周期性动态压缩应力对条件性敲除Ihh基因软骨细胞立体培养力学—生物学特性的分析

发布时间：2018-05-06 04:01

本文选题：软骨细胞 + Indian　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:利用Col2a1-Cre ERT2;Ihhfl/fl基因工程小鼠所培育的未成熟的小鼠的肋软骨细胞,使用周期性动态压缩应力(Flexcell-5000施加正弦波、0.5Hz、5k Pa、1h/d压缩应力)的方法探讨条件性敲除Ihh基因后的软骨细胞立体培养的力学生物学特性。方法:1.基因工程小鼠(Col2a1-Cre ERT2;Ihhfl/fl和Ihhfl/fl)的培育及其基因鉴定。2.取刚出生的基因工程小鼠(携带Ihhfl/fl纯合基因并且具有Col2a1-Cre ERT2基因)40只。随机的分为实验组和对照组,实验组基因小鼠给予腹腔连续注射3天TM(Tamoxifen),对照组基因小鼠给予腹腔连续注射3天等量植物油,待第6天时取小鼠肋软骨,急性消化肋软骨细胞作为本实验的细胞来源。3.用实时荧光定量PCR技术来检测Ihh基因相对表达量,计算Ihh基因的敲除率。4.将实验组与对照组急性消化分离的细胞以2×105/ml的密度混于1.5%的海藻酸钠凝胶中,利用模型制成海藻酸钠细胞盘。实验组与对照组各自分为两组:其中一组海藻酸钠细胞盘用Flexcell-5000基底加载系统施加动态压缩应力(正弦波、0.5Hz、5k Pa、1h/d)7、14、21天;另一组海藻酸钠细胞盘静态培养7、14、21天。5.分别在7、14、21天时在倒置显微镜下观察各组软骨细胞的生长状况。6.通过RT-PCR技术测定同一时间点的实验组和对照组内细胞盘的Ⅱ型胶原、Ⅹ型胶原、蛋白多糖(AGG)、基质金属蛋白酶13(MMP-13)m RNA的相对表达量。7.运用半无限体细胞力学模型与微管吸吮技术相结合来测定各组内不同时间点的细胞力学特性。结果:1.通过RT-PCR检测Ihh基因相对表达量发现:实验组基因小鼠在腹腔注射TM(Tamoxifen)后与对照组相比其Ihh基因相对表达量降低(P0.01)。对Ihh信号通路的下游基因Gli-1的相对表达量进行检测发现:实验组Gli-1基因表达降低(P0.01)(图1)。敲除效率约为64%。2.同一时间点,压缩刺激可以促进软骨细胞在海藻酸钠凝胶中的增殖,倒置显微镜观察细胞盘发现:实验组、对照组内压缩细胞盘的细胞密度随着压缩刺激天数的增长而增加,其密度明显高于同组内静态培养组;实验组(基因敲除)与对照组(未敲除)相比:敲除Ihh后的软骨细胞盘与未敲除Ihh的软骨细胞盘中的细胞密度相差不明显。3.RT-PCR检测:(1)实验组(基因敲除)与对照组(未敲除)立体培养细胞盘相比、压缩细胞盘相比:7天时,实验组的AGG、Col II相对表达量增高(P0.05);实验组内、对照组内的压缩细胞盘与立体培养细胞盘相比:压缩7天AGG、Col II相对表达量增高(P0.05);各组内不同时间点AGG、Col II相对表达量对比:压缩组7天表达量高于14天、21天(P0.05);(2)实验组(基因敲除)与对照组(未敲除)中立体培养细胞盘相比、压缩细胞盘相比:实验组的内各时间点ColⅩ、MMP-13的相对表达量较对照组内各时间点的相对表达量均降低,其中7天、21天实验组的表达水平降低明显(P0.05);实验组内、对照组内的压缩细胞盘与立体培养细胞盘相比:压缩14天时ColⅩ、MMP-13的相对表达量增高(P0.05);各组内不同时间点对比:压缩组14天ColⅩ、MMP-13的相对表达量高于7天、21天(P0.05)。4.各组内细胞瞬时模量(E0)、平衡模量(E∞)、表观黏性(μ)随着培养时间的增加而降低,其中实验组、对照组内压缩21天的E0、E∞、μ明显低于同组内压缩7、14天(P0.05),7天与14天之间无统计学差异;实验组与对照组相比较,在同一时间点实验组立体培养软骨细胞、动态压缩的软骨细胞的E0、E∞、μ均大于对照组,其中实验组内压缩21天的E0、E∞明显高于对照组内压缩21天(P0.05);实验组内与对照组内同一时间点的动态压缩软骨细胞的E0、E∞、μ均大于立体培养软骨细胞的E0、E∞、μ,其中实验组内压缩7天的软骨细胞的E0、E∞明显大于立体培养7天的软骨细胞的E0、E∞(P0.05)。结论:海藻酸钠立体培养的条件性敲除Ihh基因的小鼠肋软骨细胞在生理性动态压缩刺激7天时可以有效的提高ColⅡ和AGG的表达,抑制ColⅩ和MMP-13的表达,可以一定程度上改善软骨细胞的力学性能,然而压缩天数越长软骨细胞所分泌的基质就越少、软骨细胞力学特性也就随之减弱。
[Abstract]:Objective: To explore the mechanical and biological characteristics of the stereoscopic culture of chondrocytes after the conditional knockout of the Ihh gene by using the method of periodic dynamic compression stress (Flexcell-5000 exerts sine wave, 0.5Hz, 5K Pa, 1h/d compression stress) by using Col2a1-Cre ERT2 and Ihhfl/fl gene engineering mice. Methods: 1. Gene engineering mice (Col2a1-Cre ERT2; Ihhfl/fl and Ihhfl/fl) culture and genetic identification.2. took 40 newly born gene engineering mice (carrying Ihhfl/fl homozygous gene and Col2a1-Cre ERT2 gene). They were randomly divided into experimental and control groups. The experimental group gene mice were given a continuous injection of TM (Tamoxifen) and a control group for 3 days. The mice were injected with the same amount of vegetable oil for 3 days, and the costal cartilage was taken for sixth days. The acute alimentary costal chondrocytes were used as the source.3. of this experiment to detect the relative expression of Ihh gene by real-time fluorescence quantitative PCR technique. The knockout rate of Ihh gene was calculated by.4., and the acute digestion of the experimental group and the control group was 2 * 105. The density of /ml was mixed in 1.5% sodium alginate gel, and the sodium alginate cell disc was made by the model. The experimental group and the control group were divided into two groups. One group of sodium alginate cell plates applied the dynamic compressive stress (sine, 0.5Hz, 5K Pa, 1H /d) 7,14,21 days with the base loading system of the alginate; the other group of sodium alginate cell plates was cultured in a static culture. 