CN117599186A - 抑制laptm5的物质在aml中的制药用途 - Google Patents
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- CN117599186A CN117599186A CN202311670805.2A CN202311670805A CN117599186A CN 117599186 A CN117599186 A CN 117599186A CN 202311670805 A CN202311670805 A CN 202311670805A CN 117599186 A CN117599186 A CN 117599186A
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Abstract
本发明公开了抑制LAPTM5的物质在AML中的制药用途，涉及生物技术领域。本发明提供了抑制LAPTM5的物质在制备治疗、辅助治疗和/或预防白血病的药物中的应用。本发明还提供了抑制LAPTM5的物质在提高细胞对治疗白血病药物的敏感性中的应用。本发明发现LAPTM5是AML对AraC耐药的关键基因，靶向LAPTM5可以显著降低AML细胞对AraC的耐药性，在AML中LAPTM5和阿糖胞苷之间存在联合致死效应，LAPTM5有望成为AML中联合阿糖胞苷治疗耐药的新靶点。
Description
技术领域
本发明涉及生物技术领域，具体涉及抑制LAPTM5的物质在AML中的制药用途。
背景技术
急性髓系白血病(AML)是一种高度异质性的血液系统恶性肿瘤，以大量异常分化的髓系祖细胞在骨髓、血液或组织中克隆增殖浸润为主要特征。绝大多数患者病情急重，病程凶险，如果未进行及时治疗干预，诊断后生存期不超过1年。
AML治疗包括诱导化疗和强化巩固，预后不良的中高危患者需进行造血干细胞移植。阿糖胞苷(Cytarabine，AraC)在AML治疗中应用广泛，目前临床的一线疗法仍然是以阿糖胞苷和柔红霉素为主的“7+3”诱导疗法：将阿糖胞苷用药7天与柔红霉素等蒽环类药物用药3天相结合，可高效杀死AML中的白血病细胞，这两种药物对白血病细胞毒性主要依赖于抑制DNA合成，以及诱导DNA双链损伤断裂等机制，60％患者用药效果良好，诱导缓解治疗的完全缓解率(CR)达到80％，但远期治疗效果尚不理想，仅35％-40％小于60岁患者以及5％-15％大于60岁患者能长期生存，其余患者多数最终由于化疗耐药导致疾病持续或复发最终死于AML，化疗药物耐药是引起AML预后不良最主要的原因。因此，探究AML对AraC的耐药机制、寻找耐药关键靶点是本领域重点研究方向。
溶酶体相关跨膜蛋白5(Lysosome-associated protein transmembrane 5，LAPTM5)，是一种位于晚期核内体和溶酶体的跨膜蛋白，其编码基因位于1p34染色体。LAPTM5蛋白分子量为29kDa，包括5个跨膜区域，其N端位于溶酶体内，C端位于细胞质内，包含3个聚脯氨酸酪氨酸基序(L/PPxY)、一个泛素相互作用模序(UIM)。LAPTM5参与蛋白质由高尔基体向溶酶体的运输过程，通过与E3泛素连接酶—Nedd4相互作用，影响溶酶体和细胞质膜的分选。在肝癌研究中，LAPTM5可以通过调控细胞自噬介导化疗药物耐药，肝癌组织LAPTM5表达水平能够有效预测肝癌患者对于仑伐替尼的敏感性。另一癌症相关研究中发现LAPTM5介导了肾癌的肺特异性转移，并可以通过阻断肺源性骨形态发生蛋白(BMP)的功能以增强肾癌细胞的自我更新能力。在关于溶酶体依赖性细胞死亡途径的研究中，发现溶酶体细胞死亡调节(lysosome cell death regulator,LCDR)基因可以提高LAPTM5的稳定性，从而维持溶酶体膜的稳定性，抑制溶酶体依赖性细胞死亡，促进肿瘤细胞生存，提示LAPTM5可能是癌症研究中的潜在治疗靶点，然而LAPTM5在AML对AraC耐药中的作用未见报道，本发明首次发现LAPTM5是AML对AraC耐药的关键基因，靶向LAPTM5可以显著降低AML细胞对AraC的耐药性，提示在AML中LAPTM5和阿糖胞苷之间存在联合致死效应，LAPTM5有望成为AML中联合阿糖胞苷治疗耐药的新靶点。
