脂肪组织的体外灌注培养及其诱导成肌的研究  

In vitro perfusion culture of adipose tissue and its myogenic induction

作　　者：聂芳菲[1] 安阳[1] 李琪[1] 马建勋[1] 秦泽莲[1] Nie Fangfei;An Yang;Li Qi;Ma Jianxun;Qin Zelian(Department of Plastic Surgery, Peking University Third Hospital, Beijing 100083, China)

机构地区：[1]北京大学第三医院成形外科,100083

出　　处：《中华实验外科杂志》2019年第2期238-241,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金 (81301641、81501681);教育部博士点基金 (20130001120102).

摘　　要：目的 探讨体外灌注培养脂肪组织并诱导成肌的可能性及其机制。方法 采用生肌诱导液体外灌注培养带血管蒂的大鼠腹股沟脂肪筋膜瓣;3周后二乙酸荧光素/碘化丙锭(FAD/PI)染色法观察其组织活力;组织学染色观察组织形态学改变;免疫荧光染色观察结蛋白(Desmin)和成肌分化因子1(MyoD1)的表达;用Western blot法检测Desmin以及生肌因子5(Myf-5)和MyoD1的表达情况。 结果 FAD/PI染色显示诱导液中培养的脂肪组织在3周时仍有较好的活力。组织学观察,经诱导培养基诱导培养的脂肪组织局部有类肌样组织形成;免疫荧光染色显示有Desmin和MyoD1阳性表达。蛋白定量结果显示,与无诱导对照组(0.028 2±0.027 1)比较,诱导组中肌肉特异性蛋白Desmin(0.114 5±0.153 1)的表达略有增加,差异无统计学意义(t=0.622,P>0.05);成肌分化因子MyoD1(诱导组和无诱导组分别是0.255 2±0.103 2和0.023 9±0.024 6)的表达有增加,差异有统计学意义(t=4.877,P<0.01);Myf-5的表达量未减少(诱导组和无诱导组分别是诱导前的1.00倍和0.13倍),差异有统计学意义(t=5.215,P<0.01)。结论 本研究初步证实了采用灌注式生物反应器培养的脂肪组织可以在体外存活至少3周,并且经成肌诱导液培养局部可以转化成类肌样组织,肌肉特异性转录因子Myf-5和MyoD1可能都参与了脂肪组织的体外成肌过程。Objective To investigate the myogenic possibility and mechanism of adipose tissue block induced by in vitro perfusion.Methods Myogenic inducing liquid was perfused through the vascular pedicle to cultrue the rat inguinal fat fascia flap. After 3 weeks, the tissue viability was detected by the fluorescein diacetate and propidium iodide (FAD/PI) staining. The changes of histomorphology was observed by histological staining and immunofluorescence staining was used to observe the expression of Desmin and myogenic differentiation factor 1(MyoD1). The expression levels of Desmin, myogenic factor 5(Myf-5)and MyoD1 were detected by Western blotting.Results FAD/PI staining showed that the adipose tissue cultured in the induction fluid was still viable at 3 weeks. Histological observation showed that the adipose tissues induced by the inducing medium had formed some muscle-like tissues locally. Immunofluorescence staining showed positive expression of Desmin and MyoD1. Protein quantitative results showed that compared with non-induced group (0.028 2±0.027 1), the expression of muscle specific protein Desmin in induced group (0.114 5±0.153 1) increased slightly, yet the difference had no statistical significance (t=0.622, P>0.05). The expression level of myogenic differentiation factor MyoD1 was higher in induced group (0.255 2±0.103 2) than non-induced group (0.023 9±0.024 6) and the difference was statistically significant (t=4.877, P<0.01). The reduction of Myf-5 in non-induced group was also inhibit in the induced group (the expression level of Myf-5 were 1 time in induced group and 0.13 times in non-induced group after the induction compared to the control fat tissue group), the difference had statistical significance (t=5.215, P<0.01).Conclusion The adipose tissues cultured in the perfusion bioreactor can survive at least 3 weeks in vitro, and they can be partially transformed into muscle-like tissues. Myogenic factor Myf-5 and MyoD1 are both involved in the myogenic process of adipose tissue in vitro.

关 键 词：脂肪 诱导成肌 组织培养 体外灌注 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]