T系白血病的CAR-T治疗：进展、困境和未来出路
作者简介:
韩雅静（2000-），女，本科在读，主要从事CAR-T细胞治疗的研究，ORCID: 0009-0000-4458-2004
冯晓明: 中国医学科学院血液病医院（中国医学科学院血液学研究所）研究员，博士生导师，协和学者特聘教授，天津市131人才第一层人选。曾获国家自然科学基金优秀青年基金、天津市杰出青年基金。主要从事T细胞的基础和转化研究。在国际上首次发现Foxp1和Lkb1等分子开关对效应/调节T细胞的重要控制作用；与临床学者合作研究了多个CAR-T新策略。在Lancet Oncol、JCO、Nat Immunol、Blood等期刊上发表论文30余篇。曾获美国Wistar研究所Ching Jer Chern Memorial Award、天津市教岗先锋等荣誉
。
通讯作者:
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摘要:
免疫治疗在血液恶性肿瘤治疗领域有着举足轻重的地位，而其中嵌合抗原受体（CAR）T细胞疗法为血液恶性肿瘤免疫治疗构建了新的治疗格局，在B系血液恶性肿瘤治疗领域收获了较为满意的临床效果。而由于存在CAR-T细胞自相残杀、肿瘤细胞污染、持续性T细胞缺乏等临床相关问题，CAR-T细胞疗法在治疗急性T淋巴细胞白血病（T-ALL）时受到一定限制。因此，突破现有瓶颈，对T-ALL的CAR-T疗法进行优化，提高疗效的同时改善患者的预后，是当前的主要任务。
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关键词:
- 细胞免疫疗法 /
- CAR-T细胞疗法 /
- 急性T淋巴细胞白血病
Abstract:Tumor immunotherapy occupies a pivotal position in the field of hematological malignancies. Chimeric antigen receptor (CAR) T-cell therapy has established a new therapeutic pattern for hematological immunotherapy and achieved satisfactory clinical results in the treatment of B-lineage hematological malignancies. However, CAR T-cell therapy has some limitations in the treatment of T-cell acute lymphoblastic leukemia because of the presence of CAR T-cell fratricide, tumor cell contamination, T-cell aplasia, and other clinically relevant problems. Therefore, the current major challenge is overcoming the existing bottlenecks to optimize CAR-T therapy and improve its efficacy against T-ALL while improving the prognosis of patients.
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0 引言
子宫内膜癌是女性生殖系统常见的恶性肿瘤之一，发病率呈上升趋势，好发于绝经后女性。近年研究表明，甲壳质酶蛋白40（Chitinase-3-like Protein 1, YKL-40）可作为子宫内膜癌的候选肿瘤标志物之一，在肿瘤细胞增殖[1]、血管形成[2]和抗凋亡[3]中具有枢纽作用，但具体的生物学功能仍需进一步探索。本课题组前期临床研究也表明，YKL-40在子宫内膜癌组织和血清中的表达高于子宫肌瘤患者以及健康者[4]。
结合前期研究工作，本课题提出：降低YKL-40基因的同时可降低子宫内膜癌细胞的抗凋亡能力以及提高癌细胞对顺铂化疗的敏感度。本实验通过siRNA（small interfering RNA）技术沉默子宫内膜癌细胞中YKL-40基因的表达，采用MTT实验、流式细胞仪及Annexin V-PE/7AAD双染法检测转染前后子宫内膜癌细胞对顺铂化疗敏感度的变化，进而探讨沉默YKL-40基因对子宫内膜癌细胞增殖的影响以及YKL-40与子宫内膜癌细胞对顺铂化疗敏感度的关系，为进一步探讨其影响增殖的机制、子宫内膜癌化疗耐药机制以及提高化疗效果提供思路和研究基础。
1 材料与方法
1.1 材料
人子宫内膜癌HEC-1A细胞由广西医科大学肿瘤医学院妇瘤科张洁清教授馈赠，DMEM/F12培养液购于美国HyClone公司，胎牛血清购于美国Gibco公司。YKL-40基因引物由日本Takara公司设计并合成。Annexin V-PE/7AAD凋亡试剂盒购于美国BD公司。顺铂购自山东齐鲁药业。
1.2 细胞培养
