自然杀伤细胞联合曲妥珠单抗增强对曲妥珠单抗耐药乳腺癌细胞的杀伤作用

发布时间：2018-10-31 07:06

【摘要】：目的:曲妥珠单抗(赫赛汀#174;)是一种对HER2阳性乳腺癌十分有效的治疗方法,而耐药成为阻碍其应用的难点。自然杀伤(natural killer,NK)细胞是先天免疫的重要组成部分,并且是曲妥珠单抗诱导ADCC效应最有效的细胞。为了增强对曲妥珠单抗耐药乳腺癌的杀伤作用,本实验将曲妥珠单抗与NK细胞联合来杀伤耐药的乳腺癌细胞,同时检测相互作用的机制。方法:1.用MTT法验证耐药细胞的耐药性,同时验证曲妥珠单抗对NK细胞无增殖抑制作用。2.钙黄绿素-AM释放法检测NK细胞联合曲妥珠单抗对敏感及耐药乳腺癌细胞的协同杀伤作用,同时对比阻断Fas受体前后,两者联合杀伤的变化情况。3.流式细胞术分别检测敏感及耐药肿瘤细胞表面HER2的表达变化情况;分别检测了不同处理组NK细胞表面活化性受体NKp44、NKp46、NKG2D,抑制性受体KIR2DL1、KIR2DL2、KIR2DL3、KIR3DL1,凋亡配体Fas L的表达变化;NK细胞内部穿孔素及颗粒酶B的生成量;肿瘤细胞表面活化性配体ULBP1、ULBP2、MICA及凋亡受体Fas的表达情况;NK细胞在各实验组分泌至上清中的CCL5、IL13。4.ELISA法用于检测NK细胞在各实验组分泌至上清的IFN-α及TNF-β的生成量的变化情况。结果:1曲妥珠单抗耐药细胞的耐药性持续存在,耐药细胞表面HER2表达量明显降低。2曲妥珠单抗与NK细胞对耐药细胞单独杀伤作用均不明显,但联合后对耐药细胞的杀伤作用显著增强,且联合杀伤率随效靶比的增加而增加。进一步研究两者联合作用机制发现:曲妥珠单抗与NK细胞联合作用于肿瘤细胞后,肿瘤细胞表面的活化性配体ULBP1、ULBP2、MICA、NK细胞表面活化性受体NKp44的表达量显著增加,抑制性受体KIR2DL1,KIR2DL2,KIR2DL3及KIR3DL1的表达量明显降低;共培养环境中NK细胞相关细胞因子IFN-γ、TNF-β、CCL5、IL13均明显增加;NK细胞相关的凋亡受配体Fas及Fas L的表达被显著上调,阻断Fas受体后,联合杀伤作用明显减弱,协同作用减弱甚至消失;NK细胞内部穿孔素及颗粒酶由于其基础表达量极高,故生成量没有显著变化。结论:1.SKBR3-pool2、BT474-HR20表面的HER2表达量降低可能是其耐药的原因之一。2.HER2表达量降低不影响曲妥珠单抗介导NK细胞的ADCC效应,同时随着NK细胞效靶比的增加,协同作用增强。3.在肿瘤细胞的环境中,曲妥珠单抗能够通过以下几个方面影响NK细胞:1增加NK细胞活化性受体、活化性配体,并降低NK细胞抑制性受体的表达量来增加NK细胞的活性;2增加NK细胞的细胞因子增强NK细胞的杀伤能力;3增加NK细胞凋亡受配体Fas及Fas L的表达增强NK细胞的杀伤能力。
[Abstract]:Objective: trotozumab (Herceptin 174;) is an effective treatment for HER2 positive breast cancer, and drug resistance has become a difficulty in its application. Natural killer (natural killer,NK) cells are important components of innate immunity and are the most effective cells to induce ADCC effect by tratozumab. In order to enhance the killing effect of tratuzumab on drug-resistant breast cancer, we combined tratozumab with NK cells to kill the drug-resistant breast cancer cells, and to examine the mechanism of interaction. Methods: 1. The drug resistance of drug resistant cells was verified by MTT assay, and the inhibitory effect of trotozumab on the proliferation of NK cells was also tested. 2. 2. The synergistic killing effects of NK cells combined with tritozumab on sensitive and drug-resistant breast cancer cells were detected by calcitonin AM release assay. The changes of the combined killing of NK cells before and after blocking Fas receptor were compared. 3. The expression of HER2 on sensitive and drug-resistant tumor cells was detected by flow cytometry. The expression of activated receptor NKp44,NKp46,NKG2D, inhibitory receptor KIR2DL1,KIR2DL2,KIR2DL3,KIR3DL1, apoptotic ligand Fas L on the surface of NK cells and the amount of perforin and granzyme B in NK cells were detected. Expression of activated ligand ULBP1,ULBP2,MICA and apoptotic receptor Fas on the surface of tumor cells; The changes of IFN- 伪 and TNF- 尾 production of NK cells secreted from the supernatant of NK cells in each experimental group were detected by CCL5,IL13.4.ELISA method in the secreted supernatant of each experimental group. Results: (1) the drug resistance of the drug resistant cells was persistent, and the expression of HER2 on the surface of the resistant cells was significantly decreased. 2 the cytotoxic effects of tratuzumab and NK on the drug resistant cells were not obvious. However, the cytotoxicity of drug resistant cells was significantly increased after the combination, and the combined killing rate increased with the increase of the effective / target ratio. It was found that the expression of activated receptor NKp44 on the surface of activated ligand ULBP1,ULBP2,MICA,NK cells on tumor cells increased significantly after the combined action of tratuzumab and NK cells. The expression of KIR2DL1,KIR2DL2,KIR2DL3 and KIR3DL1 decreased significantly. IFN- 纬, TNF- 尾 and CCL5,IL13 increased significantly in co-cultured environment. The expression of Fas and Fas L, the apoptosis-associated ligand of NK cells, was upregulated significantly. After blocking the Fas receptor, the combined killing effect was obviously weakened, and the synergism was weakened or even disappeared. There was no significant change in perforin and granzyme in NK cells due to their high basal expression. Conclusion: 1. The decrease of HER2 expression on SKBR3-pool2BT474-HR20 may be one of the reasons for its drug resistance. The decrease of 2.HER2 expression does not affect the ADCC effect of NK cells mediated by tritozumab, and with the increase of NK cell response to target ratio. Synergistic enhancement. 3. In the environment of tumor cells, trotozumab can affect NK cells in the following ways: (1) increase the activity of NK cells by increasing the activated receptors, activating ligands and decreasing the expression of inhibitory receptors in NK cells; (2) increase the cytokines of NK cells to enhance the killing ability of NK cells, and increase the expression of Fas and Fas L in the apoptotic ligands of NK cells and enhance the killing ability of NK cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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