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Objective To establish a cell model based on calcium-activated chloride channel (CaCC) that could sensitively detect the second messenger Ca
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Objective To construct a high-throughput screening model for transient receptor potential vanilloid 4 (TRPV4) channel modulators based on calcium-activated chloride channels (CaCC). Methods RT-PCR was used to detect the endogenous expression of TRPV4 in Fischer rat thyroid (FRT) cells. The PCR products obtained were subjected to nucleic acid sequencing using gel-recovery technology. Western blotting was employed to detect the expression of TRPV4 protein in FRT cells. The liposome transfection method was applied to construct the FRT cell model that co-expressed anoctamin 1 (ANO1) and YFP-H148Q/ I152L. The expressions of ANO1 and YFP-H148Q/I152L in cells were identified by the inverted fluorescence microscope and the fluorescence quenching kinetics test. After adding TRPV4 activators and inhibitors, the fluorescence quenching kinetics experiment was used to test whether the model could screen TRPV4 modulators. The Fura-2 fluorescent probe method was applied to detect the calcium concentration in cells after adding TRPV4 activators; The Z' factor was calculated to evaluate the sensitivity and specificity of the cell model. Results RT-PCR and Western blotting confirmed the endogenous expression of TRPV4 in FRT cells; ANO1 was clearly expressed on the FRT cell membrane and YFP-H148Q/I152L was clearly expressed in the cytoplasm of FRT cells under the inverted fluorescence microscope. The FRT cell model co-expressing ANO1 and YFP-H148Q/I152L was successfully constructed. Fluorescence quenching kinetics experiments confirmed that the model could screen TRPV4 regulators, and the slope value of fluorescence change and the concentration of TRPV4 regulator concentration were in a dose-dependent manner. The model could sensitively detect changes in intracellular calcium concentration, and the slope value could reflect intracellular calcium concentration. The Z' factor was 0.728, which demonstrates its capacity for high-throughput screening. Conclusions We successfully constructed a high-throughput model that could screen TRPV4 modulators sensitively and efficiently.
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<p><b>OBJECTIVE</b>To explore the pathological basis of diffusion-weighted imaging (DWI) findings in hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE).</p><p><b>METHODS</b>DWI was performed in 15 patients with HCC treated by TACE within 24 - 48 hours before II-phase operation. The DWI findings of the liver lesions were analyzed and correlated with pathological findings including macroscopic observation, HE staining and immunohistochemical staining for bFGF.</p><p><b>RESULTS</b>(1) The viable tumor area showed mostly hypersignal intensity (12/15), whereas coagulative necrotic lesions showed hyposignal (8/15) or isosignal intensity (6/15). The ADC values of zones of viable tumor and necrosis in tumor were (1.42 +/- 0.16) x 10(-3) mm(2)/s and (1.58 +/- 0.18) x 10(-3) mm(2)/s, respectively. There was a significant difference of ADC values between the two zones (t = 2.618, P < 0.05). (2) There was a significant difference in ADC values of viable tumor between well and poorly differentiated tumors (t = -2.646, P < 0.05). The distinction of ADC values of the whole tumor was significant among tumors with different degree of necrosis (chi(2) = 7.236, P < 0.05). (3) A negative correlation was observed between bFGF protein expression index and ADC values of viable parts of the tumors in the study group (r = -0.552, P = 0.033).</p><p><b>CONCLUSION</b>DWI shows certain characteristic features of the HCC after TACE, and can be used to distinguish viable and necrotic tumor tissues in HCC after TACE.</p>