| 摘要: |
| 为建立鸡软骨细胞的体外分离、培养及鉴定体系，选取15 d鸡胚为试验材料，利用0.25%胰酶和0.2% II型胶原酶两步消化法消化鸡胚软骨组织，通过细胞形态学观察、软骨细胞标志蛋白Col Ⅱ的免疫荧光检测确定是否为软骨细胞；利用CCK-8法测定鸡原代软骨细胞生长曲线，观察细胞的生长规律；利用分化培养基定向诱导软骨细胞分化为成骨细胞，再通过茜素红染色鉴定软骨细胞分化能力；最后，通过荧光定量PCR(qPCR)检测成骨进程中关键基因的表达。结果表明:1)分离得到纯度较高的软骨细胞，刚分离的细胞呈球形，贴壁后细胞呈梭形或多角形。2)软骨细胞特异性标志蛋白Col Ⅱ经鉴定呈阳性，证实分离的细胞为软骨细胞。3)软骨细胞的生长曲线大致呈“S”形，细胞增殖过程经潜伏期、生长期、平台期最终达到衰亡期，符合正常的软骨细胞增殖过程。4)经诱导成骨分化后，细胞形态变为不规则状，茜素红染色可见圆形不透明钙化结节，细胞数量和密度明显增加，表明软骨细胞诱导成骨分化成功。5)成骨标志基因RUNX2(RUNX Family Transcription Factor 2)、SOX9(SRY-Box Transcription Factor 9)、NELL-1(Neural EGFL Like 1)和GDF-5(Growth Differentiation Factor 5)的mRNA表达水平极显著升高(P<0.01)。综上，本研究建立了基于两步消化法分离鸡胚软骨细胞的方法，分离得到的软骨细胞纯度较高且具有良好的增殖能力，为进一步探究软骨细胞在家禽骨骼发育和软骨类疾病治疗中的作用机制奠定基础。 |
| 关键词: 鸡 软骨细胞 分离培养 鉴定 成骨诱导分化 |
| DOI：10.11841/j.issn.1007-4333.2023.09.09 |
| 投稿时间：2022-10-04 |
| 基金项目:国家自然科学基金项目(31802028)；安徽省教育厅高校自然科学研究重点项目(2022AH050928)；高校优秀青年人才支持重点项目(gxyqZD2022017)；教育部产学研合作协同育人项目(221000488095409)；2021年省级继续教育教学改革重点项目(2021jxjy026)；安徽省重点研究与开发计划项目(202004a06020049)；2022年研究生质量工程项目(2022yjsjy03) |
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| Isolation, culture and identification of chicken primary chondrocytes |
|
JIN Sihua,HE Peili,JIA Fumin,JIA Yuqing,WANG Xin,JIANG Lijun,CHANG Liang,GUO Liping,GENG Zhaoyu*
|
| (College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China) |
| Abstract: |
| To establish an in vitro isolation, culture and identification system for chicken chondrocytes, 15-day-old chicken embryos were selected as the test material and chicken embryo cartilage tissue was digested by two-step digestion method of 0. 25% trypsin and 0. 2% type II collagenase; chondrocytes were identified by cytomorphological observation and immunofluorescence detection of the chondrocyte marker protein Col II. The growth curve of chicken primary chondrocytes was determined by CCK-8 method to observe the growth pattern of chondrocytes. The differentiation medium was used to induce the differentiation of chondrocytes into osteoblasts in a targeted manner, and then the differentiation ability of chondrocytes was identified by alizarin red staining; Finally, the relative expressions of key genes in the osteogenic process were detected by fluorescence quantitative PCR(qPCR). The results showed that: 1)Chondrocytes with high purity were isolated, and the freshly isolated cells were spherical, and the cells were shuttle-shaped or polygonal after apposition. 2)The chondrocyte-specific marker protein Col Ⅱ was identified as positive, confirming that the isolated cells were chondrocytes. 3)The growth curve of chondrocytes was roughly “S”-shaped, and the cell proliferation process went through latent phase, growth phase, plateau phase and finally reached the decay phase, which was consistent with the normal chondrocyte proliferation process. 4)After inducing osteogenic differentiation, the cell morphology became irregular and round opaque calcified nodules were visible by alizarin red staining, and the number and density of cells increased significantly, indicating that chondrocytes were successfully induced to osteogenic differentiation. 5)The marker genes of osteogenesis, RUNX2(RUNX Family Transcription Factor 2), SOX9(SRY-Box Transcription Factor 9), NELL-1(Neural EGFL Like 1)and GDF-5(Growth Differentiation Factor 5)were highly significantly increased(P<0. 01). In conclusion, a two-step digestion-based method was established for the isolation of chicken embryonic chondrocytes, and the isolated chondrocytes were of high purity and good proliferation ability, which laid the foundation for further investigation of the role of chondrocytes in poultry skeletal development and cartilage-like disease treatment. |
| Key words: chicken chondrocytes isolation and culture identification osteogenesis differentiation |
