【摘要】：目的:1、研究TRPM4(transient receptor potential M4)信號通道抑制劑—9-菲酚(9-Phenanthrenol)能否影響骨關節(jié)炎的進展,通過對軟骨細胞的肥大分化的抑制及減少軟骨組織的降解,實現(xiàn)緩解骨關節(jié)炎的目的;2、研究9-菲酚對軟骨細胞凋亡的影響,為能否治療骨關節(jié)炎提供實驗依據(jù)。方法:1、選取行膝關節(jié)置換手術截下的骨關節(jié)炎(OA)患者的脛骨平臺關節(jié)軟骨,提取軟骨細胞進行培養(yǎng)2、軟骨細胞均分為6組,一組不加藥作為對照組,其余分別加入不同量的9-菲酚使其濃度分別為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-菲酚干預1天培養(yǎng)后細胞進行RT-qPCR檢測,檢測指標為軟骨細胞肥大指標MMP-13、Runx-2及軟骨代謝相關指標Aggrecan、COL II;4、于9-菲酚干預1天培養(yǎng)后采用TUNEL染色熒光顯微鏡檢測及Annexin V和7-AAD標記流式細胞儀檢測9-菲酚對OA軟骨細胞凋亡的影響情況。結(jié)果:1、9-菲酚通過抑制TRPM4離子通道對OA軟骨細胞肥大分化和代謝影響的RT-qPCR結(jié)果:9-菲酚組在8×10-5 mol/l濃度時軟骨細胞AGG較空白對照組表達水平顯著降低(P0.05),其余濃度下均有下降趨勢。同時9-菲酚組在8×10-5 mol/l濃度時軟骨細胞COLII相對照組顯著增高(P0.05)且除5×10-6mol/l組其它組較對照組均有上升趨勢,故無法判斷9-菲酚對軟骨細胞代謝的影響;而軟骨細胞肥大指標Runx-2、MMP13的mRNA較對照組表達水平有升高趨勢,可見在mRNA水平上,9-菲酚可能促進軟骨細胞肥大。2、9-菲酚對OA軟骨細胞凋亡的影響:TUNEL染色熒光顯微鏡檢測軟骨細胞凋亡率發(fā)現(xiàn),9-菲酚濃度為2×10-5mol/l組較對照組凋亡率明顯下降(P0.05),其它濃度組與對照組比較均無統(tǒng)計學意義,但均有凋亡率下降的趨勢。Annexin V和7-AAD標記流式細胞儀檢測軟骨細胞凋亡率發(fā)現(xiàn),9-菲酚濃度為1×10-5mol/l、2×10-5mol/l、4×10-5mol/l時較對照組凋亡率均有所下降(P0.05),而在另外兩組有下降趨勢。結(jié)論:1、9-菲酚能促進OA軟骨細胞的肥大分化。2、9-菲酚能抑制OA軟骨細胞的凋亡,在OA治療方面具有積極意義。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R684.3

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