【摘要】：目的:與正常細(xì)胞比較,分析敲除Ihh基因的軟骨細(xì)胞在體外培養(yǎng)過(guò)程中的力學(xué)—生物學(xué)特性變化,明確Ihh基因在軟骨細(xì)胞力學(xué)-生物學(xué)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(jī)分為實(shí)驗(yàn)組與對(duì)照組,實(shí)驗(yàn)組腹腔注射TM(Tamoxifen),對(duì)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(xì)胞。2、實(shí)時(shí)熒光定量PCR(RT-PCR)檢測(cè)Ihh基因相對(duì)表達(dá)量,明確Ihh基因的敲除效率。3、CCK-8法和流式細(xì)胞術(shù)檢測(cè)同一時(shí)間點(diǎn)實(shí)驗(yàn)組與對(duì)照組肋軟骨細(xì)胞的增殖和凋亡(n=10)。4、RT-PCR檢測(cè)Col I、Gli-1、Col II、Col X、Agg和Mmp13的m RNA表達(dá)水平的變化(n=5)。5、微管吸吮技術(shù)及半無(wú)限體細(xì)胞力學(xué)模型測(cè)定Ihh基因敲除后肋軟骨細(xì)胞力學(xué)特性的變化(n=5)。結(jié)果:1、CCK-8檢測(cè)發(fā)現(xiàn),敲除Ihh基因后,軟骨細(xì)胞增殖效率較對(duì)照組降低,其中48h、72h最為顯著,450nm波長(zhǎng)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(xì)胞術(shù)檢分析發(fā)現(xiàn):第3天、第5天、第7天的實(shí)驗(yàn)組細(xì)胞早期凋亡率較對(duì)照組均增加(P0.05)。3、RT-PCR分析Col II和Agg的m RNA表達(dá)水平較對(duì)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(dá)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(wú)限體細(xì)胞力學(xué)模型測(cè)定軟骨細(xì)胞力學(xué)特性發(fā)現(xiàn):瞬時(shí)模量(E0)、平衡模量(E∞)、表觀黏性(μ)均相對(duì)于對(duì)照組較高(P0.05)。結(jié)論:條件性敲除軟骨細(xì)胞Ihh基因后能夠有效的提高軟骨細(xì)胞Col II和Agg的表達(dá),抑制Col X和Mmp13的表達(dá)。并且軟骨細(xì)胞力學(xué)特性明顯的增加。提示,敲除Indian hedgehog基因?qū)τ趦?yōu)化種子細(xì)胞具有一定的價(jià)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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