发明内容
本发明的术语和声明：
在本发明中，冠词“一个”、“一种”和“所述”：除非以其它方式明确地限定到一个(种)对象，否则包括复数的对象。
在本发明中，术语“蛋白质”是指至少两个共价连接的氨基酸，其包括蛋白质、多肽、寡肽和肽。该术语还包括蛋白质的表达后修饰，如糖基化、乙酰化、磷酸化等。该术语还包括对天然蛋白质或多肽的氨基酸序列的修饰如缺失、替换、插入后所得的变体。
在本发明中，术语“AraC”是指阿糖胞苷，术语嘧啶类抗代谢化疗药物，阿糖胞苷具有细胞周期特异性，对细胞S期即DNA合成期较敏感。进入人体经过激酶磷酸化后转为阿糖胞苷三磷酸和阿糖胞苷二磷酸，能抑制细胞DNA合成，干扰细胞增殖，从而达到治疗目的。阿糖胞苷主要用于急性白血病的治疗。
在本发明中，术语“耐药”，又称为抗药性，是指肿瘤细胞对于化疗药物作用的耐受性，一旦产生耐药性，药物的化疗作用就明显下降。
在本发明中，术语“治疗有效量”是指服用后足以在某种程度上缓解所治疗的疾病或病症的一个或多个症状的至少一种药剂或化合物的量。其结果可以为迹象、症状或病因的消减和/或缓解，或生物系统的任何其它所需变化。可使用诸如剂量递增试验的技术测定适合于任意个体病例中的有效量。
在本发明中，术语“药学可接受的”是指某载体、运载物、稀释剂、辅料，和/或所形成的盐通常在化学上或物理上与构成某药物剂型的其它成分相兼容，并在生理上与受体相兼容。
本发明的技术方案如下：
一方面，本发明提供了一种抑制LAPTM5的物质在制备治疗、辅助治疗和/或预防白血病的药物中的应用。
具体地，所述的抑制LAPTM5的物质为能够使得LAPTM5蛋白的表达量、含量和比活性中的至少一种发生下降。
进一步地，所述的抑制LAPTM5的物质选自靶向LAPTM5的siRNA、插入有编码靶向LAPTM5的shRNA的片段的表达质粒、插入有编码靶向LAPTM5的gRNA的片段的基因编辑载体中的一种或多种。
具体地，所述的白血病包括急性白血病和慢性白血病。
进一步地，所述的急性白血病包括急性髓系白血病和急性淋巴细胞白血病。
进一步地，所述的慢性白血病包括但不限于慢性粒细胞白血病、慢性淋巴细胞白血病、慢性粒单核细胞白血病。
再进一步地，所述的白血病为急性髓系白血病。
更进一步地，所述的急性髓系白血病包括但不限于急性粒细胞白血病、急性单核细胞白血病、急性红白血病、急性巨核细胞白血病。
更进一步地，所述的急性淋巴细胞白血病包括但不限于急性B淋巴细胞白血病、急性T淋巴细胞白血病。
优选地，所述药物的作用为如下至少一种：
(a)增加细胞对治疗白血病药物的敏感性；
(b)降低治疗白血病药物的耐药性；
(b)减少治疗白血病药物诱导的细胞凋亡。
优选地，本发明的抑制LAPTM5的物质可以和其他治疗白血病的药物联合应用。
又一方面，本发明提供了抑制LAPTM5的物质在提高细胞对治疗白血病药物的敏感性中的应用。
进一步地，所述的细胞为白血病细胞。
再进一步地，所述的白血病细胞为AML细胞。
进一步地，所述的治疗白血病的药物包括但不限于阿糖胞苷、柔红霉素、米托蒽醌、长春新碱、阿霉素、门冬酰胺酶、甲氨蝶呤。
再进一步地，所述的治疗白血病的药物为阿糖胞苷。
具体地，所述的抑制LAPTM5的物质是通过脂质代谢提高细胞对治疗白血病药物的敏感性。
又一方面，本发明提供了一种治疗、辅助治疗和/或预防白血病的药物，所述的药物中包含治疗有效量的抑制LAPTM5的物质。
具体地，所述的白血病包括急性白血病和慢性白血病。