人子宫内膜癌HEC-1A细胞株用DMEM/F12培养液（含10%胎牛血清和1%双抗）置于37℃、5%CO2培养箱中培养，细胞融合度达80%时进行传代，取对数生长期细胞进行实验。
1.3 siRNA慢病毒转染靶细胞
一条针对YKL-40的siRNA由上海汉恒公司合成。将人子宫内膜癌HEC-1A细胞分为三组：空白对照组、空载体组、siRNA实验组。siRNA实验组转染携带siRNA的慢病毒，空载体组转染只含绿色荧光蛋白（GFP）的空载病毒，空白对照组不进行转染。实验组转染的特异性siRNA序列为：GACTCTCTTTCTGTCGGA。选择细胞最佳病毒感染复数MOI值为20时进行转染，并加入8 μg/ml聚凝胺助转染。转染效率（%）=绿色荧光蛋白标记的细胞/细胞总数×100%。转染成功后用1 μg/ml嘌呤霉素筛选2~3周，耐嘌呤霉素的细胞用于后续实验。
1.4 实时荧光定量PCR实验（quantitative real-time-PCR, qRT-PCR）检测YKL-40基因沉默对子宫内膜癌细胞的沉默效率
按照TRIzol试剂使用说明书提取各组细胞RNA，根据qRT-PCR试剂盒说明书进行稀释和后续操作。反转录后cDNA扩增配置20 μl反应体系，置入ABI stepone plus实时荧光定量PCR仪进行反应，设置反应条件为：95℃ 30 s，循环1次；95℃ 5 s；60℃ 34 s，40个循环。YKL-40引物：F: 5’-ATCACCAAGGAGCCAAACATC-3’；R: 5’-GGGGAAGTAGGATAGGGGACA-3’。内参照β肌动蛋白（β-actin）引物：F: 5’-ACACTGTGCCCATCTACG-3’；R: 5’-TGTCACGCACGATTTCC-3’。根据公式2-∆∆Ct[5]（其中Ct值为循环阈值）计算各组细胞中YKL-40 mRNA的相对表达水平。实验重复三次。
1.5 MTT实验检测不同浓度梯度顺铂对各组子宫内膜癌细胞增殖能力的影响
以5×104个/孔细胞接种到96孔板，每孔100 μl。设置5~7个复孔。细胞贴壁后加入不同浓度梯度的顺铂（山东齐鲁药业），使终浓度为100、50、25、12.5、6.25、3.125和0 μmol/L，同一培养条件下培养48 h，每孔加5 mg/ml MTT溶液20 μl，继续孵育4 h后终止培养，弃上清液。每孔加150 μl DMSO（dimethyl sulfoxide），振荡10 min，使结晶物充分溶解。在酶标仪上测定各孔吸光度值（570 nm处），以药物浓度为横坐标，吸光度值为纵坐标绘制细胞生长曲线。实验重复三次。
1.6 顺铂对子宫内膜癌细胞中YKL-40基因的影响
人子宫内膜癌细胞分为两组，一组不加任何处理因素（A组），一组加入25 μmol/L顺铂（B组），继续培养48 h后用qRT-PCR实验检测两组细胞中YKL-40基因表达情况。实验过程如上。实验重复三次。
1.7 Annexin V-PE/7AAD双染法检测各组细胞凋亡情况
将各组子宫内膜癌细胞中加入25 μmol/L顺铂，继续培养48 h后用Annexin V-PE/7AAD双染法检测各组细胞凋亡情况。用胰酶分别消化经过相同浓度顺铂处理的各组癌细胞，调整细胞浓度为1×107个/毫升，各取100 μl。加入1 ml PBS洗涤细胞两次，弃上清液，按凋亡试剂盒要求将10×Binding Buffer稀释成1×Binding Buffer。各组细胞加入1×Binding Buffer 100 μl，并加入5 μl Annexin V-PE和5 μl 7AAD，避光室温孵育15 min后加入1×Binding Buffer 400 μl，1 h内置于流式细胞仪上检测各组细胞凋亡情况。实验重复三次。
1.8 统计学方法
实验数据均采用SPSS17.0软件进行分析，以（x± s）表示，两组间数据用两独立样本t检验进行分析，三组间数据采用方差分析，P < 0.05为差异有统计学意义。
2 结果
2.1 细胞转染
经慢病毒转染后，转染效率达80%以上，转染成功的细胞带有绿色荧光，表明转染成功，见图 1。
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图 1 转染siRNA实验组同一视野下明场和荧光场图(×10)Figure 1 Transfection of siRNA on same view of bright and fluorescence field (×10)A: HEC-1A cells were observed under bright field microscopy; B: on the same field, HEC-1A cells were observed under a fluorescence microscope, and siRNA with fluorescence was successfully transfected into HEC-1A cells2.2 qRT-PCR法检测各组细胞中YKL-40 mRNA表达
三组均数经SNK、LSD两两比较，实验组YKL-40 mRNA表达量明显低于空载体组（P=0.036）和空白对照组（P=0.005），而空白对照组和空载体组间差异无统计学意义（P=0.275），见图 2。
