Background The CREG is an important lysosomal protein involved in a variety of cellular functions including promoting cell differentiation,sustaining mature homeostasis, and antagonizing apoptosis.Deficiency of CREG i...Background The CREG is an important lysosomal protein involved in a variety of cellular functions including promoting cell differentiation,sustaining mature homeostasis, and antagonizing apoptosis.Deficiency of CREG in cell and tissue results in a pathologic apoptosis.The present study aimed to elucidate the mechanism of CREG regulation apoptosis.Methods We firstly generated stable NIH3T3 fibroblasts by transfection of pDS_shCREGs vectors.Furthermore, PI-Annexin V and TUNEL staining were used to identify that CREG knockdown promoted the cell apoptosis in NIH3T3 fibroblasts.Western blotting and immunofluorescence staining was used to identify the expression and localization of M6P/ IGF2R and cathepsin L in cytoplasm.Results pDS_shCREGs vector transfection produced an approximately 80%decrease in CREG levels both in the lysate and in the media.The expression and localization of M6P/IGF2R and cathepsin L in cytoplasma changed obviously associated with down-regulated of CREG.In addition,the retention and secretion of cathepsin L enhanced significantly.Using the specific inhibitor or siRNA to block cathepsin L activation attenuated the apoptosis mediated by CREG downregulation.Conclusions Our findings indicated that inhibition of CREG expression in NIH3T3 fibroblasts leads to impaired cathepsin L sorting function mediated by M6P/IGF2R and subsequently promotes pathological cell apoptosis.展开更多
AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</s...AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</sup>mol/L SA-A for 22 h.The cell viability wasassayed by[<sup>3</sup>H]proline incorporation,cellproliferation by[<sup>3</sup>H]TdR incorporation,cellcollagen synthetic rate was measured with[<sup>3</sup>H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10<sup>-4</sup>mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10<sup>-5</sup>mol/L-10<sup>-7</sup>mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10<sup>-6</sup>mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10<sup>-5</sup>mol/L-10<sup>-6</sup>mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10<sup>-5</sup>mol/L and10<sup>-6</sup>mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.展开更多
Today it is generally accepted that most bonding agents are cytotoxic. In this study the relative cytotoxicity of seven recent dentine bonding agents on mouse 3T3 fibroblast cells were investigated. Materials and Meth...Today it is generally accepted that most bonding agents are cytotoxic. In this study the relative cytotoxicity of seven recent dentine bonding agents on mouse 3T3 fibroblast cells were investigated. Materials and Methods. Near-confluent mouse 3T3 fibroblast cells were exposed to Dulbecco Modified Eagle’s Medium containing extractions from the seven different bonding agents. The cell survival rate was then determined using the standard MTT assay. Results. The cell survival rate ranking is: iBond (94%) < Gbond (78%) < Xeno V (71%) < Adper Easy Bond (63%) < Xeno V+ (61%) < Adper Scotchbond SE (33%) < XP Bond (32%). Part A of Adper Scotchbond SE had a survival rate of 35% and part B 38%. These two parts did not differ significantly. Adper Scotchbond SE and XP Bond do not differ significantly. While Xeno V+, Xeno V and Adper Easy Bond do not differ. (p < 5%;Tukey-Kramer Multiple-Comparison Test). Conclusion. All of the tested adhesive bonding agents were cytotoxic with survival rate of 3T3 cells between 94% to 31%. Of the 7 bonding agents tested iBond was found to be only slightly toxic and by far the least toxic. The two bonding agents (XP Bond and Adper Scotchbond SE) containing UDMA plus TEGDMA plus HEMA plus camphorquinone were found to be the most toxic.展开更多
Aim: To observe the apoptotic changes following exposure to EMP and to explore the possible injury mechanism in NIH - 3T3 fibroblasts. Methods: Following NIH - 3T3 cells were exposed to EMP,the proliferation and viabi...Aim: To observe the apoptotic changes following exposure to EMP and to explore the possible injury mechanism in NIH - 3T3 fibroblasts. Methods: Following NIH - 3T3 cells were exposed to EMP,the proliferation and viability of NIH - 3T3 fibroblasts were estimated by hemacytometer and MTT Measurements. Apoptotic rate was measured by flow cytometer. The imnmohistochemical SP method was used to determine bcl- 2, p53. The positively stained cells were analyzed with CMIAS- Ⅱ image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. Results: The proliferation and viability of NIH- 3T3 cells were markedly inhibited and significant apoptosis was induced following exposure to EMP.EMP could increase the number of non- adherent cells, the highest ratio (33.9%) of non- adherent cells also occurred at 6h. The As70 value of MTT decreased immediately at 1 h, 6h following the cells were exposed as compared with the control (P ＜ 0.05). The highest ratio of apoptosis was 15.07% at 6h, then decreased gradually. Down - regulation of bcl - 2 and up - regulation of p53 were induced by EMP ( P ＜ 0.05). Conclusions: EMP could promote apoptosis of NIH - 3T3 fibroblasts. EMP could also down - regulate bcl - 2 level and up - regulate p53 level in NIH - 3T3 fibroblasts. Bcl - 2 and p53 gene may take part in the process of apoptosis.展开更多
