大鼠胫骨生长板软骨细胞的体外培养及鉴定  

In Vitro Culture and Identification of Chondrocytes from Tibial Growth Plate of Rats

作　　者：潘思年[1,2] 杜敏联[1] 马华梅[1] 李燕虹[1] 

机构地区：[1]中山大学附属第一医院儿科,广东广州510080 [2]中山大学附属第三医院儿科,广东广州510630

出　　处：《中山大学学报（医学科学版）》2009年第A04期1-5,10,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省自然科学基金(06021298)

摘　　要：【目的】建立体外培养及鉴定大鼠胫骨生长板软骨细胞的方法。【方法】采用胰酶和Ⅱ型胶原酶序贯消化法对5～8只3周龄SD大鼠胫骨生长板软骨行体外分离、培养并传至第6代、观察各代软骨细胞形态,MTT法绘制细胞生长曲线,Ⅱ型胶原免疫组化染色,阿力新蓝染色检测各代软骨细胞外基质硫酸糖氨多糖(GAG)含量和结构,采用逆转录聚合酶链反应检测各代细胞Ⅱ型胶原和aggreacan mRNA表达水平。【结果】体外培养的软骨细胞随着传代次数的增加,细胞形态由原代的多角形逐渐变为长梭形;MTT比色法显示,4代以前的软骨细胞的生长曲线近似"S"形,在第4～8天细胞呈对数生长,在第9～10天达平台期,至第11天开始出现生长抑制。4代以前的软骨细胞Ⅱ型胶原免疫组化呈强阳性。原代至第6代软骨细胞的GAG含量、长链分子百分比分别为0.35±0.04,(83.0±3.6)%;0.33±0.02,(78.7±4.2)%;0.31±0.06,(77.7±2.3)%;0.30±0.05,(77.0±5.3)%;0.14±0.01,(44.3±4.0)%;0.10±0.01,(39.3±2.5)%及0.07±0.01,(28.0±2.0)%,显示从第4代后随着传代次数的增加逐渐下降(P<0.05),Ⅱ型胶原和aggreacan mRNA表达水平亦显著减少(P<0.05)。【结论】本研究所采用的软骨细胞分离和培养的方法,能在短时间内获得高纯度和高存活率的软骨细胞,第3代及以前的细胞保留了软骨细胞的表型特征,增殖较快,这一方法为更深入地从细胞水平研究儿童的生长提供了可靠有效的模式。[ Objective ] To establish a method for the culture and identification of chondrocytes from tibial growth plate of rats. [ Method ] Chondroeytes from tibial growth plate of 5 - 8 Sprague-Dawley （SD） rats aged 3 weeks by trypsin-collagenase digestion were cultured in monolayer and subcuhured to the sixth passage to observe the changes of cell morphology. The growth curve was detected by using MTT colorimetric. Immunocytochemistry and RT-PCR were performed to investigate the expression of collagen Ⅱ at the protein and mRNA level, respectively. The quantity of glycosaminoglycaus （GAGs） was examined in each passage with alcian blue precipitation, the expression of aggrecan was measured with RT-PCR method. [ Results] The primary cultured ehondrocytes was polygonal-shaped and the ehondrocytes following passages would change into long shuttle-shaped cells. MTT test showed that the growth curve before the fourth passage cells presented ＂S＂ shape, and cells were found logarithmic growth on 4^ht- 8^ht day; on 9^ht- 10^ht day, cell growth reached platform stage, and decreased at the 11^ht day. The collagen type Ⅱ immunohistochemieal staining was extensive positive in cells before the fourth passage. The amount of proteoglycan and longer chain sulfated GAGs were 0.35 ± 0.04, （83.0 ± 3.6）%;0.33 ± 0.02, （78.7 ± 4.2）%;0.31 ± 0.06, （77.7 ± 2.3）%;0.30 ± 0.05, （77.0 ± 5.3）% ;0.14 ± 0.01, （44.3 ± 4.0）% ;0.10 ± 0.01, （39.3 ± 2.5）% and 0.07 ± 0.01, （28.0 ± 2.0）% respectively and began to reduce from the fourth passage （P 〈 0.05）, the expression of collagen Ⅱ and aggreean in the mRNA level also decreased significantly （P 〈 0.05）. [Conclusion] The method to isolate and culture chondrocytes in this study can acquire pure and viable chondrocytes in short time. The cells before passage 4 maintain phenotype of chondrocyte and grow quickly. This method provides a valuable model for further study of children＇s growth at cell level.

关 键 词：胫骨生长板 软骨细胞 细胞培养 

分 类 号：R72[医药卫生—儿科]