVE To investigate the damage effect and mechanisms of cyclophosphamide(CTX)and its active metabolite derivative 4-hydroperoxycyclophosphamide(4-HC)to human neuroblas⁃toma SH-SY5Y cells.METHODS SH-SY5Y cells were treated with CTX[0(cell control),0.01,0.1,1,5,10,20,40 and 80 mmol·L^(-1)]and 4-HC[0(cell control),0.01,0.1,1,5,10,20,40 and 80μmol·L^(-1)]for 48 h.Cell confluence and morphology were observed by the IncuCyte ZOOM system.Cell viability was assessed by CCK-8 assay.Lactate dehydrogenase(LDH)release was measured by LDH assay kit.SH-SY5Y cells were treated with CTX(0,1,5,10 and 20 mmol·L^(-1))and 4-HC(0,1,5,10 and 20μmol·L^(-1))for 48 h before cell proliferation was analyzed by 5-ethynyl-2′-deoxyuridine(EdU)staining assay.Immunofluorescence was employed to assess the levels of the DNA double-strand break markerγ-H2AX and to evaluate changes in mitochondrial membrane potential.SH-SY5Y cells were treated with CTX(0,1,5 and 10 mmol·L^(-1))and 4-HC(0,1,5 and 10μmol·L^(-1))for 48 h,and the alterations in glycolysis and oxidative phosphorylation levels were analyzed using the Seahorse XFe96 Analyzer.RESULTS Compared with the cell control group,cell confluence and cell viability were significantly reduced in the CTX and 4-HC groups(P<0.01),and the half-maximal inhibitory concentrations(IC50)for CTX and 4-HC were 4.44 mmol·L^(-1) and 4.78μmol·L^(-1),respectively.The release rate of LDH was signif⁃icantly increased while the percentage of EdU+cells was significantly reduced in the CTX and 4-HC groups(P<0.01).The percentage ofγ-H2AX+cells was significantly increased and mitochondrial membrane potential significantly decreased in the CTX and 4-HC group(P<0.05).Treatment with CTX and 4-HC resulted in reduced levels of maximum glycolytic capacity,glycolytic reserve,maximal respi⁃ration,and ATP production(P<0.05).CONCLUSION CTX and 4-HC exert significant cytotoxic effects on SH-SY5Y cells by disrupting cell membrane structure,impeding cell proliferation,and reducing cell viability.The mechanisms underlying these effects may involve intracellular DNA damage,disturbance of energy metabolism and mitochondrial dysfunction.展开更多
[Objective] This study aimed to investigate the fluorosis content in the groundwater of Songnen Plain and provide a theoretical basis for the prevention and control of endemic fluorosis in Songnen Plain. [Method] The ...[Objective] This study aimed to investigate the fluorosis content in the groundwater of Songnen Plain and provide a theoretical basis for the prevention and control of endemic fluorosis in Songnen Plain. [Method] The relationship of drinking water type endemic fluorosis with the fluoride content in the soil and groundwater of Songnen Plain was analyzed. [Result] The endemic disease in this region is concentrated in the Song-Liao Plain and Hulunbuir Plateau on both sides of the Daxinganling Mountain. Severe and medium disease areas are distributed mainly in alluviallacustrine plains with dense depressions of lakes and marshes. The strata are commonly fine-grained loess and mucky soil. The groundwater is dominated by HCO3-Ca·Na and HCO3·Cl-Na·Ca types, with fluorine content of 1.80-16.00 mg/L, generally 4-6 mg/L. Light disease and non-disease areas are distributed in the alluvial-lacustrine plains with less or without depressions of lakes and marshes. The strata are mainly coarse-grained silty soil and sandy soil. The chemical types of ground water are Cl-Ca·Na and HCO 3 Cl-Na·Ca. The content of fluorine in ground water is commonly lower than 2 mg/L. [Conclusion] High fluorine groundwater formation is closely related to the geological environment.展开更多
[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively ...[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively and compare them. [Method] According to the relatively＇conserved sequences of PCV20RF1 and ORF2 registered in GenBank, two pairs of specific primers and TaqMan probes were designed and synthesized. Then the recombinant plasmids containing the whole sequences of PCV20RF1 and ORF2 were constructed to draw the standard curves through optimizing the reaction system and conditions. And thus two kinds of TaqMan real-time PCR detection methods based on the whole sequences of ORF1 and ORF2 respectively were constructed for PCV2. [Result] For the two established standard curves, the Ct values showed a good linear relationship with the loga- rithms of copy numbers of templates （F2〉0.99）. The amplification efficiency ranged from 90% to 110%. The amplifications all had a good repeatability with variation coefficients within groups all less than 5%. Moreover, the amplifications all had a good specificity. When the sequences of porcine parvovirus （PPV）, porcine