悬浮法与贴壁法提取脂肪组织分泌促成脂因子比较

Extraction of Adipogenic Secretory Factors:a Comparison Between Adherent Culture and Suspension Culture

目的 优化体外获取脂肪组织浸提液的提取方法,比较不同方法提取浸提液的成脂作用。方法 通过贴壁法和悬浮法培养脂肪组织获得浸提液,过滤、冷冻超离心浓缩浸提液,通过BCA试剂盒检测两种方式得到的浸提液分泌因子（secretory factors from adipose tissue explants,SFAE）蛋白总量及蛋白因子得率。等量蛋白成脂诱导第3代（P3）脂肪干细胞,诱导第7~13d,显微镜下观察及进行油红染色,鉴定成脂情况。结果 贴壁法和悬浮法4次提取浸提液中蛋白含量的差异无统计学意义。但悬浮培养法4次重复实验蛋白因子得率更为稳定,贴壁法每次蛋白因子得率数据相差很大,每克脂肪组织获得分泌蛋白的量最小时为7.3mg,最大可得到12.4mg,而悬浮得到的蛋白因子几乎都趋近于8.7mg。并且贴壁培养法与悬浮培养法相比,获取等量的SFAE需要T75培养瓶更多（10个）,且需手工铺匀,贴壁后才能加入培养基,而悬浮培养法仅需悬浮培养瓶1个,加入脂肪组织后即可进行培养。两种方法得到的浸提液都具有促成脂效果,在等量蛋白诱导下的成脂效果差异无统计学意义。结论悬浮和贴壁培养法得到的浸提液具有相同的促成脂能力,但是悬浮培养获得的浸提液更加省时省力且蛋白因子得率稳定,为后续研究和鉴定在SFAE中准确找到成脂因子奠定基础。

Objective To optimize the extraction method of secretory factors from adipose tissue explant （SFAE） in vitro. Methods SFAE were obtained through adherent culture （SFAE-A） and suspension culture （SFAE-S） and concentrated by filtration and centrifugation. The yield of SFAE was compared using BCA protein detection kit. P3 adipose-derived stem cells （ADSCs） were induced with equal amount of SFAE for 7-13 d, before the state of adipogenesis between suspension culture and adherent culture was compared by microscope observation and oil red O staining. Results The average amount of SFAE yielded from adherent culture and suspension culture did not show significant difference. While the yield of SFAE from suspension culture was consistent at 8.7 mg per gram of adipose tissue, the adherent culture generated an inconsistent result in the four repeat experiments, ranging from 7.3 mg to 12.4 mg per gram of adipose tissue. Moreover, ten more flasks and better distribution were needed for adherent culture to acquire an equal amount of SFAE in comparison with suspension culture. SFAE from both adherent and suspension culture promoted the adipogenesis of P3 adipose-derived stem cells. No differences on the adipogenic effect were found between the two extraction methods. Conclusion Secretory factors from adherent culture and suspension culture have the same adipogenesis effect. Suspension culture can save time and labor. The most important advantage of suspension culture is its stable yield of SFAE.