背景:巨噬细胞M1/M2极化方向的调节在组织工程应用中尤为关键,及时调控可最大程度地减少促炎、促进抗炎或组织愈合反应。目的:将慢病毒介导的miRNA-378a过表达巨噬细胞株复合胶原蛋白海绵回植入动物模型,据此检测免疫调节在体内环境中...背景:巨噬细胞M1/M2极化方向的调节在组织工程应用中尤为关键,及时调控可最大程度地减少促炎、促进抗炎或组织愈合反应。目的:将慢病毒介导的miRNA-378a过表达巨噬细胞株复合胶原蛋白海绵回植入动物模型,据此检测免疫调节在体内环境中的相关表达水平等组织修复相关的指标,进一步阐明在体内环境中miRNA-378a是否促进巨噬细胞M2极化及其对免疫调节和组织修复的作用。方法:将慢病毒介导的miRNA-378a过表达巨噬细胞株、阴性对照病毒巨噬细胞株扩增、筛选,复苏培养巨噬细胞株后与胶原蛋白海绵共培养以此构成复合体,具体分组如下:①阳性组:过表达miRNA-378a巨噬细胞-胶原蛋白海绵复合体;②阴性组:阴性对照病毒介导的miRNA-378a巨噬细胞-胶原蛋白海绵复合体;③对照组:巨噬细胞-胶原蛋白海绵;④空白对照组:胶原蛋白海绵。通过免疫荧光及扫描电镜观察各组细胞密度、表型、黏附情况,后回植入小鼠背部皮下模型,分别于造模后4,7 d处死小鼠,通过大体观察、苏木精-伊红染色、Masson染色、免疫组化分析慢病毒介导的miRNA-378a过表达巨噬细胞胶原蛋白海绵复合体中巨噬细胞极化的方向及其对机体免疫调控、组织修复的作用。结果与结论:①免疫荧光镜下观察各组巨噬细胞株确实与胶原蛋白海绵形成复合体;②扫描电镜下慢病毒介导的miRNA-378a巨噬细胞(阳性组)较其他分组细胞密度增加,细胞出现球形、椭圆形及多边形分化,具有更多的伪足;③大体观察下总体7 d愈合好于4 d,慢病毒介导的miRNA-378a过表达巨噬细胞(阳性组)无论4,7 d愈合均好于其他分组;④苏木精-伊红染色、Masson染色下,慢病毒介导的miRNA-378a过表达巨噬细胞(阳性组)具有较多量的纤维细胞、毛细血管、成纤维细胞以及胶原纤维增生;⑤免疫组化显示,慢病毒介导的miRNA-378a过表达巨噬细胞(阳性组)无论4,7 d M2极化细胞阳性率均大于其他分组;对照组及阴性组巨噬细胞无论4,7 d M2极化细胞阳性率均大于空白对照组,而对照组及阴性组之间无统计学差异;阳性组、阴性组、对照组无论4,7 d染色细胞数量均大于空白对照组,且阳性组>阴性组≈对照组>空白对照组;⑥提示在体内环境中miRNA-378a过表达巨噬细胞具有较多量的纤维细胞、毛细血管、成纤维细胞以及胶原纤维增生,对组织修复起到了正向作用,并且能促进巨噬细胞向M2型极化并抑制M1型极化,从而有助于减少机体炎症反应。展开更多
Dengue virus （DENV） remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not ...Dengue virus （DENV） remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not fully understood. Cytotoxic molecules, such as granzyme B （GrzB）, may be necessary to control viral infections. However, the exact role of GrzB during DENV infection and the mechanisms regulating GrzB expression during DENV infection are not clear. This study found that miR-27a~, miR-3Oe, and miR-378 were down-regulated in DENV-infected patients, and DENV infection in humans induced a significant up-regulation of GrzB in natural killer （NK） cells and CD8＋ T cells. Further investigation indicated that NK cells, but not CD8＋ T cells, were the major sources of GrzB, and miR-378, but not miR-27a~ or miR-3Oe, suppressed GrzB expression in NK cells. Notably, we found that overexpression of miR-378 using a miR-378 agomir in DENV-infected mice inhibited GrzB expression and promoted DENV replication. These results suggest the critical importance of miR-378 in the regulation of GrzB expression and a protective role for GrzB in controlling DENV replication in vivo. Therefore, this study provides a new insight into the immunopathogenesis mechanism of DENV infection and a biological basis for the development of new therapeutic strategies to control DENV infection.展开更多
