自然杀伤细胞联合他莫昔芬体外对乳腺癌细胞的杀伤作用及其机制
发布时间：2018-07-17 05:44
【摘要】：目的:通过体外实验观察自然杀伤(NK)细胞联合他莫昔芬(TAM)对乳腺癌细胞(BCC)的协同杀伤作用,并初步探究其作用机制。方法:1.选用表面受体表达情况不同的三种人乳腺癌细胞系,即ER+、PR+、HER-2 乳腺癌细胞系MCF-7,ER+、PR+、HER-2+乳腺癌细胞系BT-474及ER 、PR 、HER-2 乳腺癌细胞系MDA-MB-231;MTT法检测TAM对乳腺癌细胞及其NK细胞增殖抑制作用。2.钙黄绿素-AM释放法检测NK细胞联合TAM对乳腺癌细胞的杀伤作用。3.流式分析法分别检测NK表面活化性受体NKp44、NKp46、NKG2D,以及抑制性受体CD158a、CD158b、CD158b2、CD158e表达水平;乳腺癌细胞表面NK活化性配体MICA、ULBP1、ULBP2表达水平;以及NK细胞内穿孔素及颗粒酶B的释放量。4.ELISA法检测NK细胞分泌TNF-α和IFN-γ的水平变化。结果:1.TAM能够抑制MCF-7及BT-474的增殖,对MCF-7的增殖抑制作用强于BT-474,P0.05;能抑制MDA-MB-231的增殖,但抑制作用较MCF-7及BT-474弱,P0.05;对三种乳腺癌细胞的增殖抑制率具有明显的浓度-时间依赖性,P0.05。2.确定TAM最终实验浓度为5μM作用24h。TAM对NK细胞无明显增殖抑制作用,故联合实验时可与TAM同时应用。二者具有协同杀伤作用,联合组的杀伤率明显高于单独NK细胞及TAM组,P0.05。3.与TAM联合后,NK活化性受体NKp46表达水平升高,抑制性受体CD158a、CD158b、CD158b2、CD158e表达水平下降,乳腺癌细胞表面NK活化性配体MICA、ULBP1、ULBP2表达水平升高,P0.05;各组NK细胞内释放穿孔素及颗粒酶增加不明显,且基础释放量较高。4.无论有无乳腺癌细胞存在,与TAM联合后NK细胞分泌细胞因子TNF-α和IFN-γ的水平均存在不同程度升高,P0.05。结论:1.TAM能够抑制两种激素受体阳性的乳腺癌细胞的增殖,且对HER-2 细胞的抑制作用强于对HER-2+细胞;也可以抑制激素受体阴性乳腺癌细胞的增殖,但抑制作用较激素受体阳性细胞弱;对三种乳腺癌细胞的增殖抑制率具有明显的浓度-时间依赖性。2.NK细胞与TAM联合体外杀伤乳腺癌细胞具有协同作用,且随效靶比增加其协同作用随之增强。3.通过实验分析其协同作用机制可能为:1)TAM可以通过增加NK细胞活化性受体及乳腺癌细胞表面NK活化性配体,并降低NK细胞抑制性受体的表达量,进而使NK细胞的杀伤能力增加;2)TAM也能够通过增加NK细胞分泌TNF-α及IFN-γ而增强其杀伤能力。
[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- 伪 and IFN- 纬 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- 伪 and IFN- 纬 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- 伪 and IFN- 纬.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
本文编号：2129269
[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- 伪 and IFN- 纬 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- 伪 and IFN- 纬 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- 伪 and IFN- 纬.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
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