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Objective: To investigate the effects of RCCS simulated microgravity on the metabolism of the human keratinocyte cell line HaCaT. Methods: The rotary cell culture system (RCCS) was used to simulate the microgravity environment, and HaCaT cells were cultured in vitro and divided randomly into simulated microgravity group (SMG) and normal gravity group (NG). The two group HaCaT cells were collected respectively after 1 d, 2 d and 3 d culture, and the samples were analyzed by LC/MS metabolomics. The differential metabolites between the SMG and NG cells were identified with partial least squares discriminant analysis ((O)PLS-DA), and the data were input into the KEGG database for the construction and functional analysis of metabolic pathways. Results: Comparing to NG cells, after 1 d culture, there were 74 different metabolites in SMG cells, among which 16 were up-regulated and 58 were down-regulated; after 2 d culture, there were 89 different metabolites, among which 15 were up-regulated and 74 down-regulated; after 3d culture, there were 100 different metabolites, of which 23 were up-regulated and 77 were down-regulated. The differentially expressed 49 metabolites (VIP>1 and P<0.05) after 3 d were set as target metabolites, within which the sphingosine, glutamate, and docosapentaenoic acid were down-regulated, and dehydrated sorbitol was up-regulated. KEGG analysis indicated that the metabolic pathways involved were amino acid metabolism, lipid metabolism, cell proliferation and apoptosis, substance transport, catabolism, and signal transduction. Conclusion: RCCS simulated microgravity may have significant impacts on keratinocyte metabolism mainly involving metabolites such as sphingolipids and glutamate as well as the related signaling pathways.
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Objective To investigate the effects of simulated microgravity by RCCS on proliferation and cell cytoskeleton of human HaCaT keratinocyte. Methods The rotary cell culture system (RCCS) was used to simulate the microgravity environment, and human HaCaT keratinocytes were divided randomly(random number) into the simulated microgravity group (SMG) and normal gravity group (NG). HaCaT cells in the two groups were harvested respectively after 32, 36 and 42 h culture. The HaCaT cells proliferation and cycles were detected by flow cytometry, the concentration of hb-EGF in supernatant was detected by ELISA, and the cell cytoskeleton was observed after 42 hours' culture under laser confocal microscope with FITC-labeled technique. SPSS 23.0 statistical software was used for statistical analysis, and P <0.05 was considered statistically significant. Results The flow cytometry showed that the proportions of human HaCaT keratinocytes in G1 and G2/M phases were increased while the proportion of HaCaT cells in S stage was decreased significantly after 32, 36 and 42 h RCCSculture compared with those in the normal gravity group. The HaCaT cells in G1 stage were declined along with incubation time. ELISA results showed that the hb-EGF concentration in HaCaT supernatant under simulated microgravity culture for 24 and 36 h was lower than that in the normal control group (P<0.01). The laser confocal microscope revealed that the HaCaT fluorescence intensity was decreased,and there were disordered microfilaments, structural ambiguity, pseudopodia reduction and irregularshape among FITC-labeled HaCaT cells cultured 42 h in RSSC compared with the normal gravity group.Conclusions RCCS simulated microgravity environment could inhibit the cell cycle transformation and proliferation of human HaCaT keratinocyte, affect the keratinocyte-secreting function, and induce alterations of the cell cytoskeleton.
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The present study was investigated that the effects of suitable ultraviolet (UV) irradiation to the cell viability, apoptosis and expression of poly(ADP-ribose) polymerase (PARP) in HaCaT and KUMA3 cell lines that are skin keratinocyte and squamous cell carcinoma, respectively. The cell viability of UV irradiation dose appeared high in HaCaT cell line, and the percentage of apoptotic cells appeared higher in KUMA3 cell line. The cell viability in HaCaT cell line pretreated with 5 J/m(2) UV and subsequently treated with 50 J/m(2) UV was higher than that only treated with 50 J/m(2) UV. On the other hand, the percentage of apoptotic cells in KUMA3 cell line only treated with 50 J/m(2) UV was higher than that pretreated with 5 J/m(2) UV and subsequently treated with 50 J/m(2) UV. Immunocytochemical stain using PARP antibody, the most cells show positive reaction of UV irradiation in HaCaT cell line, but some negative reaction cells observe from treated with 50 J/m(2) UV in KUMA3 cell line. The expression level of PARP by western blot analysis, concomitant with generation of the 85 kDa fragment in HaCaT cell line from treated with 50 J/m(2) UV though the 116 kDa band was still retained. On the other hand, the 85 kDa fragment appeared in KUMA3 cell line from treated with 50 J/m(2) UV but the intact PARP of 116 kDa disappears in more doses. These results present that suitable UV irradiation can be effective to apoptosis in squamous cell carcinoma.
Subject(s)
Humans , Apoptosis , Blotting, Western , Carcinoma, Squamous Cell , Cell Line , Cell Survival , Hand , Keratinocytes , Poly(ADP-ribose) Polymerases , SkinABSTRACT
Aim To investigate the expression and regulation characteristics of the Substance P receptor(Neurokinin-1R, NK-1R) in human skin keratinocytes and fibroblasts. Methods HaCaT cells,a human keratinocyte cell line, and fibroblasts were cultured. The expression of NK-1R protein was examined by immunohistochemisury technique,and the mRNA level was detected by reverse transcriptase polymerase chain reaction (RT- PCR). The expression and regulation of NK-1R were measured by flow cytometry in HaCaT cells and fibroblasts treated with various stimuli and drugs. Results The expression of NK-1R existed in human keratinocyte and fibroblast, mainly located on cell membranes and cytoplasma. The NK-1R also expressed at HaCaT cells and fibroblasts transcription levels, and the mRNA levels in HaCaT cells were higher than that of fibroblasts. SP and IFN-? might upregulate the membrane expressions of NK-1R in both the two cells, while LPS might downregulate the expressions of NK-1R. Cetirizine and Spantide I can degrade the expressions of NK-1R in the two kinds of cells.Conclusions The human keratinocytes and fibroblasts can express NK- 1R at cell, protein and transcription levels, and the expression characteristics can be regulated by some inflammatory factors, it indicates the keratinocytes and fibroblasts were involved in the regulation of skin immune and NK-1R may play an important role in skin inflammation.