The objective of this study was to investigate macrophage polarization during the early stages of secondary Echinococcus granulosus sensu lato (E. granulosus s.l.) infection. We observed an early initial increase in inflammatory genes (peaking at 5-10 days) and a later rise in M (IL-4)-like genes (still rising by day 15). In addition, we showed that the induction of M (IL-4)-like genes was paralleled by an increase in expression of the transcription factor KLF4. Most of the changes observed in vivo were reproduced in vitro upon the culture of normal peritoneal macrophages with live E. granulosus s.l. protoscoleces (PSC), and that knockdown of KLF4 in this system attenuates M (IL-4) differentiation. Our results suggest that KLF4 pathway contributes to the differentiation of macrophages towards M (IL-4)-like phenotype during early stages of secondary E. granulosus s.l. infection.

目的 探讨小鼠腹腔细粒棘球蚴感染早期主要产生IL-22的CD4+T细胞Th1、Th17和Th22细胞以及IL-22R1的表达情况.方法 建立小鼠腹腔细粒棘球蚴感染模型,分别在感染后第3、6、9、12天收集外周血、小鼠脾淋巴细胞以及肝脏和肠管组织.ELISA检测外周血IFN-γ和IL-17、IL-22的蛋白表达量;qRT-PCR检测脾淋巴细胞中Th1细胞相关因子(ffng、Tbx21基因)、Th17细胞相关因子(IL-17、Rorc基因)、Th22细胞相关因子(IL-22、Ahr基因)以及肝脏和肠管组织中IL-22R1的mRNA表达水平;流式细胞术检测脾淋巴细胞中Th1细胞(CD4+ IFN-γ+)、Th17细胞(CD4+ IFN-γ-IL-22+ IL-17+)及Th22细胞(CD4+IFN-γ-IL-22+ IL-17-)的百分比情况.结果 与对照组相比,ELISA、qRT-PCR和流式细胞术检测细粒棘球蚴感染后Th1细胞、Th17细胞、Th22细胞增高:qRT-PCR检测细粒棘球蚴感染后肝脏和肠管IL-22R1的表达增高.结论 细粒棘球蚴感染小鼠早期,主要产生IL-22的CD4+T细胞Th1、Th17、Th22细胞比例以及IL-22R1表达增加,可能参与了宿主的免疫防御反应.

目的:观察细粒棘球蚴感染早期,小鼠体内脾细胞中T细胞亚群及相关细胞因子表达的改变.方法:以细粒棘球蚴感染Balb/c小鼠后收集细胞用FCM检测Th1、Th2、Treg、Th9细胞;同时用qRT-PCR检测IL-9、IL-10、IFN-γ、IL-4、PU.1和TGF-β的mRNA水平表达量;用ELISA检测血清中IL-9、IL-10、IFN-γ、1L-4、TGF-β和IL-25的含量.结果:FCM检测原头蚴组不同时间点Th1、Th2、Treg、Th9细胞的比例,差异均有统计学意义(P＜0.05);qRT-PCR检测IL-9、IL-10、IFN-γ、IL-4、PU.1和TGF-β的mRNA水平表达量在感染后升高,与对照组相比差异有统计学意义(P＜0.05);ELISA检测血清中IL-9、IL-10、IFN-γ、IL-4、TGF-β和IL-25的含量在感染后升高,与对照组相比差异有统计学意义(P＜0.05).结论:原头蚴能刺激小鼠体内脾细胞向Th1、Th2、Treg、Th9细胞分化且上调相应细胞因子的分泌,且Th9与Th2、Treg之间呈正相关,提示这些亚群细胞可能在包虫病感染的免疫逃逸中发挥一定作用.

目的 观察比较阿苯达唑粉剂和纳米化阿苯达唑在体外对细粒棘球蚴原头节作用的影响.方法 将阿苯达唑粉剂溶解于DMSO后与纳米化阿苯达唑分别配成3.13、6.25、12.5、25μg/mL浓度,按照所设分组体外培养,倒置显微镜下观察各药物处理组经伊红染色后原头节的形态及活力.ELISA试剂盒测定药物作用3、6d后原头节体内抗氧化酶NQO-1的活性变化.结果 将相同条件下阿苯达唑粉剂和纳米环阿苯达唑各浓度组原头节的活力较空白对照组降低.12.5、25μg/mL浓度组纳米化阿苯达唑酶活性变化高于阿苯达唑粉剂组.结论 阿苯达唑粉剂和纳米阿苯达唑均有不同程度的体外抗细粒棘球蚴原头节作用;纳米化阿苯达唑在体外抑制细粒棘球蚴原头节生长作用优于普通阿苯达唑粉剂,有望成为治疗细粒棘球蚴病的一种新的剂型.

Stem cell therapy is a promising treatment strategy for ischemic diseases. Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) adhere to each other in the bone marrow cavity and in in vitro cultures. We have previously demonstrated that the adhesion between MSCs and EPCs is critical for MSC self-renewal and their multi-differentiation into osteoblasts and chondrocytes. In the present study, the influence of the indirect communication between EPCs and MSCs on the endothelial differentiation potential of EPCs was investigated, and the molecular mechanisms underlying MSC-mediated EPC differentiation were explored. The effects of vascular endothelial growth factor (VEGF), which is secreted by MSCs, on EPC differentiation via paracrine mechanisms were examined via co-culturing MSCs and EPCs. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to detect the expression of genes and proteins of interest. The present results demonstrated that co-culturing EPCs with MSCs enhanced the expression of cluster of differentiation 31 and von Willebrand factor, which are specific markers of an endothelial phenotype, thus indicating that MSCs may influence the endothelial differentiation of EPCs in vitro. VEGF appeared to be critical to this process. These findings are important for the understanding of the biological interactions between MSCs and EPCs, and for the development of applications of stem cell-based therapy in the treatment of ischemic diseases.

