脊索细胞对类软骨细胞增殖及基质合成代谢的影响  

Effects of notochordal cells on proliferation and matrix anabolism of chondrocytic cells from the nu- cleus pulposus

作　　者：丁凡[1] 邵增务[1] 熊蠡茗[1] 马凯歌[1] 高飞[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《中华实验外科杂志》2012年第4期729-731,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30872610、30700841）

摘　　要：目的观察非接触共培养条件下脊索细胞对类软骨细胞增殖及生物学特性的影响。方法无菌条件下取4只4周龄13本大白兔胸腰段脊柱，获取髓核组织，酶消化法及不连续密度梯度法分离纯化的脊索细胞及类软骨细胞。取第2代类软骨细胞与脊索细胞按1：1的比例接种于共培养装置Transwell小室中，将单层培养的类软骨细胞设为对照组。培养1、3、7、10d后倒置相差显微镜下观察细胞生长状况，免疫荧光法鉴定细胞表型，细胞计数试剂盒（CCK-8）法测定细胞增殖，逆转录-聚合酶链反应（RT—PCR）检测Ⅱ型胶原、蛋白多糖的表达。结果成功分离纯化、获取脊索细胞及类软骨细胞。镜下观察见原代脊索细胞体积较大，呈圆形或椭圆形，直径10～15μm，胞质内含较多大小不等的囊泡；原代贴壁的类软骨细胞体积较脊索细胞小，呈短梭形，直径4～6μm。随着非接触共培养时间的延长，类软骨细胞增殖明显加快，以共培养7、10d明显（P〈0．05）；Ⅱ型胶原、蛋白多糖的表达量也逐渐增高。结论脊索细胞能促进髓核类软骨细胞的增殖，其可能在阻止椎间盘退变的发生中具有一定意义。Objective To investigate the effects of notochordal cells on the proliferation and matrix anabolism of chondrocytic cells in a co-culture system of no direct cellular interaction in vitro. Methods The nucleus pulposus tissues were obtained from the thoracolumbar spine of four 4-week-old Japanese white rabbits under the sterile conditions, the notochordal cells and chondrocytic cells were isolated by the colla- genase digestion and discontinuous density gradient centrifugation. The chondrocytic cells at second genera- tion and the notochord cells were co-cultured in the Transwell chamber device with a ratio of 1： 1, and the chondrocytic cells cultured in monolayer as control group. After co-culture for 1, 3, 7 and 10 days, the morphologic feature was observed under the inverted phase-contrast microscopy. The phenotype of chondro- cytic cells was confirmed by immunofluorescence, the CCK-8 was used to determine the cell proliferation, and the expression of collagen type U and aggrecan was detected by using reverse transcription-polymerase chain reaction （RT-PCR）. Results The notochord cells and chondrocytic cells were successfully isolated. The notochord cells were round or oval in shape and approximately 10-15μm in diameter, which had many cytoplasmic vesicles. While the chondrocytic ceils were smaller than the former, they displayed short spindle and had a diameter of 4-6μm. With the prolongation of co-culture durations, the growth of the chon- drocytic ceils was accelerated apparently, more obviously at 7th and 10th day （P 〈 0. 05） ; and the expression of collagen type Ⅱ and aggrecan was increased gradually. Conclusion The notochord cells could pro-mote the proliferation of the chondrocytic cells in a co-culture system and may play an important role in pre- venting the degeneration of disc.

关 键 词：脊索细胞 类软骨细胞 髓核 

分 类 号：R681.53[医药卫生—骨科学]