AIM:To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2)on human hepatic stellate cell(HSC)function. METHODS:CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences we...AIM:To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2)on human hepatic stellate cell(HSC)function. METHODS:CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz.Each vector was individually transfected into cultured LX-2 human HSCs,which were then stimulated by addition of transforming growth factor(TGF)-β1 to the culture medium.Semi- quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagenⅠ,while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA.Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS:In pTriEx2-transfected LX-2 cells,TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagenⅠ,and an increase in produced and secreted CCN2 or extracellular collagenⅠprotein levels.pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels,as well as produced and secreted CCN2 or collagenⅠprotein. Furthermore,the TGF-β1-induced increase in mRNA or protein for CCN2 or collagenⅠwas inhibited partially in pTriCCN2-Rz-transfected LX-2 cells.Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase.CONCLUSION:Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagenⅠproduction in human HSCs and regulates entry of the cells into S phase.展开更多
AIM:To determine the expression characteristics of connective tissue growth factor(CTGF/CCN2) in human hepatocellular carcinoma(HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression a...AIM:To determine the expression characteristics of connective tissue growth factor(CTGF/CCN2) in human hepatocellular carcinoma(HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS:Liver samples from 36 patients(who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1(TGF-β1) or CCN2 mRNA by in situ hybridization.Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma.Fibroblast-specific protein-1(FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells,and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining.CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS:In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci.In comparison to normal controls,CCN2 mRNA was enhanced 1.9-fold in carcinoma foci(12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma(60.27 ± 28.71 vs 6.42 ± 2.35),with concomitant expression of CCN2 and TGF-β1 mRNA in those areas.Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36(33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature.Incubation of HepG2 cells with CCN2(100 ng/mL) resulted in more of the cells transitioning into S phase(23.85 ± 2.35 vs 10.94 ± 0.23),and induced a significant migratory(4.0-fold) and invasive(5.7-fold) effect.TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.CONCLUSION:These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.展开更多
BACKGROUND: Pancreatic stellate cells(PSCs) play a critical role in the pathogenesis of pancreatic fibrosis and have emerging functions as progenitor cells,immune cells or intermediaries in pancreatic exocrine secreti...BACKGROUND: Pancreatic stellate cells(PSCs) play a critical role in the pathogenesis of pancreatic fibrosis and have emerging functions as progenitor cells,immune cells or intermediaries in pancreatic exocrine secretion. Increasing evidence has shown that desmin as an exclusive cytoskeleton marker of PSC is only expressed in part of these cells. This study was to establish a desmin-positive PSC cell line and evaluate its actions on pancreatic fibrosis,inflammation and immunity.METHODS: The presence of cytoskeletal proteins,integrin α5β1 or TLR4,was determined by immunocytochemistry while the production of desmin,collagen I,MMP-1,MMP-2,TIMP-2,or CD14 was evaluated by Western blotting. The levels of desmin,collagen I,IL-1 and IL-6 m RNA were determined by real-time quantitative PCR. The secretion of cytokines was detected by ELISA. Cell function was assessed using adhesion,migration,or proliferation assays. RESULTS: A stable activated rat PSC cell line(designated as RP-2) was established by RSV promoter/enhancer-driven SV40 large T antigen expression. RP-2 cells retained typical PSC properties,exhibited a myofibroblast-like phenotype and persistently produced desmin. The cells produced collagen I protein,matrix metalloproteinases and inhibitors thereof. RP-2 cells demonstrated typical PSC functions,including proliferation,adherence,and migration,the latter two of which occurred in response to fibronectin and were mediated byintegrin α5β1. TLR4 and its response genes including proinflammatory cytokines(IL-1,IL-6,TNF-α) and chemotactic cytokines(MCP-1,MIP-1α,Rantes) were produced by RP-2 cells and activated by LPS. LPS-induced IL-1 or IL-6 m RNA expression in this cell line was fully blocked with My D88 inhibitor.CONCLUSION: RP-2 cells provide a novel tool for analyzing the properties and functions of PSCs in the pathogenesis of fibrosis,inflammation and immunity in the pancreas.展开更多
Background: While mental health among collegiate athletes is receiving increased attention, research on factors surrounding collegiate athletes' decision to seek mental health services is limited. The goal of the ...Background: While mental health among collegiate athletes is receiving increased attention, research on factors surrounding collegiate athletes' decision to seek mental health services is limited. The goal of the present review was to analyze and synthesize the current literature concerning collegiate athletes' utilization of mental health services, including the facilitators of and barriers to use of these services.Methods: The analysis was guided and organized using a socio-ecological framework, which considered the unique context in which collegiate athletes study and perform. A total of 21 articles, published between 2005 and 2016, which concern U.S. collegiate athletes' mental health services utilization(MHSU) were selected and included for the final analysis. Conceptualizations and operationalizations of MHSU were compared and contrasted. Facilitators of and barriers to athletes MHSU were examined and summarized while appropriately considering the proximity of each factor(facilitator or barrier) to the athletes.Results: Results showed variations in conceptualizations and operationalizations of MHSU in the articles analyzed, which made interpretation and cross comparison difficult. Collegiate athletes are willing to utilize mental health services, but gender, perceived stigma, peer norms—for athletes and coaches—plus service availability impact their MHSU.Conclusion: Key stakeholders, administrators, and public health officials should partner to eliminate MHSU barriers, support facilitators, and generally empower collegiate athletes to actively manage their mental health.展开更多
