体外非接触性共培养对MSC向软骨细胞诱导分化的实验观察

目的：探讨骨髓基质干细胞（MSCs）与软骨细胞体外非接触性共培养成软骨的可行性。方法：分离、培养、扩传兔MSCs及软骨细胞，MSCs以1×10^5个/ml接种于共培养板内孔，第1代软骨细胞以1×10^4个/ml接种于共培养板外空板。48h后将内孔插如外孔中，补充全培养基覆盖于MSC细胞层上。以相同密度MSC单独培养为空白对照，观察共培养7d和14d流式细胞仪观察MSC的Ⅱ型胶原蛋白变化情况。结果：实验空白组在7d和14d，Ⅱ型胶原蛋白均阴性表达，符合MSC特性。非接触共培养1周后，Ⅱ型胶原蛋白阳性表达，与空白对照比较，P〈0.01，具有统计学意义；非接触共培养2周后，Ⅱ型胶原蛋白也阳性表达，与空白对照比较，P〈0.01，同样具有统计学意义；实验14d组与实验7d组比较，P〈0.01，具有非常显著性差异。结论：软骨微环境在MSCs成软骨分化及体内软骨形成中具有重要作用，软骨细胞能有效地诱导MSCs向软骨细胞分化并促进MSCs体内软骨形成。

Experimental observation of non-contact in vitro co-culture of the differentiation MSC toward chondrocytes

Objective： To investigate the feasibility of the bone marrow stromal stem cells （MSCs） and cartilage cells in vitro co-culture of non-contact differentiate cartilage.Methods：Isolation, culture, the proliferation of rabbit-MSCs and cartilage cells. MSCs of 1×10^5 cells/ml were inoculated culture plate, the first passage cartilage of 1×10^4 cells/ml were inoculated culture plate outer space plate. After 48 hours, the inter hole were inserted in the outer holes and then added media to coverage in the MSC cell layer. MSC with the same density as blank control cultured. Co-ultured for 7 days and 14 days, the changes of MSC type Ⅱ collagen protein were observed by flow cytometry.Results：The Experimental blank group in 7 and 14 days, type Ⅱ collagen protein were negative expression which consistent with characteristics of MSC. After non-contact co-culture one week, type Ⅱ collagen protein were expressed, and there was statistical signifi cance compared with the blank control（P〈0.01 ）, after two weeks, type Ⅱ collagen protein also positive, compared with the blank control（P〈0.01 ）, there was also statistical significance. Experimental group 14 days compared with group 7 days （P〈0.01）, there was statistical significance. Conclusion：The micro-environment of cartilage plays an important role at MSCs differentiation into chondrocytes and cartilage formation in vivo. MSCs can be effectively induced differentiation into chondrocytes and promoted cartilage formation by cartilage cells.