ABSTRACT
AIM:To explore the expression of anoctamin 1 (ANO1), one of calcium-activated chloride chan-nels ( CaCCs) , in mouse cardiomyocytes and its functional properties.METHODS:The cardiomyocytes from the myocar-dial tissues of C57BL/6 mice were isolated with enzyme and purified by the differential adherent method.The cells were stained with monoclonal anti-sarcomeric actin and Cy3 to evaluate the purity of the myocardial cells.RT-PCR was used to detect the mRNA expression of ANO1 in the mouse cardiomyocytes.The protein expression of ANO1 in the mouse cardio-myocytes was determined by Western blotting analysis.The fluorescence quenching kinetics experiment was used to identify the ion transport properties of ANO1 in the mouse cardiomyocytes.RESULTS: The results of RT-PCR confirmed that ANO1 was expressed in freshly isolated myocardial cells.The results of Western blotting clearly demonstrated the protein expression of ANO1 in primarily cultured myocardial cells.Fluorescence quenching kinetics experiment on freshly isolated single myocardial cell revealed a pronounced outward rectifying property of the ANO1.The functional properties were simi-lar to the classic CaCCs.CONCLUSION:ANO1 expression was identified in the mouse myocardial cells.The function of CaCCs was generated by ANO1, suggesting that ANO1 is the molecular basis of CaCCs.
ABSTRACT
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