摘要
目的：观察成年兔关节软骨损伤模型中能获得的关节软骨和软骨细胞数量，探讨不同浓度的Ⅱ型胶原酶在不同时间段分离软骨细胞的能力及体外培养所获得的软骨细胞的生物学活性。方法：建立成年兔关节软骨损伤模型，用0.1％-0．2％Ⅱ型胶原酶消化4小时，每小时收集消化下来的兔膝关节软骨细胞，计数细胞收获率和细胞存活率。体外培养原代及传代细胞，观察细胞形态和生长规律，绘制生长曲线，榆测细胞合成Ⅰ、Ⅱ型胶原、蛋白多糖能力。结果：成年兔膝关节软骨细胞的收获率平均为3603．6×10^4个／克，细胞存活率平均为97．3％，胶原酶浓度与细胞的获取率无显著关系，但与细胞的存活率相关，在消化时间超过3h后细胞的存活率显著降低，在培养过程中出现易老化现象，原代细胞贴壁时间为48—72h，甲苯胺蓝异染反应较强，Ⅱ型胶原免疫组化染色反应呈强阳性，Ⅰ型胶原免疫组化染色为阴性，传3代细胞形态由卵圆形、多角形变为梭形，甲苯胺蓝染色异染反应较弱，免疫组化工型胶原为强阳性而Ⅱ型胶原表达极弱。结论：0．1～0．2％Ⅱ型胶原酶消化、分批收取细胞具有较高的细胞收获率和存活率，体外培养时，从创伤后关节软骨中获得的原代细胞和1代细胞具有良好的软骨表型，第3代及以后的细胞生物学活性低下。
Objective： To evaluate the quantity of articular cartilage and ehondrocyte population isolating from adult rabbit models of impacting injury of articular cartilage and the efficiency of harvesting ehondrocyte from articular cartilage with different concentration of collagenases Ⅱ in different digestive time, and to observe the biological characteristics of the chondroeyte during cultivation in vitro. Methods： The adult rabbit model of impacting injury of articular cartilage were established. The cartilage were digested by 0.1% or 0.2 % eollagenase Ⅱ for 4 hours and the ehondrocytes were harvested per hour from the second hour on. The cell harvesting rates, viability of cultured ehondroeytes, cell growth curves and synthesis of collagen type - Ⅰ ,Ⅱ , proteoghyean were used to evaluate the effects of eolLagenase eoncentratlons and digestive time. Results： On average, the harvest rate of ehondroeyte was 3603.6 × 10^4 per gram and the viability rate of the cells was 97.3 %. The concentrations of collagenase have no significant correlation with the harvest rate of chondroeyte, but they have significant correlation with the viability rate of the cells. While the digestive time was exceeding 3 hours, the viability rate of chondroeyte decreased significantly, moreover, the cells appeared to get aged easily. The primary condrocytes had need 48 to 72 hours to adhere, and had shown strong heteroehromia to toluldlne blue, positive immunohistoehemieal stain of collagen type - Ⅱ and negative stain of collagen type - Ⅰ .The third passage chondroeytes became spindle shape from elliptic and multi- angle shape, with negative stain of collagen type - Ⅱ and slight heterochromia to toluidine blue. But the stain of collagen type - I had shown strong positive. Coaclnsion. Digesting the cartilage by 0.1% or 0.2% collagenase Ⅱ and harvesting the cells every hours can obtain high number of chondrocytes and fine viability rates of the cells. The primary and first passage chondrocytes obtained from the impacting injuryed articular cartilage had fine biological activity during cultivation in vitro, and the chondrocytes after the third passiging had lost their special biological activity.
出处
《华西医学》
CAS
2007年第1期108-110,共3页
West China Medical Journal
关键词
软骨细胞
胶原酶
软骨创伤
细胞培养
成年兔
chondrocyte
collagenase
impacting injury
cell culture
adult rabbit
