体外间接共培养兔膝关节软骨单位对软骨细胞生物学特性的影响

发布时间：2018-04-30 10:32

本文选题：骨关节炎 + 软骨单位　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:通过Transwell间接共培养技术研究兔膝关节软骨单位对软骨细胞生物学特性的影响,为组织工程技术修复损伤软骨选取良好种子细胞提供依据。方法:用不同消化酶将2月龄新西兰兔膝关节软骨分别消化成软骨细胞和软骨单位。按2×105个细胞/孔浓度接种于Transwell双层细胞培养板。实验组:软骨单位接种于上室,软骨细胞接种于下室;对照组:下室接种软骨细胞,上室未接种。分别在2、4、6、8天提取各组Transwell下室软骨细胞进行指标检测。早期凋亡率检测:Annexin V和7-AAD标记流式细胞仪检测,TUNEL染色荧光显微镜检测;增殖率检测:PCNA标记流式细胞仪检测,Ed U染色荧光显微镜检测;相关基因相对表达量检测:PCR技术检测AGG、Col-II、MMP-13基因相对表达量。结果:Annexin V和7-AAD标记流式细胞仪检测Transwell下室软骨细胞早期凋亡率发现,在实验早期(第2、4天)实验组与对照组Transwell下室软骨细胞早期凋亡率差异不明显,差别没有统计学意义(p0.05)。在实验第6天、第8天实验组软骨细胞早期凋亡率明显低于对照组(P0.05)。TUNEL染色荧光显微镜检测Transwell下室软骨细胞凋亡率发现,实验组和对照组软骨细胞凋亡率没有显著差异,但有实验组软骨细胞凋亡率低于对照组的趋势。PCNA标记流式细胞仪检测Transwell下室软骨细胞增殖率发现,在实验早期(第2、4天)实验组与对照组Transwell下室软骨细胞增值率差异不明显,差别没有统计学意义(p0.05)。在实验第6天、第8天实验组软骨细胞增殖率明显高于对照组(P0.05)。Ed U染色荧光显微镜检测Transwell下室软骨细胞增殖率发现,第6天实验组软骨细胞增殖率明显高于对照组(P0.05)。PCR技术检测Transwell下室软骨细胞相应基因表达量发现,在实验早期(第2、4天)实验组与对照组Transwell下室软骨细胞AGG、Col-II,MMP-13基因表达较为相似,实验组与对照组差异不明显,差别没有统计学意义(P0.05)。在实验后期AGG、Col-II基因相对表达量在第6天、第8天时实验组明显高于对照组(P0.05),MMP-13表达量在第8天时对照组明显高于实验组(P0.05)。结论:1.软骨单位作为关节软骨的基本功能单位,能够更长时间有效抵抗或延缓软骨细胞凋亡、维持软骨细胞增殖及正向调节软骨基质相关物质的基因表达。2.软骨单位有望在组织工程中作为种子细胞用于骨关节炎关节软骨缺损的修复。
[Abstract]:Aim: to study the effect of articular cartilage units on the biological characteristics of chondrocytes in rabbit knee joint by Transwell indirect co-culture technique, and to provide a basis for selecting good seed cells for repairing damaged cartilage by tissue engineering technique. Methods: the articular cartilage of 2 month old New Zealand rabbits was digested with different digestive enzymes to form chondrocytes and chondrocytes respectively. Transwell bilayer cell culture plate was inoculated with 2 脳 105 cells / well concentration. Experimental group: chondrocytes were inoculated in superior chamber and chondrocytes were inoculated in inferior chamber, while in control group, chondrocytes were inoculated in lower chamber without inoculation. The ventricular chondrocytes were extracted from each group of Transwell for 8 days. Early apoptotic rate was detected by flow cytometry and 7-AAD labeled flow cytometry, and proliferation rate was detected by flow cytometry with TUNEL-stained fluorescence microscope, proliferation rate by flow cytometry labeled with 7-AAD and fluorescence microscope with Ed U staining. The relative expression of MMP-13 in AGGN Col-III was detected by PCR. Results the early apoptosis rate of ventricular chondrocytes under Transwell was detected by flow cytometry with 7-AAD and 7% Annexin V. It was found that there was no significant difference in early apoptosis rate between experimental group and control group in the early stage of experiment (2 ~ 4 days), but there was no significant difference between experimental group and control group in early apoptosis rate of ventricular chondrocytes under Transwell (p 0.05). The early apoptotic rate of chondrocytes in the experimental group was significantly lower than that in the control group after 6 days and 8 days. The apoptosis rate of chondrocytes in the experimental group was not significantly different from that in the control group. However, the apoptosis rate of chondrocytes in experimental group was lower than that in control group. The proliferative rate of chondrocytes under Transwell was detected by flow cytometry. It was found that there was no significant difference in the proliferation rate of chondrocytes between experimental group and control group under Transwell at the early stage of experiment (2 ~ 4 days). The difference was not statistically significant (P 0.05). On the 6th and 8th day, the proliferation rate of chondrocytes in the experimental group was significantly higher than that in the control group. On the 6th day, the proliferation rate of chondrocytes in the experimental group was significantly higher than that in the control group (P 0.05). The expression of the corresponding genes in the chondrocytes under Transwell was similar to that in the experimental group and the control group at the early stage of the experiment (on the 2th day), and the expression of AGG- Col-IIMP-13 in the chondrocytes of the experimental group was similar to that of the control group. There was no significant difference between the experimental group and the control group (P 0.05). At the end of the experiment, the relative expression of AGGG Col-II gene was significantly higher in the experimental group than that in the control group on the 6th day and the 8th day. The expression of MMP-13 in the experimental group was significantly higher than that in the control group on the 8th day. The expression of MMP-13 in the experimental group was significantly higher than that in the control group on the 8th day. Conclusion 1. As the basic functional unit of articular cartilage, chondrocytes can effectively resist or delay the apoptosis of chondrocytes for a longer time, maintain the proliferation of chondrocytes and positively regulate the gene expression of cartilage matrix related substances. Cartilage units are expected to be used as seed cells in tissue engineering for the repair of osteoarthritis articular cartilage defects.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687

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