circovirus type 1 （PCV1）, swine pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus （PRRSV） were used as templates, the target sequence was not amplified. The amplifications also had a high sensitivity. The ORF1 detection method could reach 1.0x10T copies/;ul, and the ORF2 detection method could reach 1.0×10^2 copies/μl. The two established real-time PCR detection methods were used to detect the 80 clinical samples respectively. The results showed the magnitudes of 72 amplified samples were basically consistent between the 2 detection methods, while the magnitudes of the other 8 amplified samples were inconsistent. Then the 8 samples were detected with SYBR Green I real-time PCR method established based on the sequence of PCV2-1ike factor P1 by Wen et aL The PCV2-1ike factor P1 was amplified in all the 8 samples, indicating the 8 samples were all infected with PCV2-1ike factor P1. [Conclusion] The ORFl-based detection method has a higher accuracy, and it can be used for the rapid detection of PCV2.展开更多
[ Objective ] The aim of the study was to construct associated DNA vaccine of PRRS （Porcine reproductive and respiratory syndrome） and PCV-2 （Porcine circovirus type 2） disease and study its immunogenicity. [ Meth...[ Objective ] The aim of the study was to construct associated DNA vaccine of PRRS （Porcine reproductive and respiratory syndrome） and PCV-2 （Porcine circovirus type 2） disease and study its immunogenicity. [ Method] In_ this study, the ORF5 gene of PRRSV isolated in Liaoning was cloned into plRES-neo expression vector, and the neo gene of plRES-neo expression vector was substituted by the ORF2 gene of the PCV-2 Mongolia strain to construct the recombinant expression vector. The expression in BHK cells was detected through Western blot and IFA. Then the ELISA antibody level and the number of spleen T lymphocytes were detected after Balb/c mice were immunized with this DNA vaccine. E Result] The recombinant plasmid plRES-ORF2-ORF5 was constructed successfully and could express the target proteins in BHK cells, as indicated by Western blot and IFA. There was no significant difference in ELISA antibody between plRES-ORF2-ORF5 immunized group and inactived vaccine immunized groups, while the number of spleen T lymphocytes induced by DNA vaccine was higher than that induced by inactived vaccine. [ Conclusion] The recombinant plasmid plRES-ORF2-ORF5 should induce good humoral immune response and cellular immune response in mice, providing the conditions for better prevention and control of PRRS and PCV-2 disease.展开更多
文摘OBJECTIVE To investigate the damage effect and mechanisms of cyclophosphamide(CTX)and its active metabolite derivative 4-hydroperoxycyclophosphamide(4-HC)to human neuroblas⁃toma SH-SY5Y cells.METHODS SH-SY5Y cells were treated with CTX[0(cell control),0.01,0.1,1,5,10,20,40 and 80 mmol·L^(-1)]and 4-HC[0(cell control),0.01,0.1,1,5,10,20,40 and 80μmol·L^(-1)]for 48 h.Cell confluence and morphology were observed by the IncuCyte ZOOM system.Cell viability was assessed by CCK-8 assay.Lactate dehydrogenase(LDH)release was measured by LDH assay kit.SH-SY5Y cells were treated with CTX(0,1,5,10 and 20 mmol·L^(-1))and 4-HC(0,1,5,10 and 20μmol·L^(-1))for 48 h before cell proliferation was analyzed by 5-ethynyl-2′-deoxyuridine(EdU)staining assay.Immunofluorescence was employed to assess the levels of the DNA double-strand break markerγ-H2AX and to evaluate changes in mitochondrial membrane potential.SH-SY5Y cells were treated with CTX(0,1,5 and 10 mmol·L^(-1))and 4-HC(0,1,5 and 10μmol·L^(-1))for 48 h,and the alterations in glycolysis and oxidative phosphorylation levels were analyzed using the Seahorse XFe96 Analyzer.RESULTS Compared with the cell control group,cell confluence and cell viability were significantly reduced in the CTX and 4-HC groups(P<0.01),and the half-maximal inhibitory concentrations(IC50)for CTX and 4-HC were 4.44 mmol·L^(-1) and 4.78μmol·L^(-1),respectively.The release rate of LDH was signif⁃icantly increased while the percentage of EdU+cells was significantly reduced in the CTX and 4-HC groups(P<0.01).The percentage ofγ-H2AX+cells was significantly increased and mitochondrial membrane potential significantly decreased in the CTX and 4-HC group(P<0.05).Treatment with CTX and 4-HC resulted in reduced levels of maximum glycolytic capacity,glycolytic reserve,maximal respi⁃ration,and ATP production(P<0.05).CONCLUSION CTX and 4-HC exert significant cytotoxic effects on SH-SY5Y cells by disrupting cell membrane structure,impeding cell proliferation,and reducing cell viability.The mechanisms underlying these effects may involve intracellular DNA damage,disturbance of energy metabolism and mitochondrial dysfunction.
