微重力环境下肢体废用性萎缩对于软骨细胞外基质成分和表型分化的作用研究

Effects of Limb Disuse Atrophy on Extracellular Matrix Composition and PhenotypicDifferentiation of Chondrocytes in Microgravity Environment

研究微重力环境下肢体废用性萎缩对于软骨细胞外基质成分及其表型分化的作用,为寻找适当的机械负荷平衡骨关节炎造成的细胞外基质的降解,维持软骨细胞表型提供新思路。方法:使用胰酶及Ⅱ型胶原酶消化提取大鼠膝关节软骨细胞,传代培养至第3代。将细胞分为4组:正常重力软骨细胞组、正常重力关节炎软骨细胞组(关节炎软骨细胞采用10 ng/ml IL-1β诱导48小时造模)、微重力软骨细胞组、微重力关节炎软骨细胞组。微重力环境的2组细胞采用双向多样本回转器(2D-RWVs)培养,正常重力组不施加旋转,培养72小时。期间利用倒置显微镜观察各组软骨细胞的排列和形态差异。培养完成后,使用Western blot法检测各组软骨细胞Ⅱ型胶原、基质金属蛋白酶-13(Matrix metalloproteinase-13,MMP13)的表达。结果:从软骨细胞的形态学来看,正常重力软骨细胞集落样生长,多层重叠,可见典型铺路石样改变。微重力环境下软骨细胞形态不规则,排列紊乱,界限模糊,典型铺路石样表现缺如。Western blot结果显示,无论是正常软骨还是关节炎软骨,MMP-13及II型胶原的相对蛋白水平均为微重力组明显高于正常地面组(P<0.05)。结论:微重力环境能够维持软骨细胞的表型,获得分化良好的软骨细胞。微重力环境在分子层面上调了MMP-13及II型胶原的合成与分泌。

To investigate the effects of limb disuse atrophy on extracellular matrix components and phenotypic differentiation of chondrocytes in microgravity environment, providing a new idea for finding appropriate mechanical load balancing extracellular matrix degradation caused by osteoarthritis and maintaining chondrocyte phe-notype. Methods:The knee chondrocytes were digested and extracted by trypsin and collagenase typeⅡand cultured to the 3rd generation. The cells were divided into four groups:normal gravity chondrocytes group, normal gravity ar-thritis chondrocytes group (arthritic chondrocytes were modeled by 10ng/ml IL-1βinduction for 48h), microgravity chondrocytes group, and microgravity arthritis chondrocytes group . The two groups of cells in microgravity environ-ment were cultured with bidirectional multitext rotary (2D-RWVs), and the normal gravity group was cultured for 72 hours without rotation . The arrangement and morphological differences of chondrocytes in each group were ob-served by inverted microscope . After culture, the expression of typeⅡcollagen and MMP13 in chondrocytes of each group was detected by Western blot. Result:According to the morphology of chondrocytes, normal chondrocytes grow like colonies, with multiple layers overlapping and typical paver-like changes can be seen . In microgravity environ-ment, chondrocytes have irregular morphology, disordered arrangement, blurred boundaries, and lack of typical pav-ing stones . Western blot results showed that the relative protein levels of MMP-13 and type II collagen in both nor-mal and arthritic cartilage in microgravity group were significantly higher than those in normal ground group ( P <0. 05). Conclusion:Microgravity environment can maintain the phenotype of chondrocytes and obtain well-differen-tiated chondrocytes. Microgravity environment up-regulates the synthesis and secretion of MMP-13 and type II col-lagen at the molecular level.