本文選題：軟骨細(xì)胞 + Indian��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【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軟骨細(xì)胞,使用周期性動態(tài)壓縮應(yīng)力(Flexcell-5000施加正弦波、0.5Hz、5k Pa、1h/d壓縮應(yīng)力)的方法探討條件性敲除Ihh基因后的軟骨細(xì)胞立體培養(yǎng)的力學(xué)生物學(xué)特性。方法:1.基因工程小鼠(Col2a1-Cre ERT2;Ihhfl/fl和Ihhfl/fl)的培育及其基因鑒定。2.取剛出生的基因工程小鼠(攜帶Ihhfl/fl純合基因并且具有Col2a1-Cre ERT2基因)40只。隨機(jī)的分為實(shí)驗(yàn)組和對照組,實(shí)驗(yàn)組基因小鼠給予腹腔連續(xù)注射3天TM(Tamoxifen),對照組基因小鼠給予腹腔連續(xù)注射3天等量植物油,待第6天時(shí)取小鼠肋軟骨,急性消化肋軟骨細(xì)胞作為本實(shí)驗(yàn)的細(xì)胞來源。3.用實(shí)時(shí)熒光定量PCR技術(shù)來檢測Ihh基因相對表達(dá)量,計(jì)算Ihh基因的敲除率。4.將實(shí)驗(yàn)組與對照組急性消化分離的細(xì)胞以2×105/ml的密度混于1.5%的海藻酸鈉凝膠中,利用模型制成海藻酸鈉細(xì)胞盤。實(shí)驗(yàn)組與對照組各自分為兩組:其中一組海藻酸鈉細(xì)胞盤用Flexcell-5000基底加載系統(tǒng)施加動態(tài)壓縮應(yīng)力(正弦波、0.5Hz、5k Pa、1h/d)7、14、21天;另一組海藻酸鈉細(xì)胞盤靜態(tài)培養(yǎng)7、14、21天。5.分別在7、14、21天時(shí)在倒置顯微鏡下觀察各組軟骨細(xì)胞的生長狀況。6.通過RT-PCR技術(shù)測定同一時(shí)間點(diǎn)的實(shí)驗(yàn)組和對照組內(nèi)細(xì)胞盤的Ⅱ型膠原、Ⅹ型膠原、蛋白多糖(AGG)、基質(zhì)金屬蛋白酶13(MMP-13)m RNA的相對表達(dá)量。7.運(yùn)用半無限體細(xì)胞力學(xué)模型與微管吸吮技術(shù)相結(jié)合來測定各組內(nèi)不同時(shí)間點(diǎn)的細(xì)胞力學(xué)特性。結(jié)果:1.通過RT-PCR檢測Ihh基因相對表達(dá)量發(fā)現(xiàn):實(shí)驗(yàn)組基因小鼠在腹腔注射TM(Tamoxifen)后與對照組相比其Ihh基因相對表達(dá)量降低(P0.01)。對Ihh信號通路的下游基因Gli-1的相對表達(dá)量進(jìn)行檢測發(fā)現(xiàn):實(shí)驗(yàn)組Gli-1基因表達(dá)降低(P0.01)(圖1)。敲除效率約為64%。2.同一時(shí)間點(diǎn),壓縮刺激可以促進(jìn)軟骨細(xì)胞在海藻酸鈉凝膠中的增殖,倒置顯微鏡觀察細(xì)胞盤發(fā)現(xiàn):實(shí)驗(yàn)組、對照組內(nèi)壓縮細(xì)胞盤的細(xì)胞密度隨著壓縮刺激天數(shù)的增長而增加,其密度明顯高于同組內(nèi)靜態(tài)培養(yǎng)組;實(shí)驗(yàn)組(基因敲除)與對照組(未敲除)相比:敲除Ihh后的軟骨細(xì)胞盤與未敲除Ihh的軟骨細(xì)胞盤中的細(xì)胞密度相差不明顯。3.RT-PCR檢測:(1)實(shí)驗(yàn)組(基因敲除)與對照組(未敲除)立體培養(yǎng)細(xì)胞盤相比、壓縮細(xì)胞盤相比:7天時(shí),實(shí)驗(yàn)組的AGG、Col II相對表達(dá)量增高(P0.05);實(shí)驗(yàn)組內(nèi)、對照組內(nèi)的壓縮細(xì)胞盤與立體培養(yǎng)細(xì)胞盤相比:壓縮7天AGG、Col II相對表達(dá)量增高(P0.05);各組內(nèi)不同時(shí)間點(diǎn)AGG、Col II相對表達(dá)量對比:壓縮組7天表達(dá)量高于14天、21天(P0.05);(2)實(shí)驗(yàn)組(基因敲除)與對照組(未敲除)中立體培養(yǎng)細(xì)胞盤相比、壓縮細(xì)胞盤相比:實(shí)驗(yàn)組的內(nèi)各時(shí)間點(diǎn)ColⅩ、MMP-13的相對表達(dá)量較對照組內(nèi)各時(shí)間點(diǎn)的相對表達(dá)量均降低,其中7天、21天實(shí)驗(yàn)組的表達(dá)水平降低明顯(P0.05);實(shí)驗(yàn)組內(nèi)、對照組內(nèi)的壓縮細(xì)胞盤與立體培養(yǎng)細(xì)胞盤相比:壓縮14天時(shí)ColⅩ、MMP-13的相對表達(dá)量增高(P0.05);各組內(nèi)不同時(shí)間點(diǎn)對比:壓縮組14天ColⅩ、MMP-13的相對表達(dá)量高于7天、21天(P0.05)。4.各組內(nèi)細(xì)胞瞬時(shí)模量(E0)、平衡模量(E∞)、表觀黏性(μ)隨著培養(yǎng)時(shí)間的增加而降低,其中實(shí)驗(yàn)組、對照組內(nèi)壓縮21天的E0、E∞、μ明顯低于同組內(nèi)壓縮7、14天(P0.05),7天與14天之間無統(tǒng)計(jì)學(xué)差異;實(shí)驗(yàn)組與對照組相比較,在同一時(shí)間點(diǎn)實(shí)驗(yàn)組立體培養(yǎng)軟骨細(xì)胞、動態(tài)壓縮的軟骨細(xì)胞的E0、E∞、μ均大于對照組,其中實(shí)驗(yàn)組內(nèi)壓縮21天的E0、E∞明顯高于對照組內(nèi)壓縮21天(P0.05);實(shí)驗(yàn)組內(nèi)與對照組內(nèi)同一時(shí)間點(diǎn)的動態(tài)壓縮軟骨細(xì)胞的E0、E∞、μ均大于立體培養(yǎng)軟骨細(xì)胞的E0、E∞、μ,其中實(shí)驗(yàn)組內(nèi)壓縮7天的軟骨細(xì)胞的E0、E∞明顯大于立體培養(yǎng)7天的軟骨細(xì)胞的E0、E∞(P0.05)。結(jié)論:海藻酸鈉立體培養(yǎng)的條件性敲除Ihh基因的小鼠肋軟骨細(xì)胞在生理性動態(tài)壓縮刺激7天時(shí)可以有效的提高ColⅡ和AGG的表達(dá),抑制ColⅩ和MMP-13的表達(dá),可以一定程度上改善軟骨細(xì)胞的力學(xué)性能,然而壓縮天數(shù)越長軟骨細(xì)胞所分泌的基質(zhì)就越少、軟骨細(xì)胞力學(xué)特性也就隨之減弱。
[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.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R68

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