【摘要】：目的探討構(gòu)建可注射型組織工程脂肪的可行性,為臨床軟組織缺損修復(fù)及美容填充提供簡(jiǎn)便易行的方法。方法取脂肪抽吸術(shù)獲取的脂肪組織,采用酶消化法分離培養(yǎng)獲得人脂肪來(lái)源干細(xì)胞(human adipose-derived stem cells,h ADSCs),進(jìn)行細(xì)胞形態(tài)觀察、流式細(xì)胞術(shù)及成脂誘導(dǎo)鑒定。用攜帶肝細(xì)胞生長(zhǎng)因子(hepatocyte growth factor,HGF)和綠色熒光蛋白(green fluorescent protein,GFP)的慢病毒HGF-GFP-LVs,以不同感染復(fù)數(shù)(multiplicity of infection,MOI)(分別為10、30、50、100)轉(zhuǎn)染h ADSCs,計(jì)算轉(zhuǎn)染效率以確定最適MOI。取SPF級(jí)6周齡單純T細(xì)胞缺陷BALB/c雌性裸鼠10只,將經(jīng)過(guò)最適MOI HGF-GFP-LVs轉(zhuǎn)染并成脂誘導(dǎo)的h ADSCs與纖維蛋白膠混合注入裸鼠背部右側(cè)皮下(轉(zhuǎn)染組),單純成脂誘導(dǎo)的h ADSCs與纖維蛋白膠混合注入背部左側(cè)皮下(未轉(zhuǎn)染組),單純纖維蛋白膠注入頸后正中皮下(空白對(duì)照組),每點(diǎn)注射0.4 m L。移植后12周處死裸鼠,取出移植物,行大體觀察、濕重測(cè)量、HE染色、GFP熒光標(biāo)記檢測(cè)及免疫熒光染色觀察,評(píng)價(jià)種子細(xì)胞的體內(nèi)成脂能力以及移植物血管新生情況。結(jié)果經(jīng)鑒定所培養(yǎng)細(xì)胞為h ADSCs。MOI為10和30時(shí)HGFGFP-LVs轉(zhuǎn)染率較低,MOI為50和100時(shí)轉(zhuǎn)染率較高(均80%),確定最適MOI為50。移植12周,轉(zhuǎn)染組和未轉(zhuǎn)染組均獲得外觀類似脂肪組織的新生物,濕重分別為(32.30±4.06)mg和(25.27±3.94)mg,差異有統(tǒng)計(jì)學(xué)意義(t=3.929,P=0.001);空白對(duì)照組未見(jiàn)新生物。轉(zhuǎn)染組新生脂肪細(xì)胞數(shù)為(126.93±5.36)個(gè)/視野,顯著高于未轉(zhuǎn)染組的(71.36±4.52)個(gè)/視野(t=30.700,P=0.000)。熒光顯微鏡下可見(jiàn)部分單房脂肪細(xì)胞發(fā)出網(wǎng)狀綠色熒光。免疫熒光染色示轉(zhuǎn)染組血管密度為(16.37±2.76)個(gè)/視野,顯著高于未轉(zhuǎn)染組的(9.13±1.68)個(gè)/視野(t=8.678,P=0.000)。結(jié)論以h ADSCs為種子細(xì)胞,經(jīng)慢病毒轉(zhuǎn)染HGF基因并成脂誘導(dǎo)后與纖維蛋白膠支架復(fù)合可在體內(nèi)成功構(gòu)建成熟脂肪,能促進(jìn)移植后的血管新生,從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 鹵4.06) mg and (25.27 鹵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 鹵5.36) / visual field, which was significantly higher than that in the untransfected group (71.36 鹵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 鹵2.76) / visual field, which was significantly higher than that in the non-transfected group (9.13 鹵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.
【作者單位】： 南昌大學(xué)第二附屬醫(yī)院整形美容科;
【基金】：江西省自然科學(xué)基金資助項(xiàng)目(20151BAB205034) 江西省科技支撐計(jì)劃資助項(xiàng)目(2010BSA15100) 南昌大學(xué)研究生創(chuàng)新專項(xiàng)基金(cx2016333)~~
【分類號(hào)】：R318.08

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