骨髓间充质干细胞外泌体通过Notch信号通路促进移植脂肪血管化的实验研究

Exosomes derived from bone marrow mesenchymal stem cells promote angiogenesis of fat grafting through Notch signaling pathway

目的探讨骨髓间充质干细胞外泌体(BMSC-Exo)通过调控Notch信号通路促进移植脂肪血管化的机制。方法获取1例2020年8月在空军军医大学第一附属医院整形外科行腹部脂肪抽吸术的30岁健康女性的废弃脂肪,处理后将脂肪颗粒分别移植于12只Balb/c裸鼠背部皮下左、右两侧,每侧注射0.5 ml,建立脂肪移植模型。左侧设为外泌体组,每周以50μg的BMSC-Exo溶于100μl PBS溶液中,移植后即刻在脂肪周围多点局部注射,然后每周注射1次,连续4次;右侧设为对照组,于同一时间注射同等剂量的PBS溶液。观测项目:(1)分别在术后4、8周获取2组脂肪移植物组织,用电子天平称其湿重并计算保留率。(2)术后8周,通过HE染色、免疫荧光检测脂肪移植物中脂滴包被蛋白A(Perilipin A)阳性细胞,观察2组脂肪细胞的结构和完整性;(3)术后8周,免疫组织化学法检测脂肪移植物中CD31阳性细胞、计算微血管密度,检测Notch1、Notch4阳性细胞、计算阳性细胞面积比例;(4)术后8周,实时荧光定量PCR检测脂肪移植物中Notch1、Notch4、VEGF mRNA相对表达量。应用Graphad Prism 8.0分析数据,组间比较采用t检验,P<0.05表示差异有统计学意义。结果(1)术后4、8周,对照组脂肪湿重保留率分别为(41.15±9.68)%和(34.30±6.45)%,外泌体组分别为(61.77±10.98)%和(55.93±5.89)%,均高于对照组(P<0.05)。(2)术后8周,外泌体组较对照组中脂肪细胞结构完整性更好,具有较多的新生毛细血管;外泌体组中有较多的完整Perilipin A阳性脂肪细胞,而对照组中则较少。(3)对照组微血管密度为(4.67±0.57)个,显著小于外泌体组的(13.00±2.00)个(P<0.05);对照组每视野下Notch1和Notch4阳性细胞面积比例均低于外泌体组[(0.53±0.28)%vs.(1.67±0.11)%,(0.11±0.06)%vs.(1.20±0.24)%,P<0.05];(4)外泌体组Notch1、Notch4、VEGF mRNA相对表达量分别为1.53±0.20、1.50±0.26、2.56±0.55,均比对照组(1.00+0.00)明显上调(P<0.05)。结论BMSC-Exo可能通过上调VEGF mRNA的表达水平激活Notch信号通路,从而促进移植脂肪血管新生,提高移植脂肪保留率。

Objective The study aimed to investigate the effect of exosomes derived from bone marrow mesenchymal stem cells(BMSC-Exo)in promoting angiogenesis of fat grafts by regulating Notch signaling pathway.Methods Fat from the abdomen of a 30-year-old healthy female who received liposuction in the Department of Plastic Surgery of the First Affiliated Hospital of Air Force Military Medical University in August 2020 was collected.Fat transplantation models were established using 12 BALB/c nude mice,with 0.5 ml of processed fat particles injected into the left and right sides of the backs.100μl BMSC-Exo(50μg)was injected into the left side of the back of nude mice once per week for 4 weeks after fat grafting,which was the exosome group.The right side of the back was injected with 100μl of phosphate-buffered saline at the same time points for 4 weeks after fat grafting,which was the control group.The following items were observed:(1)Fat grafts were collected and weighed at 4 weeks and 8 weeks after transplantation,the retention rate was calculated.(2)HE staining and immunofluorescence staining of Perilipin A was used to visualize the structure and integrity of adipocytes at 8 weeks after transplantation.(3)At 8 weeks after transplantation,immunohistochemical staining was used to detect CD31 positive cells,calculate micro vessel density,detect Notch1 and Notch4 positive cells and calculate the proportion of positive area.(4)At 8 weeks after transplantation,the relative expressions levels of Notch1,Notch4 and VEGF mRNA in fat grafts were detected by real-time fluorescence quantitative PCR.The data were analyzed by Graphpad Prism 8.0.T-test was used for comparison between groups.P<0.05 indicated the difference was statistically significant.Results(1)At 4 and 8 weeks after transplantation,the retention rates of adipose tissue wet weight in the control group were(41.15±9.68)%and(34.30±6.45)%respectively.The results in the exosome group were(61.77±10.98)%and(55.93±5.89)%,which were higher than those in the control group(P<0.05).(2)At 8 weeks after transplantation,the structural integrity of adipocytes in the exosome group was better than the control group,with more neovascularization.There were more intact Perilipin A positive adipocytes in the exosome group and less in the control group.(3)At 8 weeks after transplantation,the density of micro vessels in the control group was 4.67±0.57,which was significantly lower than that in the exosome group(13.00±2.00,P<0.05).The proportion of Notch1 and Notch4 positive area per visual field in the control group was lower than that in the exosome group[(0.53±0.28)%vs.(1.67±0.11)%,(0.11±0.06)%vs.(1.20±0.24)%,P<0.05].(4)The mRNA expression levels of Notch1,Notch4 and VEGF were 1.53±0.20,1.50±0.26 and 2.56±0.55 in the exosome group,which were significantly higher than those in the control group(1.00+0.00)(P<0.05).Conclusions BMSC-Exo may promote angiogenesis and improve retention rate of transplanted fat grafts through the elevation of VEGF expression and the activation of Notch signaling pathway.