成体半月板细胞体外分区培养研究_医学论文.docVIP

成体半月板细胞体外分区培养研究_医学论文 成体半月板细胞体外分区培养研究_医学论文 【摘要】 目的 分区培养成体半月板细胞，观察不同区域的半月板细胞生长特性以及生长因子对其的影响。方法 无菌切取1月龄雄性山羊双后肢半月板，剪碎后D液漂洗，Ⅱ型胶原酶振荡消化，滤网过滤后离心收集细胞。接种6孔细胞培养板，每孔1×105个，37 ℃、体积分数为0.05的CO2、饱和湿度培养。绘制生长曲线，台盼蓝染色测细胞活力。细胞爬片行苏木精伊红及Ⅱ型胶原免疫组化染色。RT检测aggrecan的表达。用流式细胞仪检测细胞周期的分布。结果 山羊成体半月板细胞自36 h后开始贴壁，外侧区细胞以长梭形为主，内侧区以多角形为主，混合区细胞形态为混合型。台盼蓝拒染试验测细胞活力约为92％。群体倍增时间约为47.4 h。加入人胰岛素样生长因子Ⅰ和碱性成纤维细胞生长因子后细胞增殖加快，多角形细胞增多；细胞爬片行Ⅱ型胶原免疫组化染色证实在转染细胞胞浆中有棕黄色颗粒。RT检测结果显示目的条带与aggrecan的预期大小相符。流式细胞仪检测加入生长因子后的半月板细胞S期比例增加，而G1期细胞减少。结论 不同区域的半月板细胞有形态差异。加入生长因子后可促进其生长、增殖。 【关键词】 半月板 胫骨 胰岛素样生长因子 成纤维细胞生长因子2 ［ABSTRACT］ObjectiveTo investigate the morphology of cultured meniscal fibrochondrocytes in different areas and the influence of growth factor on the cells. MethodsThe menisci were taken from both hindlimbs of onemonthold male goat under sterile condition, cut into pieces, rinsed with DHanks, then digested with type Ⅱ collagenase. Fibrochondrocytes were collected and cultured at 37 ℃ with 5% CO2 under saturated humidity. Trypan blue stain and immunohistochemistry stain of type Ⅱ collagen were applied to determine the vitality of the cells. Reverse transcription polymerase chain reaction (RTPCR) was used to study the expression of aggrecan and flow cytometry to analyze the distribution of the cell cycle. ResultsMeniscal fibrochondrocytes adhered to the disk 36 hours after inoculation. The peripheral cells were mostly fusiformed, the central cells polygonal and the cells in between mixed. Trypan blue failed to stain the cells. After adding hIGFⅠ and bFGF to the medium, more polygonal cells were found in the peripheral region. A flow cytometry study showed that the cell proliferation was accelerated: percentage of meniscal chondrocytes in the S stage increased, while in G1 stage decreased. Immunoreactive product of type Ⅱ collagen was detected in the cultured cells by immunohistochemistry and the existence of aggrecan band proved by RTPCR. ConclusionThe morphology of cultured meniscal fibrochondrocytes was different i