發(fā)布時(shí)間：2018-09-01 10:07

【摘要】：目的·構(gòu)建可用于發(fā)現(xiàn)調(diào)節(jié)維甲酸受體(retinoic acid receptors,RARα)蛋白穩(wěn)定性的化合物的細(xì)胞篩選體系。方法·對(duì)pMSCV質(zhì)粒進(jìn)行改造,插入綠色熒光蛋白(EGFP)-RARα融合基因和紅色熒光蛋白(Ds Red)基因,兩者之間以內(nèi)部核糖體結(jié)合位點(diǎn)(IRES)序列分隔;構(gòu)建成功的質(zhì)粒穩(wěn)定轉(zhuǎn)染急性早幼粒細(xì)胞白血病細(xì)胞NB4,流式細(xì)胞術(shù)分析全反式維甲酸、丙戊酸鈉、阿糖胞苷、來(lái)那度胺、依托泊苷、孟魯司特納及藤黃酸處理細(xì)胞后EGFP與DsRed的熒光信號(hào)的變化,間接反映RARα蛋白表達(dá)水平;并通過Western blotting實(shí)驗(yàn)進(jìn)一步驗(yàn)證陽(yáng)性化合物對(duì)RARα蛋白水平的影響。結(jié)果·成功構(gòu)建了蛋白穩(wěn)定性雙熒光篩選體系,并發(fā)現(xiàn)丙戊酸鈉可有效使RARα蛋白的表達(dá)水平穩(wěn)定。結(jié)論·雙熒光篩選系統(tǒng)可用于調(diào)節(jié)RARα蛋白穩(wěn)定性的化合物的篩選。丙戊酸鈉是一個(gè)新的穩(wěn)定RARα的化合物。該方法對(duì)建立穩(wěn)定其他蛋白的化合物篩選系統(tǒng)具有參考價(jià)值。
[Abstract]:Objective to construct a cell screening system that can be used to identify compounds that regulate the stability of retinoic acid receptor (retinoic acid receptors,RAR 偽. Methods pMSCV plasmid was modified and inserted into (EGFP) RAR 偽 fusion gene and red fluorescent protein (Ds Red) gene, separated by internal ribosomal binding site (IRES) sequence. NB4, flow cytometry analysis of all trans retinoic acid, sodium valproate, cytarabine, linalamine, etoposide was successfully constructed and transfected into acute promyelocytic leukemia cells by flow cytometry. The changes of fluorescence signals of EGFP and DsRed after treated with mongolu and luteinic acid indirectly reflected the expression level of RAR 偽 protein, and the effect of positive compounds on RAR 偽 protein level was further verified by Western blotting experiment. Results the double fluorescence screening system of protein stability was successfully constructed, and it was found that sodium valproate could effectively stabilize the expression of RAR 偽 protein. Conclusion double fluorescence screening system can be used to screen compounds that regulate the stability of RAR 偽 protein. Sodium valproate is a new stable compound of RAR 偽. This method has reference value for the establishment of screening system for stabilizing other proteins.
【作者單位】： 上海交通大學(xué)醫(yī)學(xué)院病理生理學(xué)教研室細(xì)胞分化與凋亡教育部重點(diǎn)實(shí)驗(yàn)室;上海市瑞金康復(fù)醫(yī)院;
【基金】：國(guó)家自然科學(xué)基金(81570118) 上海市科委資助項(xiàng)目(15401901800) 國(guó)家大學(xué)生創(chuàng)新性實(shí)驗(yàn)計(jì)劃項(xiàng)目(201110248075)~~
【分類號(hào)】：R3411
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本文編號(hào)：2216870