血清浓度对脂肪成体干细胞向软骨细胞分化的影响

Effect of concentrations of serum on differentiation of adipose-derived adult stem cells into chondrocytes

[目的]比较不同浓度的血清,在转化生长因子-β1(TGF-β1)存在的条件下,对脂肪成体干细胞(ad-ipose-derived adult stem cells,ADASCs)向软骨细胞分化的影响,探讨较为合适的血清浓度。[方法]取成年新西兰兔颈部脂肪组织,剪碎后通过Ⅰ型胶原酶消化,得到脂肪成体干细胞;稳定传代后,分别用含1%FBS和10%FBS的软骨诱导液干预ADASCs 2周,采用MTT检测方法对细胞增殖活性进行比较,应用甲苯胺蓝染色及Ⅱ型胶原免疫组化染色进行软骨细胞鉴定,利用Leica病理图像软件分析两组间Ⅱ型胶原免疫组化染色后的灰度,比较两组细胞分化的效果。[结果]MTT检测显示10%FBS组细胞增殖活性高于1%FBS组(P<0.05),两组细胞的甲苯胺蓝染色及Ⅱ型胶原免疫组化染色均为阳性,并以10%FBS组更为显著;灰度分析提示10%FBS组Ⅱ型胶原表达量高于1%FBS组(P<0.05)。[结论]体外研究表明,含10%FBS的软骨诱导液更有利于脂肪成体干细胞向软骨细胞方向增殖和分化,这将为作者采用这种新型的干细胞修复软骨缺损做好前期的准备。

[ Objective] To study the differentiation of adipose-derived adult stem cells （ADASCs） into chondrocytes induced by TGF-β1 in the presence of different concentrations of serum in vitro. [ Method ] Adipose tissue-derived adult stem cells were isolated from rabbit＇s subcutaneous adipose tissue using enzymatic digestion. The adipose tissue was minced and digested with collagenase type Ⅰ, and the released cells were collected by density centrifugation and then placed in culture. After being passaged three times, ADASCs were induced to differentiate into chondrocytes by chondrogenic culture media containing 1% or 10% of FBS for 2 weeks. MTT, alkaline phosphatase toluidine blue staining and type Ⅱ collagen immunohistochemistry staining were then tested in both groups. Gray value was analyzed by Leica patho-image analysis system to observe if there was any difference in differentiative status between the two groups. [ Result] MTT showed that the cells of 10% FBS group had higher proliferation activity than those of 1% FBS group （P 〈 0. 05）. Alkaline phosphatase toluidine blue staining and type II collagen immunohistochemistry staining were showed positive in both groups, but they were more significant in 10% FBS group. The analysis of gray value showed that 10% FBS group expressed more type Ⅱ collagen in the cells＇ matrix （P 〈 0. 05 ）. [ Conclusion] In vitro, the chondrogenic culture media which contained 10% FBS is more useful in promoting the proliferation and differentiation of ADASCs into chondrocytes, which will contribute to the development of new kind of stem cells for repair of cartilage defects.