Background:Few published studies have examined child passenger safety practices across countries.This study compared the prevalence and associated factors of child passenger restraint use among children,aged 0 to 17 i...Background:Few published studies have examined child passenger safety practices across countries.This study compared the prevalence and associated factors of child passenger restraint use among children,aged 0 to 17 in the state of Iowa in the United States,and the city of Shantou in China.Methods:Child restraint use observations were conducted in Iowa and in Shantou in 2012,respectively,among child passengers.Observations in Iowa were conducted at randomly selected gas stations,while in Shantou observations were completed at randomly selected schools or medical clinics.Research observers approached the driver,observed restraint use,and collected brief survey data.Results:A total of 3049 children from Iowa and 3333 children aged 0 to 17 years from Shantou were observed.For children aged 0 to 3 years,only 0.1% were compliantly restrained in Shantou as compared to 95.9% in Iowa.The proportion of children who were compliantly restrained in Shantou increased with age,but generally decreased with age in Iowa.In Shantou,36.0% of children aged 0 to 3 were sitting in the front seat as compared to only 1.7% of children of the same age in Iowa.Driver seat belt use was significantly associated with child restraint in both Iowa and Shantou;the association was stronger in Iowa than Shantou for aH age groups.Conclusions:A significantly higher prevalence of children who were not appropriately restrained was observed in Shantou than in Iowa.Our findings support the need of mandatory child safety restraint use legislation in China.展开更多
Piwi-interacting RNAs(piRNAs of 26-32 nt in length)regulate gene expression,epigenetics,and transcriptional and post-transcriptional processes.1 In humans,piRNAs are expressed in germline and somatic tissues,and gener...Piwi-interacting RNAs(piRNAs of 26-32 nt in length)regulate gene expression,epigenetics,and transcriptional and post-transcriptional processes.1 In humans,piRNAs are expressed in germline and somatic tissues,and generally have a uracil at the 5'-end(position+1),an adenine at the+10 position,and are 2'-0 methylated at the 3'-end.2 Onice piRNAs form the RNA-induced silencing complex(RISC)with PIWl proteins,they serve as guides to find their complementary sequences of the target mRNA,thus initiating their degradation.The Wuhan patient's genome is approximately 265 nucleotides(nts)in its 3'-UTR;piRNAs-like sequences have been reported in this region.3 Our objective was to search,through bioinformatics,for mRNA sequences that were homologous to the reverse complementary of the previously reported piRNA-like sequences from the 3'-UTR of SARS-Cov-2,3 and identify the possible interaction between them.展开更多
Immunomodulatory signaling imposes tight regulations on metabolic programs within immune cells and consequentially determines immune response outcomes.Although the glucocorticoid receptor(GR)has been recently implicat...Immunomodulatory signaling imposes tight regulations on metabolic programs within immune cells and consequentially determines immune response outcomes.Although the glucocorticoid receptor(GR)has been recently implicated in regulating the function of myeloid-derived suppressor cells(MDSCs),whether the dysregulation of GR in MDSCs is involved in immune-mediated hepatic diseases and how GR regulates the function of MDSCs in such a context remains unknown.Here,we revealed the dysregulation of GR expression in MDSCs during innate immunological hepatic injury(IMH)and found that GR regulates the function of MDSCs through modulating HIF1α-dependent glycolysis.Pharmacological modulation of GR by its agonist(dexamethasone,Dex)protects IMH mice against inflammatory injury.Mechanistically,GR signaling suppresses HIF1αand HIF1α-dependent glycolysis in MDSCs and thus promotes the immune suppressive activity of MDSCs.Our studies reveal a role of GR-HIF1αin regulating the metabolism and function of MDSCs and further implicate MDSC GR signaling as a potential therapeutic target in hepatic diseases that are driven by innate immune cell-mediated systemic inflammation.展开更多
基金Supported by National Natural Scientific Foundation No.30872236 to Run-Ping Gao and NIH 5R01AA016003 to David R Brigstock
文摘AIM:To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2)on human hepatic stellate cell(HSC)function. METHODS:CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz.Each vector was individually transfected into cultured LX-2 human HSCs,which were then stimulated by addition of transforming growth factor(TGF)-β1 to the culture medium.Semi- quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagenⅠ,while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA.Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS:In pTriEx2-transfected LX-2 cells,TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagenⅠ,and an increase in produced and secreted CCN2 or extracellular collagenⅠprotein levels.pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels,as well as produced and secreted CCN2 or collagenⅠprotein. Furthermore,the TGF-β1-induced increase in mRNA or protein for CCN2 or collagenⅠwas inhibited partially in pTriCCN2-Rz-transfected LX-2 cells.Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase.CONCLUSION:Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagenⅠproduction in human HSCs and regulates entry of the cells into S phase.
