ABSTRACT
Objective: To establish a detection method based on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that can sensitively detect the second messenger cyclic AMP (cAMP) in the cytoplasm. Methods: The eukaryotic expression vectors of CFTR and YFP-H148Q / I152L were constructed respectively. FRT cells co-expressing CFTR and YFP-H148Q / I152L were obtained by liposome transfection. The expression of CFTR and YFP-H148Q / I152L in FRT cells was observed by an inverted fluorescence microscopy, and flow cytometry was used to detect the purity of cells; The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CFTR modulators was verified by the fluorescence quenching kinetics experiments. The radioimmunoassay was used to detect the cAMP concentration in cytoplasm after adding CFTR activator. Results: The results of the inverted fluorescence microscope showed that CFTR was expressed in the cell membrane and YFP-H148Q / I152L was expressed in the cytoplasm of FRT cells. The FRT cell model stably co-expressing ANO1 and YFP-H148Q / I152L was successfully constructed. The model could screen CFTR modulators, and the slope of fluorescence change and the concentration of CFTR modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the cAMP concentration in the cytoplasm. The cell model could sensitively detect the intracellular cAMP concentration. Conclusion: The cell model could efficiently and sensitively detect the second messenger cAMP concentration in the cytoplasm, and it provided a simple and efficient method for the study of other targets associated cAMP signal.
Subject(s)
Cyclic AMP , Cystic Fibrosis Transmembrane Conductance Regulator , Cytoplasm , Second Messenger SystemsABSTRACT
OBJECTIVE@#To evaluate the early clinical efficacy and safety of vesselplasty for the treatment of spinal metastases complicated by posterior wall destruction of vertebral body.@*METHODS@#The clinical data of 19 patients(21 segments) with spinal metastases complicated by posterior wall destruction of vertebral body treated from January 2016 to January 2017 were retrospectively analyzed. There were 15 males and 4 females, aged 40 to 85 years old with a mean of (66.00±10.25) years . All patients had severe low back pain before the operation, which were diagnosed by CT as damage-type metastatic tumor of the vertebral posterior wall. All patients were treated by vesselplasty technique. Nineteen vertebrae received percutaneous unilateral pedicle puncture and two vertebrae received percutaneous bilateral pedicle puncture. VAS, ODI were recorded before operation, 1 d and 3 d after operation respectively. X-ray and CT scan were used to observe bone cement leakage and complications.@*RESULTS@#All the operations were successful and postoperative pain was significantly relieved. Postoperative VAS score and ODI of the two groups were significantly improved (<0.05). A small amount of bone cement leakage occurred in one vertebral body, which was a vertebral venous plexus leakage, but no clinical symptoms after operation.@*CONCLUSION@#Vesselplasty for the treatment of spinal metastases complicated by posterior wall destruction of vertebral body can significantly reduce the symptoms of thoracolumbar back pain, improve the quality of life, reduce the incidence of bone cement leakage, and has high clinical efficacy and safety.
