线粒体在压力诱导椎间盘细胞凋亡和能量代谢障碍中的作用及其实验研究

发布时间：2018-06-26 10:36

本文选题：椎间盘细胞 + 压力　；参考：《华中科技大学》2011年博士论文

【摘要】：第一部分兔椎间盘细胞气压加压培养模型的建立目的建立兔椎间盘细胞体外气压加压培养模型,观察在持续静压力培养12h、24h和36h后兔椎间盘细胞生物学行为改变,评价该细胞加压模型的优缺点。方法将体外培养的兔纤维环和髓核细胞随机分为对照组(不加压)、加压12h组、24h组和36h组,在1MPa压力下干预后观察各组细胞形态学变化、增殖活性和细胞基质(COAⅠ、Ⅱ及AGG)表达改变。结果加压培养后,倒置相差显微镜下纤维环和髓核细胞均有不同程度密度减低和细胞体积减小,细胞呈长梭形变。加压24h和36h可见明显纤维环和髓核细胞增殖活性抑制；髓核细胞加压时间12h后即出现AGG的mRNA表达下降,而加压时间24h后出现COAⅡ的mRNA表达下降；纤维环细胞加压时间24h后COAⅠ、Ⅱ的mRNA均出现不同程度的表达下降。结论该细胞气压加压培养装置可短期内模拟机械应力导致的兔椎间盘细胞退变改变,为实验室实施体外压力细胞实验提供了一种简单、安全的方案。第二部分线粒体凋亡途径参与过度压力诱导的兔椎间盘细胞的凋亡目的探讨过度机械压力刺激诱导体外培养的兔纤维环和髓核细胞凋亡的主要信号途径。方法将体外培养的兔椎间盘纤维环和髓核细胞给予1MPa的持续机械压力干预12h、24h和36h,另设不加压的对照组,各4组。各组纤维环和髓核细胞达到加压时间后,流式细胞仪检测细胞凋亡,荧光显微镜、荧光分光光度计和激光共聚焦显微镜检测细胞线粒体膜电位平均相对荧光强度,酶标仪检测caspase-8,9,3活性。结果流式细胞仪结果显示纤维环细胞加压24h组、36h组和髓核细胞加压12h组、24h组和36h组均出现明显细胞凋亡增加；荧光显微镜、荧光分光光度计和激光共聚焦显微镜结果显示,加压24h组和36h组的纤维环和髓核细胞以绿色荧光为主,红色荧光明显减少,细胞线粒体膜电位均明显下降；酶标仪结果显示髓核细胞组加压后caspase-8,9,3活性均升高,而纤维环细胞组加压后caspase-9活性明显增强,caspase-8活性无明显变。结论1MPa静压力加压时间24h作为过度机械压力可以明显诱导椎间盘纤维环和髓核细胞的凋亡。机械压力诱导的纤维环细胞凋亡信号途径主要是由线粒体依赖的凋亡途径参与的,而线粒体依赖途径和DISC途径(非线粒体依赖途径)二者则均参与了髓核细胞的凋亡。第三部分过度压力对兔内外层纤维环细胞线粒体功能和超微结构的影响目的研究过度机械压力对体外藻酸盐串珠培养的兔椎间盘内外层纤维环细胞线粒体功能和超微结构的影响。方法将体外藻酸盐串珠培养的兔纤维环细胞分为四组：OA1组为外层纤维环细胞,不加压；OA2组为外层纤维环细胞,1MPa压力干预24h；IA1组为内层纤维环细胞,不加压；IA2组,内层纤维环细胞,1MPa压力干预24h。加压完成后,荧光酶标仪检测ATP产量,荧光显微镜和荧光分光光度计检测线粒体膜电位和活性氧簇的释放,分光光度计检测4种线粒体呼吸链酶复合物活性和透射电镜观察线粒体超微结构。结果兔纤维环细胞经1MPa压力作用24h后,与各对照组相比,内外层纤维环细胞均可见ATP产量明显减少,活性氧簇释放增加,线粒体膜电位显著下降,细胞线粒体超微结构呈现损伤的病理改变；外层纤维环细胞呼吸链酶复合物Ⅰ、Ⅲ和Ⅳ活性相比对照组显著下降,而内层纤维环细胞呼吸链酶复合物仅Ⅲ和Ⅳ活性相比对照组明显下降。外层纤维环细胞的线粒体损伤作用更为明显。结论过度压力负荷损害椎间盘细胞线粒体的能量代谢功能,并使线粒体形态结构发生明显病理改变,这可能是从能量代谢角度研究椎间盘退变的重要机制。
[Abstract]:Part one establishment of rabbit air pressure culture model for intervertebral disc cells
Objective to establish an in vitro pressure culture model of rabbit disc cells in vitro, and to observe the biological behavior changes of the intervertebral disc cells after continuous static pressure culture of 12h, 24h and 36h, and evaluate the advantages and disadvantages of the pressure model of the cells.
Methods rabbit fibrorings and nucleus pulposus cells were randomly divided into control group (non pressurized), pressure 12h group, 24h group and 36h group. The changes of cell morphology, proliferation activity and expression of cell matrix (COA I, II and AGG) were observed under the pressure of 1MPa.
Results after pressure culture, the density of the rings and nucleus pulposus cells in the inverted phase contrast microscope were reduced to varying degrees and the cell volume decreased, and the cells showed long spindle deformation. The proliferation activity of the fibrous ring and nucleus pulposus cells was obviously inhibited by the pressure 24h and 36h, and the mRNA expression of AGG decreased after the compression time of the nucleus pulposus cells, and the pressure time was 24h after 12h. The expression of mRNA in COA II decreased, and the expression of mRNA in COA I and II decreased in different degrees after 24h compression.
Conclusion the cell pressure pressure culture device can simulate the degeneration and change of the intervertebral disc cells in rabbits in the short term. It provides a simple and safe way for the laboratory to carry out the in vitro pressure cell experiment.
The second part of mitochondrial apoptotic pathway is involved in the apoptosis of rabbit intervertebral disc cells induced by overpressure.
Objective to investigate the main pathway of apoptosis induced by overmechanical pressure stimulation in rabbit fibroblast and nucleus pulposus cells in vitro.
