不同方法诱导兔脂肪干细胞向软骨细胞分化的对比研究

Comparative study on chondrogenic differentiation of adipose-derived stem cells induced by different methods

目的 分别用细胞共培法和诱导液培养法诱导脂肪干细胞（adipose-derived stemcells,ADSCs）向软骨细胞分化,比较两种方法的诱导效果.方法 分离培养新西兰大白兔ADSCs和软骨细胞,分别培养至第2代用于实验.根据实验目的将实验分为三组：（1）对照组：将ADSCs接种于6孔板,普通高糖培养液培养;（2）细胞共培组：将软骨细胞和ADSCs分别接种于Transwell6孔板的上层和下层,普通高糖培养液共同培养;（3）诱导液培养组：将ADSCs接种于6孔板,用软骨诱导液进行培养.倒置显微镜观察各组ADSCs的形态变化,14d后行甲苯胺蓝染色、Ⅱ型胶原免疫组化,实时荧光定量PCR测定软骨细胞相关基因（蛋白聚糖、Ⅱ型胶原、SOX9）的表达情况.结果 细胞共培组和诱导液培养组ADSCs逐渐变为软骨样细胞形态,甲苯胺蓝、Ⅱ型胶原免疫组化均为阳性,其中诱导液培养组染色更深,对照组为阴性.荧光定量PCR结果显示Ⅱ型胶原、蛋白聚糖、SOX9的基因转录水平细胞共培组（1.427±0.066,2.128±0.076,1.082±0.081）和诱导液培养组（3.438±0.108,4.297±0.147,2.929±0.090）明显高于对照组（0.040±0.021,0.128±0.038,0.098±0.022） （P ＜0.05）,且诱导液培养组高于细胞共培组（P＜0.05）.结论 细胞共培法和诱导液培养法均可诱导ADSCs向软骨细胞定向分化,诱导液培养法诱导效果更好.

Objective To compare the effect of coculture with chondrocytes or culture with induction medium on chondrogenic differentiation of adipose-derived stem cells （ADSCs）.Methods ADSCs and chondrocytes were isolated from rabbits and cells of passage 2 were collected.The study contained 3 groups：ADSCs were cultured with common high glucose culture medium in a 6-well plate in control group,chondrocytes and ADSCs were separately seeded on the upper and lower layers of Transwell six-well plate and cultured with common high glucose culture medium in coculture group,ADSCs were cultured with induction medium in a 6-well plate in induction medium group.ADSCs were observed under inverted contrast phase microscope for morphological changes and harvested 14 days after incubationfor toluidine blue staining and collagen type Ⅱ immunohistochemistry staining.Quantitative real-time PCR was used to detect and quantify chondrocyte-specific genes （collagen type Ⅱ,aggrecan and SOX9）.Results ADSCs in coculture group and induction medium group showed a chondrocytes-like morphology.Toluidine blue staining and immunohistochemistry staining were positive in coculture group and induction medium group with relatively deeper staining in induction medium group,while the results were negative in control group.Quantitative real-time PCR indicated that the transcriptional levels of collagen type Ⅱ,aggrecan and SOX9 in coculture group （1.427 ± 0.066,2.128 ± 0.076 and 1.082 ± 0.081） were lower than those in induction medium group （3.438 ± O.108,4.297 ± 0.147 and 2.929 ± 0.090,P 〈 0.05）,but all were higher than those in control group （0.040 ± 0.021,0.128 ± 0.038 and 0.098± 0.022,P 〈0.05）.Conclusion ADSCs can differentiate into cartilage-like cells after co-culturing with chondrocytes or culturing with induction medium,but induction medium has better differential effect.