进一步地，所述的急性白血病包括急性髓系白血病和急性淋巴细胞白血病。
进一步地，所述的慢性白血病包括但不限于慢性粒细胞白血病、慢性淋巴细胞白血病、慢性粒单核细胞白血病。
再进一步地，所述的白血病为急性髓系白血病。
优选地，所述药物中还包括药学上可接受的载体，所述药学上可接受的载体选自稀释剂、粘合剂、表面活性剂、润滑剂、填充剂、崩解剂、稳定剂中的至少一种。
再进一步优选地，所述稀释剂包括但不限于淀粉、乳糖、葡萄糖、氯化钠、尿素。
再进一步优选地，所述粘合剂包括但不限于糊精、蔗糖、阿拉伯胶、乙基纤维素、聚乙烯醇、预胶化淀粉、麦芽糖糊精、聚乙二醇、羧甲基纤维素、聚乙烯比咯烷酮、明胶、羟丙基纤维素和羟丙基甲基纤维素。
再进一步优选地，所述表面活性剂包括但不限于聚氧化乙烯山梨聚糖脂肪酸酯、硬脂酸单甘油酯、十二烷基硫酸钠、十六烷醇。
再进一步优选地，所述润滑剂包括但不限于单硬脂酸甘油酯、滑石粉、硬脂酸锌、硬脂富马酸钠、聚乙二醇、单月桂蔗糖酸酯、聚乙二醇、月桂醇硫酸钠、月桂醇硫酸镁、聚氧乙烯单硬脂酸酯、十二烷基硫酸镁。
再进一步优选地，所述填充剂包括但不限于木糖醇、麦芽糖、山梨醇、乳糖、蔗糖、糊精、甘露醇、葡萄糖、淀粉、海藻酸钠、赤藓糖、海带多糖粉末、微晶纤维素、琼脂粉末、碳酸钙和碳酸氢钠。
再进一步优选地，所述崩解剂包括但不限于羧甲基淀粉钠、交联乙烯吡咯烷酮、低取代羟丙基甲基、交联羧甲基纤维素钠。
再进一步优选地，所述稳定剂包括但不限于人类血清蛋白、L-氨基酸、糖及纤维素衍生物。
优选地，所述的药物可口服给药、非胃肠道给药、通过吸入喷雾给药、局部给药、直肠给药、鼻给药、颊给药或通过植入的贮药装置给药。本发明药物可含有任何常用的无毒可药用载体、辅料或赋形剂。
优选地，所述的药物的剂型包括片剂、胶囊剂、颗粒剂、丸剂、滴丸剂、糖浆剂、粉剂、散剂、栓剂、滴剂、气雾剂、乳剂、注射剂和混悬剂。
具体地，本发明提供了一种体外非诊断或非治疗目的地提高白血病细胞对治疗白血病药物的敏感性的方法，所述的方法为向白血病细胞施用抑制LAPTM5的物质。
本发明的有益效果为：
本发明首次发现LAPTM5是AML对AraC耐药的关键基因，靶向LAPTM5可以显著降低AML细胞对AraC的耐药性，提示在AML中LAPTM5和阿糖胞苷之间存在联合致死效应，LAPTM5有望成为AML中联合阿糖胞苷治疗耐药的新靶点。
附图说明
图1为活细胞/死细胞双染实验检测各组细胞经AraC处理后的细胞凋亡情况。
图2为活细胞/死细胞双染实验结果的定量统计。
图3为WB检测经AraC处理后的各组细胞CASPASE3和PARP蛋白的表达情况。
图4为CCK8法检测各组细胞对AraC的IC50值。
图5为qPCR法检测各组细胞LAPTM5 mRNA相对表达量。
图6为CCK8法检测各组细胞对AraC的IC50值。
图7为WB检测经药物处理后的各组细胞CASPASE3和PARP蛋白的表达情况。
图8为qPCR法检测各组细胞LAPTM5 mRNA相对表达量。
图9为CCK8法检测各组细胞对AraC的IC50值。
图10为WB检测经AraC处理后的各组细胞CASPASE3和PARP蛋白的表达情况。
图11为流式细胞术检测AraC处理后的凋亡细胞比例。
图12为流式细胞术检测凋亡细胞比例的定量统计。
图13为活细胞/死细胞双染实验检测各组细胞经AraC处理后的细胞凋亡情况。
图14为活细胞/死细胞双染实验实验结果的定量统计。
图15为Scramble组和shLAPTM5组肿瘤体积变化曲线图。
图16为Scramble组和shLAPTM5组H&E、KI67染色结果图。
图17为Scramble组和shLAPTM5组KI67染色结果统计图。
图18为HL60 Arac-R scramble组和Arac-R shLAPTM5组肿瘤体积变化曲线图。
图19为HL60 Arac-R scramble组和Arac-R shLAPTM5组H&E、KI67染色结果图。
