条件性敲除软骨细胞Indian hedgehog力学—生物学特性变化的研究
发布时间：2019-03-16 15:32
【摘要】：目的:与正常细胞比较,分析敲除Ihh基因的软骨细胞在体外培养过程中的力学—生物学特性变化,明确Ihh基因在软骨细胞力学-生物学特性维持作用。方法:1、取刚出生的Ihhfl/fl纯合并具有Col2a1-Cre ERt2的基因工程小鼠40只,随机分为实验组与对照组,实验组腹腔注射TM(Tamoxifen),对照组腹腔注射等量的植物油,连续注射5天后,急性分离肋软骨细胞。2、实时荧光定量PCR(RT-PCR)检测Ihh基因相对表达量,明确Ihh基因的敲除效率。3、CCK-8法和流式细胞术检测同一时间点实验组与对照组肋软骨细胞的增殖和凋亡(n=10)。4、RT-PCR检测Col I、Gli-1、Col II、Col X、Agg和Mmp13的m RNA表达水平的变化(n=5)。5、微管吸吮技术及半无限体细胞力学模型测定Ihh基因敲除后肋软骨细胞力学特性的变化(n=5)。结果:1、CCK-8检测发现,敲除Ihh基因后,软骨细胞增殖效率较对照组降低,其中48h、72h最为显著,450nm波长吸光度分别为(0.534±0.15)、(0.722±0.13)(p0.05)和(0.758±0.14)、(0.946±0.17)(p0.05)。2、流式细胞术检分析发现:第3天、第5天、第7天的实验组细胞早期凋亡率较对照组均增加(P0.05)。3、RT-PCR分析Col II和Agg的m RNA表达水平较对照组分别增高4.2倍(P0.01)和3.2倍(P0.05),Col X和Mmp13的m RNA表达水平分别降低3.6倍(P0.01)和2.6倍(P0.05)。4、微管吸吮及半无限体细胞力学模型测定软骨细胞力学特性发现:瞬时模量(E0)、平衡模量(E∞)、表观黏性(μ)均相对于对照组较高(P0.05)。结论:条件性敲除软骨细胞Ihh基因后能够有效的提高软骨细胞Col II和Agg的表达,抑制Col X和Mmp13的表达。并且软骨细胞力学特性明显的增加。提示,敲除Indian hedgehog基因对于优化种子细胞具有一定的价值。
[Abstract]:Aim: to analyze the changes of mechanical-biological characteristics of chondrocytes with knockout of Ihh gene in vitro compared with normal cells, and to determine the role of Ihh gene in maintaining the mechanical-biological characteristics of chondrocytes. Methods: 1. Forty newly born Ihhfl/fl mice with Col2a1-Cre ERt2 were randomly divided into experimental group and control group. The experimental group was injected intraperitoneally with the same amount of vegetable oil as the control group, and the control group was injected intraperitoneally with the same amount of vegetable oil. After 5 days of continuous injection, the costal chondrocytes were isolated. 2. The relative expression of Ihh gene was detected by real-time fluorescence quantitative PCR (RT-PCR), and the knockout efficiency of Ihh gene was determined. 3. CCK- 8 method and flow cytometry were used to detect the proliferation and apoptosis of costal chondrocytes in experimental group and control group at the same time (n = 10). 4. RT-PCR was used to detect Col I, glia 1, Col II,Col X, and so on. The changes of m RNA expression levels of Agg and Mmp13 were determined by micropipette aspiration technique and semi-infinite somatic cell mechanics model. The changes of mechanical properties of costal chondrocytes after Ihh gene knockout were measured (n = 5). Results: 1. After knockout of Ihh gene, the proliferation efficiency of chondrocytes was lower than that of control group (48 h, 72 h). The absorbance of 450nm was (0.534 卤0.15), respectively. (0.722 卤0.13) (p0.05) and (0.758 卤0.14), (0.946 卤0.17) (p0.05). The expression of m-RNA in Col II and Agg were increased 4.2 times (P0.01) and 3.2-fold (P0.05), respectively, on the 7th day in the experimental group compared with the control group (P0.05), and the expression of mRNA in RT-PCR was 4.2-fold and 3.2-fold higher than that in the control group (P0.05). The expression of m RNA in Col X and Mmp13 decreased 3.6 times (P0.01) and 2.6 fold (P0.05), respectively. 4. The mechanical properties of chondrocytes were measured by micropipette aspiration and semi-infinite somatic cell mechanical model. The results showed that the instantaneous modulus (E0) of chondrocytes was determined by microtubule aspiration and semi-infinite somatic cell mechanical model. The equilibrium modulus (E 鈭，
本文编号：2441643
[Abstract]:Aim: to analyze the changes of mechanical-biological characteristics of chondrocytes with knockout of Ihh gene in vitro compared with normal cells, and to determine the role of Ihh gene in maintaining the mechanical-biological characteristics of chondrocytes. Methods: 1. Forty newly born Ihhfl/fl mice with Col2a1-Cre ERt2 were randomly divided into experimental group and control group. The experimental group was injected intraperitoneally with the same amount of vegetable oil as the control group, and the control group was injected intraperitoneally with the same amount of vegetable oil. After 5 days of continuous injection, the costal chondrocytes were isolated. 2. The relative expression of Ihh gene was detected by real-time fluorescence quantitative PCR (RT-PCR), and the knockout efficiency of Ihh gene was determined. 3. CCK- 8 method and flow cytometry were used to detect the proliferation and apoptosis of costal chondrocytes in experimental group and control group at the same time (n = 10). 4. RT-PCR was used to detect Col I, glia 1, Col II,Col X, and so on. The changes of m RNA expression levels of Agg and Mmp13 were determined by micropipette aspiration technique and semi-infinite somatic cell mechanics model. The changes of mechanical properties of costal chondrocytes after Ihh gene knockout were measured (n = 5). Results: 1. After knockout of Ihh gene, the proliferation efficiency of chondrocytes was lower than that of control group (48 h, 72 h). The absorbance of 450nm was (0.534 卤0.15), respectively. (0.722 卤0.13) (p0.05) and (0.758 卤0.14), (0.946 卤0.17) (p0.05). The expression of m-RNA in Col II and Agg were increased 4.2 times (P0.01) and 3.2-fold (P0.05), respectively, on the 7th day in the experimental group compared with the control group (P0.05), and the expression of mRNA in RT-PCR was 4.2-fold and 3.2-fold higher than that in the control group (P0.05). The expression of m RNA in Col X and Mmp13 decreased 3.6 times (P0.01) and 2.6 fold (P0.05), respectively. 4. The mechanical properties of chondrocytes were measured by micropipette aspiration and semi-infinite somatic cell mechanical model. The results showed that the instantaneous modulus (E0) of chondrocytes was determined by microtubule aspiration and semi-infinite somatic cell mechanical model. The equilibrium modulus (E 鈭，
本文编号：2441643
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