【作者】 王向玲；

【导师】 纪春岩；

【作者基本信息】 山东大学 ， 内科学血液病， 2009， 博士

【摘要】 背景泛素-蛋白酶体（Ubiquitin-proteasome pathway,UPP）是哺乳动物细胞内主要的蛋白水解酶体系,参与和调控细胞的增殖、分化和凋亡。蛋白酶体主要由20S催化颗粒和19S调节颗粒两部分组成,其活性状态对维持细胞的正常功能起重要作用。蛋白酶体抑制剂可通过抑制蛋白酶体活性进而干扰和影响细胞功能,尤其对肿瘤细胞生长有明显的抑制作用。蛋白酶体抑制剂硼替佐米（bortezomib,velcade）是近年研究较多的一种蛋白酶体抑制剂,目前已被较广泛地应用于多种恶性肿瘤特别是多发性骨髓瘤的治疗。除了直接诱导肿瘤细胞凋亡外,硼替佐米尚可提高肿瘤细胞对自然杀伤（natural killer,NK）细胞杀伤的敏感性。然而,近年来研究发现硼替佐米对免疫细胞如T细胞和树突细胞有一定的毒性作用,如诱导活化的T细胞凋亡,抑制树突细胞活化T细胞的功能等。NK细胞是先天免疫细胞,在抗感染和抗肿瘤免疫中起着致关重要的作用。目前,有关硼替佐米对NK细胞功能调节作用的研究甚少。目的研究蛋白酶体抑制剂硼替佐米对正常人外周血NK细胞活性和功能的影响,探讨硼替佐米诱导NK细胞凋亡机制;观察联合应用硼替佐米与IL-12对多发性骨髓瘤SCID鼠皮下移植瘤生长及体内NK细胞杀伤活性的影响,研究IL-12与硼替佐米协同抗肿瘤作用以及IL-12抵抗硼替佐米对NK细胞的毒性作用。方法1.体外研究硼替佐米对人外周血自然杀伤细胞活性和功能的影响1.1硼替佐米对人外周血淋巴细胞或LAK细胞活性的影响:采用淋巴细胞分离液分离正常人外周血单个核细胞,去除贴壁细胞后获取悬浮细胞即外周血淋巴细胞（PBLs）。体外应用白介素-2（IL-2）诱导PBLs 72h产生淋巴因子激活的杀伤（LAK）细胞。将PBLs、LAK细胞进行体外培养,给予不同浓度的硼替佐米处理12、24、48h,采用AnnexinV（AV）和propidium iodide（PI）双标进行流式细胞术检测细胞凋亡情况。1.2硼替佐米对人外周血自然杀伤细胞活性和功能的影响:采用NK细胞阴性选择试剂盒分离纯化正常人NK细胞,流式细胞术检测NK纯度,将NK细胞进行体外培养,给予不同浓度的硼替佐米处理12、24h,或联合应用GSH（氧自由基清除剂）或zVAD（全Caspase抑制剂）预处理,AV和PI双标进行流式细胞术检测细胞凋亡;荧光测定半胱天冬酶caspase-3活性的变化;Mitotracker Red荧光染色法测定线粒体膜电位的改变;细胞内染色法检测穿孔素的表达;细胞表面染色检测NK细胞膜活化性受体的表达;分别以K562细胞和P815细胞作为靶细胞采用标准的4h⁵¹Cr释放试验检测NK细胞的细胞毒作用或杀伤功能。2.硼替佐米对骨髓瘤小鼠自然杀伤细胞活性和功能的影响2.1建立SCID鼠多发性骨髓瘤皮下移植瘤模型:对数生长期的RPMI 8226细胞,调整细胞数至1×10⁸/ml,取0.1 ml含10⁷个细胞接种于5-6周龄雌性SCID鼠前肢处背部皮下。14d后实体瘤长至直径大于0.5cm时进行药物干预。2.2荷瘤小鼠体内注射NS、硼替佐米和/或IL-12抑瘤效果观察、NK细胞数变化及脾细胞杀伤功能SCID鼠皮下成瘤后随机分成NS组、IL-12组（0.4ug/只）、硼替佐米组（0.75mg/kg）和硼替佐米+IL-12序贯治疗组,分别给予腹腔注射IL-12,尾静脉注射硼替佐米和NS,观察荷瘤小鼠瘤体的生长抑制情况;干预前后取外周血做血常规及流式检测NK细胞比例,末期取脾细胞悬液做LDH释放实验检测NK细胞的杀伤功能。结果:1.体外研究硼替佐米对人外周血自然杀伤细胞活性和功能的影响1.1硼替佐米诱导PBLs和LAK细胞凋亡流式检测结果显示:硼替佐米明显诱导PBLs凋亡,细胞凋亡率随药物浓度和作用时间增加而升高;低剂量硼替佐米（4.7ng/ml）处理PBLs细胞12h后,PBLs部分发生凋亡,作用48h后,超过80%的PBLs出现调亡。硼替佐米同样以时间和剂量依赖方式诱导LAK细胞凋亡。1.2硼替佐米诱导NK细胞凋亡,并抑制其杀伤活性。此研究中所用NK细胞的纯度均在90%以上。硼替佐米以时间-剂量依赖方式显著诱导NK细胞凋亡,4.7ng/ml和18.8ng/ml硼替佐米处理NK细胞12h、24h后细胞的总凋亡率显著升高。18.8ng/ml硼替佐米处理NK细胞12h、24h,凋亡率明显高于4.7ng/ml组,二者比较差异有显著性统计学意义（12h时P＜0.01,24h时P＜0.001）,呈剂量依赖关系。同样浓度硼替佐米（4.7ng/ml或18.8ng/ml）处理NK细胞,24h细胞凋亡率明显高于12h处理组（P＜0.0001）,呈时间依赖关系。为进一步研究其诱导NK细胞凋亡的机制,我们检测了Caspase-3活性。结果表明硼替佐米诱导NK细胞Caspase-3活性增高,硼替佐米（18.8ng/ml）处理24h的NK细胞其Caspase-3活性较未处理组升高了约4倍。有趣的是GSH（氧自由基清除剂）或zVAD（全Caspase抑制剂）预处理完全阻断了Caspase-3的活化,同时发现GSH预处理组细胞24h的早期和总凋亡率较硼替佐米（4.7ng/ml）单用组明显下降（P＜0.05）,然而zVAD预处理组细胞的总凋亡率未显著减少,这一发现提示Caspase-3的活化是ROS（reactive oxygen species活性氧簇）依赖机制。为进一步确定线粒体在硼替佐米诱导的NK细胞凋亡中的作用,我们利用Mitotracker Red标记结合流式细胞仪技术检测了线粒体膜电位的变化,结果显示4.7ng/ml、18.8ng/ml硼替佐米处理NK细胞12h、24h后,Mitotracker Red的平均荧光强度明显低于未处理组（P＜0.01）。GSH预处理有效的阻止了大部分线粒体膜电位的耗散。相反,zVAD无线粒体膜电位的保护作用,提示硼替佐米诱导NK细胞凋亡机制中线粒体膜电位的耗散早于caspase-3的活化,同时说明ROS介导的线粒体损伤在硼替佐米诱导的NK细胞凋亡机制中起重要作用。NK细胞内穿孔素及NK细胞膜表面活化性受体的表达方面的研究结果显示:对照组与硼替佐米处理组NK细胞内穿孔素的表达以及活化性受体（NKG2D、NKp30、DNAM-1）表达水平无明显差异。然而,硼替佐米显著抑制了NKp46的表达,其抑制作用与使用的药物剂量成依赖关系。此外,Bay-11阻断NF-κB活性后NKp46表达水平降低,提示NF-κB可能参与了NKp46的表达调节,硼替佐米所诱导的NKp46的表达下调部分是通过其阻断NF-κB活性的功能实现的。NKp46是重要的NK细胞活化性受体之一,为了研究NKp46表达的下调对NK细胞杀伤功能的影响,我们采用铬51释放试验检测NK细胞的重向细胞毒作用。结果显示:低剂量硼替佐米（4.7ng/ml）处理NK细胞12小时后,在anti-NKp46抗体存在的条件下NK细胞对P815细胞的杀伤作用明显下降。同对照组比较,在效靶比20:1和10:1时,二者之间的杀伤水平有显著差异。Bay-11也同样抑制了NK细胞裂解P815细胞的能力。然而,硼替佐米并未影响NK细胞对K562细胞的杀伤能力,提示硼替佐米特异性、选择性地干扰NKp46活化途径介导的NK细胞杀伤作用。2.硼替佐米对骨髓瘤小鼠体内自然杀伤细胞活性和功能影响2.1 44只SCID鼠皮下移植RPMI 8226细胞后2周均达到成瘤标准,NS和IL-12治疗组小鼠皮下肿瘤以时间依赖方式生长,虽IL-12治疗组肿瘤生长轻微受抑,相比NS组,二者无显著差异。而硼替佐米治疗组及硼替佐米联合IL-12治疗组小鼠肿瘤生长明显受到抑制,肿瘤体积明显缩小（P＜0.01）。