图20为HL60 Arac-R scramble组和Arac-R shLAPTM5组H&E、KI67染色结果统计图。
图21为Scramble组、HL60 Arac-R scramble组和Arac-R shLAPTM5组脂滴染色结果图。
图22为Scramble组、HL60 Arac-R scramble组和Arac-R shLAPTM5组脂滴染色结果统计图。
图23为Scramble组、HL60 Arac-R scramble组和Arac-R shLAPTM5组PLIN2蛋白免疫荧光结果图。
图24为Scramble组、HL60 Arac-R scramble组和Arac-R shLAPTM5组PLIN2蛋白免疫荧光结果统计图。
组别说明：图1-图4中，WT：HL60敏感细胞株；Arac-R：HL60阿糖胞苷耐药细胞株。
图5-图14中，Scramble：空载病毒载体感染的HL60敏感细胞株；Sh1：敲低LAPTM5基因病毒载体感染的HL60敏感细胞株1；Sh2：敲低LAPTM5基因病毒载体感染的HL60敏感细胞株2；Sh3：敲低LAPTM5基因病毒载体感染的HL60敏感细胞株3；Arac-R scramble：空载病毒载体感染的HL60 Arac-R细胞株；R-sh1：敲低LAPTM5基因病毒载体感染的HL60 Arac-R细胞株1；R-sh2：敲低LAPTM5基因病毒载体感染的HL60 Arac-R细胞株2；R-sh3：敲低LAPTM5基因病毒载体感染的HL60 Arac-R细胞株3。
图15-图20中，Scramble：皮下移植Scramble细胞株的小鼠；和Scramble一组的ShLAPTM5：皮下移植Sh3细胞株的小鼠；Arac-R scramble：皮下移植Arac-R scramble细胞株的小鼠；和Arac-R scramble一组的ShLAPTM5：皮下移植R-sh1细胞株的小鼠。
图21-图24中，Scramble：空载病毒载体感染的HL60敏感细胞株；Arac-Rscramble：空载病毒载体感染的HL60 Arac-R细胞株；ShLAPTM5：R-sh1细胞株。
上述附图中，*p<0.05；**p<0.01；***p<0.001；ns表示没有显著性差异。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例，进一步阐明本发明，但下述实施例仅为本发明的优选实施例，并非全部。基于实施方式中的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得其它实施例，都属于本发明的保护范围。下述实施例中，若无特殊说明，所用的操作方法均为常规操作方法，所用设备均为常规设备，各个实施例所用设备材料均相同。
实施例1构建AML AraC耐药细胞株
为研究抑制LAPTM5是否可以影响肿瘤耐药细胞对AraC的敏感性，首先使用药物浓度递增法在体外构建了人早幼粒白血病细胞HL60的AraC耐药细胞株(以下简称耐药细胞株、HL60 Arac-R)，蛋白免疫印迹(Western blot,WB)和CCK8实验结果表明成功构建了耐药细胞株。
1、试剂：AraC(购自于Selleck Chemicals公司,S1648)、Cell Counting Kit-8(CCK-8,购自于APExBIO Technology公司,K1018)、ACTB(P30002),CASP3(TA7022),PARP(T40050)抗体均购于Abmart公司。
