TRPM4通道抑制剂9-菲酚对OA软骨细胞作用的研究

发布时间：2018-07-14 21:08

【摘要】：目的:1、研究TRPM4(transient receptor potential M4)信号通道抑制剂—9-菲酚(9-Phenanthrenol)能否影响骨关节炎的进展,通过对软骨细胞的肥大分化的抑制及减少软骨组织的降解,实现缓解骨关节炎的目的;2、研究9-菲酚对软骨细胞凋亡的影响,为能否治疗骨关节炎提供实验依据。方法:1、选取行膝关节置换手术截下的骨关节炎(OA)患者的胫骨平台关节软骨,提取软骨细胞进行培养2、软骨细胞均分为6组,一组不加药作为对照组,其余分别加入不同量的9-菲酚使其浓度分别为5×10-6mol/l、l×10-5 mol/l、2×10-5mol/l、4×10-5 mol/l、8×10-5 mol/l。3、于9-菲酚干预1天培养后细胞进行RT-qPCR检测,检测指标为软骨细胞肥大指标MMP-13、Runx-2及软骨代谢相关指标Aggrecan、COL II;4、于9-菲酚干预1天培养后采用TUNEL染色荧光显微镜检测及Annexin V和7-AAD标记流式细胞仪检测9-菲酚对OA软骨细胞凋亡的影响情况。结果:1、9-菲酚通过抑制TRPM4离子通道对OA软骨细胞肥大分化和代谢影响的RT-qPCR结果:9-菲酚组在8×10-5 mol/l浓度时软骨细胞AGG较空白对照组表达水平显著降低(P0.05),其余浓度下均有下降趋势。同时9-菲酚组在8×10-5 mol/l浓度时软骨细胞COLII相对照组显著增高(P0.05)且除5×10-6mol/l组其它组较对照组均有上升趋势,故无法判断9-菲酚对软骨细胞代谢的影响;而软骨细胞肥大指标Runx-2、MMP13的mRNA较对照组表达水平有升高趋势,可见在mRNA水平上,9-菲酚可能促进软骨细胞肥大。2、9-菲酚对OA软骨细胞凋亡的影响:TUNEL染色荧光显微镜检测软骨细胞凋亡率发现,9-菲酚浓度为2×10-5mol/l组较对照组凋亡率明显下降(P0.05),其它浓度组与对照组比较均无统计学意义,但均有凋亡率下降的趋势。Annexin V和7-AAD标记流式细胞仪检测软骨细胞凋亡率发现,9-菲酚浓度为1×10-5mol/l、2×10-5mol/l、4×10-5mol/l时较对照组凋亡率均有所下降(P0.05),而在另外两组有下降趋势。结论:1、9-菲酚能促进OA软骨细胞的肥大分化。2、9-菲酚能抑制OA软骨细胞的凋亡,在OA治疗方面具有积极意义。
[Abstract]:Objective to investigate whether 9-Phenanthrenol (9-Phenanthrenol), a signal channel inhibitor of TRPM4 (transient receptor potential M4, can affect the progression of osteoarthritis by inhibiting the hypertrophic differentiation of chondrocytes and reducing the degradation of chondrocytes. Objective to alleviate osteoarthritis and study the effect of 9-phenanthroline on the apoptosis of chondrocytes in order to provide experimental evidence for the treatment of osteoarthritis. Methods: 1. Chondrocytes were extracted from osteoarthritis (OA) patients after knee arthroplasty and cultured for 2 years. Chondrocytes were divided into 6 groups. The others added different amounts of 9- phenanthroline to 5 脳 10-6 mol / L 1 脳 10 -5 mol / L 2 脳 10 ~ (-5) mol / L ~ (-1) 4 脳 10 ~ (-5) mol / L ~ (10 ~ (-5) mol / L ~ (-1), respectively. The cells were treated with 9-phenanthrene for one day and then detected by RT-qPCR. Chondrocyte hypertrophy index MMP-13 Runx-2 and chondrocyte metabolism related index Aggrecanconicol III4 were detected by Tunel staining fluorescence microscope and Annexin V and 7-AAD labeled flow cytometry after 1-day culture with 9-phenanthroline. The effect of apoptosis. Results the RT-qPCR results showed that the expression level of agg in chondrocytes in the control group was significantly lower than that in the control group at the concentration of 8 脳 10 ~ (-5) mol/l (P0.05). At the same time, at the concentration of 8 脳 10 ~ (-5) mol/l, the chondrocytes in the chondrocyte phase II phase control group were significantly increased (P0.05) and the other groups except the 5 脳 10-6mol/l group had an upward trend, so it was impossible to judge the effect of 9-phenanthrene on the chondrocyte metabolism. The expression of Runx-2mMP13 mRNA in chondrocyte hypertrophy was higher than that in control group. It can be seen that 9- phenanthrene may promote the apoptosis of chondrocyte hypertrophy on OA chondrocytes at the mRNA level; the apoptosis rate of chondrocytes detected by fluorescence microscope with 7% Tunel staining was higher than that of the control group with concentration of 2 脳 10-5mol/l. There was no significant difference between the other concentration groups and the control group (P0.05). But the apoptotic rate of chondrocytes was decreased by Annexin V and 7-AAD labeled flow cytometry. When the concentration of phenanthroline was 1 脳 10 ~ (-5) mol / L ~ (-1) 2 脳 10 ~ (-5) mol / L ~ (4) 脳 10-5mol/l, the apoptosis rate of chondrocytes decreased (P0.05). Conclusion 1: 1 phenanthrene can promote the hypertrophic differentiation of OA chondrocytes. 2 phenanthrene can inhibit the apoptosis of OA chondrocytes, which is of positive significance in the treatment of OA.
【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】：R684.3

【参考文献】