大鼠髁突软骨细胞冻存复苏后的生物学特性观察

Observation of biological characteristics on rat condylar chondrocytes after frozen-thawing

目的观察低温冻存1月后,不同年龄大鼠髁突软骨细胞的生物学特性。方法体外培养出生后1天、7天、14天、28天共4组SD大鼠髁突软骨细胞。对冻存复苏后的细胞进行形态学观察;甲苯胺蓝染色、Ⅱ型胶原免疫组化染色检测其对糖胺聚糖(GAG)、Ⅱ型胶原的合成能力;台盼蓝拒染试验测定细胞成活率;MTT法观察细胞增殖能力;实时定量-聚合酶链式反应(real time-PCR)检测冻存后细胞增殖核抗原(PCNA)基因表达情况。结果低温冻存后,各年龄组大鼠髁突软骨细胞仍能保持软骨细胞特有的形态及特性;细胞成活率均大于90%;生长曲线近似"S"型;冻存后各年龄组细胞pcna基因表达较冻存前无显著统计学差异(P>0.05)。结论低温冻存1月后的大鼠髁突软骨细胞成活率高,并保持了其生物学特性及增殖活性。

Objective To study the biological characteristics of rat condylar chondrocytes after 1 month frozenthawing. Methods The 1, 7, 14 and 28 day-old SD rat condylar chondrocytes were cultured in vitro. The morphology of frozen-thawed chondrocytes was observed, and their ability to synthesizing glycosaminoglycans （GAG） and type II collagen was determined by toluidine blue stain and type II collagen immunohistochemistry reaction.Viability and the proliferation activity were detected by trypan blue exclusion method, MTT assay. Otherwise, real time-PCR was performed by using proliferation cell nuclear antigen （PCNA） specific primers to detect its gene expresion. Results The frozenthawed condylar chondrocytes could still keep the morhoplogy and biological characteristics of cartilage chondrocytes specially; Cell viability was over 90%. The growth curve of condylar ehondrocytes resembled ＂S＂ in shape. Proliferation cell nuclear antigen （PCNA） gene expression changed little between the before- and after-cryopresevation condylar chondroctyes （P 〉0.05）. Conclusion Rat condylar chondrocytes after 1 month frozen-thawing still maintain their biological characteristics, proliferation activity and keep high viability .