2、检测HL60细胞对AraC的IC50(半抑制浓度)值：在IC50检测实验中，设置的AraC浓度梯度范围为：0-20uM。同时设置对照组(PBS代替AraC)和空白组(未铺板细胞)。取生长状态良好的HL60细胞，离心去上清后用适量PBS重悬制成细胞悬液，使用细胞计数板计数。根据细胞计数结果，并按照96孔板每个孔铺板8000个细胞的标准计算后，用培养基补足体积制得含有特定细胞数量的细胞悬液。然后向96孔板中每个孔加入90ul细胞悬液和10ul药物溶液，96孔板四周的未处理孔中加入100ul PBS以减少边缘溶剂蒸发带来的影响，将96孔板放置在细胞培养箱培养。培养48h后，每孔加入10ul CCK-8试剂，37℃避光孵育1h后使用酶标仪在波长450nm下检测吸光度，最后使用GraphPad Prism计算HL60细胞对AraC的IC50值。
3、构建耐药细胞：采用上述检测得到的HL60细胞对AraC的IC50值的百分之一(浓度为50nM)做为初始诱导浓度。加入药物处理细胞24h后，换成新鲜培养基继续培养24h，随后根据细胞状态，选择使用相同药物浓度或者更高药物浓度处理细胞。
4、验证耐药细胞：
每隔2周对诱导培养后的细胞进行1次IC50检测。细胞耐药的标准：IC50值达到敏感细胞IC50值的10倍(5uM)。
当诱导细胞的IC50值达到耐药标准后，检测药物处理下的细胞凋亡水平：用AraC(5uM)同时处理HL60野生型细胞和耐药细胞，使用WB检测细胞凋亡指标：Caspase-3、PARP蛋白的表达变化情况。后续的细胞实验中同样选用5uM做为处理浓度。
WB具体操作方法如下：收集经药物处理后的细胞，离心去上清，细胞沉淀用PBS洗一遍，根据细胞数量加入适量RIPA裂解液(含蛋白酶抑制剂)，冰上裂解30min，每10min混匀一次。12000rpm，4℃，10min离心后，转移上清至新的EP管。经蛋白定量后与蛋白上样缓冲液混合以制备电泳样品。蛋白样品经12％的SDS-PAGE分离，转移到0.2um PVDF膜上，随后进行5％脱脂牛奶封闭、一抗过夜孵育、二抗室温孵育。通过化学发光成像系统采集信号，最后使用ImagJ和GraphPad Prism软件对结果进行定量分析。
同时通过活细胞/死细胞双染实验检测AML细胞系对药物敏感程度，双染实验具体操作方法如下：取生长状态良好的细胞，离心去上清，经PBS洗涤1次后，用PBS重悬并用细胞计数板进行细胞计数。不同组的细胞需以相同细胞密度(10000颗细胞/孔)铺于孔板中，并加入相同浓度(5uM)的AraC，且保持药物处理时间一致。药物处理24h后，收集细胞至EP管中，离心去上清，细胞沉淀用1x的Assay Buffer洗涤1次，加入适量1x的Assay Buffer重悬，每1x105个细胞加入1ul Calcein-AM和3ul PI试剂，置于细胞培养箱，避光孵育20min后离心去上清，PBS洗涤1次后，再次用PBS重悬，使用荧光显微镜拍照，记录数据。
实验结果：
如图1-图4所示，体外培养体系中，细胞持续处于药物压力下，逐渐表现出明显的耐药表型，具体表现为：在相同浓度的药物处理下，Arac-R细胞株的凋亡蛋白cleavedcaspase3和cleaved PARP水平较敏感细胞株的明显减少，且耐药细胞的IC50值达到敏感细胞IC50值的10倍(5uM)
实施例2AML AraC耐药细胞株中LAPTM5检测
1、实验试剂：TRIzol裂解液(购自于Vazyme公司,R401-01)、逆转录试剂盒和SYBRGreen qPCR SuperMix均购于北京全式金公司、LAPTM5抗体(购自于Abcepta公司,AP10077a)。
2、检测耐药细胞LAPTM5 mRNA水平包括以下步骤：收集细胞，加入1ml TRIzol裂解液充分裂解细胞。加入200ul氯仿，充分混匀后静置5min，随后设置转速12000rpm，离心15min，转移最上层澄清液体至新的EP管。加入等体积异丙醇混匀后，冰上静置5min，随后设置转速12000rpm，离心10min，去上清，使用75％乙醇洗涤沉淀2次。得到的RNA沉淀用适量DEPC水溶解，测定RNA浓度。使用逆转录试剂盒将RNA逆转录成cDNA。随后使用SYBR体系进行qPCR。使用引物序列如下：
