【作者】 张丽平；

【导师】 叶韵斌；

【作者基本信息】 福建医科大学 ， 免疫学， 2008， 硕士

【摘要】 【目的】分析T-reg细胞在乳腺癌病人中的表达情况及其与NK细胞、T细胞亚群之间的关系;探讨体外富集的T-reg对NK细胞杀伤乳腺癌细胞的影响;探讨NKG2D-MICA在NK细胞杀伤乳腺癌中的作用;观察T-reg对NKG2D-MICA作用的影响。【方法】1、流式细胞术检测乳腺癌患者外周血T-reg的比例;探讨乳腺癌患者TGF-β1的变化。2、应用LDH法检测NK细胞对四种乳腺癌细胞株杀伤率活性。3、ELISA检测培养上清液中IFN-γ和TGF-β1的水平4、荧光定量PCR检测四种乳腺癌细胞株MICA-mRNA的表达。5、应用流式细胞术检测四种乳腺癌细胞株表面及胞内MICA的表达;6、应用免疫组化技术和免疫荧光技术检测四种乳腺癌细胞株MICA的分布情况。7、应用FCM检测NK细胞表面的NKG2D的变化。【结果】1、乳腺癌病人T-reg的表达:乳腺癌病人和健康志愿者外周血中T-reg占CD4+T细胞总数的百分比分别是(7.5±3.0)%和(5.1±1.5)%,前者高于后者(P<0.01)。2、当NK细胞与乳腺癌细胞效靶比为10:1时,对MCF-7、MDA-MB-435s、SKBr-3和MDA-MB-231的杀伤活性分别为(40.7±3.0)%、(15.5±3.1)%、(29.7±1.8)%以及59.1±2.3)%;加入T-reg的数量为1/5的NK细胞时,杀伤活性分别降至(1.9±0.2)%、(5.7±0.4)%、(3.6±2.8)%以及(3.9±0.2)%;加入T-reg的数量为1/10的NK细胞时,杀伤活性分别降至(7.6±1.6)%、(7.3±1.0)%、(6.6±2.5)%以及(19.7±1.1)%。3、ELISA检测NK细胞分泌的IFN-γ结果显示:在无T-reg存在时,NK细胞与MCF-7、MDA-MB-435s、SKBr-3和MDA-MB-231共培养时IFN-γ浓度分别为(221.5±2.6)pg/ml、(264.4±5.5)pg/ml、(754.6±19.0)pg/ml以及(423.0±14.6)pg/ml;加入T-reg的数量为1/5的NK细胞时,IFN-γ浓度分别为(70.7±8.5)pg/ml、(14.6±0.9)pg/ml、(128.3±4.0)pg/ml以及(6.0±1.0)pg/ml;加入T-reg的数量为1/10的NK细胞时,IFN-γ浓度分别为(216.6±4.8)pg/ml、(251.7±2.2)pg/ml、(535.0±8.5)pg/ml以及(52.57±3.4)pg/ml。4、ELISA检测培养上清液中T-reg分泌的TGF-β1结果显示:在无T-reg存在时,NK细胞与MCF-7、MDA-MB-435s、SKBr-3和MDA-MB-231共培养时TGF-β1浓度分别为(13.4±0.2)pg/ml、(11.3±0.8)pg/ml、(62.9±1.3)pg/ml以及(6.2±0.5)pg/ml;加入T-reg的数量为1/5的NK细胞时,TGF-β1浓度分别为(27.2±0.3)ng/ml、(12.2±1.0)ng/ml、(77.6±1.0)ng/ml以及(9.1±0.1)ng/ml;加入T-reg的数量为1/10的NK细胞时,TGF-β1浓度分别为(46.8±1.6)ng/ml、(15.8±0.9)ng/ml、(87.8±2.3)ng/ml以及(10.9±0.6)ng/ml。5、MICA的表达与分布:(1)荧光定量PCR结果显示:MDA-MB-231、SKBr-3、MCF-7和MDA-MB-435s四种细胞株MICAmRNA相对于GAPDH的相对含量分别为5.56×10-3、2.53×10-3、1.68×10-3和5.99×10-5。(2)流式细胞术检测4种乳腺癌细胞株MICA表达,结果显示MDA-MB-231表达量为(61.5±4.5)%、MCF-7为(56.5±4.7)%,而SKBr-3和MDA-MB-435s几乎不表达MICA分子;穿孔后MDA-MB-231和MCF-7这两株细胞株MICA的表达量与穿孔前相比增加不明显,而MDA-MB-435s和SKBr-3这两株细胞株MICA的表达量分别为(26.4±2.5)%和(20.7±1.3)%。(3)免疫组化证实MDA-MB-231细胞和SKBr-3细胞MICA在细胞膜和细胞浆都有表达,MCF-7细胞MICA在细胞浆和核膜都有表达,MDA-MB-435s细胞MICA主要在细胞浆表达;免疫荧光证实MDA-MB-231细胞和SKBr-3细胞MICA在细胞膜和细胞浆都有表达,MCF-7细胞MICA主要表达在细胞膜上,MDA-MB-435s细胞MICA主要在细胞浆表达。NK对不同的乳腺癌细胞株的杀伤结果表明,乳腺癌细胞株MICA表达部位不同,NK细胞对其杀伤率不同。【结论】T-reg细胞对NK细胞杀伤乳腺癌的作用其机制可能与T-reg分泌的细胞因子TGF-β1有关。MICA在乳腺癌细胞膜上表达越多,NK细胞对该细胞的杀伤率越高。更多还原

