【摘要】：目的:通過體外實驗觀察自然殺傷(NK)細(xì)胞聯(lián)合他莫昔芬(TAM)對乳腺癌細(xì)胞(BCC)的協(xié)同殺傷作用,并初步探究其作用機(jī)制。方法:1.選用表面受體表達(dá)情況不同的三種人乳腺癌細(xì)胞系,即ER+、PR+、HER-2 乳腺癌細(xì)胞系MCF-7,ER+、PR+、HER-2+乳腺癌細(xì)胞系BT-474及ER 、PR 、HER-2 乳腺癌細(xì)胞系MDA-MB-231;MTT法檢測TAM對乳腺癌細(xì)胞及其NK細(xì)胞增殖抑制作用。2.鈣黃綠素-AM釋放法檢測NK細(xì)胞聯(lián)合TAM對乳腺癌細(xì)胞的殺傷作用。3.流式分析法分別檢測NK表面活化性受體NKp44、NKp46、NKG2D,以及抑制性受體CD158a、CD158b、CD158b2、CD158e表達(dá)水平;乳腺癌細(xì)胞表面NK活化性配體MICA、ULBP1、ULBP2表達(dá)水平;以及NK細(xì)胞內(nèi)穿孔素及顆粒酶B的釋放量。4.ELISA法檢測NK細(xì)胞分泌TNF-α和IFN-γ的水平變化。結(jié)果:1.TAM能夠抑制MCF-7及BT-474的增殖,對MCF-7的增殖抑制作用強(qiáng)于BT-474,P0.05;能抑制MDA-MB-231的增殖,但抑制作用較MCF-7及BT-474弱,P0.05;對三種乳腺癌細(xì)胞的增殖抑制率具有明顯的濃度-時間依賴性,P0.05。2.確定TAM最終實驗濃度為5μM作用24h。TAM對NK細(xì)胞無明顯增殖抑制作用,故聯(lián)合實驗時可與TAM同時應(yīng)用。二者具有協(xié)同殺傷作用,聯(lián)合組的殺傷率明顯高于單獨NK細(xì)胞及TAM組,P0.05。3.與TAM聯(lián)合后,NK活化性受體NKp46表達(dá)水平升高,抑制性受體CD158a、CD158b、CD158b2、CD158e表達(dá)水平下降,乳腺癌細(xì)胞表面NK活化性配體MICA、ULBP1、ULBP2表達(dá)水平升高,P0.05;各組NK細(xì)胞內(nèi)釋放穿孔素及顆粒酶增加不明顯,且基礎(chǔ)釋放量較高。4.無論有無乳腺癌細(xì)胞存在,與TAM聯(lián)合后NK細(xì)胞分泌細(xì)胞因子TNF-α和IFN-γ的水平均存在不同程度升高,P0.05。結(jié)論:1.TAM能夠抑制兩種激素受體陽性的乳腺癌細(xì)胞的增殖,且對HER-2 細(xì)胞的抑制作用強(qiáng)于對HER-2+細(xì)胞;也可以抑制激素受體陰性乳腺癌細(xì)胞的增殖,但抑制作用較激素受體陽性細(xì)胞弱;對三種乳腺癌細(xì)胞的增殖抑制率具有明顯的濃度-時間依賴性。2.NK細(xì)胞與TAM聯(lián)合體外殺傷乳腺癌細(xì)胞具有協(xié)同作用,且隨效靶比增加其協(xié)同作用隨之增強(qiáng)。3.通過實驗分析其協(xié)同作用機(jī)制可能為:1)TAM可以通過增加NK細(xì)胞活化性受體及乳腺癌細(xì)胞表面NK活化性配體,并降低NK細(xì)胞抑制性受體的表達(dá)量,進(jìn)而使NK細(xì)胞的殺傷能力增加;2)TAM也能夠通過增加NK細(xì)胞分泌TNF-α及IFN-γ而增強(qiáng)其殺傷能力。
[Abstract]:Aim: to observe the synergistic killing effect of natural killer (NK) cells combined with tamoxifen (TAM) on breast cancer cells (BCC) in vitro and to explore its mechanism. Method 1: 1. Three human breast cancer cell lines with different surface receptor expression were selected. The breast cancer cell line BT-474 and the breast cancer cell line MDA-MB-231 were detected by MTT assay. The inhibitory effect of TAM on the proliferation of breast cancer cells and their NK cells was determined by MTT assay. The cytotoxicity of NK cells combined with TAM on breast cancer cells was detected by calcitonin-AM release assay. Flow cytometry was used to detect the expression of NK activated receptor NKp44-NKp46-NKG2D and inhibitory receptor CD158b ~ + CD158b ~ (2 +) CD158b ~ (2 +), and the expression of NK activated ligand MICAULBP1 / ULBP2 on breast cancer cells. The release of perforin and granzyme B in NK cells. 4. The levels of TNF- 偽 and IFN- 緯 secreted by NK cells were detected by Elisa. Results: 1. TAM could inhibit the proliferation of MCF-7 and BT-474, the inhibitory effect of TAM on MCF-7 was stronger than that of BT-474P0.05, the inhibition of MDA-MB-231 was weaker than that of MCF-7 and BT-474, and the inhibition rate of MCF-7 and BT-474 was obviously dose-time dependent (P0.05.2). The final concentration of TAM was 5 渭 M for 24 h. TAM had no obvious inhibitory effect on NK cell proliferation, so the combination of TAM and TAM could be used at the same time. The killing rate of the combined group was significantly higher than that of the single NK cell and TAM group (P0.05.3). The expression of NK activated receptor NKp46 was increased in combination with TAM, the expression level of inhibitory receptor CD158bnc CD158b ~ (2 +) CD158b ~ (2) was decreased, and the expression of NK activated ligand MICAULBP1 / ULBP2 was increased in breast cancer cells (P < 0.05), but the release of perforin and granzyme in NK cells was not significantly increased in each group. And the basal release amount was higher. 4. In combination with TAM, the levels of TNF- 偽 and IFN- 緯 in NK cells increased in varying degrees with or without breast cancer cells. Conclusion: 1. Tam can inhibit the proliferation of two steroid-receptor positive breast cancer cells, and inhibit the proliferation of HER-2 cells more strongly than HER-2 cells, but also inhibit the proliferation of steroid-receptor negative breast cancer cells. But the inhibitory effect was weaker than that of hormone receptor positive cells, and the inhibition rate of proliferation of three kinds of breast cancer cells was obviously concentration-time dependent. 2. NK cells had synergistic effect with TAM combination on killing breast cancer cells. The synergistic effect increased with the increase of the target ratio. The synergistic mechanism of Tam may be: 1) TAM can increase NK cell activated receptor and NK activated ligand on breast cancer cell surface, and decrease the expression of NK cell inhibitory receptor. TAM could also enhance the cytotoxicity of NK cells by increasing the secretion of TNF- 偽 and IFN- 緯.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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