CN106065402B - Nok突变体及其在肿瘤诊断、治疗和药物筛选中的用途 - Google Patents
Nok突变体及其在肿瘤诊断、治疗和药物筛选中的用途 Download PDFInfo
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- CN106065402B CN106065402B CN201610244757.4A CN201610244757A CN106065402B CN 106065402 B CN106065402 B CN 106065402B CN 201610244757 A CN201610244757 A CN 201610244757A CN 106065402 B CN106065402 B CN 106065402B
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Abstract
本发明提供了NOK突变体及其在肿瘤诊断、治疗和药物筛选中的用途。具体地，本发明提供一种分离的核酸、含有所述分离核酸的载体、所述载体稳定转染的细胞系以及所述分类的核酸在肿瘤诊断、治疗和药物筛选平台中的应用。所述分离的核酸在一定程度上抑制细胞的增殖以及细胞锚定非依赖生长特性。
Description
技术领域
本发明涉及癌基因的突变体及其在疾病诊断和治疗中的用途，特别涉及NOK突变体及其在肿瘤诊断、治疗和药物筛选中的用途。
背景技术
受体型酪氨酸激酶(RPTKs)是一类酶蛋白，它们调控着机体内多种多样的生物学活动，包括细胞的增殖、分化、细胞的周期调控、胚胎形成、血管生成和代谢等(1-3)。由于它们在维持正常细胞的生理活动中有重要作用，因此它们的活性总是受到严格的调控。但是，当RPTKs的正常活性被打乱后，机体会发生非常严重的功能紊乱，如疾病的发生，甚至癌症(4-8)。在经典的受体型酪氨酸激酶活化的过程中，配体首先识别并结合受体特异性的胞外结构域，之后可促使受体同源或者异源二聚体的形成。在胞内所形成的受体二聚体中，其中的一个受体可以通过发挥其激酶的特性磷酸化与之结合的另一个受体，通过相互的磷酸化最终导致受体复合物的活化。而二聚体中发生了磷酸化的位点则可以作为锚定位点，通过这些锚定位点招募胞内关键性的效应蛋白或者接头蛋白，这些效应或者接头蛋白一般都包含有Src同源的(SH2)和磷酸化酪氨酸结合的结合域(PTB)，从而进一步激活不同的下游信号通路(10)。
NOK是最新被发现的受体型酪氨酸激酶，它可能是该家族中独特的一员(11,12)。前期的研究表明NOK是一个强致癌基因，具有导致肿瘤发生和转移的特性(11,13)。通过对人类癌症基因组的系统性分析，发现V395I的突变与人脑星型胶质母细胞瘤的发生相关(4)。与NOK相比，STYK1仅仅只有一个氨基酸的差别，即由203位的脯氨酸替换为了亮氨酸(14)。STYK1这个基因是学者Ye等在人的胚胎脑cDNA文库中首次克隆得到。
发明内容
NOK基因是最新被鉴定的包含激酶结构域的癌基因，它属于受体型酪氨酸激酶家族中非典型的一类受体蛋白，并具有潜在的致癌特性。NOK在多种肿瘤中其表达明显上升，比如肺癌，乳腺癌，结肠癌等等。尽管如此，目前很少有关于NOK突变与其癌症发生的相关性报道。
在本研究中，我们制备了NOK的两个定点突变子，分别为P203L和V395I，其核酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示。NOK(P203L)也被称为丝氨酸/苏氨酸/酪氨酸激酶1(STYK1)，是NOK的另外一个别名。而V395I突变是在人脑星型胶质母细胞瘤细胞中发现的。通过本文研究发现，这两个突变子均没有削弱NOK的激酶活性，但是其中的突变V395I抑制了NOK的自身磷酸化。尽管在整体上这两个突变对NOK介导的信号通路有抑制作用，但是它们对ERK、Akt和STAT等信号分子的调控在不同细胞中如在HEK293T细胞中与在HeLa细胞和BaF3稳定细胞系中就存在差异。而且，这两个突变都能够显著性地抑制细胞的增殖能力。同时，这两个突变可以充分抑制BaF3稳定细胞系对IL-3的依赖性生长特性及锚定非依赖生长特性。
本发明一方面涉及分离的核酸，其中所述核酸是癌基因突变体，所述癌基因优选为NOK基因，所述突变体优选为NOK基因的P203L或V395I突变体，其中P203L的点突变使nok蛋白的氨基酸203位由P变成L，而V395I突变体点突变使nok蛋白的氨基酸395位由P变成L。所述突变体的核酸序列如SEQ ID NO:1或SEQ ID NO:2分别所示。
