發(fā)布時(shí)間：2018-07-14 21:08

【摘要】：目的:1、研究TRPM4(transient receptor potential M4)信號(hào)通道抑制劑—9-菲酚(9-Phenanthrenol)能否影響骨關(guān)節(jié)炎的進(jìn)展,通過(guò)對(duì)軟骨細(xì)胞的肥大分化的抑制及減少軟骨組織的降解,實(shí)現(xiàn)緩解骨關(guān)節(jié)炎的目的;2、研究9-菲酚對(duì)軟骨細(xì)胞凋亡的影響,為能否治療骨關(guān)節(jié)炎提供實(shí)驗(yàn)依據(jù)。方法:1、選取行膝關(guān)節(jié)置換手術(shù)截下的骨關(guān)節(jié)炎(OA)患者的脛骨平臺(tái)關(guān)節(jié)軟骨,提取軟骨細(xì)胞進(jìn)行培養(yǎng)2、軟骨細(xì)胞均分為6組,一組不加藥作為對(duì)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(yù)1天培養(yǎng)后細(xì)胞進(jìn)行RT-qPCR檢測(cè),檢測(cè)指標(biāo)為軟骨細(xì)胞肥大指標(biāo)MMP-13、Runx-2及軟骨代謝相關(guān)指標(biāo)Aggrecan、COL II;4、于9-菲酚干預(yù)1天培養(yǎng)后采用TUNEL染色熒光顯微鏡檢測(cè)及Annexin V和7-AAD標(biāo)記流式細(xì)胞儀檢測(cè)9-菲酚對(duì)OA軟骨細(xì)胞凋亡的影響情況。結(jié)果:1、9-菲酚通過(guò)抑制TRPM4離子通道對(duì)OA軟骨細(xì)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(shí)軟骨細(xì)胞AGG較空白對(duì)照組表達(dá)水平顯著降低(P0.05),其余濃度下均有下降趨勢(shì)。同時(shí)9-菲酚組在8×10-5 mol/l濃度時(shí)軟骨細(xì)胞COLII相對(duì)照組顯著增高(P0.05)且除5×10-6mol/l組其它組較對(duì)照組均有上升趨勢(shì),故無(wú)法判斷9-菲酚對(duì)軟骨細(xì)胞代謝的影響;而軟骨細(xì)胞肥大指標(biāo)Runx-2、MMP13的mRNA較對(duì)照組表達(dá)水平有升高趨勢(shì),可見(jiàn)在mRNA水平上,9-菲酚可能促進(jìn)軟骨細(xì)胞肥大。2、9-菲酚對(duì)OA軟骨細(xì)胞凋亡的影響:TUNEL染色熒光顯微鏡檢測(cè)軟骨細(xì)胞凋亡率發(fā)現(xiàn),9-菲酚濃度為2×10-5mol/l組較對(duì)照組凋亡率明顯下降(P0.05),其它濃度組與對(duì)照組比較均無(wú)統(tǒng)計(jì)學(xué)意義,但均有凋亡率下降的趨勢(shì)。Annexin V和7-AAD標(biāo)記流式細(xì)胞儀檢測(cè)軟骨細(xì)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(shí)較對(duì)照組凋亡率均有所下降(P0.05),而在另外兩組有下降趨勢(shì)。結(jié)論:1、9-菲酚能促進(jìn)OA軟骨細(xì)胞的肥大分化。2、9-菲酚能抑制OA軟骨細(xì)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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R684.3

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