动态负荷力对软骨前体干细胞甲状旁腺激素相关肽的影响及细胞外基质蛋白组研究

Effect of dynamic pressure on parathyroid hormone-related peptide and proteomics of extraceilular matrix in precartilaginous stem cells of rat

目的探讨在动态负荷压力对体外软骨前体干细胞(PSC)甲状旁腺激素相关肽(PTHrP)的影响,寻找在细胞外基质呈差异性表达的蛋白质。方法体外培养大鼠软骨PSC,并用磁性细胞分选系统分离纯化,所得PSC行不同强度(30、60、90 mm Hg)和持续时间(2、6、24 h)的压力刺激,设未刺激组为对照组,采用逆转录PCR检测各组细胞的PTHrP水平;随机将软骨前体干细胞分成实验组与对照组,实验组以90 mm Hg强度的压力持续24 h处理软骨前体干细胞,对照组不予处理,提取两组蛋白质行双向电泳,以基质辅助激光解吸/离子化飞行时间质谱仪鉴定2组细胞呈差异表达的蛋白质。结果 PSC在动态压力培养环境下,各时间点PTHrP表达水平均明显高于对照组(P<0.01);PTHrP mRNA的表达水平随时间增加而升高;同一时间点,压力越大,PTHrP mRNA表达水平越高。质谱鉴定在压力作用下的实验组与对照组呈差异表达共6种蛋白质,分别为脯氨酰羟化酶α亚单位、丙酮酸激酶、L-乳酸脱氢酶、脯氨酰羟化酶β亚单位、细胞骨架蛋白、烯醇酶。实验组的所有差异性蛋白表达均强于对照组。结论压力能促进体外培养的大鼠软骨前体干细胞PTHrP的表达,压力作用下软骨细胞外基质有6个关键蛋白呈差异表达,其在软骨改建中可能起重要作用。

Objective To explore the effect of dynamic pressure on parathyroid hormone related pep-tide （PTHrP）in cultured cartilage precursor stem cell （PSC）,and find the differential expression of pro-teomics in extracellular matrix. Methods PSC of cartilage were isolated from rats,then purified by magnetic activated cell sorting system and cultured in vitro.The PSC were subjected to different intensities of compressive pressure （30,60,90 mm Hg）for 2,6 and 24 h. Unstimulated cells were used as control group. PTHrP mRNA levels of PSC in each group were detected by RT-PCR. Then PSC of cartilage were randomly divided in-to experimental group and control group. Experimental group were treated with 90 mm Hg pressure for 24 h.Extracted protein was subjected to two-dimensional electrophoresis （2-DE）,and matrix-assisted laser desorp-tion /ionization time of flight mass spectrometry （MALDI-TOF-MS）was used to identify proteins that different-ly expressed between two groups. Results PTHrP levels of PSC of cartilage in the experimental group were significantly increased and higher than that of control group at each time point （P＆lt;0.01）. PTHrP mRNA ex-pression was up-regulated with time increased. At the same time point,the PTHrP mRNA expression was high-er with the greater pressure. By MALDI-TOF-MS,there were 6 proteins showed significant difference between experimental group and control group under pressure. They were prolyl hydroxylase alpha-subunit,pyruvate ki-nase,L-lactate dehydrogenase A,prolyl hydroxylase beta-subunit and destrin,enolase. Conclusion Appro-priate dynamic compressive stress can effectively promote PTHrP expression in PSC of cartilage. There were 6 proteins that are expressed differently in extracellular matrix under pressure. PTHrP mechanical signal trans-duction may play an important role in the process of cartilage remodeling.