7,14,21 days.5. observed the growth of cartilage cells in each group under the inverted microscope at 7,14,21 days,.6. was used to determine the type II collagen, collagen, proteoglycan (AGG), and the relative expression of matrix metalloproteinase 13 (MMP-13) m RNA by RT-PCR technique, and the relative expression of matrix metalloproteinase (MMP-13) m RNA by semi infinite body thin. Cell mechanics model and microtube sucking technique were combined to determine the cellular mechanical properties at different time points in each group. Results: 1. the relative expression of Ihh gene was detected by RT-PCR. The relative expression of Ihh gene in the experimental group was lower than that of the control group (P0.01) after TM (Tamoxifen) was injected into the abdominal cavity of the experimental group (P0.01). The lower swimming base of the Ihh signaling pathway was found. The relative expression of Gli-1 showed that the expression of Gli-1 gene was reduced (P0.01) in the experimental group (P0.01). The knockout efficiency was about 64%.2. at the same time point. The compression stimulation could promote the proliferation of chondrocytes in the alginate gel. The cell disc of the experimental group was observed by the inverted microscope: the density of the cell disk in the control group with the pressure The density of the contraction stimulation days increased, and its density was significantly higher than that in the static culture group in the same group. Compared with the control group, the experimental group (gene knockout) compared with the control group (not knockout): the difference between the cartilage cell disc after knocking Ihh and the cell density in the cartilage cell disk that did not knock out Ihh was not obvious.3.RT-PCR test: (1) the experimental group (gene knockout) and the control group (not knockout) stand. Compared with the body plate, compared with the compressed cell disk, the relative expression of AGG and Col II in the experimental group was increased at 7 days (P0.05). In the experimental group, the compressed cell disc in the control group was compared with the stereoscopic culture cell disc: the compression 7 days AGG, the relative expression of Col II increased (P0.05); the relative expression of Col II in the different time points in each group was compared: the compression group was 7 days The expression level was higher than 14 days, 21 days (P0.05); (2) compared with the stereoscopic culture cell disc in the control group, the compression cell disc was compared with the control group (Col). The relative expression of MMP-13 in the experimental group was lower than that in the control group at each time point, and the expression level of the experimental group was reduced in 7 days and 21 days. In the experimental group, the compression cell disc in the control group was compared with the stereoscopic culture cell disc: the relative expression of MMP-13 increased (P0.05) at 14 days of compression and the relative expression of MMP-13 was higher (P0.05). The relative expression of MMP-13 was higher than 7 days in the compression group at 14 days, and the instantaneous modulus (E0) and the equilibrium modulus (E infinity) of the cells within the 21 days (P0.05).4.. The apparent viscosity (MU) decreased with the increase of the culture time. In the experimental group, E0, E, and Mu were obviously lower than 7,14 days in the same group (P0.05) for 21 days in the control group, and there was no statistical difference between the 7 days and the 14 days. The experimental group was compared with the control group in the same time point in the experimental group of cartilage cells and the E0 of the dynamically compressed cartilage cells. E infinity and micron were greater than those in the control group, in which the E0 of 21 days in the experimental group was significantly higher than that in the control group for 21 days (P0.05), and the E0, E, and mu of the dynamic compression cartilage cells in the same time point in the experimental group were larger than the E0, E, and mu of the cartilage cells in the stereoscopic culture. The E0, E infinity of the cartilage cells in the experimental group for 7 days was obvious, and the E infinity was obvious The E0 and E infinity (P0.05) of the chondrocytes of 7 days is greater than that in the stereoscopic culture. Conclusion: the conditioned response of Ihh gene in the costal knockout of Ihh gene can effectively improve the expression of Col II and AGG, inhibit the expression of Col and MMP-13, and improve the mechanical properties of cartilage cells to a certain extent. However, the longer the compression days, the smaller the matrix secreted by chondrocytes, and the weaker the mechanical properties of chondrocytes.

【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】：R68

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