基金Supported by National Natural Scientific Foundation,No. 30872236,81070370,to Gao RPNIH 5R01AA016003,to Brigstock DR
文摘AIM:To determine the expression characteristics of connective tissue growth factor(CTGF/CCN2) in human hepatocellular carcinoma(HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS:Liver samples from 36 patients(who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1(TGF-β1) or CCN2 mRNA by in situ hybridization.Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma.Fibroblast-specific protein-1(FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells,and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining.CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS:In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci.In comparison to normal controls,CCN2 mRNA was enhanced 1.9-fold in carcinoma foci(12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma(60.27 ± 28.71 vs 6.42 ± 2.35),with concomitant expression of CCN2 and TGF-β1 mRNA in those areas.Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36(33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature.Incubation of HepG2 cells with CCN2(100 ng/mL) resulted in more of the cells transitioning into S phase(23.85 ± 2.35 vs 10.94 ± 0.23),and induced a significant migratory(4.0-fold) and invasive(5.7-fold) effect.TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.CONCLUSION:These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.
基金support by grants from the National Natural Science Foundation of China(81070370 and 81270544)
文摘BACKGROUND: Pancreatic stellate cells(PSCs) play a critical role in the pathogenesis of pancreatic fibrosis and have emerging functions as progenitor cells,immune cells or intermediaries in pancreatic exocrine secretion. Increasing evidence has shown that desmin as an exclusive cytoskeleton marker of PSC is only expressed in part of these cells. This study was to establish a desmin-positive PSC cell line and evaluate its actions on pancreatic fibrosis,inflammation and immunity.METHODS: The presence of cytoskeletal proteins,integrin α5β1 or TLR4,was determined by immunocytochemistry while the production of desmin,collagen I,MMP-1,MMP-2,TIMP-2,or CD14 was evaluated by Western blotting. The levels of desmin,collagen I,IL-1 and IL-6 m RNA were determined by real-time quantitative PCR. The secretion of cytokines was detected by ELISA. Cell function was assessed using adhesion,migration,or proliferation assays. RESULTS: A stable activated rat PSC cell line(designated as RP-2) was established by RSV promoter/enhancer-driven SV40 large T antigen expression. RP-2 cells retained typical PSC properties,exhibited a myofibroblast-like phenotype and persistently produced desmin. The cells produced collagen I protein,matrix metalloproteinases and inhibitors thereof. RP-2 cells demonstrated typical PSC functions,including proliferation,adherence,and migration,the latter two of which occurred in response to fibronectin and were mediated byintegrin α5β1. TLR4 and its response genes including proinflammatory cytokines(IL-1,IL-6,TNF-α) and chemotactic cytokines(MCP-1,MIP-1α,Rantes) were produced by RP-2 cells and activated by LPS. LPS-induced IL-1 or IL-6 m RNA expression in this cell line was fully blocked with My D88 inhibitor.CONCLUSION: RP-2 cells provide a novel tool for analyzing the properties and functions of PSCs in the pathogenesis of fibrosis,inflammation and immunity in the pancreas.
文摘Background: While mental health among collegiate athletes is receiving increased attention, research on factors surrounding collegiate athletes' decision to seek mental health services is limited. The goal of the present review was to analyze and synthesize the current literature concerning collegiate athletes' utilization of mental health services, including the facilitators of and barriers to use of these services.Methods: The analysis was guided and organized using a socio-ecological framework, which considered the unique context in which collegiate athletes study and perform. A total of 21 articles, published between 2005 and 2016, which concern U.S. collegiate athletes' mental health services utilization(MHSU) were selected and included for the final analysis. Conceptualizations and operationalizations of MHSU were compared and contrasted. Facilitators of and barriers to athletes MHSU were examined and summarized while appropriately considering the proximity of each factor(facilitator or barrier) to the athletes.Results: Results showed variations in conceptualizations and operationalizations of MHSU in the articles analyzed, which made interpretation and cross comparison difficult. Collegiate athletes are willing to utilize mental health services, but gender, perceived stigma, peer norms—for athletes and coaches—plus service availability impact their MHSU.Conclusion: Key stakeholders, administrators, and public health officials should partner to eliminate MHSU barriers, support facilitators, and generally empower collegiate athletes to actively manage their mental health.
