【摘要】：目的:與正常細胞比較,分析敲除Ihh基因的軟骨細胞在體外培養(yǎng)過程中的力學—生物學特性變化,明確Ihh基因在軟骨細胞力學-生物學特性維持作用。方法:1、取剛出生的Ihhfl/fl純合并具有Col2a1-Cre ERt2的基因工程小鼠40只,隨機分為實驗組與對照組,實驗組腹腔注射TM(Tamoxifen),對照組腹腔注射等量的植物油,連續(xù)注射5天后,急性分離肋軟骨細胞。2、實時熒光定量PCR(RT-PCR)檢測Ihh基因相對表達量,明確Ihh基因的敲除效率。3、CCK-8法和流式細胞術檢測同一時間點實驗組與對照組肋軟骨細胞的增殖和凋亡(n=10)。4、RT-PCR檢測Col I、Gli-1、Col II、Col X、Agg和Mmp13的m RNA表達水平的變化(n=5)。5、微管吸吮技術及半無限體細胞力學模型測定Ihh基因敲除后肋軟骨細胞力學特性的變化(n=5)。結果:1、CCK-8檢測發(fā)現(xiàn),敲除Ihh基因后,軟骨細胞增殖效率較對照組降低,其中48h、72h最為顯著,450nm波長吸光度分別為(0.534±0.15)、(0.722±0.13)(p0.05)和(0.758±0.14)、(0.946±0.17)(p0.05)。2、流式細胞術檢分析發(fā)現(xiàn):第3天、第5天、第7天的實驗組細胞早期凋亡率較對照組均增加(P0.05)。3、RT-PCR分析Col II和Agg的m RNA表達水平較對照組分別增高4.2倍(P0.01)和3.2倍(P0.05),Col X和Mmp13的m RNA表達水平分別降低3.6倍(P0.01)和2.6倍(P0.05)。4、微管吸吮及半無限體細胞力學模型測定軟骨細胞力學特性發(fā)現(xiàn):瞬時模量(E0)、平衡模量(E∞)、表觀黏性(μ)均相對于對照組較高(P0.05)。結論:條件性敲除軟骨細胞Ihh基因后能夠有效的提高軟骨細胞Col II和Agg的表達,抑制Col X和Mmp13的表達。并且軟骨細胞力學特性明顯的增加。提示,敲除Indian hedgehog基因對于優(yōu)化種子細胞具有一定的價值。
[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 鹵0.15), respectively. (0.722 鹵0.13) (p0.05) and (0.758 鹵0.14), (0.946 鹵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，

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