硼替佐米单用组荷瘤小鼠瘤体显著减少,但停止应用硼替佐米后,观察到小鼠肿瘤再生长。为明确是否IL-12具有协同硼替佐米的抗肿瘤效应,给予IL-12序贯治疗结果显示,同药物单用组比较,硼替佐米联合IL-12治疗导致肿瘤再生长延迟,并且瘤体继续缩小。提示IL-12联合硼替佐米治疗较单药组有更强的抗肿瘤效果。IL-12可增加硼替佐米抗瘤效应,二者联合有协同作用。硼替佐米抗骨髓瘤同时是否会影响荷瘤小鼠体内NK细胞的数量和功能,治疗前后取外周血做血常规及流式染色检测结果显示:硼替佐米处理组治疗后NK细胞数明显低于治疗前（P＜0.01）,联合IL-12序贯治疗后NK细胞数仍低于治疗前,此结果提示硼替佐米在体内具有抑制NK细胞增殖和或诱导NK细胞凋亡的作用。NS组、IL-12组治疗前后NK细胞数无明显变化。LDH释放实验检测显示经硼替佐米治疗后的荷瘤小鼠NK细胞杀伤靶细胞的功能明显低于NS组和IL-12单用组（P＜0.01）,联合IL-12序贯治疗后其杀伤活性显著提高。这提示IL-12虽不能阻止NK细胞数量的减少,但对硼替佐米治疗所造成的NK细胞杀伤功能缺陷具有保护作用。结论1.硼替佐米主要过ROS依赖及非依赖机制造成线粒体膜电位的耗散,导致人PBLs、LAK细胞、NK细胞凋亡。2.硼替佐米可以降低静止期NK细胞表面NKp46的表达,并抑制NKp46活化途径所介导的NK细胞杀伤功能。3.硼替佐米可减少骨髓瘤小鼠体内NK细胞数,影响其杀伤功能,联合IL-12治疗有协同抗肿瘤作用,同时可部分解救硼替佐米对NK细胞的毒性作用。更多还原

【Abstract】 Backgroud Ubiquitin-proteasome pathway is the major proteolytic system in the mammal cells, involved in cell proliferation , differentiation and apoptosis. 26S proteasome is a ATP-dependent proteolytic complex that ubiquitously exists in the cytoplasm and nucleus of almost all eukaryotic cells. 26S proteasome consisting of a 20S core catalytic cylindrical complex capped at both ends by 19S regulatory subunits is responsible for the degradation of the proteins regulating cell proliferation, differentiation and apoptosis. Bortezomib, a specific and potent 26S proteasome inhibitor, has recently received much attention by hematologists/oncologists due to its potent apoptosis induction proterty in tumor cells. It was first approved by FDA of America to treat multiple myeloma （MM）, but now it has been applied to treat other hematological malignancies such as lymphoma, and certain types of solid rumors. More interestingly, Bortezomib also is shown to sensitize tumor cells to NK cell mediated-lysis through TRAIL and/or Fas/Fas ligand pathways. Apart from its treatment effects on tumors, toxic effects of bortezomib on immune-competent cells such as T cells and dendritic cells have also been revealed. Recent studies have demonstrated that proteasome inhibition by bortezomib induced apoptosis in activated human T cells and suppressed the expression of activation-associated cell surface receptors and cytokines in T cells and DCs, implicating that proteasome inhibition by bortezomib may affect function of T cells and DCs. However, to date little is known about its regulatory effects of proteasome inhibition by bortezomib on natural keller （NK） cells.Objective The aim of the study was to investigate the effects of bortezomib on natural killer cell survival and function, to explore underling mechanisms of bortezomib mediated apoptosis of resting NK cells; To investigate its inhibitory effect on NK cell-mediated antitumor activity in a mouse model of MM. Combination therapy with bortezomib and IL-12 was designed to test whether IL-12 had a synergistic antitumor effect with bortezomib in vivo and protected NK cells from bortezomib treatment-associated damage.Methods1. To investigate the effects of bortezomib on NK cells survival and function in vitro.1.1 The effects of bortezomib on PBLs or LAK cells survival Human peripheral blood mononuclear cells （PBMCs） from blood donors were separated by Ficoll-Paque gradient centrifugation. Peripheral blood lymphocytes （PBLs） were harvested after incubation of PBMCs in a plastic flask for 1h. To generate LAK cells, PBLs were cultured in the