Human-ACTIN:
上游引物：CATGTACGTTGCTATCCAGGC(SEQ ID NO:1)
下游引物：CTCCTTAATGTCACGCACGAT(SEQ ID NO:2)
Human-LAPTM5:
上游引物：GCGTCTTGTTGTTCATCGAGC(SEQ ID NO:3)
下游引物：CGATCCTGAGGTAGCCCAT(SEQ ID NO:4)
qPCR的反应体系为20ul；反应体系比例：DEPC水:上游引物:下游引物:待测cDNA＝10:7:1:1。
反应程序：94℃30秒，94℃5秒，60℃15秒，72℃10秒，设置40个循环。
3、检测耐药细胞LAPTM5蛋白水平：蛋白样品制备方法同实施例一。WB中使用10％SDS-PAGE分离蛋白样品，其余实验操作同实施例一。
试验结果：
如图3所示，和敏感株细胞相比，耐药细胞株高表达LAPTM5，表明LAPTM5可能和AML细胞对AraC耐药有关。
实施例3构建AML LAPTM5敲低稳转细胞系
1、试剂：聚凝胺(Polybrene,Sigma-Aldrich,MFCD00133397)、pLV3-U6-Laptm5(human)-shRNA-GFP-Puro和pLV3-U6-GFP-Puro质粒均购于淼灵生物公司。LAPTM5敲低质粒引物序列如下：
shRNA1-F：
GGTGCTACAGATTGATCAAGTTTCAAGAGAACTTGATCAATCTGTAGCACC(SEQ ID NO:5)。
shRNA1-R：
GGTGCTACAGATTGATCAAGTTCTCTTGAAACTTGATCAATCTGTAGCACC(SEQ ID NO:6)。
shRNA2-F：
GCCATCTACCATGTGATCATGTTCAAGAGACATGATCACATGGTAGATGGC(SEQ ID NO:7)。
shRNA2-R：
GCCATCTACCATGTGATCATGTCTCTTGAACATGATCACATGGTAGATGGC(SEQ ID NO:8)。
shRNA3-F：
GCCTTCATCACTGTCCTTATCTTCAAGAGAGATAAGGACAGTGATGAAGGC(SEQ ID NO:9)。
shRNA3-R：
GCCTTCATCACTGTCCTTATCTCTCTTGAAGATAAGGACAGTGATGAAGGC(SEQ ID NO:10)。敏感细胞株和耐药细胞株分别敲低LAPTM5用的质粒相同。
2、获取目的质粒病毒液：HEK 293T细胞以合适的细胞密度铺板，待细胞密度达到60-70％，更换无血清培养基。同时按照慢病毒包装说明书(快速慢病毒包装试剂盒购自上海雅酶生物医药科技有限公司，货号为GY101)，将目的质粒、病毒相关质粒、转染试剂一起加入HEK 293T细胞培养体系中，转染6小时后换含20％血清的培养基继续培养48h。
3、感染目的细胞：HL60细胞提前以40-50％密度铺板于6孔板中。收集HEK 293T细胞上清，经0.45um滤膜过滤后加入到HL60细胞中，同时加入聚凝胺(Polybrene,8ug/ml)提高感染效率。目的细胞经病毒感染12h后，离心去上清，更换含10％血清的新鲜培养基。继续培养24h后，加入0.1ug/ml嘌呤霉素进行抗性筛选。
4、验证稳转细胞系：经嘌呤霉素筛选后的细胞，通过qPCR和WB检测LAPTM5基因和蛋白表达水平，检测方法同实施例2。
试验结果：
如图5-图10所示，成功构建AML LAPTM5敲低稳转细胞系。
实施例4抑制LAPTM5增加了AML细胞对AraC的敏感性
1、试剂：Calcein-AM/PI活细胞/死细胞双染试剂盒购于索莱宝公司、Annexin V-APC/DAPI Apoptosis Kit购于Elabscience公司。
