CAJ | 학술논문

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周龄日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、10 d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 ～ 15 μm,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 ～6 μm.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、10 d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.
목적 관찰비접촉공배양조건하척색세포대류연골세포증식급생물학특성적영향.방법 무균조건하취4지4주령일본대백토흉요단척주,획취수핵조직,매소화법급불련속밀도제도법분리순화적척색세포급류연골세포.취제2대류연골세포여척색세포안1:1적비례접충우공배양장치Transwell소실중,장단층배양적류연골세포설위대조조.배양1、3、7、10 d후도치상차현미경하관찰세포생장상황,면역형광법감정세포표형,세포계수시제합(CCK-8)법측정세포증식,역전록-취합매련반응( RT-PCR)검측Ⅱ형효원、단백다당적표체.결과 성공분리순화、획취척색세포급류연골세포.경하관찰견원대척색세포체적교대,정원형혹타원형,직경10 ～ 15 μm,포질내함교다대소불등적낭포；원대첩벽적류연골세포체적교척색세포소,정단사형,직경4 ～6 μm.수착비접촉공배양시간적연장,류연골세포증식명현가쾌,이공배양7、10 d명현(P＜0.05)；Ⅱ형효원、단백다당적표체량야축점증고.결론 척색세포능촉진수핵류연골세포적증식,기가능재조지추간반퇴변적발생중구유일정의의.
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 collagenase digestion and discontinuous density gradient centrifugation.The chondrocytic cells at second generation 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 chondrocytic cells was confirmed by immunofluorescence,the CCK-8 was used to determine the cell proliferation,and the expression of collagen type Ⅱ 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 10-15 μm in diameter,which had many cytoplasmic vesicles.While the chondrocytic cells were smaller than the former,they displayed short spindle and had a diameter of 4-6 μm.With the prolongation of co-culture durations,the growth of the chondrocytic cells 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 promote the proliferation of the chondrocytic cells in a co-culture system and may play an important role in preventing the degeneration of disc.