Objective:3,4-Oxo-isopropylidene-shikimic acid(ISA),a derivative of shikimic acid,has exhibited ameliorative effect on cognitive impairment in experimental animal models of dementia.This study investigated the effect ...Objective:3,4-Oxo-isopropylidene-shikimic acid(ISA),a derivative of shikimic acid,has exhibited ameliorative effect on cognitive impairment in experimental animal models of dementia.This study investigated the effect of ISA on lipid accumulation and adipokine secretion during differentiation of 3T3-L1 fibroblasts to adipocytes.Methods:3T3-L1 cells were cultured and treated with ISA(50e800 mM)from days 3e8.Lipid accumulation and triglyceride content were measured.Gene expression of adipokines(adiponectin,leptin,and resistin),CCAAT/enhancer binding protein(C/EBP)b,C/EBP a and peroxisome proliferator-activated receptor g(PPAR g)and PPAR target genes,including adipocyte fatty acid binding protein(aP2)and fatty acid synthase(FAS)were investigated.Results:ISA promoted 3T3-L1 fibroblast differentiation to adipocytes and increased triglyceride content by 26%.On mechanistic levels,ISA increased expressions of C/EBP b,PPAR g,C/EBP a,aP2 and FAS.Moreover,ISA stimulated expressions of adipokines secreted by adipocytes,including adiponectin,leptin,and resistin.Conclusions:These findings demonstrated that ISA promoted adipogenesis by up-regulating expressions of C/EBP b,PPAR g,C/EBP a,aP2 and FAS,and also stimulated adipokines during adipocyte differentiation.Further study should clarify the relationship between stimulation of adipokines and cognitive enhancing effect of ISA.展开更多
In vitro responses of human primary pulp cells (HPCs) and 3T3 mouse fibroblasts to six contempo-rary commercial dental restoratives were evaluated using the WST-1 assay. The results show that Fuji II is not cytotoxic ...In vitro responses of human primary pulp cells (HPCs) and 3T3 mouse fibroblasts to six contempo-rary commercial dental restoratives were evaluated using the WST-1 assay. The results show that Fuji II is not cytotoxic to both cells. Fuji II LC is not cyto-toxic to HPCs but cytotoxic to 3T3 cells, indicating that 3T3 cells are more vulnerable to 2-hydroxyethyl methacrylate (HEMA) than HPCs. Vitremer is very cytotoxic probably due to having diphenyliodonium chloride and HEMA in it. Z100 is very cytotoxic probably due to having triethylene glycol dimethacry-late (TEGDMA) in it. P60 is cytotoxic but less cyto-toxic than Z100 probably due to no TEGDMA in it. Durelon is the most cytotoxic among the six materials studied probably due to the high cytotoxicity of zinc ions. Additionally, the cytotoxcity of the tested mate-rials was found to be dose-dependent.展开更多
文摘Background The CREG is an important lysosomal protein involved in a variety of cellular functions including promoting cell differentiation,sustaining mature homeostasis, and antagonizing apoptosis.Deficiency of CREG in cell and tissue results in a pathologic apoptosis.The present study aimed to elucidate the mechanism of CREG regulation apoptosis.Methods We firstly generated stable NIH3T3 fibroblasts by transfection of pDS_shCREGs vectors.Furthermore, PI-Annexin V and TUNEL staining were used to identify that CREG knockdown promoted the cell apoptosis in NIH3T3 fibroblasts.Western blotting and immunofluorescence staining was used to identify the expression and localization of M6P/ IGF2R and cathepsin L in cytoplasm.Results pDS_shCREGs vector transfection produced an approximately 80%decrease in CREG levels both in the lysate and in the media.The expression and localization of M6P/IGF2R and cathepsin L in cytoplasma changed obviously associated with down-regulated of CREG.In addition,the retention and secretion of cathepsin L enhanced significantly.Using the specific inhibitor or siRNA to block cathepsin L activation attenuated the apoptosis mediated by CREG downregulation.Conclusions Our findings indicated that inhibition of CREG expression in NIH3T3 fibroblasts leads to impaired cathepsin L sorting function mediated by M6P/IGF2R and subsequently promotes pathological cell apoptosis.
文摘AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</sup>mol/L SA-A for 22 h.The cell viability wasassayed by[<sup>3</sup>H]proline incorporation,cellproliferation by[<sup>3</sup>H]TdR incorporation,cellcollagen synthetic rate was measured with[<sup>3</sup>H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10<sup>-4</sup>mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10<sup>-5</sup>mol/L-10<sup>-7</sup>mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10<sup>-6</sup>mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10<sup>-5</sup>mol/L-10<sup>-6</sup>mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10<sup>-5</sup>mol/L and10<sup>-6</sup>mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.
