Objective To obtain the lung adenocarcinoma initiating cells from the A 549 cell line based on paclitaxel treatment combination with serum-free cultivation and to validate spared cells can represent tumor initiating cells (TICs) .Methods After dis-sociated by trypsogen ,about 106 /mL cells were suspended in serum-free medium supplemented with 0 .4% bovine serum albumin (BSA) ,insulin ,basic fibroblast growth factor (bFGF) ,human recombinant epidermal growth factor (EGF) and obtained spheroid cells .At the second passage ,paclitaxel was added at a concentration of 100 nmol/L for 48 h and then replaced with completely fresh medium once or twice per week until new spheroids emerged .Results The subpopulation of cells that survived serum-free cultiva-tion and paclitaxel treatment could highly express the cluster of differentiation 133/cluster of differentiation (CD133/CD326) mo-lecular markers and have features of stemness including differentiation ,high expression of cancer stem cells (CSCs)-associated genes and stronger capability of tumorigenesis .Conclusion The survived subpopulation that highly express the CD 133/CD326 molecu-lar markers presenting the characteristics of stemness in vitro and in vivo ,and could be used in future researches of biological functions .

Objective To study the role of TLR2 and TLR4 signal transduction in RAW264.7 monocyte-macrophages stimulated by Aspergillus fumigatus conidia,and to investigate the expression of TLR2 signal transduction after silencing gene of TLR4.Methods Macrophages were randomly divided into normal group ( N group),normal+stimulated with Aspergillus fumigatus conidia ( N +Af group ),normal + transfected with TLR4-siRNA [ TLR4 (RNAi) group ],normal+transfected with TLR4-siRNA +stimulated with Aspergillus fumigatus conidia[ TLR4(RNAi) +Af group].RT-PCR and Western blot were used to assay expression levels of TLR2,TLR4,MyD88 mRNA and pro-inflammatory cytokines TNF-α protein when macrophages were stimulated 12 h by Aspergillus fumigatus conidia after tranfected 24 h with TLR4-siRNA by technology of RNAi.Results ( 1 ) Compared with N group,the expression of TLR2,TLR4,MyD88 mRNA and TNF-αprotein in N+Af group significantly increased before silencing gene of TLR4.(2) Silencing efficiency of macrophates was up to 83％ after transfected with TLR4-siRNA.(3)The expression of TLR2,MyD88 mRNA in TLR4 (RNAi) group significantly decreased contrast with normal group.Meanwhile the expression of TLR2,MyD88 mRNA and TNF-α protein also obviously reduced in TLR4(RNAi) +Af group when compared with N +Af group.Compared with TLR4 (RNAi) group,the expression of MyD88 mRNA in TLR4 (RNAi) +Af group significantly increased.However,the expression of TLR2 mRNA and TNF-α protein have no significant change after silencing gene of TLR4.Conclusion Signaling pathway of TLR2 and TLR4 in macrophages was activated by given stimulus of Aspergillusfumigatus conidia and exerted the effect of anti-Aspergillus fumigatus spores stimulation through the release of pro-inflammatory cytokines TNF-α.Meanwhile,silencing gene of TLR4 down-regulate the effect of TLR2 signal transduction in RAW264.7 cells to anti-Aspergillus fumigatus conidia stimulation,and it found that TLR4 played an more important role by contrast with TLR2.

Objective To study the activation of TLRs/NF-κB signal pathway and production of different functional cytokines during invasive pulmonary aspergillosis( IPA) , in order to probe the pathogene-sis of IPA. Methods Mouse were randomly divided into normal, normal + inoculated with Aspergillus fumigatus( normal inoculation group), and immune suppression + inoculation with Aspergillus fumigatus (IPAmodel group) , the mouse were killed at different time points after inhaling Aspergillus fumigatus spores by nose. Removing the lung tissue in a sterile manner and making pathological section respectively, counting Aspergillus fumigatus colony, dynamiclly detecting the expression of TLR2, TLR4 mRNA, variation of NF-κB p65 protein, pro-inflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokines IL-10 levels in the lung tissue by RT-PCR and Western blot method during Aspergillus fumigatus infection in mouse. Results (1) When it's 72 h after inhaling Aspergillus fumigatus by nose, IPA model emerged severe lung tissue inflammation, and generated a large number of hyphae, meanwhile, burthen of Aspergillus fumigatus was higher than normal inoculation group at each time point. (2)Compared with the normal inoculation group, IPA group whose TLR2 mRNA was low expression at early stage of infection (24 h), and emerged high expression at late stage of infection (120 h, 144 h); and TLR4 mRNA has been at a state of low expression in the infection process; NF-κB p65 suddenly increased at early stage of infection(24 h) and then continued to decline. (3) After infected by Aspergillus fumigatus in normal mouse, proinflammatory cytokine TNF-α, IL-1β in lung exhibited high expression at the early stages of infection, and the highest expression levels appeared at 48 h or 72 h, then decreased and recovered to normal level. And the expression level of anti-inflammatory cytokine IL-10 rised at late stage of infection; The IP A mouse released a lot of anti-inflammatory cytokine IL-10 at early stage of infection, which significantly reduced at late stage, and released pro-inflammatory cyto-kines TNF-α, IL-1β at slow and low level. Conclusion The abnormal activation of TLRs/NF-β signaling pathway caused the loss of dynamic balance between pro-inflammatory cytokines and anti-inflammatory cytokines, leading to the occurrence and development of IPA.