携带肝细胞生长因子基因慢病毒转染人脂肪来源干细胞复合纤维蛋白胶支架构建可注射型组织工程脂肪的实验研究

发布时间：2018-10-18 08:11

【摘要】：目的探讨构建可注射型组织工程脂肪的可行性,为临床软组织缺损修复及美容填充提供简便易行的方法。方法取脂肪抽吸术获取的脂肪组织,采用酶消化法分离培养获得人脂肪来源干细胞(human adipose-derived stem cells,h ADSCs),进行细胞形态观察、流式细胞术及成脂诱导鉴定。用携带肝细胞生长因子(hepatocyte growth factor,HGF)和绿色荧光蛋白(green fluorescent protein,GFP)的慢病毒HGF-GFP-LVs,以不同感染复数(multiplicity of infection,MOI)(分别为10、30、50、100)转染h ADSCs,计算转染效率以确定最适MOI。取SPF级6周龄单纯T细胞缺陷BALB/c雌性裸鼠10只,将经过最适MOI HGF-GFP-LVs转染并成脂诱导的h ADSCs与纤维蛋白胶混合注入裸鼠背部右侧皮下(转染组),单纯成脂诱导的h ADSCs与纤维蛋白胶混合注入背部左侧皮下(未转染组),单纯纤维蛋白胶注入颈后正中皮下(空白对照组),每点注射0.4 m L。移植后12周处死裸鼠,取出移植物,行大体观察、湿重测量、HE染色、GFP荧光标记检测及免疫荧光染色观察,评价种子细胞的体内成脂能力以及移植物血管新生情况。结果经鉴定所培养细胞为h ADSCs。MOI为10和30时HGFGFP-LVs转染率较低,MOI为50和100时转染率较高(均80%),确定最适MOI为50。移植12周,转染组和未转染组均获得外观类似脂肪组织的新生物,湿重分别为(32.30±4.06)mg和(25.27±3.94)mg,差异有统计学意义(t=3.929,P=0.001);空白对照组未见新生物。转染组新生脂肪细胞数为(126.93±5.36)个/视野,显著高于未转染组的(71.36±4.52)个/视野(t=30.700,P=0.000)。荧光显微镜下可见部分单房脂肪细胞发出网状绿色荧光。免疫荧光染色示转染组血管密度为(16.37±2.76)个/视野,显著高于未转染组的(9.13±1.68)个/视野(t=8.678,P=0.000)。结论以h ADSCs为种子细胞,经慢病毒转染HGF基因并成脂诱导后与纤维蛋白胶支架复合可在体内成功构建成熟脂肪,能促进移植后的血管新生,从而提高移植物的存活率。
[Abstract]:Objective to explore the feasibility of constructing injectable tissue engineered fat and to provide a simple and convenient method for clinical soft tissue defect repair and cosmetic filling. Methods Human adipose-derived stem cells (human adipose-derived stem cells,h ADSCs),) were isolated from adipose tissue by liposuction and cultured by enzyme digestion for cell morphology observation, flow cytometry and lipogenesis identification. The transfection efficiency of lentivirus HGF-GFP-LVs, carrying hepatocyte growth factor (hepatocyte growth factor,HGF) and green fluorescent protein (green fluorescent protein,GFP) was calculated by calculating the transfection efficiency of lentivirus HGF-GFP-LVs, with different infective plural (multiplicity of infection,MOI (10 ~ 30 ~ 50 ~ 100) in order to determine the optimal MOI.. Ten SPF grade 6-week-old BALB/c female nude mice with simple T cell deficiency were selected. The optimal MOI HGF-GFP-LVs transfection and lipogenic induction of h ADSCs and fibrin glue were injected into the right side of the back of nude mice (transfection group), and the pure adipogenic h ADSCs and fibrin glue were injected into the left subcutaneous of the back (untransfected group). ), the fibrin glue was injected into the middle subcutaneous of the neck (blank control group), and 0.4 mL per point. Twelve weeks after transplantation, nude mice were killed and grafts were taken out. The grafts were observed by gross observation, wet weight measurement, HE staining, GFP fluorescence labeling and immunofluorescence staining to evaluate the adipogenic ability of seed cells and the angiogenesis of the grafts. Results the transfection efficiency of HGFGFP-LVs was lower when h ADSCs.MOI was 10 and 30, and when MOI was 50 and 100, the transfection efficiency was higher (80%), and the optimal MOI was 50%. After 12 weeks of transplantation, both the transfected group and the untransfected group obtained new organisms with a similar appearance to adipose tissue, the wet weight of which was (32.30 卤4.06) mg and (25.27 卤3.94) mg, respectively (t = 3.929, P0. 001), but no new organisms were found in the blank control group. The number of new adipocytes in the transfected group was (126.93 卤5.36) / visual field, which was significantly higher than that in the untransfected group (71.36 卤4.52) / visual field (t 30.700). Under the fluorescence microscope, some monocytic adipocytes emit reticular green fluorescence. The blood vessel density in the transfected group was (16.37 卤2.76) / visual field, which was significantly higher than that in the non-transfected group (9.13 卤1.68) / visual field (t = 8.678). Conclusion using h ADSCs as seed cells, lentivirus transfection of HGF gene and lipogenic induction combined with fibrin gel scaffold can successfully construct mature fat in vivo, promote angiogenesis after transplantation, and improve the survival rate of grafts.
【作者单位】：南昌大学第二附属医院整形美容科;
【基金】：江西省自然科学基金资助项目(20151BAB205034) 江西省科技支撑计划资助项目(2010BSA15100) 南昌大学研究生创新专项基金(cx2016333)~~
【分类号】：R318.08

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