ObjectiveTo study the effect of Triton X-100 promoting liposome-mediated bone morphogenetic protein 2 (BMP-2) gene transfection of rat bone marrow mesenchymal stem cells (BMSCs). MethodsBMSCs were separated and cultured from the femur and tibia of healthy Wistar rats (8-week-old, male). The 3rd passage BMSCs identified by detecting the surface antigen were used to transfect. The optimum concentration of Triton X-100 for liposome mediated gene transfection was determined with ELISA meter by the way of MTT. In optimum concentration of Triton X-100, liposome mediated BMP-2 gene was transfected to BMSCs. The experiment was divided into 3 groups according to types of trasfection agents:BMSCs were transfected with Triton X-100+liposome+BMP-2 (experimental group), with liposome+ BMP-2 (conventional transfection group), and untransfected BMSCs served as blank control group. After 48 hours of transfecting, the green fluorescent protein (GFP) in cells was detected through inverted fluorescence microscope. After 72 hours of transfection, real-time fluorescence quantitative PCR was applied to measure the mRNA expression of BMP-2. Results0.01% Triton X-100 was determined to be the optimum concentration for not only making the BMSCs maintain vitality, but also achieving a certain effect on BMSCs. After trasfecting for 48 hours, GFP was observed through inverted fluorescence microscope in the experimental group and conventional transfection group, but was not observed in the blank control group. After trasfecting for 72 hours, the relative BMP-2 mRNA expression level was 5.94±0.12 in the experimental group, and was 4.99±0.08 in the conventional transfection group, showing significant difference (t=360.28, P=0.02). The transfection efficiency was increased by 19% in the experimental group. Conclusion0.010%Triton X-100 can promote the liposome mediated BMP-2 gene transfection of rat BMSCs, and can improve the transfection efficiency.
Abstract: Objective To evaluate a new type of treatment that reduces calcification of glutaraldehydetreated bovine jugular venous conduit (BJVC). Methods Fresh bovine jugular veins were treated with glutaraldehyde, followed by Triton X-100 and epoxy chloropropane (EC+Tr group). We compared the group’s appearance, histology, shrinkage temperature, tensile strength, and elongation at break with those of a fresh group, and with a group treated with glutaraldehyde only (GA group). We then implanted the EC+Tr and GA group BJVCs subcutaneously into the backs of SD rats and left them for eight weeks (n=8). The morphologic properties and inflammatory response of the test specimens were evaluated by HE staining. The tissue calcium content was determined by atomic absorption spectrophotometer. Results The shrinkage temperature, tensile strength, and elongation at break of the EC+Tr group were significantly higher than those of the fresh group (86.15±0.92 ℃ vs. 69.94±0.92 ℃,t=35.239, P=0.000; 5.31±0.14 mPa vs.3.15±0.95 mPa,t=6.362, P=0.000; 265.11%±27.80% vs. 16521%±25.06%,t=7.550, P=0.000) and of the GA group (86.15±0.92 ℃ vs. 82.73±1.28 ℃, t=6.137, P=0.000; 5.31±0.14 mPa vs. 4.52±0.56 mPa,t=3.871, P=0.002; 265.11%±27.80% vs.237.85%±17.41%,t=2.351,P=0.034). The tissue structure of the subcutaneously implanted EC+Tr veins remained intact;degradation was slight and they contained few inflammatory cells. The calcium content of the EC+Tr group was lower than that of the GA group (51.22±2.69 μg/mg vs. 73.24±3.82 μg/mg, t=11.545，P=0.000). Conclusion Treatment with Triton X-100 and epoxy chloropropane modification with glutaraldehydetreated bovine jugular venous conduit was an effective way to prepare BJVC that avoided calcification.