【摘要】：目的檢測腫瘤壞死因子α(TNF-α)對MCF-7和MDA-MB-231人乳腺癌細(xì)胞增殖的影響及神經(jīng)酰胺(Cer)的介導(dǎo)作用。方法首先確定TNF-α的最佳處理劑量和時間,免疫熒光細(xì)胞化學(xué)染色結(jié)合共聚焦顯微鏡技術(shù)檢測細(xì)胞Cer含量的變化,采用50 ng/mL TNF-α單獨或聯(lián)合ASM抑制劑地昔帕明(Des)、神經(jīng)鞘氨醇激酶1(SPHK1)抑制劑N,N,-二甲基鞘氨醇(DMS)處理乳腺癌細(xì)胞,檢測其在TNF-α所致細(xì)胞增殖變化中的作用。采用MTT法檢測細(xì)胞增殖;Western blot法檢測酸性鞘磷脂酶(ASM)、SPHK1及凋亡與增殖相關(guān)蛋白Bcl2、Bax、增殖細(xì)胞核抗原(PCNA)表達(dá)。結(jié)果 TNF-α可抑制MCF-7細(xì)胞增殖,促進(jìn)MDA-MB-231細(xì)胞增殖,且具劑量和時間依賴性;在MCF-7細(xì)胞,TNF-α可顯著增加ASM蛋白表達(dá),對SPHK1蛋白表達(dá)無明顯影響,而在MDA-MB-231細(xì)胞,可同時增加ASM和SPHK1蛋白表達(dá);TNF-α作用后,MCF-7細(xì)胞Cer含量顯著增加,ASM抑制劑Des預(yù)處理可抑制此作用;TNF-α作用可降低MDA-MB-231細(xì)胞Cer含量,此作用可被SPHK1抑制劑DMS所阻止;Des可抑制TNF-α作用所導(dǎo)致的MCF-7細(xì)胞增殖降低、Bcl2表達(dá)降低、Bax表達(dá)增加;DMS可抑制TNF-α作用所導(dǎo)致的MDA-MB-231細(xì)胞PCNA和細(xì)胞增殖指數(shù)的高表達(dá)。結(jié)論 TNF-α通過調(diào)節(jié)ASM和SPHK1蛋白的表達(dá)水平調(diào)控Cer生成,影響MCF-7和MDA-MB-231人乳腺癌細(xì)胞的增殖。
[Abstract]:Objective to investigate the effect of tumor necrosis factor 偽 (TNF- 偽) on the proliferation of MCF-7 and MDA-MB-231 human breast cancer cells and the mediated effect of ceramide (Cer). Methods the optimal treatment dose and time of TNF- 偽 were first determined. The changes of Cer content in cells were detected by immunofluorescence cytochemical staining combined with confocal microscopy. 50 ng/mL TNF- 偽 was used alone or in combination with ASM inhibitor desipramine (Des),. Breast cancer cells were treated with neurinosinolkinase-1 (SPHK1) inhibitor, NNN-dimethyl-sphingosine (DMS), and their role in the proliferation of breast cancer cells induced by TNF- 偽 was examined. The expression of Bcl2,Bax, proliferating cell nuclear antigen (PCNA) and apoptosis and sphingomyelinase (ASM), SPHK1) were detected by MTT assay by; Western blot assay. Results TNF- 偽 inhibited the proliferation of MCF-7 cells and promoted the proliferation of MDA-MB-231 cells in a dose-and time-dependent manner. In MCF-7 cells, TNF- 偽 could significantly increase the expression of ASM protein, but had no effect on the expression of SPHK1 protein, while in MDA-MB-231 cells, it could increase the expression of ASM and SPHK1 simultaneously. After the treatment of TNF- 偽, the content of Cer in MCF-7 cells increased significantly, which was inhibited by Des pretreatment with ASM inhibitor Des, and the Cer content of MDA-MB-231 cells was decreased by TNF- 偽, which was blocked by DMS, a SPHK1 inhibitor. Des inhibited the proliferation of MCF-7 cells induced by TNF- 偽, decreased the expression of Bcl2 and increased the expression of Bax, and DMS inhibited the high expression of PCNA and proliferation index of MDA-MB-231 cells induced by TNF- 偽. Conclusion TNF- 偽 affects the proliferation of MCF-7 and MDA-MB-231 human breast cancer cells by regulating the expression of ASM and SPHK1 protein.
【作者單位】： 空軍軍醫(yī)大學(xué)西京醫(yī)院甲乳血管外科;
【基金】：國家自然科學(xué)基金面上項目(81470120,81672593)
【分類號】：R737.9

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