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Objective To investigate whether Liuwei Dihuang Pills enhances the antigen cross-presenting ability of dendritic cell(DC)by increasing gap junctional intercellular communication(GJIC),and to explore the mechanisms involved.Methods Western Blot and immunofluorescence were used to observe the effects of Liuwei Dihuang Pills-containing serum on the expression and membrane localisation of gap junction protein connexin43(Cx43)in mouse melanoma cells(B16);Calcein-AM/DiI fluorescence tracer assay was used to observe the effects of Liuwei Dihuang Pills-containing serum on the function of GJIC in B16 cells;flow cytometry was used to observe the role of GJIC in the enhancement of DC antigen presenting ability by Liuwei Dihuang Pills-containing serum;and propidium iodide(PI)/Hoechst staining assay was used to observe the immunocidal effect of CD8+ T-lymphocytes.Results Western Blot and immunofluorescence experiments showed that Liuwei Dihuang Pills-containing serum led to the up-regulation of Cx43 expression;fluorescence tracer experiments proved that the GJIC function of B16 cells was significantly enhanced by Liuwei Dihuang Pills-containing serum;flow cytometry analyses showed that the DC antigen-presenting ability was enhanced by Liuwei Dihuang Pills-containing serum;and the results of PI/Hoechst staining showed that the immuno-killing effect of CD8+T-cells was more significant after the intervention of Liuwei Dihuang Pills-containing serum in B16-OVA.Conclusion Liuwei Dihuang Pills improve the GJIC function by up-regulating the Cx43 expression of melanoma cells,and then enhance the cross-presenting ability of DCs thus activating stronger CD8+ T-cell immunocidal responses.
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Aim To explore the effect of connexin 43 ( Cx 4 3 ) on acquired gefitinib-resistance in human non small cell lung cancer ( NSCLC ) . Methods HCC827 GR, a gefitinib-resistant ( GR) NSCLC cell lines from their parental cells was established by gradually in-creasing the concentration of gefitinib. Gefitinib effica-cy in HCC827 and HCC827 GR cells was detected by MTT assay. Expression of Cx43 mRNA in HCC827 and HCC827 GR cells was determined by RT-PCR. The protein expressions of Cx43 and phospho-Akt ( p-Akt) in these cells were detected by Western blot. The func-tional gap junction intercellular communication ( GJIC ) was measured by parachute assay. The cellular locali-zation of Cx43 protein was evaluated by immunofluores-cence staining. Results MTT assay showed less ge-fitinib cytotoxicity in HCC827 GR cells than that in their parental cells with IC50 of (10. 84 ± 0. 021) μmol ·L-1 versus (0. 07 ± 0. 019) μmol·L-1 , respective-ly. Moreover, both mRNA and protein expressions of Cx43 in HCC827 GR cells were significantly lower than those in HCC827 cells ( P<0. 05 ) . However, the p-Akt protein in HCC827 GR cells was obviously higher than that in HCC827 cells ( P<0. 05 ) . Furthermore, treatment with LY294002 caused a significant reduced p-Akt expression, but a significant increased Cx43 ex-pression in HCC827 GR cells. Moreover, no detecta-ble GJIC was found in HCC827 and their GR cells with or without RA ( a well-defined GJIC enhancer ) treat-ment. Immunofluorescence staining clearly showed that Cx43 protein accumulated in the cytoplasm of HCC827 and their GR cells. Conclusion The down-regulation of Cx43 expression in cytoplasm of HCC827 GR cells may contribute to the acquired gefitinib resistance in NSCLC cells by GJIC-independent activation of PI3 K/Akt signaling pathway.
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Objective:To investigate Cx43 expression and gap junctional intercellular communication(GJIC) in ectopic and eutopic endometrial stromal cells in endometriosis(EMs),and to explore the influence of aberrant GJIC in stromal cells on pathogenesis of EMs. Methods:The stromal cells were isolated from samples including 24 ectopic endometriotic tissues located in ovaries,41 eutopic endometria with endometriosis and 30 normal endometria. The endometrial stromal cells models were established in vitro by being cultured in manual conditions mimicked with estrogen and progesterone. Laser scanning confocal microscopy(LSCM) was used to determine the expression of Cx43 protein and the function of GJIC in three groups stromal cells. Results:The success rate of isolation and culture of endometriotic stromal cells was 45.8%(11/24);of eutopic endometrial stromal cells with EMs was 92.7(38/41);of normal endometrial stromal cells was 93.3%(28/30). The purities of ectopic and eutopic endometrial stromal cells were 95% and 98% respectively. The level of Cx43 protein and the function of GJIC in stromal cells from ectopic endometrial tissues were much lower than those from the other two groups,the highest level of Cx43 protein and the function of GJIC were observed in normal endometrial stromal cells group,and the differences among these groups were significant (P﹤0.01). Conclusions:(1) It will be helpful to establish models of normal,ectopic and eutopic endometrial stromal cells in vitro simultaneously when investigating the pathgenesis of EMs. (2)Downregulation of Cx43 expression and aberrant function of GJIC are related to pathogenesis of EMs. Regulation of Cx43 or GJIC in endometrial stromal cells is implied to be a potential strategy to treat EMs.
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Objective To investigate the gene expression of connexin43(Cx43) and its effect on gap junction intercellular communication(GJIC) of acute leukemia bone marrow stromal cells(ALBMSCs).Methods After ALBMSCs were transfected by recombinant adenovirus Ad-Cx43-GFP,the expression of report gene GFP and the transfection efficiency were monitored by fluorescent microscopy.RT-PCR,Western blot and immunocytochemical method were used to detect the mRNA and protein expressions of Cx43.Dye transfer procedure was performed to examine the GJIC function.Results After transfected by Ad-Cx43-GFP for 24 h,the expression of GFP in ALBMSCs was detected by fluorescent microscopy and the transfection efficiency was(82.7?2.16)%;The mRNA and protein expressions of Cx43 in ALBMSCs transfected by Ad-Cx43-GFP were higher than those not transfected by Ad-Cx43-GFP(P