ABSTRACT
BACKGROUND: RIPA Buffer exhibits different extraction efficiencies of proteins of cells and tissues, which is not appropriate for all samples. OBJECTIVE: To achieve an optimal lysis buffer for skeletal muscle protein extraction in mice of acidosis, and to provide basis for studies on skeletal muscle atrophy. METHEDS: Twenty male healthy C57BL/6 mice, aged 3 months, weighting 25-30 g, were provided by Laboratory Animal Center of Shanxi Medical University. The mice were sacrificed after anesthesia, and the gastrocnemius muscle of lower extremity was isolated. There were two groups: acidosis group was given 10 g of feed mixed with 0.4 mol/L hydrochloric acid (10 mL) , and control group received 10 g of feed mixed with same volume of water, for 7 consecutive days. The effect of RIPA Buffer, Original Buffer and JP Buffer on the skeletal muscle protein extraction in mice of acidosis was compared. The expression levels of AKT, p-AKT (Thr308) , rpS6 and p-rpS6 (Ser235/236) were detected by western blot assay. GLUT4 mRNA expression was examined by RT-qPCR. RESULTS AND CONCLUSION: (1) Different buffers generated different protein-yields. The protein yield was highest in JP Buffer, but the target protein signal was not high. The protein yield was low in RIPA Buffer. Original Buffer could extract sufficient proteins, and had clear band detected by western blot assay. (2) Western blot assay scores in Original Buffer were higher than those of other two buffers. (3) Western blot assay results showed that the extent of phosphorylation in both groups showed no significant changes. (4) GLUT4 mRNA expression level examined by RT-qPCR showed no significant changes in both groups. (5) These results indicate that Original Buffer is optimal lysate of skeletal muscle protein extraction. Inactivated AKT signaling pathway is seen in the short-term hydrochloric acid-induced acidosis group, so whether lengthening acidosis time can activate the signaling pathway. Selecting the optimal lysis buffer for different samples is premise to ensure western blot assay results.
ABSTRACT
Objective To evaluate 18F-FDG hPET/CT in the diagnosis of postoperative recurrence and metastasis of colorectal cancer. Methods GE HAWKEYE coincidence SPECT was carried out in 81 colorectal cancer patients with suspected recurrence or metastasis after intravenous injection of 259 ~ 298 MBq (7-8 mCi) 18F-FDG. The acquired data were reconstructed using iterative algorithm and attenuation-corrected X-ray. The results were compared with the final diagnosis established by histological examination of resected specimens、and clinical follow-up. Results The sensitivity, specificity, positive predictive value (PPV)and negative predictive value(NPV)was 93% (57/61)、80% (16/20)、93% (57/61)、80% (16/20) for 18F-FDG hPET/CT respectively. For conventional CT the sensitivity, specificity, PPV and NPV was 67% (37/55)、73% (19/26)、84% (37/44)、51% (19/37) respectively; 18F-FDG hPET/CT detected 91 recurrent or metastatic lesions whereas only 46 lesions were detected by conventional CT in 65 patients. Conclusions 18 F-FDG hPET/CT has unique value in the diagnosis of recurrence and metastasis in postoperative colorectal cancer patients which was superior to conventional CT. Combined 18 F-FDG coincidence imaging with localizing CT improves the detection and localization of postoperative recurrence and/or metastasis in colorectal cancer patients.