文摘Background:Few published studies have examined child passenger safety practices across countries.This study compared the prevalence and associated factors of child passenger restraint use among children,aged 0 to 17 in the state of Iowa in the United States,and the city of Shantou in China.Methods:Child restraint use observations were conducted in Iowa and in Shantou in 2012,respectively,among child passengers.Observations in Iowa were conducted at randomly selected gas stations,while in Shantou observations were completed at randomly selected schools or medical clinics.Research observers approached the driver,observed restraint use,and collected brief survey data.Results:A total of 3049 children from Iowa and 3333 children aged 0 to 17 years from Shantou were observed.For children aged 0 to 3 years,only 0.1% were compliantly restrained in Shantou as compared to 95.9% in Iowa.The proportion of children who were compliantly restrained in Shantou increased with age,but generally decreased with age in Iowa.In Shantou,36.0% of children aged 0 to 3 were sitting in the front seat as compared to only 1.7% of children of the same age in Iowa.Driver seat belt use was significantly associated with child restraint in both Iowa and Shantou;the association was stronger in Iowa than Shantou for aH age groups.Conclusions:A significantly higher prevalence of children who were not appropriately restrained was observed in Shantou than in Iowa.Our findings support the need of mandatory child safety restraint use legislation in China.
基金supported by the Faculty of Medicine of the UABJO,Oaxaca,Mexico,and by the National Technology of Mexico(TecNM)and CONACYT(BP-PA-2021050723-4900732-959110).
文摘Piwi-interacting RNAs(piRNAs of 26-32 nt in length)regulate gene expression,epigenetics,and transcriptional and post-transcriptional processes.1 In humans,piRNAs are expressed in germline and somatic tissues,and generally have a uracil at the 5'-end(position+1),an adenine at the+10 position,and are 2'-0 methylated at the 3'-end.2 Onice piRNAs form the RNA-induced silencing complex(RISC)with PIWl proteins,they serve as guides to find their complementary sequences of the target mRNA,thus initiating their degradation.The Wuhan patient's genome is approximately 265 nucleotides(nts)in its 3'-UTR;piRNAs-like sequences have been reported in this region.3 Our objective was to search,through bioinformatics,for mRNA sequences that were homologous to the reverse complementary of the previously reported piRNA-like sequences from the 3'-UTR of SARS-Cov-2,3 and identify the possible interaction between them.
基金This research is supported by grants from the National Natural Science Foundation for General Programs of China(31671524,31171407 and 81273201,GL)the Key Basic Research Project of the Science and Technology Commission of Shanghai Municipality(12JC1400900,GL)+2 种基金the Innovation Program of Shanghai Municipal Education Commission(14ZZ009,GL)the Excellent Youth Foundation of Chinese Academy of Sciences(KSCX2-EW-Q-7,GL)R21AI117547,1R01AI114581,V2014-001 from the V-foundation,and 128436-RSG-15-180-01-LIB from the American Cancer Society(RW).
文摘Immunomodulatory signaling imposes tight regulations on metabolic programs within immune cells and consequentially determines immune response outcomes.Although the glucocorticoid receptor(GR)has been recently implicated in regulating the function of myeloid-derived suppressor cells(MDSCs),whether the dysregulation of GR in MDSCs is involved in immune-mediated hepatic diseases and how GR regulates the function of MDSCs in such a context remains unknown.Here,we revealed the dysregulation of GR expression in MDSCs during innate immunological hepatic injury(IMH)and found that GR regulates the function of MDSCs through modulating HIF1α-dependent glycolysis.Pharmacological modulation of GR by its agonist(dexamethasone,Dex)protects IMH mice against inflammatory injury.Mechanistically,GR signaling suppresses HIF1αand HIF1α-dependent glycolysis in MDSCs and thus promotes the immune suppressive activity of MDSCs.Our studies reveal a role of GR-HIF1αin regulating the metabolism and function of MDSCs and further implicate MDSC GR signaling as a potential therapeutic target in hepatic diseases that are driven by innate immune cell-mediated systemic inflammation.
