目的通过观察胃癌源性的外泌体对HLA-DR^(^(neg))leukocytes中Dicer1、PTEN基因表达的影响,探讨肿瘤源性外泌体调控机体免疫抑制功能的机制。方法提取胃癌患者血清外泌体并鉴定,免疫磁珠法分选HLA-DR^(neg)leukocytes,吖啶橙染色后的外...目的通过观察胃癌源性的外泌体对HLA-DR^(^(neg))leukocytes中Dicer1、PTEN基因表达的影响,探讨肿瘤源性外泌体调控机体免疫抑制功能的机制。方法提取胃癌患者血清外泌体并鉴定,免疫磁珠法分选HLA-DR^(neg)leukocytes,吖啶橙染色后的外泌体与CFSE染色后的HLA-DR^(neg)leukocytes在37℃,5%CO_2条件下共培养12 h;采用QRT-PCR和Western blot方法分别检测共培养后胃癌患者与健康对照组中Dicer1、PTEN基因和蛋白的表达。结果成功分离胃癌患者外周血外泌体;同时观察到外泌体被HLA-DR^(neg)leukocytes有效摄取。QRT-PCR检测结果显示Dicer1 m RNA、PTEN m RNA的相对表达量分别是健康对照组的0.46±0.19、0.48±0.27倍(P<0.05);Western blot结果显示Dicer1、PTEN蛋白表达量是健康对照组的0.15±0.11、0.33±0.19倍(P<0.05)。结论胃癌来源的外泌体可以抑制HLA-DR^(neg)leukocytes中Dicer1、PTEN基因的表达,为今后进一步研究肿瘤来源的外泌体的免疫调控作用提供线索。展开更多
AIM:To investigate the continuous hepatic histopathological processes which occur in response to the loss of Dicer1.METHODS:We generated a hepatocyte-selective Dicer1 knockout mouse and observed the gradual hepatic hi...AIM:To investigate the continuous hepatic histopathological processes which occur in response to the loss of Dicer1.METHODS:We generated a hepatocyte-selective Dicer1 knockout mouse and observed the gradual hepatic histopathological changes in the mutant liver.Immunohistochemistry and Western blotting were performed to detect Dicer1 expression.We performed hematoxylin and eosin staining,Periodic acid-Schiff staining,Oil Red O staining,and Masson's trichrome staining to detect histological changes in Dicer1-deficient livers.Ki67 immunohistochemistry,terminal deoxynucleotidyl transferase-mediated d UTP nickend labeling assay,and Western blotting were used to determine hepatocyte proliferation and apoptosis.Serum biochemistry,cytokine assays,and flow cytometric analysis were performed to quantity liver necrosis and inflammation.Fibrogenic markers were determined by Western blotting and q PCR.CK19,CD133,and OV6 immunofluorescence were used to observe liver progenitor cells.Immunofluorescence and q PCR were performed to reveal embryonic gene expression.We also performed histological staining and Western blotting to analyze hepatocellular carcinoma(HCC) development.RESULTS:Dicer1 inactivation resulted in significant architecture disorganization and metabolism disruptionin the liver.Dicer1 disruption impaired hepatocyte survival and resulted in profound cell apoptosis and continuous necrosis.In contrast to previous reports,the mutant liver exhibited chronic inflammation and progressive fibrosis,and could not be repopulated by Dicer1-positive cells.In addition,extensive activation of hepatic progenitor cells was observed.Primary HCC was observed as early as 4 mo after birth.CONCLUSION:Hepatic loss of Dicer1 results in complex chronic pathological processes,including hepatocyte death,inflammatory infiltration,chronic fibrosis,compensatory proliferation,progenitor activation,and spontaneous hepatocarcinogenesis.展开更多
文摘目的通过观察胃癌源性的外泌体对HLA-DR^(^(neg))leukocytes中Dicer1、PTEN基因表达的影响,探讨肿瘤源性外泌体调控机体免疫抑制功能的机制。方法提取胃癌患者血清外泌体并鉴定,免疫磁珠法分选HLA-DR^(neg)leukocytes,吖啶橙染色后的外泌体与CFSE染色后的HLA-DR^(neg)leukocytes在37℃,5%CO_2条件下共培养12 h;采用QRT-PCR和Western blot方法分别检测共培养后胃癌患者与健康对照组中Dicer1、PTEN基因和蛋白的表达。结果成功分离胃癌患者外周血外泌体;同时观察到外泌体被HLA-DR^(neg)leukocytes有效摄取。QRT-PCR检测结果显示Dicer1 m RNA、PTEN m RNA的相对表达量分别是健康对照组的0.46±0.19、0.48±0.27倍(P<0.05);Western blot结果显示Dicer1、PTEN蛋白表达量是健康对照组的0.15±0.11、0.33±0.19倍(P<0.05)。结论胃癌来源的外泌体可以抑制HLA-DR^(neg)leukocytes中Dicer1、PTEN基因的表达,为今后进一步研究肿瘤来源的外泌体的免疫调控作用提供线索。
基金Supported by National Key Clinical Project,and National Sciencesand Technology Major Project of China,No.2012ZX10002-017
文摘AIM:To investigate the continuous hepatic histopathological processes which occur in response to the loss of Dicer1.METHODS:We generated a hepatocyte-selective Dicer1 knockout mouse and observed the gradual hepatic histopathological changes in the mutant liver.Immunohistochemistry and Western blotting were performed to detect Dicer1 expression.We performed hematoxylin and eosin staining,Periodic acid-Schiff staining,Oil Red O staining,and Masson's trichrome staining to detect histological changes in Dicer1-deficient livers.Ki67 immunohistochemistry,terminal deoxynucleotidyl transferase-mediated d UTP nickend labeling assay,and Western blotting were used to determine hepatocyte proliferation and apoptosis.Serum biochemistry,cytokine assays,and flow cytometric analysis were performed to quantity liver necrosis and inflammation.Fibrogenic markers were determined by Western blotting and q PCR.CK19,CD133,and OV6 immunofluorescence were used to observe liver progenitor cells.Immunofluorescence and q PCR were performed to reveal embryonic gene expression.We also performed histological staining and Western blotting to analyze hepatocellular carcinoma(HCC) development.RESULTS:Dicer1 inactivation resulted in significant architecture disorganization and metabolism disruptionin the liver.Dicer1 disruption impaired hepatocyte survival and resulted in profound cell apoptosis and continuous necrosis.In contrast to previous reports,the mutant liver exhibited chronic inflammation and progressive fibrosis,and could not be repopulated by Dicer1-positive cells.In addition,extensive activation of hepatic progenitor cells was observed.Primary HCC was observed as early as 4 mo after birth.CONCLUSION:Hepatic loss of Dicer1 results in complex chronic pathological processes,including hepatocyte death,inflammatory infiltration,chronic fibrosis,compensatory proliferation,progenitor activation,and spontaneous hepatocarcinogenesis.