presence of IL-2 for 72h. PBLs or LAK cells were cultured in a 24-well plate in the presence or absence of bortezomib at different concentrations defined for 12h, 24h, and/or 48h, and the apoptotic cells were quantified by dual labeling of recombinant Annexin V（AV） and propidium iodide （PI）, and flow cytometry（FCM）.1.2 The effects of bortezomib on NK cells survival and function. Primary resting natural killers were purified from peripheral blood mononuclear cells of health donors by negative selection. Purity of these NK cells was determined by FCM. Cells were cultured in a 24-well plate in the presence, absence of bortezomib or combined with GSH or zVAD at different concentrations defined for 12h or 24h,respectively. The percentages of apoptotic cells were quantified by dual labeling of AV and PI , and flow cytometry. Caspase-3-like activity was measured by fluoroscan II plate reader; Functional mitochondria were labeled by Mitotracker Red, mitochondria membrane potential were detected by FCM; Intracelluar staining for perforin and cell surface staining for NK cells activation receptors were assayed by FCM. NK cell cytolytic activity against K562 cell and P815 cell were evaluated using a standard chromium-51 release assay.2. The effects of bortezomib on NK cells survival and function in vivo2.1 The establishment of SCID mouse model bearing MM tumor （ RPMI 8226; a human multiple myeloma cell line）: The five-to-six week old female SCE) mice were injected subcutaneously with 1×10⁷ logarithmically growing RPMI 8226 cells to right forelegs back. Treatment began about 14 days post-inoculation when tumor reached 0.5cm in diameter.2.2 To investigate inhibitory effects of antitumor activity, quantify NK cell numbers in blood and to evaluate killing activity of spleen cells in the mouse model bearing tumor treated with NS, bortezomib and/or IL-12. The mice were randomly divided into NS, IL-12, bortezomib and bortezomib + IL-12 groups when tumor formed. NS, bortezomib （0.75mg/kg） and/or IL-12（0.4ug/each） were administered intravenously or intraperitonealy, respectively. The tumor growth （tumor mass） was monitored after the beginning of treatment. The absolute numbers of NK cells in peripheral blood were calculated by blood routine and FCM before and after treatment. The cytotoxicity of NK cells in a spleen was evaluated through LDH release assay.Result1. Study the effects of bortezomib on NK cells survival and function in vitro.1.1 Bortezomib induced PBLs and LAK cell apoptosis: The results show that bortezomib induced apoptosis in primary PBLs in a dose and/or incubation time-dependent manner. When exposure to the lowest dose of bortezomib for 12h, the apoptotic cells were found in PBLs. When prolonged the incubation time to 48h, bortezomib induced over 80% of apoptosis. Moreover, LAK cells were also markedly compromised to apoptosis induced by bortezomib, as compared with untreated group （P<0.05）.1.2 Bortezomib induced NK cell apoptosis and suppressed NK cell killing activity. Our results demonstrated that Bortezomib markedly induced apoptosis in NK cells in a time-and dose-dependent manner. The percentages of total apoptotic cells were significantly increased in the NK cells treated by bortezomib with the dose of 4.7ng/ml and 18.8ng/ml