2、通过活细胞/死细胞双染实验检测AML细胞系对药物敏感程度，双染实验操作同实施例一。
3、检测AML细胞系IC50：检测AML LAPTM5敲低细胞的IC50，IC50检测方法同实施例一。
4、通过WB检测AML细胞系凋亡水平：检测AML LAPTM5敲低细胞的凋亡水平，WB实验操作同实施例一。
5、通过流式检测AML细胞系凋亡水平：取生长状态良好的细胞，离心去上清，经PBS洗涤1次后，用PBS重悬并用细胞计数板进行细胞计数。不同组的细胞需以相同细胞密度(10000颗细胞/孔)铺于孔板中，并加入相同浓度(5uM)的AraC，且保持药物诱导凋亡时间一致。诱导处理12h后，收集细胞至EP管中，离心去上清，PBS重悬后进行细胞计数，每组均取1x105个重悬的细胞，离心去上清，加入100ul的1x Annexin V Binding Buffer重悬细胞，再加入2.5ul Annexin V-APC和2.5ul DAPI试剂，室温避光孵育15min。染色结束后，加入400ul 1xAnnexin V Binding Buffer稀释样本，通过流式细胞仪检测。
试验结果：
结果如图5-图14所示，在野生型细胞株和耐药细胞株中抑制LAPTM5，均可以促进AraC诱导的细胞凋亡，抑制LAPTM5增加了AML细胞系对阿糖胞苷的敏感性。更值得关注的是，在耐药细胞株中抑制LAPTM5后，阿糖胞苷诱导的细胞凋亡效应要强于在野生株中抑制LAPTM5。这提示了在AML中，LAPTM5和阿糖胞苷之间存在联合致死效应。
实施例5SCID小鼠皮下荷瘤实验结果验证抑制LAPTM5可抑制AML进展
所有动物实验均得到中山大学实验动物伦理委员会批准，并按照委员会批准的指导方法进行，伦理批准编号：SYSU-IACUC-2023-001717。
1、实验分组及皮下瘤种植方案：24只4周龄雄性SCID小鼠，随机分为4组，每组分为两笼饲养，每天更换洁净的饮用水、饲料及垫料。具体操作如下：
组1—HL60 Scramble组：共7只小鼠。皮下瘤种植：首先固定小鼠，用动物专用剃毛刀剔除每只小鼠右侧背侧部位的被毛。酒精棉球消毒皮肤，用1ml注射器将HL60 Scramble细胞以0.1ml体积，1×106个细胞/只鼠，接种到小鼠背部右翼。饲养期间每天观察并记录小鼠的体重，体表、体温、饮食饮水和肿瘤体积变化等情况，并选择合适时间节点对小鼠背部拍照。
组2—HL60 shLAPTM5组：共7只小鼠。皮下瘤种植操作步骤、观察记录动物方法同组1。本组皮下种植的细胞种类为HL60目的基因敲除细胞。
组3—HL60 Arac-R scramble组：共5只小鼠。皮下瘤种植操作步骤、观察记录动物方法同组1。本组皮下种植的细胞种类为HL60耐药细胞。
组4—HL60 Arac-R shLAPTM5组：共5只小鼠。皮下瘤种植操作步骤、观察记录动物方法同组1。本组皮下种植的细胞种类为HL60耐药后目的基因敲除细胞。
2、给药方案：阿糖胞苷提前用注射用生理盐水配制，现用现配，并通过0.22um滤膜除菌。当组3和组4的小鼠的皮下瘤生长至可触及时，经尾静脉给药阿糖胞苷，给药剂量为12mg/kg，给药时间为每日1次，连续给药7天。给药期间每天观察并记录小鼠的体重，体表、体温、饮食饮水和肿瘤生长等情况。
3、处理肿瘤组织：当组1和组2肿瘤生长至第30天，而给药组经药物处理1周后，对小鼠背部肿瘤组织取材、拍照。肿瘤组织经过4％多聚甲醛液浸泡、石蜡包埋、切片等步骤后，进行后续的苏木精-伊红、免疫组织化学染色，均采用本领域的常规操作方法。
试验结果：
结果如图15-图20所示，体内实验中，敲低细胞系的成瘤率显著下降，肿瘤生长缓慢，肿瘤增殖标志物明显减少。耐药细胞株的小鼠荷瘤实验表明，抑制LAPTM5的同时联合阿糖胞苷治疗进一步抑制了AML的进展。这更加提示LAPTM5可以做为联合阿糖胞苷治疗耐药的新靶点。
实施例6LAPTM5可能通过脂滴代谢调控AML细胞对AraC耐药
