【摘要】：目的本實(shí)驗(yàn)主要在細(xì)胞水平探討親環(huán)蛋白A是否通過調(diào)控自噬水平促進(jìn)氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)誘導(dǎo)RAW264.7單核巨噬細(xì)胞的活化與凋亡。方法體外培養(yǎng)小鼠巨噬細(xì)胞RAW264.7,應(yīng)用蛋白質(zhì)印跡法(Western blot)檢測親環(huán)蛋白A、β-actin、LC3、beclin-1、bcl-2、ABCA1及CD36的蛋白表達(dá);ELISA法檢測細(xì)胞培養(yǎng)上清中親環(huán)蛋白A的蛋白含量;應(yīng)用小干擾RNA沉默親環(huán)蛋白A的表達(dá),并應(yīng)用western blot檢測沉默效果;應(yīng)用電鏡觀察細(xì)胞內(nèi)自噬體的形成;應(yīng)用GFP-RFP-LC3病毒感染細(xì)胞觀察自噬流強(qiáng)弱;應(yīng)用油紅染色觀察細(xì)胞內(nèi)脂質(zhì)沉積;應(yīng)用流式細(xì)胞術(shù)(Flow Cytometry,FCM)檢測細(xì)胞表面CD80、CD86及MHCII的陽性率;應(yīng)用Tunel法檢測細(xì)胞凋亡率。結(jié)果(1)ox-LDL(100mg/L)刺激24h后RAW264.7細(xì)胞親環(huán)蛋白A蛋白水平顯著升高,在6h達(dá)到高峰(p0.05);細(xì)胞培養(yǎng)上清中親環(huán)蛋白A蛋白水平在24h內(nèi)隨時間梯度上升(p0.05)。(2)應(yīng)用轉(zhuǎn)染小干擾RNA沉默親環(huán)蛋白A表達(dá)后,再予ox-LDL刺激0,6,12及24h后,親環(huán)蛋白A沉默組中自噬相關(guān)蛋白LC3II/I水平較陰性對照組顯著降低(p0.05);Beclin-1水平?jīng)]有在兩組間沒有顯著差異;電鏡觀察,親環(huán)蛋白A沉默組細(xì)胞內(nèi)自噬體顯著降低于陰性對照組(p0.05)。(3)預(yù)先感染GFP-RFP-LC3腺病毒后,再用ox-LDL干預(yù)24h,親環(huán)蛋白A沉默組的細(xì)胞中GFP—RFP+光斑數(shù)(自噬溶酶體)較陰性對照組顯著升高(p0.05)。(4)ox-LDL干預(yù)24h后,親環(huán)蛋白A沉默組的ABCA1蛋白水平顯著升高,CD36蛋白水平顯著降低,與陰性對照組相比(p0.05);親環(huán)蛋白A沉默組細(xì)胞內(nèi)脂質(zhì)沉積較陰性對照組顯著降低(p0.05)。(5)ox-LDL干預(yù)24h后,Cy PA沉默組細(xì)胞CD80、CD86及MHCII陽性率較陰性對照組顯著降低(p0.05),而當(dāng)提前使用自噬抑制劑3-MA預(yù)刺激1h后再予ox-LDL干預(yù)后,兩組間CD86及MHCII的無顯著差異(p0.05)。(6)Ox-LDL干預(yù)24h后,親環(huán)蛋白A沉默組細(xì)胞凋亡率較陰性對照組顯著降低(p0.05),而當(dāng)提前使用自噬抑制劑3-MA預(yù)刺激1h后再予ox-LDL干預(yù)后,兩組間細(xì)胞凋亡率無顯著差異(p0.05)。結(jié)論(1)ox-LDL可以刺激RAW264.7細(xì)胞親環(huán)蛋白A表達(dá)升高并分泌到細(xì)外。(2)親環(huán)蛋白A促進(jìn)ox-LDL誘導(dǎo)RAW264.7細(xì)胞的自噬,抑制自噬流。(3)親環(huán)蛋白A促進(jìn)ox-LDL干預(yù)后RAW264.7細(xì)胞內(nèi)脂質(zhì)沉積。(4)親環(huán)蛋白A通過調(diào)控自噬促進(jìn)ox-LDL誘導(dǎo)RAW264.7細(xì)胞的活化及凋亡。
[Abstract]:Aim to investigate whether cyclin A can promote the activation and apoptosis of RAW264.7 mononuclear macrophages by regulating autophagy level and promoting oxidized low density lipoprotein (oxidized low density lipoprotein,ox-LDL). Methods the expression of cyclin A, 尾-actin,LC3,beclin-1,bcl-2,ABCA1 and CD36 in murine macrophages cultured in vitro was detected by Western blot (Western blot). ELISA method was used to detect the protein content of cyclophile protein A in the supernatant of cell culture, small interference RNA was used to silence the expression of cyclin A and western blot was used to detect the silencing effect, and electron microscope was used to observe the formation of autophagy. The intensity of autophagy was observed by GFP-RFP-LC3 virus infection, the lipid deposition was observed by oil red staining, and the positive rates of CD80,CD86 and MHCII on cell surface were detected by flow cytometry (Flow Cytometry,FCM). Apoptosis rate was detected by Tunel assay. Results (1) after ox-LDL (100mg/L) stimulation for 24 h, the level of cyclin A protein in RAW264.7 cells increased significantly and reached its peak at 6 h (p0.05). The level of cyclin A protein in the supernatant of cell culture increased with time gradient (p0.05). (2) within 24 hours. After transfection of small interfering RNA, the expression of cyclin A was silenced, and then stimulated by ox-LDL for 12 and 24 hours. The level of autophagy associated protein LC3II/I in the silencing group was significantly lower than that in the negative control group (p0.05). There was no significant difference in Beclin-1 level between the two groups. Electron microscope observation showed that the intracellular autophagy in the cyclin A silencing group was significantly lower than that in the negative control group (p0.05). (3) after preinfection with GFP-RFP-LC3 adenovirus, and then treated with ox-LDL for 24 h. The number of GFP-RFP spots (autophagy lysosomes) in the cells of the cyclophile A silencing group was significantly higher than that of the negative control group (p0.05). (4) ox-LDL for 24 h, and the ABCA1 protein level in the cyclin A silencing group was significantly higher than that in the negative control group (p0.05). (4). The level of CD36 protein was significantly lower than that of negative control group (p0. 05). The intracellular lipid deposition in cyclophile A silencing group was significantly lower than that in the negative control group (p0.05). (5). The positive rates of CD80,CD86 and MHCII in the, Cy PA silencing group were significantly lower than those in the negative control group after 24 hours of ox-LDL intervention (p0.05). However, there was no significant difference in CD86 and MHCII between the two groups after pretreatment with autophagy inhibitor 3-MA for 1 h and then ox-LDL for 24 h (p0.05). (6) Ox-LDL intervention. The apoptosis rate of cyclin A silencing group was significantly lower than that of negative control group (p0.05), but there was no significant difference in apoptosis rate between the two groups after pretreatment with 3-MA for 1 hour and then with ox-LDL (p0.05). Conclusion (1) ox-LDL can stimulate the expression of cyclin A in RAW264.7 cells and secrete to the outside. (2) Cyclophile A can promote the autophagy of RAW264.7 cells induced by ox-LDL. Inhibition of autophagy. (3) Cyclophile A promoted lipid deposition in RAW264.7 cells after ox-LDL intervention. (4) Cyclophile A promoted the activation and apoptosis of RAW264.7 cells induced by ox-LDL by regulating autophagy.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R54

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