for 12h and 24h. Bortezomib treatment for 12 and 24h at the dose of 18.8ng/ml induced more pronounced apoptosis in NK cells than 4.7ng/ml, respectively （ P<0.01 for 12h and P<0.001 for 24h, respectively）. When the same dose of bortezomib （4.7ng/ml or 18.8ng/ml） was applied, 24h treatment triggered more apoptosis in NK cells than 12h treatment （P<0.0001）.Our results showed that bortezomib at dose of 18.8ng/ml induced about 4 fold increasement of caspase-3 activity. GSH almost completely abolished caspase activity of NK cells induced by bortezomib, moreover the percentages of early and total apoptotic cells in NK cells induced by bortezomib for 24h in the presence of GSH were significantly decreased as compared with those of bortezomib（4.7ng/ml） alone group （P<0.05 ） . Blocking caspase activation by zVAD did not effectively block total apoptosis in NK cells induced by bortezomib. These data indicated that caspase activation is ROS-dependent, and ROS generation contributes to apoptosis in NK cells induced by bortezomib.Mitochondria play pivotal roles in the process of apoptosis. To determine a role of mitochondria in the bortezomib-induced apoptosis in NK cells, mitochondrial membrane potential （MMP） was measured by Mitotracker Red. The results showed that bortezomib markedly induced the loss of Mitotracker Red fluorescence intensity in NK cells in a dose-dependent manner. Statistical analysis showed that the differences in mean fluorescence intensity of Mitotracker Red between bortezomib （4.7ng/ml or 18.8ng/ml）-treated and untreated NK cell were significant. Furthermore, our studies showed that GSH partially and significantly prevented the loss of MMP induced by bortezomib （4.7ng/ml）. In contrast, zVAD did not show any protective effects on MMP, suggesting that the dissipation of MMP is upstream of caspase activation in bortezomib-treated NK cells.To explore whether bortezomib affects NK cells function, intracellular staining of perforin and cell surface staining of NK cell activation receptors were quantified by flow cytometry. The results showed that expression of intracellular perforin on bortezomib-treated NK cells was not significantly different from untreated NK cells. Moreover expression of DNAM-1, NKG2D and NKp30 was compared between bortezomib treated and untreated NK cells, and no difference was found between these two groups. By contrast, NKp46 expression in NK cells was significantly reduced upon treatment with bortezomib in a dose-dependent manner. We further found blocking NF-kB activity by a NF-kB blocker Bay-11 induced significant loss of NKp46 expression on NK cells, indicating that NF-kB may be involved in the regulation of NKp46 expression and bortezomib-induced decrease in NKp46 expression may be in part attributable to its blocking effects on NF-kB activity. It is known that NKp46 is one of the important activating receptors for NK cell function. To investigate functional consequence of decreased NKp46 expression on NK cells induced by bortezomib, redirected NK cell cytotoxicity was assayed by ⁵¹Cr release test. The results showed that the lysis of P815 cells in the presence of anti-NKp46 antibody by the NK cells exposed to a low dose of bortezomib （4.7ng/ml） for 12h was significantly decreased when compared