ABSTRACT
The rotary cell culture system(RCCS)was used to simulate the microgravity environment, and FRTL-5 cells were divided into simulated microgravity group(SMG)and normal gravity group(NG). FRTL-5 cells were harvested after treatment for 6, 12, 24, and 36 h, the cell viability was measured by MTT assay, and the cells cycles were detected by flow cytometry. The ultrastructure of FRTL-5 cells was observed under laser confocal microscope with FITC-labeled technique. The MTT assay showed that the proliferation of FRTL-5 cells was significantly inhibited after RCCS treatment for 6, 12, 24, and 36h compared with NG(P<0.05), in which the most obvious effect was observed at 24h. The flow cytometry showed that the proportion of FRTL-5 cells at G1 stage in RCCS group was increased significantly after 6, 12, 24, and 36h compared with NG(P<0.05), while the proportion of FRTL-5 cells at S stage was decreased significantly(P<0.05)except that cultured with RCCS for 6 h. The proportion of FRTL-5 cells at G2/M stage was decreased in early phase(6-12 hours)of RCCS culture, with the lowest at 12h and transient increase at 24h of RCCS culture. The laser confocal microscope revealed that there were local microfilament depolymerization, tension fibers decrease, structure disorder, cellular pseudopodia reduction, and irregular shape among FITC-labeled FRTL-5 cells cultured with RCCS for 36h. (Chin J Endocrinol Metab, 2018, 34: 598-601)
ABSTRACT
AIM:To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thy-roid follicular epithelial ( FRT) cells and its electrophysiologic properties .METHODS: The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection .In order to ob-tain the high efficiency of gene transfection and expression , the quantity and ratio of lipid/DNA complexes were optimized . The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence .The expression and location of TMEM 16A in the FRT cells were observed under an in-verted fluorescence microscope .TMEM16A protein was associated with calcium-dependent chloride current , as measured with halide-sensitive fluorescent protein and patch-clamp technique .RESULTS: The results of double digestion and se-quencing indicated that TMEM16A was cloned into pUB6/V5.The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A.The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM 16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L.CONCLUSION:The protein expression of TMEM16A in the FRT cells was observed.TMEM16A is the molecular identity of calcium-activated chloride channels .