文摘Today it is generally accepted that most bonding agents are cytotoxic. In this study the relative cytotoxicity of seven recent dentine bonding agents on mouse 3T3 fibroblast cells were investigated. Materials and Methods. Near-confluent mouse 3T3 fibroblast cells were exposed to Dulbecco Modified Eagle’s Medium containing extractions from the seven different bonding agents. The cell survival rate was then determined using the standard MTT assay. Results. The cell survival rate ranking is: iBond (94%) < Gbond (78%) < Xeno V (71%) < Adper Easy Bond (63%) < Xeno V+ (61%) < Adper Scotchbond SE (33%) < XP Bond (32%). Part A of Adper Scotchbond SE had a survival rate of 35% and part B 38%. These two parts did not differ significantly. Adper Scotchbond SE and XP Bond do not differ significantly. While Xeno V+, Xeno V and Adper Easy Bond do not differ. (p < 5%;Tukey-Kramer Multiple-Comparison Test). Conclusion. All of the tested adhesive bonding agents were cytotoxic with survival rate of 3T3 cells between 94% to 31%. Of the 7 bonding agents tested iBond was found to be only slightly toxic and by far the least toxic. The two bonding agents (XP Bond and Adper Scotchbond SE) containing UDMA plus TEGDMA plus HEMA plus camphorquinone were found to be the most toxic.
基金supported by the project foundation of the 9th-Five Year Plan of the Logistics Department of the Chinese PLA (No. 39770697)
文摘Aim: To observe the apoptotic changes following exposure to EMP and to explore the possible injury mechanism in NIH - 3T3 fibroblasts. Methods: Following NIH - 3T3 cells were exposed to EMP,the proliferation and viability of NIH - 3T3 fibroblasts were estimated by hemacytometer and MTT Measurements. Apoptotic rate was measured by flow cytometer. The imnmohistochemical SP method was used to determine bcl- 2, p53. The positively stained cells were analyzed with CMIAS- Ⅱ image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. Results: The proliferation and viability of NIH- 3T3 cells were markedly inhibited and significant apoptosis was induced following exposure to EMP.EMP could increase the number of non- adherent cells, the highest ratio (33.9%) of non- adherent cells also occurred at 6h. The As70 value of MTT decreased immediately at 1 h, 6h following the cells were exposed as compared with the control (P ＜ 0.05). The highest ratio of apoptosis was 15.07% at 6h, then decreased gradually. Down - regulation of bcl - 2 and up - regulation of p53 were induced by EMP ( P ＜ 0.05). Conclusions: EMP could promote apoptosis of NIH - 3T3 fibroblasts. EMP could also down - regulate bcl - 2 level and up - regulate p53 level in NIH - 3T3 fibroblasts. Bcl - 2 and p53 gene may take part in the process of apoptosis.
基金Mukogawa Women’s University Short-term Student Exchange Program,Science Foundation for The Excellent Youth Scholars of Beijing University of Chinese Medicine(No.2012-QNJSZX006)Natural Science Foundation of Beijing Municipality(No.7144222).
文摘Objective:3,4-Oxo-isopropylidene-shikimic acid(ISA),a derivative of shikimic acid,has exhibited ameliorative effect on cognitive impairment in experimental animal models of dementia.This study investigated the effect of ISA on lipid accumulation and adipokine secretion during differentiation of 3T3-L1 fibroblasts to adipocytes.Methods:3T3-L1 cells were cultured and treated with ISA(50e800 mM)from days 3e8.Lipid accumulation and triglyceride content were measured.Gene expression of adipokines(adiponectin,leptin,and resistin),CCAAT/enhancer binding protein(C/EBP)b,C/EBP a and peroxisome proliferator-activated receptor g(PPAR g)and PPAR target genes,including adipocyte fatty acid binding protein(aP2)and fatty acid synthase(FAS)were investigated.Results:ISA promoted 3T3-L1 fibroblast differentiation to adipocytes and increased triglyceride content by 26%.On mechanistic levels,ISA increased expressions of C/EBP b,PPAR g,C/EBP a,aP2 and FAS.Moreover,ISA stimulated expressions of adipokines secreted by adipocytes,including adiponectin,leptin,and resistin.Conclusions:These findings demonstrated that ISA promoted adipogenesis by up-regulating expressions of C/EBP b,PPAR g,C/EBP a,aP2 and FAS,and also stimulated adipokines during adipocyte differentiation.Further study should clarify the relationship between stimulation of adipokines and cognitive enhancing effect of ISA.
文摘In vitro responses of human primary pulp cells (HPCs) and 3T3 mouse fibroblasts to six contempo-rary commercial dental restoratives were evaluated using the WST-1 assay. The results show that Fuji II is not cytotoxic to both cells. Fuji II LC is not cyto-toxic to HPCs but cytotoxic to 3T3 cells, indicating that 3T3 cells are more vulnerable to 2-hydroxyethyl methacrylate (HEMA) than HPCs. Vitremer is very cytotoxic probably due to having diphenyliodonium chloride and HEMA in it. Z100 is very cytotoxic probably due to having triethylene glycol dimethacry-late (TEGDMA) in it. P60 is cytotoxic but less cyto-toxic than Z100 probably due to no TEGDMA in it. Durelon is the most cytotoxic among the six materials studied probably due to the high cytotoxicity of zinc ions. Additionally, the cytotoxcity of the tested mate-rials was found to be dose-dependent.
