CKIP-1对氟致成骨细胞凋亡的调控作用研究

The casein kinase 2 interacting protein-1 (CKIP-1) plays an important role in regulation of bone formation and muscle differentiation. However, whether and how CKIP-1 activity is regulated on osteoblast apoptosis induced by fluoride remains largely unclear. The objective of this program is to evaluate the regulation of CKIP-1 on osteoblast apoptosis induced by fluoride. Currently, our research group has studied the effect of fluorine on cytokine expression and apoptosis of osteoblasts for a long time, it was proved that herein we have obtained good groundwork for the osteoblasts related research. The CKIP-1 has recently been demonstrated to express positive regulatory action for apoptosis of osteoblasts. So, we combined these research findings together to clarify the role of CKIP-1 when it join in the apoptosis of osteoblast caused by fluorine using RNAi assay. The contents of our design hereby are as following: (1) cultivation and identification of osteoblasts of ovine in vitro; (2) determination of both mRNA and protein expression of CKIP-1 during the process of apoptosis caused by fluoride using real time-PCR and western-blot; (3) determining the proliferation vitality and observing the morphology characteristics of osteoblasts by MTT assay and electron microscope; (4) analyzing the generation cycle and apoptosis of osteoblasts using flow cytometry; (5) detecting the changes of free radical produced by osteoblasts during the process of apoptosis, using electron paramagnetic resonance spectrometer. Finally, we concluted that the promoting action of CKIP-1 for fluorine-causing apoptosis of osteoblast can be clearly demonstrated. The interference of CKIP-1 expression might be chosen as an effective tool for skeletal fluorosis.

近年来，本研究团队通过分子生物学和细胞生物学方法进行了一系列的氟对成骨细胞（OB）细胞因子表达和OB凋亡的影响研究，具备良好的研究基础。而CKIP-1因子于近期被证实对成骨细胞凋亡具有正向调控作用。因此本研究旨在通过RNAi技术干扰成骨细胞CKIP-1基因的表达，进行CKIP-1对体外氟致成骨细胞增殖凋亡的调控作用研究。研究内容包括：①建立OB原代培养模型并进行鉴定；②实时荧光定量PCR和Western-blot对氟致OB凋亡过程中CKIP-1基因在 mRNA和蛋白水平的表达；③用MTT法、电镜技术研究OB凋亡过程中其增殖活力和形态学的变化；④流式细胞术分析OB增殖周期和凋亡的变化；⑤利用电子顺磁共振波谱仪测定OB凋亡过程中自由基的变化。通过以上研究，阐明CKIP-1在氟致成骨细胞增殖凋亡过程中发挥的作用，从而为CKIP-1经过干扰表达技术应用于临床防治氟骨症提供依据。

氟致成骨细胞出现凋亡，CKIP-1基因对成骨细胞凋亡具有正向调控作用。但是，CKIP-1对氟致成骨细胞凋亡的调控机理仍不清楚。本研究旨在通过RT-PCR、Western-blot、电镜技术和流式细胞仪等研究氟致成骨细胞凋亡过程中CKIP-1基因mRNA和蛋白水平表达；细胞增殖活力和形态改变；凋亡和自由基的变化。结果表明，小鼠CKIP-1基因特异性引物为CKIP-1-1，退火温度为58℃。CKIP-1的mRNA表达结果显示，三对siRNA-CKIP-1在沉默CKIP-1 24h和48h后，对CKIP-1mRNA相对表达量极显著的下降。优化Western Blot条件后，NaF对成骨细胞（MC3T3-E1）CKIP-1mRNA基因和蛋白表达的结果显示：1d，NaF浓度为10-5mol/L时，CKIP-1 mRNA的表达量极显著高于对照组；2d，各个NaF处理组mRNA的表达量极显著高于对照组，不同浓度间对mRNA的表达量未见明显差异。试验的第1天，10-4、5×10-4、5×10-5和10-5 mol/L组CKIP-1蛋白表达量极显著高于对照组；2d，10-3-5×10-5 mol/L组蛋白表达量显著高于对照组；3d和4d，各NaF处理组的蛋白表达量均极显著高于对照组。即NaF在1d、2d、3d和4d可以引起CKIP-1蛋白表达量的升高，并且在10-4-10-5 mol/L浓度NaF范围内，CKIP-1蛋白表达量与NaF浓度间没有显著的浓度依懒性。SiRNA沉默CKIP-1的沉默效率结果显示：1d、2d、3d和4d，siRNA沉默CKIP-1的效率分别达到50.9%、96.4%、52.1%和60.5%，其中以第2d的沉默效率最高，即siRNA沉默组CKIP-1 mRNA表达量极显著低于阴性对照组。沉默CKIP-1后NaF对MC3T3-E1CKIP-1基因表达的结果显示：对于1d和2d，siRNA沉默CKIP-1效率达到58.1%和58.5%，即对照组的mRNA表达量都极显著低于阴性对照组；在1d，10-3mol/L浓度NaF处理组mRNA的相对表达量极显著高于对照组；在2d，10-4mol/L浓度NaF处理组mRNA的相对表达量显著高于对照组。上述研究表明，CKIP-1在氟致成骨细胞凋亡过程中发挥促进作用，从而为CKIP-1干扰表达技术应用于临床防治氟骨症提供依据。