Résumé
OBJECTIVE@#To establish a simple, low-cost and time-saving method for primary culture of mature white adipocytes from mice.@*METHODS@#Mature white adipocytes were isolated from the epididymis and perirenal area of mice for primary culture using a modified mature adipocyte culture method or the ceiling culture method. The morphology of the cultured mature adipocytes was observed using Oil Red O staining, and the cell viability was assessed with CCK8 method. The expression of PPARγ protein in the cells was detected with Western blotting, and the mRNA expressions of CD36, FAS, CPT1A and FABP4 were detected using RT-qPCR.@*RESULTS@#Oil Red O staining showed a good and uniform morphology of the adipocytes in primary culture using the modified culture method, while the cells cultured using the ceiling culture method exhibited obvious morphological changes. CCK8 assay showed no significant difference in cell viability between freshly isolated mature white adipocytes and the cells obtained with the modified culture method. Western blotting showed that the freshly isolated adipocytes and the cells cultured for 72 h did not differ significantly in the expression levels of PPARγ protein (P=0.759), which was significantly lowered in response to treatment with GW9662 (P < 0.001). GW9662 treatment of the cells upregulated mRNA expressions of CD36 (P < 0.001) and CPT1A (P=0.003) and down-regulated those of FAS (P=0.001) and FABP4 (P < 0.001).@*CONCLUSION@#We established a convenient and time-saving method for primary culture mature white adipocytes from mice to facilitate further functional studies of mature adipocytes.
Sujets)
Mâle , Souris , Animaux , Adipocytes blancs/métabolisme , Récepteur PPAR gamma/métabolisme , ARN messager , Différenciation cellulaire , Cellules 3T3-L1Résumé
<p><b>OBJECTIVE</b>To research the mechanism of dopamine (DA) controlled memory in mice.</p><p><b>METHODS</b>Mice received i.p. injection of scopolamine (0.3 mg/kg, SCOP 0.3, and 3.0mg/kg, SCOP 3.0, respectively, n = 10) and saline (NS, n = 10) for 60 days in experiment 1. Memory of mice was detected by dark avoidance behavior in the 53" d and the 60"' d. Animals were sacrificed after the memory test; brain tissues were processed for Fos-ir and TH-ir by immunohistochemistry. Mice were divided into four groups according results of expri-ment 1, they received i.p. injection of apomorphine (0.1 mg/kg, APO 0.1, 0.5 mg/kg, APO 0.5, and 2.0 mg/kg, APO2.0 respectively, n = 10).</p><p><b>RESULTS</b>Memory was inhibited in mice injected scopolamine 3.0 mg/kg. Latency was significantly less than in NS group, only 1/ 4 that of NS group (P > 0.05). The number of mistake of SCOP 3.0 group increased about four times than that of NS group (P > 0.05). But there was no difference of latency and number of mistake between SCOP 0.3 and NS group in expriment 1. Scopolamine-induced memory deficit was associated with decreased cellular activation, indicated by Fos immunoreactive (ir) staining, in NAcc CA1 and CA3 (P < 0.05), and also associated with decreases in the number of cells labeled for tyrosine hydroxylase (TH-ir), the rate limiting enzyme for dopamine conversion (P < 0.01) and the number of cells co-labeled for TH-ir/Fos-ir (P <0.01) in the ventral tegmental area(VTA), apomorphine lessened scopolamine-induced memory deficit in experiment 2. The number of cells co-labeled for TH-ir/Fos-ir (P <, 0.05) was increased in VTA after apomorphine treatment.</p><p><b>CONCLUSION</b>Apomorphine lessened scopolamine-induced memory deficit in mice by increasing DA activities in VTA.</p>