本文選題：食欲素受體 + 膽囊收縮素受體�。� 參考：《中國(guó)生物化學(xué)與分子生物學(xué)報(bào)》2017年08期

【摘要】：食欲素1受體(orexin 1 receptor,OX1R)與膽囊收縮素2受體(cholecystokinin receptor,CCK2R)在結(jié)腸癌細(xì)胞中高表達(dá),且異常表達(dá)的OX1R、CCK2R與其配體誘導(dǎo)的結(jié)腸癌細(xì)胞增殖密切相關(guān),但具體機(jī)制尚不清楚。以前的研究證實(shí),OX1R與CCK1R在HT-29細(xì)胞中能以二聚體的形式發(fā)揮作用。本文利用多種(熒光)共振能量轉(zhuǎn)移技術(shù)(FRET)結(jié)合免疫共沉淀(Co-IP),進(jìn)一步研究活細(xì)胞中OX1R與CCK2R是否發(fā)生相互作用。生物發(fā)光能量共振轉(zhuǎn)移(BRET)結(jié)果顯示,在控制供體(OX1R-Rluc)量不變,而逐漸增加受體(CCK2R-e YFP)轉(zhuǎn)染量時(shí),與無(wú)刺激的(對(duì)照)細(xì)胞比較,食欲素或胃泌素刺激HEK293T細(xì)胞5 min,BRET信號(hào)伴隨受體表達(dá)量的增加而增加,并達(dá)到最大值。采用熒光共振能量轉(zhuǎn)移技術(shù)在HEK293T細(xì)胞中,能夠檢測(cè)到OX1R-e YFP與CCK2Re CFP明顯的FRET信號(hào)。同時(shí),受體漂白FRET(ap FRET)結(jié)果揭示,在同時(shí)表達(dá)OX1R-e YFP和CCK2R-e CFP的細(xì)胞膜特定區(qū)域,進(jìn)行受體蛋白(OX1R-e YFP)完全光漂白、破壞了受體-供體之間的相互作用和能量傳遞后,由于供體(CCK2R-e CFP)熒光強(qiáng)度比漂白前明顯增強(qiáng),其熒光共振能量轉(zhuǎn)移效率(FREPe)明顯增加,是對(duì)照轉(zhuǎn)染細(xì)胞的3.7倍(P0.05)。此外,基因轉(zhuǎn)染結(jié)合Co-IP結(jié)果顯示,僅有在共轉(zhuǎn)染HA-OX1R與Myc-CCK2R的HEK293T細(xì)胞提取液的免疫沉淀物中,可同時(shí)檢出HA-OX1R、Myc-CCK2R融合蛋白,而在未轉(zhuǎn)染或單轉(zhuǎn)Myc-CCK2R的細(xì)胞提取液沉淀物中,卻不能同時(shí)檢出兩種融合蛋白。以上結(jié)果表明,在活細(xì)胞生理?xiàng)l件下,OX1R可與CCK2R相互作用,這為進(jìn)一步探討二者相互作用在結(jié)腸癌細(xì)胞增殖中的作用及相關(guān)信號(hào)通路提供了新的線索。
[Abstract]:Orexin 1 receptor (OX1R) and cholecystokinin receptor (CCK2R) were highly expressed in colon cancer cells, and the abnormal expression of OX1R- CCK2R was closely related to the proliferation of colon cancer cells induced by its ligand, but the specific mechanism was not clear. Previous studies have confirmed that OX1R and CCK1R can act as dimers in HT-29 cells. In this paper, a variety of (fluorescence) resonance energy transfer techniques (fret) combined with immunoprecipitation Co-IPP were used to further study the interaction between OX1R and CCK2R in living cells. The results of Bioluminescence Energy Resonance transfer (BRET) showed that when the amount of OX1R-Rluca was controlled and the amount of CCK2R-e YFPtransfected was increased gradually, it was compared with that of unstimulated (control) cells. After stimulation of orexin or gastrin, the BRET signal of HEK293T cells increased with the increase of receptor expression at 5 min, and reached the maximum value. Fluorescence resonance energy transfer technique was used to detect the FRET signals of OX1R-e YFP and CCK2Re CFP in HEK293T cells. At the same time, the results of receptor bleaching FRET(ap fret revealed that the receptor protein OX1R-e YFP-induced complete photobleaching of specific regions of the cell membrane expressing OX1R-e YFP and CCK2R-e CFP at the same time, which destroyed the receptor-donor interaction and energy transfer. The fluorescence intensity of donor CCK2R-e CFP was significantly higher than that before bleaching, and the fluorescence resonance energy transfer efficiency (FREP) of CCK2R-e CFP was significantly increased, which was 3.7 times as much as that of control transfected cells (P0.05). In addition, the results of gene transfection combined with Co-IP showed that HA-OX1Rnc-CCK2R fusion protein could be detected at the same time in the immunoprecipitation of HEK293T cell extracts cotransfected with Myc-CCK2R, while in the untransfected or single-transfected Myc-CCK2R cell extracts, HA-OX1Rnc-CCK2R fusion protein could be detected at the same time. However, two fusion proteins could not be detected at the same time. These results suggest that OX1R can interact with CCK2R under living cell physiological conditions, which provides a new clue to further study the role of OX1R in colon cancer cell proliferation and related signal pathways.
【作者單位】： 濟(jì)寧醫(yī)學(xué)院神經(jīng)生物研究所;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(No.3097108;No.816712276;No.31271243;No.81070961) 山東省自然科學(xué)基金項(xiàng)目(No.ZR2014HL404) 山東省高�？萍加�(jì)劃項(xiàng)目(No.JY2015KJ004;No.2015-57-6)~~
【分類號(hào)】：R735.35

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