Résumé
Objective To investigate the effects and the mechanism of thrombospondin 4 (Thbs4) gene-edited bone marrow mesenchymal stem cells (BMSCs) transplantation on vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in diabetic rats with hind limb ischemia. Methods Thirty Sprague-Dawley rats were randomly divided into the model group, BMSCs treatment group and Thbs4-BMSCs treatment group on average. After constructing the typeⅡdiabetic rat model with hind limb ischemia, 100μl normal saline, BMSCs suspension and Thbs4-BMSCs suspension (cell number: 2×106) were locally injected into the ischemic injury area of rats for the model group, BMSCs group and Thbs4-BMSCs group, respectively. The rats were sacrificed on the 14th day after stem cell transplantation, and the muscle tissues near the ischemic area were collected. The relative expression of VEGF and p-Smad2/3 protein was detected by Western Blot. The Ang-1 protein expression was detected by immunofluorescence staining. The levels of related genes were detected by qRT-PCR, and the von Willebrand Factor (vWF) protein expression was detected by immunohistochemistry staining. Results The relative expression levels of VEGF, Ang-1 and vWF protein in the Thbs4-BMSCs group were significantly higher than those in the model group and BMSCs group (VEGF protein:P<0.01 and P<0.05). The mRNA expression of VEGF and Ang-1 were significantly up-regulated, the differences were statistically significant(VEGF mRNA:all P<0.01;Ang-1:P<0.01 and P<0.05). The expression of p-Smad2/3 protein in the Thbs4-BMSCs group was significantly higher than that in the model group and the BMSCs treatment group (all P<0.01). The expression of p-Smad2/3 protein was significantly decreased after the addition of p-Smad2/3 pathway inhibitor, the differences were statistically significant (P<0.05). Conclusions Thbs4-BMSCs transplantation can effectively promote angiogenesis in diabetic rats with hind limb ischemia, and the effect of angiogenesis may be related to the activation of Smad2/3 signaling pathway.
Résumé
Objective: To establish capillary zone electrophoresis method for determination of sildenafil citrate (Viagra) content in its troche. Methods： Using tetrandrine as internal standard(IS), the electrophoretic separation was achieved with 25 mmol/L borate (pH=7.89) running buffer. And a voltage of 14 kV was applied to the 40 cm×75 μm(i.d) capillary. The analytes were introduced into capillary by siphon (1 s) and determined with on-column monitoring at 214 nm. Results：The determination could be completed within 4 min and the minimum concentration of detection was 5 μg/ml.The analytical results of sildenafil citrate samples demonstrated a good linear relationship within the range of 24-480 μg/ml.The relative standard deviations (RSD) of intra-day was less than 1.58% and that of inter-day was less than 2.46%.The present recoveries were between 95%-105％. Conclusion：The CZE method is accurate, simple, rapid and reliable, so it can be applied to the determination of sildenafil citrate content.
Résumé
Objective To determine diosgenin content in Jingangteng soft capsule by RP-HPLC.Methods A RP-HPLC was performed on a Shim-Pak VP-ODS C18column(5?m,4.6 mm? 250 mm),the mobile phase consisting of acetonitri le-H2O(87∶13) and the detected wavelength at 209 nm.Results A good line arity was obtained in the range 0.0595～ 14.875 ? g(r=0.99999) for diosgenin. The average recovery and RSD were 99.4 % and 2.0 % respectively(n=5).Concl usion The method is simple,rapid,stable and reliable,and can be used to determine diosgenin content in Jingangteng soft capsule.