| Objective:An engineering laboratory model of C57 BL mice was established,and FGF/FGFR inhibitor(LY2874455)was used to explore the mechanism of its effect on FGF-2-induced adipogenesis in tissue engineering chamber,so as to provide laboratory basis for clinical combined application of fat and FGF2.Methods:1.Construction of engineered pedicled fat flap: the fat flap with vascular pedicle on one side of the groin of mice was trimmed and placed into the tissue engineering chamber,which was fixed and sutured for 42 days.2.Detection of FGF2 concentration of exudate in tissue engineering chamber: the model of tissue engineering chamber was established in 20 mice.The exudate samples were taken at 1 day,3 days,7 days,14 days,28 days and 56 days respectively(3 mice were randomly detected at each time point).The concentration of FGF2 was measured by ELISA kit.3.Study on the mechanism of FGF/FGFR inhibitor(LY2874455)on the regeneration of engineered fat flap: 30 mice were randomly divided into two groups:experimental group(50n M-LY2874455 group,n = 15)and control group(DMSO group,n = 15).Experimental group: the tissue engineering chamber model was established,and the 1ml syringe was used to fill the tissue engineering chamber with 50 n M FGF/FGFR inhibitor(LY2874455).In the control group,the tissue engineering chamber model was established and the DMSO of 50 n M was filled with 1ml syringe.The samples of the two groups were taken at 7 days,14 days,28 days and 56 days respectively(3 at each time point).The specimens were weighed and detected by HE,WB,immunofluorescence and immunohistochemistry.Results:The construction of tissue engineering pedicled vascular fat flap was successful.1.The concentration of FGF2 in the exudate of tissue engineering chamber showed a bell-shaped change: it increased gradually from 11.47 ±7.07pg/ml on the first day to the peak on the 14 th day(51.69 ±1.71pg/ml,),which was statistically significant compared with that on the first day(p < 0.05).Then it decreased to 10.92 ±2.94 pg/ml,on the 56 th day,which was significantly higher than that on the 14 th day(p < 0.001).2.FGF/FGFR inhibitor(LY2874455)inhibited fat regeneration in engineering chamber: the fat mass of DMSO group and 50 n M-LY2874455 group was 10.71±0.43 mg and 8.19 ±1.11 mg respectively on the 7th day after operation,and there was no significant difference between the two groups on the 14 th,28th and 56 th day after operation,p < 0.05.The results showed that there was no significant difference in fat mass between the two groups on the 14 th,28th and 56 th day after operation.With the passage of time,the fat mass of DMSO group increased gradually,from10.71 ±0.43 mg to 20.09 ±5.0mg,which was statistically significant.However,the fat mass in 50 n M-LY2874455 group was basically the same,and there was no significant increase in fat mass.3.FGF/FGFR inhibitor(LY2874455)inhibited FGFR receptor expression,FGFR phosphorylation and proliferation of adipose stem cells: fluorescence results showed that FGF/FGFR inhibitor(LY2874455)significantly decreased the phosphorylation intensity of FGFR1(Tyr653 / Tyr654)in this group,and phosphorylated FGFR1(Tyr653 / Tyr654)was strongly expressed in DMSO group on the 14 th day.In the early stage(within 14 days),we could observe the expression of CD34+ and Ki-67+ around the blood vessels in the DMSO group.A small amount of expression was found on the28 th and 56 th day,and it was significantly more than that in the 50 n M-LY2874455 group at each time point.After the addition of 50 n M-LY2874455 inhibitor,the expression of FGFR1 was decreased,and the phosphorylation intensity of FGFR1(Tyr653/Tyr654)was significantly decreased.4.FGF/FGFR inhibitor(LY2874455)inhibited the expression of ERK in engineered pedicled adipose tissue: the expression of ERK in DMSO group increased significantly,while the overall level of ERK1/2 in 50 n M-LY2874455 group had no significant change,and it was lower than that in DMSO group at different time points,which was statistically significant(p < 0.001).5.FGF/FGFR inhibitor(LY2874455)inhibited the adipogenic differentiation of adipose stem cells with pedicled vascular fat flap: In the early stage(14 days),the positive percentage of C/EBPb expression increased from 3.80 ±0.08% on the 7th day to 5.64 ±0.13%,peaked on the 14 th day,and gradually decreased to 1.10 ±0.12% in the late stage(14 days later).The percentage of positive expression in PPARγ group gradually increased with the passage of time,from 4.19 ±0.15% on the 7th day to 7.74±1.75% on the 56 th day.On the 56 th day,the expression of PPARγ in the DMSO group was significantly higher than that in the 50 n M-LY2874455 group(4.33 ±0.18%),and had statistical significance(p < 0.001).Conclusion:1.The construction of fat engineering chamber is successful and repeatable.2.FGF2 promoted the proliferation of adipose stem cells in the early stage(within14 days)and adipogenic differentiation in the late stage(14 days)after the implantation of engineered pedicled fat flap.That is,in the early stage of adipose regeneration in adipose engineering chamber(within 14 days),high concentration of FGF2 acts on FGFR receptors on adipose stem cells to activate ERK signal pathway to participate in inducing the proliferation of adipose stem cells in tissue engineering chamber.in the later stage of adipose engineering(14 days later),the concentration of FGF2 decreased gradually,and adipose regeneration was mainly adipose differentiation and spontaneous differentiation.3.FGF-2 is a necessary factor to induce fat regeneration in adipose tissue engineering laboratory. |
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