【Abstract】 Objective To analyze the expression of T-reg in peripheral blood from patients with breast cancer and its relationship with NK cell and the subset of T cell; explore the effect of T-reg cell on cytotoxicity of NK cell against breast cancer cell in vitro and analyze the possible underlying mechanism; explore the role of NKG2D-MICA on cytotoxicity of NK cell against breast cancer cell; observe the effect on the NKG2D-MICA by T-reg cell.Methods 1. Flow cytometry(FCM)were evaluated the proportion of T-reg、NK cell and the subset of T cell;and detect the expression of Membrane-bound TGF-β1 on T-reg. 2. LDH assay was manipulated to test the cytotoxicity of NK cell on breast cancer cells.3. ELISA was performed to examine the level of IFN-γsecreted by NK cell in supernatant and TGF-β1 secreted by T-reg cell.4. RQ-PCR was used to identify the expression of MICA-mRNA on breast cancer cell lines. 5. FCM were performed to identify the expression of MICA on the cell surface and intracell of breast cancer cell lines、Immunohistochemistry(IH) and immunofluorescence (IF) were used to detect the distribution of MICA on breast cancer cell lines. 6. FCM were used to detect the expression of NKG2D on NK cells.Results 1. The expression of T-reg in patients with breast cancer:The proportion of T-reg in patients with breast cancer was higher than that of volunteers ((7.5±3.0)% Vs(5.1±1.5)% ) P<0.01. 2. The effect of T-reg on cytotoxicity of NK cells:when the ratio of NK and breast cancer cell was 10:1,the cytotoxicity of NK cell was (40.7±3.0)%、(15.5±3.1)%、(29.7±1.8)% and (59.1±2.3)% on MCF-7、MDA-MB-435s、SKBr-3 and MDA-MB-231 respectively;the cytotoxicity of NK cell decreased to(1.9±0.2)%、(5.7±0.4)%、(3.6±2.8)% and(3.9±0.2)% respectively while co-culture with 1/5 T-reg cell;and (7.6±1.6)%、(7.3±1.0)%、(6.6±2.5)% and(19.7±1.1)% respectively while co-culture with 1/10 T-reg cell.3.ELISA detect IFN-γsecreted by NK cell display: when the ratio of NK and breast cancer cell was 10:1, the concentration of IFN-γwas (221.5±2.6)pg/ml、(264.4±5.5)pg/ml、(754.6±19.0)pg/ml and(423.0±14.6)pg/ml on MCF-7、MDA-MB-435s、SKBr-3 and MDA-MB-231 respectively; the concentration of IFN-γdecreased to (70.7±8.5)pg/ml、(14.6±0.9)pg/ml、(128.3±4.0)pg/ml and(6.0±1.0)pg/ml respectively while co-culture with 1/5 T-reg cell;the concentration of IFN-γchange to(216.6±4.8)pg/ml、(251.7±2.2)pg/ml、(535.0±8.5)pg/ml and(52.57±3.4)pg/ml respectively while co-culture with 1/10 T-reg cell.4. ELISA detect TGF-β1 secreted by T-reg cell in the supernatant display: when the ratio of NK and breast cancer cell was 10:1, the concentration of TGF-β1 was(13.4±0.2)pg/ml、(11.3±0.8)pg/ml、(62.9±1.3)pg/ml and(6.2±0.5)pg/ml on MCF-7、MDA-MB-435s、SKBr-3 and MDA-MB-231 respectively; the concentration of TGF-β1 increased to (27.2±0.3)ng/ml、(12.2±1.0)ng/ml、(77.6±1.0)ng/ml and(9.1±0.1)ng/ml respectively while co-culture with 1/5 T-reg cell; the concentration of TGF-β1 increse to(46.8±1.6)ng/ml、(15.8±0.9)ng/ml、(87.8±2.3)ng/ml and(10.9±0.6)ng/ml respectively while co-culture with 1/10 T-reg cell.5.The distribution and expressin of MICA:(1)RQ-PCR discover: Relative content of MICA mRNA of MDA-MB-231、SKBr-3、MCF-7 and MDA-MB-435s was 5.56×10-3、2.53×10-3、1.68×10-3and 5.99×10-5 respectively;(2) MICA was not detected on the surface of MDA-MB-435s and SKBr-3,(61.5±4.5)% on MDA-MB-231 and(56.5±4.7)% on MCF-7 by FCM;after cell perforating,the percentage of MICA was (26.4±2.5)% and(20.7±1.3)% on the MDA-MB-435s and SKBr-3 respectively, but the percentage of MICA had not changes for the MDA-MB-231 and MCF-7; (3)Immunohistochemistry(IH) expreriments confirm that MICA express on the membrane and in the cytoplasm of the MDA-MB-231 and SKBr-3, MICA express in the cytoplasm and nuclear membrane of the MCF-7, and in the cytoplasm of the MDA-MB-435s;Immunofluorescence(IF) expreriments confirm that MICA express on the membrane and in the cytoplasm of the MDA-MB-231 and SKBr-3, MICA express on the membrane of the MCF-7,and in the cytoplasm of the MDA-MB-435s.With the different location of MICA,cytotoxicity of NK cells is different.Conclusion T-reg cells may inhibit the cytotoxicity of NK cell, which was associated with the secretion of TGF-β1. The membrane MICA could increase the cytotoxicity of NK cell on breast cancer cell.更多还原