microRNA-15a模拟物对人关节软骨细胞增殖与凋亡的影响

目的：近年来研究表明，关节软骨细胞凋亡在骨关节炎发病过程中起到了重要的作用，本文旨在探讨microma-15a模拟物对于原代人膝关节软骨细胞增殖与凋亡的影响。方法：取人外伤性截肢后的膝关节软骨，采用双酶消化法分离获得人膝关节软骨细胞，并进行体外培养，通过甲苯胺蓝染色和II型胶原免疫细胞化学染色进行软骨细胞鉴定。将培养的软骨细胞传代后取第l代细胞，分为实验组和对照组，实验组采用mir．15a模拟物（has．mir-15amimics）转染软骨细胞，上调软骨细胞内mir-15a的表达量；对照组分为阴性对照组、空白对照组。采用MTT法测定细胞增殖曲线，流式细胞仪测定细胞凋亡率。结果：原代细胞中细胞呈多角形、圆形与梭型，贴壁生长；甲苯胺蓝染色胞质呈深蓝色，II型胶原染色胞质呈黄褐色，为特异性染色。经统计学分析，实验组与对照组相比增殖速率明显下降（P〈0．05）。实验组凋亡率（7．13％±0．57）与阴性对照组凋亡率（2．66％±0．15）相比明显增高（P〈0．05）。结论：采用双酶消化法成功分离并培养具有生物学特性的原代人膝关节软骨细胞，通过转染mir-15a模拟物外源性增加关节软骨细胞内mir．15a表达量可显著促进其凋亡并抑制其增殖，为阐明骨关节炎发病机制提供了新的理论依据，为’临床治疗提供了新的靶点。

Effects of MicroRNA-15a mimics On Priliferation and Apoptosis of Human Articular Chondrocytes

Objective： Recent researches suggest that apoptosis of articular chondrocytes have an important role in the pathogene- sis of osteoarthritis, and this experiment is aimed to investigate the effects of microRNA-15a on the proliferation and apoptosis of human articular chondrocytes. Methods： Human articular cartilage was obtained from human injury knee joints under sterile condition and pri- mary chondrocytes were isolated and cultured by sequential enzymatic digestion at 37℃. The chondrocytes were biologically confirmed by toluidine blue staining and immunocytochemical staining of collagen type II. Chondrocytes were divided into three groups：experi- mental group, negative control group and blank group. In experimental group, chondrocytes of the first passage were transfected with has-mir-15a mimics to upregulate the expression ofmir-15a while scramble oligonucleotides were transtected into chondrcytes as nega- tive control. The apoptosis was measured by flow cytometry. MTT assay was used to detect the proliferation of chondocytes. Results： The chondrocytes were successfully isolated and showed round, spindle and multiangle shape after 36 hours culture. Toluidine blue staining and immunocytochemical staining of collagen type II were positive. Has-mir-15a mimics inhibited the proliferation of primary human articular chondrocytes compared with two control groups （P 〈0.05）. The apoptosis ratio of control groups was lower than experiment group （P〈0.05）. Conclusion： Active primary human articular chondrocytes were successfully obtained and cultured. The apoptosis was promoted and the proliferation was inhibited after mir-15a mimics was transfected into chondrocytes. The mir-15a plays a critical role in the proliferation and apoptosis in human articular chondrocytes, which may give us new insights for understanding the complex mecha- nisms of osteoarthritic pathogenesis.