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图 2 qRT-PCR法检测各组细胞YKL-40 mRNA表达含量Figure 2 Relative expression of YKL-40 mRNA detected by qRT-PCR*: P < 0.05, compared with blank control group and mock-treatment group. Bars indicated the mean±standard deviation from each experiment; 1: blank control group; 2: mock-treatment group; 3: experimental group2.3 顺铂对子宫内膜癌HEC-1A细胞YKL-40基因降低前后细胞生长的影响
MTT实验显示：加入不同浓度梯度的顺铂培养细胞48 h后，细胞的生长受到明显抑制，实验组细胞转染siRNA后比空白对照组和空载体组细胞生长抑制更显著（P < 0.05），但空白对照组和空载体组差异无统计学意义（P > 0.05），见图 3。
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图 3 不同浓度顺铂处理后各组细胞生长抑制曲线Figure 3 Cell growth inhibition curves after different concentrations of cisplatin treatment**: P < 0.01, compared with blank control group and mock-treatment group. We determined that the effects of siRNA on chemosensitivity of EC HEC-1A cells by MTT assay. Cells proliferative ability in the experimental group was significantly inhibited, compared with those in the blank control and the mock-treatment groups when treated with 25μmol/L cisplatin. In this concentration, the chemosensitivity of HCE-1A cells to cisplatin was the highest by silencing YKL-40 gene2.4 顺铂对子宫内膜癌细胞中YKL-40基因的影响
qRT-PCR结果显示：子宫内膜癌细胞中加入相同浓度的顺铂处理48 h后，B组（处理后）细胞的YKL-40基因表达相对含量高于A组（处理前），细胞中YKL-40基因表达上调，差异有统计学意义（P=0.000），见图 4。
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图 4 加入顺铂前后子宫内膜癌细胞YKL-40 mRNA的表达含量变化Figure 4 Relative expression of YKL-40 mRNA before and after cisplatin treatment detected by qRT-PCR**: P < 0.01, compared with group A; A: blank cells; B: the cells treated with cisplatin. Bars indicated the mean±standard deviation from each experiment2.5 沉默YKL-40基因对子宫内膜癌HEC-1A细胞顺铂敏感度的影响
流式细胞实验显示：相同浓度顺铂处理各组细胞后，三组均数经SNK、LSD两两比较，实验组细胞的总凋亡率（38.07±4.88）均明显高于空白对照组（13.3±1.01）（P=0.000）和空载体组（12.5±0.17）（P=0.000），而空白对照组和空载体组比较差异无统计学意义（P=0.776），见图 5。
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图 5 流式细胞术检测转染后各组细胞凋亡图Figure 5 Apoptosis of each group after transfection detected by FCMA: blank control group; B: mock-treatment group; C: experimental group; HEC-1A cells apoptosis rate was increased after the inhibition of YKL-40 expression (P=0.000)3 讨论
YKL-40最早被发现于软骨细胞和平滑肌细胞[6]，是哺乳动物甲壳质酶蛋白之一，但是缺乏甲壳质酶活性，它由各种细胞产生，包括肿瘤细胞和炎性细胞等[7]。YKL-40在多种恶性肿瘤[8-10]、炎性疾病[11]的组织以及血清中表达增高。在妇科恶性肿瘤如卵巢癌[12]和子宫内膜癌[13]组织和血清中的表达也高于正常组织。血清以及组织中高表达的YKL-40是各种肿瘤发生、发展的独立危险因子[10]。
