Objective To investigate the expression of anoctamin 1 in Chinese hamster ovary cells (CHO)and to analyze the functional properties of its ion channel,and to provide experimental basis for study on the physiological function of calcium-activated chloride channel.Methods The whole sequence of anoctamin 1 was obtained by PCR technique and was subcloned into pcDNA3.1 to construct the expression vector pcDNA3.1-anoctamin 1 was transfected into CHO by liposome-mediated transfection and the CHO stably expressing anoctamin 1 were aquired by selection with zeocin. The expression and distribution of anoctamin 1 in CHO were measured by RT-PCR technique and inverted fluorescence microscope.The functional properties of anoctamin 1 were measured with halide-sensitive fluorescence proteins YFP-H148Q/I152L.The PBS buffer solution with calcimycin and high concentration of iodine ion was used as experimental group,andthe PBS buffer solution without calcimycin and high concentration of iodine ion was used as control group.Results The results of digestion and sequencing confirmed that anoctamin 1 was cloned into pcDNA3.1 by electrophoresis and blast. The results of RT-PCR and inverted fluorescence microscope indicated that anoctamin 1 was expressed in CHO. The results of I- influx as measured by halide-sensitive fluorescence proteins YFP-H148Q/I152L showed that anoctamin 1 had the more functional properties of trans-epithelial transporting I-,and the transporting speed in experimental group was higher than that in control group (P<0.05).Conclusion Anoctamin 1 can be expressed highly in the CHO;Anoctamin 1 expressed in CHO has the characteristics of calcium-activated chloride channel.

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 .

OBJECTIVE To predict the quality marker(Q-Marker) of Aurantii Fructus Immaturus in Wendan decoction (WDD) based on fingerprint and network pharmacology. METHODS Establish the fingerprint of Aurantii Fructus Immaturus and WDD reference samples, use Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software(2012A Edition)for similarity analysis and common component identification; use network pharmacology to screen the related targets and key pathways of common components, and establish the composition-target-pathway network. Predict the potential Q-marker of expectorant stop vomiting, clear gallbladder and stomach of Aurantii Fructus Immaturus in WDD, and determine its content. RESULTS The fingerprints of 15 batches of Aurantii Fructus Immaturus and 15 batches of WDD reference samples were established, and the similarity of fingerprints between 15 batches of Aurantii Fructus Immaturus and WDD reference sample >0.9. Five chemical components were identified, which were naringin, hesperidin, neohesperidin, nobiletin and tangerine. Network pharmacologic screen analysis to getTP53, MAPK8, EGFR, JUN, MMP9 and other 32 core targets of the five compounds and Endocrine resistance, pathways in cancer, and 10 other key pathways. Construct the "component-target-pathway" network diagram. It was predicted that naringin, hesperidin, neohesperidin, nobiletin and tangerine were Q-markers of Aurantii Fructus Immaturus in WDD. The contents of naringin, hesperidin, neohesperidin, nobiletin, tangerine in WDD were not less than 0.011 7%, 0.025 3%, 0.025 7%, 0.010 4%, 0.010 7%. CONCLUSION Q-Marker prediction analysis of Aurantii Fructus Immaturus in WDD provides a reference for establishing a complete WDD quality evaluation system, and also lays a foundation for elucidating the mechanism of its pharmacodynamic material basis.