TGF-β1，bFGF，IGF联合诱导兔脂肪基质干细胞向软骨细胞分化的实验研究

Experimental study on chondrogenic differentiation of rabbit adipose-derived stem cells treated with TGF-β1,bFGF and IGF

[目的]分离培养兔脂肪基质干细胞(adipose-derived stem cells,ADSCs),观察在TGF-β1,bFGF,IGF联合诱导下其生物学特性及向软骨细胞定向分化的能力。[方法]取4个月龄新西兰大白兔颈背部脂肪组织,分离、培养ADSCs。选取第3代,分为两组,实验组用软骨诱导液(含TGF-β1、bFGF、IGF)对细胞进行培养,对照组用普通高糖培养液培养,倒置显微镜观察两组细胞形态,MTT测定其增殖情况;14 d后行组织学观察(包括甲苯胺蓝染色、Ⅱ型胶原免疫组化),并实时荧光定量PCR检测两组细胞Ⅱ型胶原、蛋白聚糖、SOX 9基因的表达情况。[结果]实验组细胞逐渐变为软骨样细胞形态,增殖速度明显加快,14 d后实验组甲苯胺蓝染色、Ⅱ型胶原免疫组化阳性,对照组为阴性,荧光定量PCR结果显示实验组Ⅱ型胶原、蛋白聚糖、SOX 9的基因转录水平明显高于对照组。[结论]在TGF-β1,bFGF,IGF联合诱导条件下ADSCs增殖迅速,可高表达Ⅱ型胶原、蛋白聚糖、SOX 9,向软骨细胞定向分化。

[ Objective] Adipose -derived stem cells （ADSCs） were isolated and cuhured with TGF-β1, bFGF, IGF to observe their biological characteristics and investigated the chondrogenic ability. [ Methods ] Adipose tissue was resected from the back of neck of the 4 - month - old New Zealand white rabbits, and then ADSCs were isolated and cultured. ADSCs at pas- sage 3 were used in experiment which contained two groups. ADSCs were treated with chondrogenic medium （supplemented with 10 ng/ml TGF-β1, 10 ng/ml bFGF, 100 ng/ml IGF） and common high glucose culture medium （control group）, and har- vested after 14 days. The morphology changes of the induced ADSCs were observed by inverted contrast phase microscope and their growth state were detected by MTr. The GAG and Col II synthesized by the induced ADSCs were detected with toluidine blue staining and immunohistochemistry staining. The mRNA quantities of Col II, aggrecan and SOX 9 expressed by the induced ADSCs were detected with RT - PCR. [ Results ] ADSCs in the experimental group gradually became chondrocytes in morpholo- gy and proliferated rapidly. Forteen days later, toluidine blue staining and immunohistochemistry staining were positive in the ex- perimental group, while they were negative in the control group. The result of fluorescence quantitative PCR indicated that the transcriptional levels of collagen II , aggrecan and SOX 9 in the experimental group were significantly higher than those in the control group. [ Conclusion] ADSCs treated with certain concentration of TGF - -β1, bFGF and IGF proliferate rapidly, have higher expression of Col II, aggrecan and SOX 9, and can differentiate into chondrocytes.