Objective:To investigate the expression levels of PPARα/γin RAW264. 7 cells in the early stages of co-cultivation with Echinococcus granulosus in vitro. Methods:RAW264. 7 cells were co-cultured with E. granulosus and collected at 12,24,36,48, 72 h. The mRNA levels of PPAR-γ,PPAR-α,M1 macrophages-associated cytokines including TNF-α,MCP-1 and IL-1β,and M2 mac-rophages-associated cytokines including Arg-1,TGF-β and Fizz-1 were detected by qRT-PCR. The protein levels of Arg-1 and MR were analyzed by ELISA. Results:The expression levels of PPAR-γ, PPAR-αand the M2 macrophages-associated cytokines including Arg-1,TGF-β,Fizz-1 and MR were significantly increased,especially at 72 h (P<0. 05). M1 macrophages-associated cytokines including TNF-α,MCP-1 and IL-1β were decreased at 72h although increased at first. Conclusion:During the early stages of co-cultivation with Echinococcus granulosus in vitro, the levels of PPAR-γ/α are up-regulated in RAW264. 7 cells, which may drive macrophage polarization and play a role in the immune escape.

目的:通过对胚胎鼠跖骨周围芽生的管状结构面积进行量化,探讨加入血管形成模型的相对最适合的EPCs细胞量,并为EPCs联合胚胎鼠跖骨血管形成模型奠定实验基础.方法:改良差时贴壁培养法提取EPCs,与胎鼠跖骨共培养14天后,进行CD31免疫染色,用IPP对染色阳性面积进行量化分析.结果:不同EPCs细胞数量处理组的胎鼠跖骨,可根据其周围芽生的管状结构面积的量化来分析模型中EPCs的活性;加入数量为5×104个EPCs时,胎鼠周围芽生的管状结构的面积最大(P＜0.05).结论:初步构建了EPCs联合胚胎鼠跖骨血管生成实验模型,并探讨了模型中EPCs的最适数量.

目的探讨原头蚴可能通过刺激巨噬细胞分泌KLF4而促使其向M2分化。方法原头蚴与RAW264.7细胞共培养,分别在12、36、48、84 h收集细胞和培养上清液,q RT-PCR检测KLF4、M1（Arg-1,Fizz-1,MR）、M2（TNF-α、IL-6、MCP-1）巨噬细胞因子的表达;ELISA检测相关因子分泌的含量。结果实验组（84 h）的KLF4 m RNA水平较对照组明显升高。随着感染时间延长,M2巨噬细胞的m RNA表达量及分泌含量呈上升趋势,M1巨噬细胞的m RNA表达量及分泌含量呈先升后降趋势。结论原头蚴刺激RAW264.7细胞,可能通过分泌某些抗原、蛋白,诱导巨噬细胞KLF4高表达而促使其向M2分化,抑制巨噬细胞的功能,从而逃逸宿主的免疫杀伤。

目的 观察细粒棘球绦虫幼虫原头蚴及囊液对体外培养小鼠脾细胞产生IL-22的影响.方法 取Balb/c小鼠脾细胞,分别加入不同浓度原头蚴或囊液共培养48 h,检测上清液IL-22表达量及细胞IL-22 mRNA的相对表达量.同浓度原头蚴或囊液分别在0、12、24、36和48 h收集细胞,流式细胞术检测CD4+IL-22+T细胞比例变化.RPMI 1640培养基与脾细胞共培养设为对照组.结果 与对照组相比,ELISA和qRT-PCR检测显示在原头蚴为1 000/ml和囊液蛋白质量浓度为2.05 mg/ml时脾细胞产生IL-22增加;流式细胞术检测原头蚴组及囊液组CD4+IL-22+T细胞比例逐渐增加.结论 原头蚴及囊液均可促进小鼠脾细胞产生IL-22增加,提示IL-22可能参与宿主防御细粒棘球蚴感染.

Objective:To observe the effect of protoscolex on Th subsets and correlative cytokine in mice spleen cells in vitro.Methods:Co-culture spleen cells from BALB/c mice with protoscolices,then IL-4,IFN-γand TGF-βproduction in cell culture supernatants were analyzed by ELISA.The percentage of Th subsets were detected by Flow Cytometry analysis.Results:Secretion levels of IL-4 and TGF-βwere significantly increased in spleen cells at different time point in co-culture system with protoscolices.Ratios of Th2 and Treg cells were also significantly increased in co-culture system at different time points than the control groups.However,there was no statistical significance for ratio of Th1 cells at different time points.Conclusion:The protoscolex can increase the ratios of Th2 cells and Treg cells from spleen cells.Secretion levels of IL-4 and TGF-βwere also increased in spleen cells co-cultured with protosco-lices.The results suggest that these Th cell subsets play a role in the immune escape of the hydatid disease.