基金Supported by Chinese Geological Investigation Bureau Financial Support Project(1212010634701)~~
文摘[Objective] This study aimed to investigate the fluorosis content in the groundwater of Songnen Plain and provide a theoretical basis for the prevention and control of endemic fluorosis in Songnen Plain. [Method] The relationship of drinking water type endemic fluorosis with the fluoride content in the soil and groundwater of Songnen Plain was analyzed. [Result] The endemic disease in this region is concentrated in the Song-Liao Plain and Hulunbuir Plateau on both sides of the Daxinganling Mountain. Severe and medium disease areas are distributed mainly in alluviallacustrine plains with dense depressions of lakes and marshes. The strata are commonly fine-grained loess and mucky soil. The groundwater is dominated by HCO3-Ca·Na and HCO3·Cl-Na·Ca types, with fluorine content of 1.80-16.00 mg/L, generally 4-6 mg/L. Light disease and non-disease areas are distributed in the alluvial-lacustrine plains with less or without depressions of lakes and marshes. The strata are mainly coarse-grained silty soil and sandy soil. The chemical types of ground water are Cl-Ca·Na and HCO 3 Cl-Na·Ca. The content of fluorine in ground water is commonly lower than 2 mg/L. [Conclusion] High fluorine groundwater formation is closely related to the geological environment.
基金Supported by National Natural Science Foundation of China(31302071)Special Fund for Agro-scientific Research in the Public Interest(201303046)+1 种基金Jiangsu Agricultural Science and Technology Innovation Fund(CX(14)2045)"333 High-level Personnel Training Project"of Jiangsu Province(BRA2012194)~~
文摘[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively and compare them. [Method] According to the relatively＇conserved sequences of PCV20RF1 and ORF2 registered in GenBank, two pairs of specific primers and TaqMan probes were designed and synthesized. Then the recombinant plasmids containing the whole sequences of PCV20RF1 and ORF2 were constructed to draw the standard curves through optimizing the reaction system and conditions. And thus two kinds of TaqMan real-time PCR detection methods based on the whole sequences of ORF1 and ORF2 respectively were constructed for PCV2. [Result] For the two established standard curves, the Ct values showed a good linear relationship with the loga- rithms of copy numbers of templates （F2〉0.99）. The amplification efficiency ranged from 90% to 110%. The amplifications all had a good repeatability with variation coefficients within groups all less than 5%. Moreover, the amplifications all had a good specificity. When the sequences of porcine parvovirus （PPV）, porcine circovirus type 1 （PCV1）, swine pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus （PRRSV） were used as templates, the target sequence was not amplified. The amplifications also had a high sensitivity. The ORF1 detection method could reach 1.0x10T copies/;ul, and the ORF2 detection method could reach 1.0×10^2 copies/μl. The two established real-time PCR detection methods were used to detect the 80 clinical samples respectively. The results showed the magnitudes of 72 amplified samples were basically consistent between the 2 detection methods, while the magnitudes of the other 8 amplified samples were inconsistent. Then the 8 samples were detected with SYBR Green I real-time PCR method established based on the sequence of PCV2-1ike factor P1 by Wen et aL The PCV2-1ike factor P1 was amplified in all the 8 samples, indicating the 8 samples were all infected with PCV2-1ike factor P1. [Conclusion] The ORFl-based detection method has a higher accuracy, and it can be used for the rapid detection of PCV2.
文摘[ Objective ] The aim of the study was to construct associated DNA vaccine of PRRS （Porcine reproductive and respiratory syndrome） and PCV-2 （Porcine circovirus type 2） disease and study its immunogenicity. [ Method] In_ this study, the ORF5 gene of PRRSV isolated in Liaoning was cloned into plRES-neo expression vector, and the neo gene of plRES-neo expression vector was substituted by the ORF2 gene of the PCV-2 Mongolia strain to construct the recombinant expression vector. The expression in BHK cells was detected through Western blot and IFA. Then the ELISA antibody level and the number of spleen T lymphocytes were detected after Balb/c mice were immunized with this DNA vaccine. E Result] The recombinant plasmid plRES-ORF2-ORF5 was constructed successfully and could express the target proteins in BHK cells, as indicated by Western blot and IFA. There was no significant difference in ELISA antibody between plRES-ORF2-ORF5 immunized group and inactived vaccine immunized groups, while the number of spleen T lymphocytes induced by DNA vaccine was higher than that induced by inactived vaccine. [ Conclusion] The recombinant plasmid plRES-ORF2-ORF5 should induce good humoral immune response and cellular immune response in mice, providing the conditions for better prevention and control of PRRS and PCV-2 disease.
