人腹壁脂肪组织的体外灌注培养及其诱导成肌的实验研究  

Experimental study on the in vitro perfusion culture of human abdominal 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,Belting 100083,China)

机构地区：[1]北京大学第三医院成形外科,100083

出　　处：《中华整形外科杂志》2018年第7期565-569,共5页Chinese Journal of Plastic Surgery

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

摘　　要：目的观察体外灌注培养人脂肪组织块及其诱导成肌的可行性。方法采用体外灌注培养法体外培养人腹壁来源的脂肪组织；1、3、5、7周后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；用免疫组化和Westernblot法检测生肌相关分子Myf-5、myoDl，以及肌肉特异性蛋白Desmin的表达情况。结果经生肌诱导液培养的脂肪组织在7周时仍以脂肪组织的外观为主；未加诱导液的基础培养基中培养7周时血管蒂已掉落，难以继续培养。FAD／PI染色显示诱导液中培养的组织在7周时仍有较好的活力，但基础培养液中的组织在5周时活力已明显下降。组织学观察，肌肉对照组织阳性表达Myf-5、myoDl和Desmin；脂肪对照组织中的部分间质及血管内皮细胞阳性表达Myf-5，不表达myoDl，在个别血管的平滑肌细胞阳性表达Desmin；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，阳性表达Myf一5、myoDl和Desmin；基础培养液培养的脂肪组织未见类肌组织形成，目的分子的表达情况与脂肪对照组织相似。Westernblot法显示肌肉组织中Myf-5和Desmin的表达量均显著高于其他各组（P〈0．05）；Myf-5在同一时间点诱导组中的表达量略高于无诱导组，但差异无统计学意义；Desmin在诱导1周时的表达量显著高于肌肉对照以外的其他各组（P〈0．05），诱导3、5周时的表达量均略高于同一时间点无诱导组的表达量，但差异无统计学意义；myoDl在目标分子量的位置各组中均未检测到阳性条带。结论研究初步证实了体外采用灌注式生物反应器培养的人源脂肪组织可以存活长达7周，并且经成肌诱导液培养局部可以转化成类肌样组织。Objective To investigate the feasibility of in vitro perfusion culture of human adipose tissue and its induction into muscle tissue. Methods Human abdominal adipose tissue were cultured in vitro by perfusion culture. After 1, 3, 5 or 7 weeks, FAD/PI staining was used to detect the tissue vitality. Histological staining was used to observe the changes of its histomorphology. Protein expressions of myogenic molecules Myf-5 and myoD1 as well as muscle specific protein Desmin were measured by immunohistochemistry and Western blot assay. Results The adipose tissues cultured in myogenic induction media were still in the appearance of adipose tissue at 7 weeks. While in the basal medium without inducing, vascular pedicles shed after 7 weeks and could not continue to be cultured. FAD/PI staining showed that the tissue cultured in the induction media remained viable at 7 weeks, while the viability of the tissue in the basal culture medium decreased significantly at 5 weeks. Histologically, Myf- 5, myoD1 and Desmin were all positively expressed in muscle tissues, while in adipose tissues, somemesenchymal and vascular endothelial cells expressed Myf-5 but not myoD1, and only separate vascular smooth muscle cells expressed Desmin. Interestingly, in adipose tissues cultured in myogenic induction medium, partial muscle-like tissue formed, evidenced as positive expression of Myf-5, myoD1 and Desmin. There was no muscle-like tissue formation in adipose tissue cultured in basal medium and the expression patterns were similar to that of the control group. Western blot results showed that the expression levels of Myf-5 and Desmin in muscle tissue were significantly higher than that of the other groups （P 〈 0.05 ）. The expression level of Myf-5 was slightly higher in inducing group than that in non-inducing group at the same time points, but the difference was not statistically significant. Similarly, the expression of Desmin was higher in inducing group than that in non-inducing group, yet there was statistically significant

关 键 词：组织培养 体外灌注 脂肪 诱导成肌 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]