with controls at E.T ratios of 20:1 and 10:1. Moreover, blocking NF-kB activation by Bay-11 for 12h substantially inhibited NK cell-mediated lysis of P815 cells in the presence of anti-NKp46 antibody. By contrast, NK cells treated by bortezomib for 12h were able to kill K562 target cells as efficiently as the untreated NK cells. These data implied that the bortezomib-induced impairment of NK cell killing mediated through the NKp46 activation pathway.2. The effect of bortezomib on NK cells survival and function in vivo （aninal study） .2.1 Tumor formed in all the mice 14 days after implantation. After given different treatments, growth inhibition on the xenografted tumors was monitored and compared. Our results demonstrated that tumor volumes in the mice that treated with NS and IL-12 increased in a time-dependent manner, A slight suppression on tumor growth from IL-12-treated mice was observed when compared with NS-treated mice, however difference in tumor mass between these two groups did not reach significance. The tumor volumes of SCID mice were markedly reduced after treatment with bortezomib alone and bortezomib plus IL-12 as compared with control group （P<0.01）, respectively. Bortezomib treatment alone induced markedly reduction of RPMI 8226 tumors in all of the treated mice. However, tumor re-growth was observed after termination of bortezomib treatment. As expected, treatment with bortezomib plus IL-12 resulted in a significance delay of tumor re-growth as compared with either Bortezomib or IL-12 alone treated mice. These data indicated a synergistic anti-MM effect of IL-12 and bortezomib.To determine whether bortezomib affected the numbers and function of NK cells in these mice in vivo, the numbers of NK cells in peripheral blood of the mice before and after the treatment were quantified by blood routine test and FCM. The results showed that NK cell number was markedly decreased after bortezomib treatment and combined treament of Bortezomib with IL-12 did not result in an increasement of NK cell number. In addition, NK cell numbers after NS and IL-12-treated mice were not significantly different from the mice before treatment. Cytotoxictity of spleen NK cells from mice treated by Bortezomib alone was significantly decreased when compared with the control and IL-12 alone -treated mice （P<0.01） , as determined by LDH release assay. In contrast, combined treatment of bortezomib with IL-12 resulted in a significance increasement of NK cell killing activity, suggesting that IL-12 may restore NK cell cytolytic activity deficiency induced by Bortezomib.Conclusion1. Bortezomib induces apoptosis in PBLs, LAK cells, primary resting natural killer cells in a dose- and time dependent manner, ROS-dependent and independent pathways are implicated in the apoptosis machinery of resting NK cells induced by bortezomib.2. Bortezomib decreased NKp46 receptor expression on primary resting NK cells, resulting in decreased NK cell-mediated lysis of target cells through the NKp46 activation pathway.3. Bortezomib reduces absolute numbers of NK cells in peripheral blood, and affects NK cell cytotoxicity in SCID mice. IL-12 showed a synergistic antitumor activity with bortezomib and restored NK cell killing defects induced by bortezomib.更多还原