RESUMO
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 .
RESUMO
AIM: It is found that some active priciples of ginseng can enhance and activate body immune system, and have many bioactivities such as anti-tumor, anti-aging and anti-radiation. This study examined the effect of ginsenoside-Rh2 (GS-Rh2) on proliferation and apoptosis in human myeloid leukemia cell strain HL60, and analyzed the dose- and time-dependent manners of GS-Rh2. METHODS: Experiments were performed at the Department of Clinical Laboratory of Jilin Medical College from July to August in 2006. ①Rh2 was purchased from Hongjiu Biotech Co., Ltd. (batch number 050801), and prepared into 50 g/L stock solution by dissolving in pH 7.4 phosphate buffer saline. The HL60 cell strain was purchased from Shanghai Institute of Cell Biology of Chinese Academy of Sciences. ②HL60 cells in logarithmic growth phase were inoculated into 3?108 L-1 cell suspension. After the cells were cultured for 6 hours, 100 ?L GS-Rh2 at different concentrations (5,10,20,40,80 mg/L) was added respectively. After the cells were administrated for 48 hours, cell inhibition ratio (IR) was evaluated by MTT colorimetric assay. The 50% inhibitory concentration (IC50) was worked out. HL60 cell was acted with this concentration for different time (6, 12, 24, 48 and 72 hours), cell inhibition ratio (IR) at different time points was evaluated by MTT colorimetric assay, and compare it to the control. After the IC50 of GS-Rh2 acted for 48 hours, HL60 cells were observed with an inverted microscope. HL60 cell was stained by Giemsa, and the typical apoptosis cells were discovered. RESULTS: ①Dose-effect relationship: When the concentration of GS-Rh2 was 5,10,20,40 mg/L, the IR of GS-Rh2 to the growth of HL60 cells was increased gradually in obviously dose-dependent manner. The IR was similar between 80 mg/L and 40 mg/L. After the cells were administrated for 48 hours, the IC50 value was 13.0 mg/L. ②Time-effect relationship: After the concentration of IC50 of GS-Rh2 (13.0 mg/L) acting on HL60 line at different time points (6, 12, 24, 48 and 72 hours), cell IR was increased gradually (F=9.32,P