摘要
目的:观察以海藻酸钠为载体的成年羊软骨细胞移植前体外短期培养的生物学性状。方法:实验于2004-07/2005-11在解放军总医院骨科研究所完成。①取成年山羊关节软骨,酶消化法得到原代软骨细胞,体外培养扩增,选用培养的第2,3代羊关节软骨细胞与12g/L海藻酸钠混合,种植密度为2×109L-1,将102mmol/L氯化钙溶液滴入软骨细胞藻酸钠悬液,形成藻酸钙软骨细胞凝胶,用软骨细胞培养液培养。②3,6,912d,分别取凝胶,测定DNA、蛋白聚糖含量,并行苏木精-伊红、藩红花O染色及Ⅱ型胶原免疫组化染色。结果:①藻酸钙凝胶中细胞的光镜形态学观察:软骨细胞在藻酸钙中呈丛状或球状生长,细胞在整个培养过程维持球形状态。②复合软骨细胞的藻酸钙凝胶的组织形态学及免疫组化测定结果:木精-伊红染色可见随培养时间的延长,细胞群增多变大;藩红花O染色显示软骨细胞周围存在不断增大的橙红染区,提示软骨细胞分泌蛋白聚糖含量持续增加。Ⅱ型胶原免疫组化染色阳性。③软骨细胞藻酸钙凝胶培养过程中DNA及蛋白聚糖含量的测定:随时间的延长,DNA的量逐渐增加,说明藻酸钙中的软骨细胞在不断增殖。藻酸钙中软骨细胞分泌的特异性基质蛋白聚糖随时间的延长而增多,象征着软骨细胞表型的稳定。结论:成年羊软骨细胞在藻酸钙中短期培养生长增殖良好,藻酸钙能保留羊软骨细胞表型,具备植入体内的条件。
AIM： To observe the biological characteristics of chondrocytes of adult goats short time cultured in alginate before transplantation in vitro. METHODS： The experiment was carried out in the Laboratory of the Institute of Orthopaedics, General Hospital of Chinese PLA between July 2004 and November 2005. ①Chondroeytes from the joints of adult goats were obtained using collagenase enzyme. After expansion in vitro, the second or third generation chondrocytes were selected to mix with 12 g/L low viscosity alginate solution in a cell density of 2×10^9 L^-1 The alginatechondrocyte suspension was allowed to gel by carefully adding 102 mmoL/L CaCI2 and cultured in chondrocyte medium. ②The cell in gel were taken at the end of 3, 6, 9and 12 days： respectively after culture, and were used for evaluation the content of DNA, proteoglycan and staining with HE, Safranin ＂O＂ and immunohistochemistry of type Ⅱ collagen . RESULTS： ① Morphological observation of cells cultured in alginate by light microscope： In alginate, many chondroeytes clusters or round appeared. maintained their round shape throughout the culture period. ②Histological and immunohistochemical characterization of chondrocytes cells cultured in alginate： HE staining indicated an increase in the number of chondrocytes clusters with the prolongation of time. Safranin ＂O＂ staining around the cells ~ncreased area and in intensity throughout the culture time, indicating the continuous increased quantity of proteoglycan. Positive staining was noted in immunohistochemistry of type Ⅱ collagen. ③Quantitative assay for DNA and proteoglycan of ehondrocytes cells cultured in alginate： The increased DNA and proteoglycan production with the prolongation of time, which were secreted specially by chondrocytes cells cultured in alginate, demonstrate a continuous proliferation and stable phenotype ofchondrocytes. CONCLUSION： The chondrocytes from the adult goats short time cultured in alginate can keep good proliferation. The alginate can keep phenotype of chondrocytes of goats, can be implanted in vivo.
出处
《中国临床康复》
CSCD
北大核心
2006年第1期61-63,共3页
Chinese Journal of Clinical Rehabilitation
基金
国家高技术研究发展计划资助项目(2002AA205021)~~