Methods the intervertebral disc and nucleus pulposus cells cultured in vitro were given the continuous mechanical pressure of 1MPa, 12h, 24h and 36h, and the non pressurized control group was set up in 4 groups. The cell apoptosis was detected by flow cytometry, fluorescence microscopy, fluorescence spectrophotometer and laser confocal microscope after the compression time was reached. The mean relative fluorescence intensity of mitochondrial membrane potential was detected, and caspase-8,9,3 activity was detected by enzyme labelling apparatus.
Results the results of flow cytometry showed that the 24h group was pressurized in the fibrous ring cells, the 36h group and the nucleus pulposus cells were pressurized in the 12h group, and the apoptosis increased in both 24h and 36h groups. Fluorescence microscopy, fluorescence spectrophotometer and laser confocal microscopy showed that the fibrous ring and nucleus pulposus cells of group 24h and 36h group were mainly green fluorescence, red The cell mitochondrial membrane potential of the cells decreased obviously, and the results of the enzyme labeling showed that the activity of caspase-8,9,3 in the nucleus pulposus group increased and the activity of caspase-9 increased obviously after the compression of the fibrous ring cell group, and the activity of caspase-8 was not significantly changed.
Conclusion the apoptosis of the intervertebral disc and nucleus pulposus cells can be induced by the excessive mechanical pressure of 1MPa 24h. The apoptotic signaling pathway induced by mechanical pressure is mainly involved in the mitochondrial dependent apoptosis pathway, while the mitochondrial dependent pathway and the DISC pathway (non mitochondrial dependent pathway) are the two ones. All of them were involved in the apoptosis of nucleus pulposus cells.
The third part is the effect of excessive pressure on mitochondrial function and ultrastructure of rabbit's inner and outer fibrosus cells.
Objective to study the effects of excessive mechanical pressure on mitochondrial function and ultrastructure of rabbit intervertebral disc in vitro.
Methods rabbit fibroring cells cultured in vitro alginate beads were divided into four groups: OA1 group was outer layer of fibrous ring cells, non pressurization, OA2 group was outer layer of fibrous ring cells, 1MPa pressure intervened 24h, IA1 group was inner layer of fibrous ring cells, non pressurization, IA2 group, inner fibrous ring cells, 1MPa pressure intervention 24h. pressure completion, fluorescent enzyme labeling instrument detected AT The P yield, the fluorescence microscope and the fluorescence spectrophotometer were used to detect the mitochondrial membrane potential and the release of the active oxygen cluster. The spectrophotometer was used to detect the activity of 4 mitochondrial respiratory chain enzyme complexes and the ultrastructure of the mitochondria.
Results after the 1MPa pressure action of 24h, the output of ATP in the inner and outer fibroring cells was significantly reduced, the release of active oxygen cluster increased, the mitochondrial membrane potential decreased significantly, the mitochondrial ultrastructure showed a pathological change in the ultrastructure of the mitochondria, and the respiratory chain enzyme complex in the outer layer of fibrous ring cells I, III and IV live. Compared with the control group, the respiratory chain enzyme complex in the inner layer was significantly lower than that in the control group. The damage of the mitochondria in the outer fibrous ring cells was more obvious.
Conclusion overstress damage can damage the energy metabolism of the mitochondria of the intervertebral disc cells and make the morphologic structure of the mitochondria pathological changes. This may be an important mechanism for the study of the degeneration of intervertebral disc from the angle of energy metabolism.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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