文摘背景:巨噬细胞M1/M2极化方向的调节在组织工程应用中尤为关键,及时调控可最大程度地减少促炎、促进抗炎或组织愈合反应。目的:将慢病毒介导的miRNA-378a过表达巨噬细胞株复合胶原蛋白海绵回植入动物模型,据此检测免疫调节在体内环境中的相关表达水平等组织修复相关的指标,进一步阐明在体内环境中miRNA-378a是否促进巨噬细胞M2极化及其对免疫调节和组织修复的作用。方法:将慢病毒介导的miRNA-378a过表达巨噬细胞株、阴性对照病毒巨噬细胞株扩增、筛选,复苏培养巨噬细胞株后与胶原蛋白海绵共培养以此构成复合体,具体分组如下:①阳性组:过表达miRNA-378a巨噬细胞-胶原蛋白海绵复合体;②阴性组:阴性对照病毒介导的miRNA-378a巨噬细胞-胶原蛋白海绵复合体;③对照组:巨噬细胞-胶原蛋白海绵;④空白对照组:胶原蛋白海绵。通过免疫荧光及扫描电镜观察各组细胞密度、表型、黏附情况,后回植入小鼠背部皮下模型,分别于造模后4,7 d处死小鼠,通过大体观察、苏木精-伊红染色、Masson染色、免疫组化分析慢病毒介导的miRNA-378a过表达巨噬细胞胶原蛋白海绵复合体中巨噬细胞极化的方向及其对机体免疫调控、组织修复的作用。结果与结论:①免疫荧光镜下观察各组巨噬细胞株确实与胶原蛋白海绵形成复合体;②扫描电镜下慢病毒介导的miRNA-378a巨噬细胞(阳性组)较其他分组细胞密度增加,细胞出现球形、椭圆形及多边形分化,具有更多的伪足;③大体观察下总体7 d愈合好于4 d,慢病毒介导的miRNA-378a过表达巨噬细胞(阳性组)无论4,7 d愈合均好于其他分组;④苏木精-伊红染色、Masson染色下,慢病毒介导的miRNA-378a过表达巨噬细胞(阳性组)具有较多量的纤维细胞、毛细血管、成纤维细胞以及胶原纤维增生;⑤免疫组化显示,慢病毒介导的miRNA-378a过表达巨噬细胞(阳性组)无论4,7 d M2极化细胞阳性率均大于其他分组;对照组及阴性组巨噬细胞无论4,7 d M2极化细胞阳性率均大于空白对照组,而对照组及阴性组之间无统计学差异;阳性组、阴性组、对照组无论4,7 d染色细胞数量均大于空白对照组,且阳性组>阴性组≈对照组>空白对照组;⑥提示在体内环境中miRNA-378a过表达巨噬细胞具有较多量的纤维细胞、毛细血管、成纤维细胞以及胶原纤维增生,对组织修复起到了正向作用,并且能促进巨噬细胞向M2型极化并抑制M1型极化,从而有助于减少机体炎症反应。
文摘Dengue virus （DENV） remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not fully understood. Cytotoxic molecules, such as granzyme B （GrzB）, may be necessary to control viral infections. However, the exact role of GrzB during DENV infection and the mechanisms regulating GrzB expression during DENV infection are not clear. This study found that miR-27a~, miR-3Oe, and miR-378 were down-regulated in DENV-infected patients, and DENV infection in humans induced a significant up-regulation of GrzB in natural killer （NK） cells and CD8＋ T cells. Further investigation indicated that NK cells, but not CD8＋ T cells, were the major sources of GrzB, and miR-378, but not miR-27a~ or miR-3Oe, suppressed GrzB expression in NK cells. Notably, we found that overexpression of miR-378 using a miR-378 agomir in DENV-infected mice inhibited GrzB expression and promoted DENV replication. These results suggest the critical importance of miR-378 in the regulation of GrzB expression and a protective role for GrzB in controlling DENV replication in vivo. Therefore, this study provides a new insight into the immunopathogenesis mechanism of DENV infection and a biological basis for the development of new therapeutic strategies to control DENV infection.