3.1 YKL-40对子宫内膜癌增殖和凋亡的影响
研究表明，血清以及组织中YKL-40的表达可作为监测子宫内膜癌复发和预后的标志。YKL-40的表达与肿瘤分期相关，与患者的年龄、性别无关[13]。YKL-40在各种恶性肿瘤中具有促进细胞增殖、血管形成以及抗凋亡作用。本实验的前期研究表明[14]，应用siRNA抑制YKL-40基因在子宫内膜癌中的表达，子宫内膜癌细胞的增殖和侵袭能力降低，表明YKL-40可能在子宫内膜癌中发挥促进细胞增殖和转移的作用。与胆管癌、前列腺癌、脑胶质瘤等体外肿瘤细胞中的研究结果一致[15-17]。体内动物实验也表明，基因干扰后其成瘤体积小于干扰前[2]。其机制可能与以下几个信号相关：在胆管癌细胞中，YKL-40促进细胞增殖与AKT/ERK通路相关[15]；在胶质瘤细胞中，YKL-40促进细胞增殖可能与细胞外调节激酶ERK1/2（extracellular regulated kinase 1/2）通路相关[18]；在卵巢癌的研究中可通过诱导Mcl-1抗凋亡基因，抑制卵巢癌细胞的凋亡[5]；还有研究表明，YKL-40参与肿瘤细胞耐药机制可能与信号转导和转录激活因子3（STAT3）信号通路相关[19]。以上说明YKL-40在子宫内膜癌增殖和凋亡中发挥重要作用。
3.2 顺铂（化疗）对YKL-40表达的影响
本研究通过制备特异性YKL-40 siRNA慢病毒重组载体，转染到子宫内膜癌细胞中，可有效抑制YKL-40基因表达，有效地阻断YKL-40的活性。相同浓度的顺铂处理子宫内膜癌细胞，子宫内膜癌细胞中YKL-40基因的表达水平增加，提示在外界不良刺激条件下，YKL-40可能与子宫内膜癌增殖和凋亡相关。与van Linde[20]的研究一致，用ELISA的方法检测化疗后肿瘤患者血清中YKL-40的表达增高（P=0.0002）。且耐药性上皮性卵巢癌中YKL-40的水平高于化疗敏感的上皮性卵巢癌，YKL-40也是卵巢癌新辅助化疗的监测指标[21]。但是在Xu等[22]研究中，化疗后患者血清YKL-40的表达水平反而降低。在一项乳腺癌的研究中描述了新辅助化疗前后血清中YKL-40的表达情况：化疗敏感组化疗后YKL-40的水平降低；而化疗不敏感组化疗后YKL-40的水平较化疗前增高[23]。化疗后肿瘤患者YKL-40的表达水平可能是肿瘤对化疗病理反应，可能与肿瘤患者的预后密切相关。由此看出，在外界不良刺激下（比如化疗药物），YKL-40的表达增高可能是应激性增加，YKL-40也可能是一种抗凋亡因子，是一种化疗预后的监测指标。
3.3 YKL-40对顺铂化疗敏感度的影响
化疗前高表达的YKL-40是肿瘤化疗的独立预后因子[24]。YKL-40与耐药肿瘤细胞株的化疗敏感度密切相关，降低耐药细胞株中YKL-40的含量可提高肿瘤化疗药物的敏感度[19]。同时，卵巢癌患者血清中高表达的YKL-40增加了其二线化疗药物耐受的风险[25]。然而在一项非小细胞肺癌的研究中却表明，化疗前YKL-40的水平与化疗敏感度无关[26]，化疗前后YKL-40的中位水平差异无统计学意义（P=0.62）。本研究表明，YKL-40 siRNA可增加子宫内膜癌细胞的化疗敏感度。用流式细胞仪测定发现，实验组细胞的凋亡率明显高于对照组，可能提示YKL-40与子宫内膜癌铂类药物耐药有关，抑制子宫内膜癌细胞YKL-40基因后，子宫内膜癌对铂类药物的敏感度增加，说明YKL-40可能是子宫内膜癌细胞铂类耐药的一种病理反应产物，其在子宫内膜癌细胞增殖和抗凋亡中具有重要作用。同时提示，子宫内膜癌细胞对化疗药物的耐药性可能与YKL-40水平相关。化疗前血清中YKL-40水平增高，则对化疗的敏感度低、预后差[22]。但YKL-40参与肿瘤耐药以及化疗敏感度的机制尚不明确。Boisen等[27]对卵巢癌的研究表明，YKL-40可能作为肿瘤化疗预后的评估指标。在胶质瘤细胞的研究也获得类似的结果，通过沉默YKL-40基因，在癌细胞的迁移能力明显降低的同时对铂类化疗药物的敏感度增加[17]。YKL-40基因沉默能使卵巢癌细胞总体凋亡率增加，表明其在卵巢癌细胞中可能起到抗凋亡的作用[5]。癌细胞的研究中[16, 19, 28-29]，体外通过特异性siRNA片段沉默YKL-40基因也可有效抑制增殖能力。
本文从体外细胞实验验证了通过YKL-40基因沉默可提高子宫内膜癌细胞对铂类化疗药物的敏感度，且YKL-40基因具有抗凋亡作用。研究结果为子宫内膜癌的临床化疗效果提供了新的思路，表明YKL-40可能作为子宫内膜癌治疗的潜在靶点进行更加深入的研究。
Competing interests: The authors declare that they have no competing interests.利益冲突声明：所有作者均声明不存在利益冲突。作者贡献：韩雅静：文献检索、论文构思、撰写及修改赵丽平、唐凯婷、牛卿、潘静：论文审校及修改冯晓明：论文选题、撰写指导及修改 -
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