1、试剂：AIE脂滴黄色探针试剂盒购于生工生物工程(上海)股份有限公司。
2、检测AML细胞系脂滴水平：首先准备染料工作液：取1μl AIE Lipid dropletsYellow Probe储备液加入到1ml PBS缓冲液中，得到终浓度10μM的AIE Lipid dropletsYellow Probe工作液。收集细胞，加入适量的工作液后，置于细胞培养箱中孵育30min，PBS洗涤3次，使用共聚焦荧光显微镜观察，激发波长设为488nm；收集波长550-650nm的信号。
3、通过免疫荧光实验(Immunofluorescent,IF)检测AML细胞系脂滴标志物—PLIN2蛋白表达水平：收集细胞，用PBS洗涤一次。使用细胞涂片离心机离心机将悬浮细胞固定在载玻片上。弃去上清，加入4％多聚甲醛，在室温下固定15min，然后吸出多聚甲醛并用PBS洗涤3次，每次5min。向载玻片中加入0.5％ Triton X-100，在室温下孵育10分钟，弃去上清，用PBS洗涤3次，每次5分钟，然后加入10％山羊血清，室温下封闭45min。吸出封闭液，用PBST洗涤1次，加入PLIN2一抗，4度摇床过夜。吸出一抗，用PBST溶液洗涤3次，每次洗涤5分钟，加入荧光二抗，室温下避光孵育1小时。最后，小心滴加抗荧光淬灭封片剂，尽快使用显微镜观察。
试验结果：
结果如图21-图24所示，与野生型细胞相比，耐药细胞的脂滴水平显著增加，药物处理后，耐药细胞的脂滴水平明显下降，而LAPTM5受到抑制时，表现为敲低LAPTM5细胞的耐药表型减弱、脂滴蛋白PLIN2水平无明显变化，这提示LAPTM5可能通过脂滴代谢调控AML细胞对Arac耐药。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.抑制LAPTM5的物质在制备治疗、辅助治疗和/或预防白血病的药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述的抑制LAPTM5的物质为能够使得LAPTM5蛋白的表达量、含量和比活性中的至少一种发生下降。
3.根据权利要求1所述的应用，其特征在于，所述的白血病包括急性白血病和慢性白血病。
4.根据权利要求3所述的应用，其特征在于，所述的白血病为急性髓系白血病。
5.抑制LAPTM5的物质在提高细胞对治疗白血病药物的敏感性中的应用。
6.根据权利要求5所述的应用，其特征在于，所述的细胞为白血病细胞；所述的白血病细胞为急性髓系白血病细胞。
7.根据权利要求5所述的应用，其特征在于，所述的治疗白血病药物选自阿糖胞苷、柔红霉素、米托蒽醌、长春新碱、阿霉素、门冬酰胺酶、甲氨蝶呤中的一种或多种。
8.根据权利要求7所述的应用，其特征在于，所述的治疗白血病药物为阿糖胞苷。
9.根据权利要求5所述的应用，其特征在于，所述的抑制LAPTM5的物质是通过脂质代谢提高细胞对治疗白血病药物的敏感性。
10.一种治疗、辅助治疗和/或预防白血病的药物，其特征在于，所述的药物中包含治疗有效量的抑制LAPTM5的物质。
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| SHAOYAN HU ETAL.: "Overexpression of Lysosomal-Associated Protein Transmembrane 5 (LAPTM5) Deceases Autophagy Activity Via Reducing the Lysosomal pH Value", 《BLOOD》, vol. 124, no. 21, 31 December 2014 (2014-12-31), pages 1 - 4 * |
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