脂肪干細胞的分離提取、鑒定及與脂肪顆粒的混合移植研究

袁志堅+何文涓（等）

[摘要] 目的 分離提取脂肪干細胞（ADSCs），對其進行成脂、成骨的鑒定，并與脂肪顆粒混合移植，觀察移植后的脂肪組織的存活情況。 方法 取大鼠腹股溝脂肪，磷酸鹽緩沖液清洗，0.1%Ⅰ型膠原酶37℃消化1 h，離心，取沉淀，用含10%血清的DMEM培養基進行培養、傳代，取第3代ADSCs用于成脂、成骨鑒定；另取第3代ADSCs用于移植，分為兩組：ADSCs（密度為5×107/mL）0.9 mL+脂肪顆粒0.6 mL組、脂肪顆粒1.5 mL組，分別移植于大鼠背部兩側，共移植24只大鼠，分別于2周、1、3個月時脫頸椎處死8只大鼠，再取出背部脂肪組織。 結果 大鼠ADSCs成脂、成骨誘導后，經油紅O、茜素紅染色呈陽性，移植2周、1個月時，兩組取出的脂肪組織的體積變化不大；但移植3個月后，取背部脂肪組織，稱重：脂肪顆粒1.5 mL組平均重量為（0.4551±0.0024）g，而ADSCs（密度為5×107/mL）0.9 mL+脂肪顆粒0.6 mL組的平均重量為（0.7798±0.0033）g，兩組脂肪組織的重量差異有統計學意義（P < 0.05）。 結論 原代分離、培養的ADSCs生長狀況良好，具有向成脂、成骨分化的能力，與脂肪顆粒混合移植后組織的存活率高，并能夠改善脂肪顆粒單獨移植時的液化吸收情況。

[關鍵詞] 脂肪干細胞；成脂誘導；成骨誘導；移植

[中圖分類號] R329 [文獻標識碼] A [文章編號] 1673-7210（2014）02（a）-0009-05

Isolation， culture and transplantation of rat adipose-derived stem cells

YUAN Zhijian1 HE Wenjuan1 ZHOU Hong1 JIANG Meiling2

1.Wuxi Higher Health Vocational School， Jiangsu Province， Wuxi 214028， China； 2.China Pharmaceutical University， Jiangsu Province， Nanjing 210009， China

[Abstract] Objective To isolate and culture the rat adipose-derived stem cells （ADSCs） and to identificate its multipotent differentiation ability and to observe the survival of transplantation of ADSCs with adipose compared with adipose in the back of the rats. Methods Adipose tissue were obtained from inguinal region and washed in PBS， then digested with 0.1% collagenase type Ⅰ at 37℃ for 1 h followed by centrifugation and ADSCs pellet was resuspended in DMEM with 10% fetal bovine serum （FBS） and seeded in flasks， cells at passages 3 were used for adipogenic differentiation and osteogenic differentiation， both sides of twenty-four rats were received subcutaneously 5×107/mL ADSCs resuspended in 0.9 mL of DMEM medium+0.6 mL of adipose and 1.5 mL of adipose， took out the adipose tissue of eight rats after two weeks， one month and three months respectively. Results ADSCs stained positively for oil red-O and alizarin red， two weeks and one month later， the weight of adipose had no difference with each other， however， after three months， the average weight of 1.5 mL of adipose group and the 5×107/mL ADSCs resuspended in 0.9 mL of DMEM medium+0.6 mL of adipose group were （0.4551±0.0024） g and （0.7798±0.0033） g respectively， thus， there were significant differences between the two groups （P < 0.05）. Conclusion ADSCs is in good condition and has adipogenic and osteogenic differentiation ability， ADSCs with adipose can improve the survival and the liquefied of the adipose after transplantation when compared with adipose alone.