本发明的另一方面涉及包含NOK基因突变体的载体，优选为包含NOK基因突变体P203L或V395I突变体载体，所述载体用于瞬时转染或稳定转染细胞系。
本发明为了进一步研究，构建了NOL、P203L和V395I的瞬时转染表达载体和稳定转染表达载体，例如pCDNA3.0-NOK、pCDNA3.0-STYK1和pCDNA3.0-V395I、pCDH-CMV-MCS-EF1-CD511B-STYK1-HA和pCDH-CMV-MCS-EF1-CD511B-NOK(V395I)-HA。
本发明提供使用所述分离核酸转染的细胞系。所述细胞系为瞬时转染细胞系或稳定转染细胞系，优选自人胚肾细胞系HEK293T、人宫 颈癌细胞系HeLa、小鼠髓单核白血病细胞系WEHI-3B、小鼠前B细胞系BaF3。
本发明还提供使用所述分离核酸稳定转染的细胞系，所述细胞系优选为小鼠前B细胞系BaF3。
本发明的又一方面涉及上述细胞系在肿瘤的药物筛选平台中的应用。
本发明的还一方面涉及所述分离的核酸在制备用于治疗肿瘤的药物中的用途，所述肿瘤选自NOK过表达的肿瘤，优选自肺癌，乳腺癌，结肠癌、宫颈癌、肾细胞癌、白血病等，更优选自宫颈癌、肾细胞癌、髓单核白血病，更优选自人宫颈癌细胞HeLa、小鼠前B细胞BaF3。
根据本发明的研究，与NOK基因相比，P203L或V395I突变降低了NOK过表达细胞的增殖性和非依赖性生长性。因此，具有P203L或V395I突变的肿瘤具有更好的预后。因此，本发明提供分离的核酸的探针在制备用于肿瘤诊断和/或预后试剂盒中的用途，所述肿瘤选自NOK过表达的肿瘤，优选自肺癌，乳腺癌，结肠癌、宫颈癌、肾细胞癌、白血病等，更优选自宫颈癌、肾细胞癌、髓单核白血病。
总而言之，我们的研究结果表明，NOK基因P203和V395这两个氨基酸残基位点对NOK所介导的有丝分裂信号传导起着非常重要的作用，并且这两个位点的氨基酸替换可能选择性地影响着某些特定组织中促有丝分裂信号的传递。
附图说明
图1.NOK基因结构及体细胞突变。
图2A-图2C.NOK及其突变体激酶活性检测。图2A是NOK及其突变体(STYK1及V395I)的自主磷酸化活性检测。瞬时转染HEK293T细胞48小时后，用抗HA抗体免疫沉淀。转膜后用抗酪氨酸磷酸化抗体(anti p-Tyr)杂交。图上半部为裂解液检测NOK及其突变体过表达情况，图下半部为NOK及其突变体的自主磷酸化检测。图2B和图2C是NOK及其突变体的激酶活性检测。将等量NOK、STYK1及V395I质粒DNA瞬时转染293T细胞，48小时后收取细胞并用裂解液裂解。取5微克裂解液与等量多肽底物进行孵育，用试剂盒检测NOK及其突变体的酪氨 酸激酶活性。
图3A-图3D.在HEK293T和HeLa细胞中进行NOK介导的增值信号通路检测。在HEK293T细胞(图3A和图3B)和HeLa细胞(图3C和图3D)中分别过表达NOK及其突变体并检测它们对RAS/MAPK,PI3K/Akt及STAT信号通路的影响。用NOK及其突变体瞬时转染细胞，48小时后收取细胞并进行细胞裂解。用ERK,磷酸化ERK(p-ERK)(Tyr204),AKT,磷酸化AKT(p-AKT)(Thr 308),STAT1,磷酸化STAT1(p-STAT1)(Tyr705),STAT3,磷酸化STAT3(p-STAT3)(Ser727),STAT5,磷酸化STAT5(p-STAT5)(Tyr694)抗体检测相关蛋白表达。结果代表了三次相似的独立检测结果。
图4A-图4B.在BaF3细胞中进行NOK介导的增值信号通路检测。在BaF3过表达NOK及其突变体并检测它们对RAS/MAPK,PI3K/Akt及STAT信号通路的影响。用NOK及其突变体瞬时转染细胞，48小时后收取细胞并进行细胞裂解。图4A用ERK,磷酸化ERK(p-ERK)(Tyr204),AKT,磷酸化AKT(p-AKT)(Thr 308),STAT1,图4B使用磷酸化STAT1(p-STAT1)(Tyr705),STAT3,磷酸化STAT3(p-STAT3)(Ser727),STAT5,磷酸化STAT5(p-STAT5)(Tyr694)抗体检测相关蛋白表达。结果代表了三次相似的独立检测结果。
图5A-图5C.NOK及其突变体对细胞增殖的影响。在HEK293T细胞(图5A)及HeLa细胞(图5B)中过表达NOK及其突变体。细胞计数后，以每孔～1×104细胞的密度铺96孔板。转染48小时后进行cck8检测。将1×105细胞/孔NOK及其突变体的BaF3稳定细胞系接种96孔板，培养1、2、3天后用cck8方法检测细胞增殖变化(图5C)。图示代表了三次相似的独立检测结果。
图6A-图6C.NOK及其突变体对锚定非依赖性生长的影响。NOK及其突变体(STYK1及V395I)的BaF3稳定细胞系在琼脂糖培养基中的集落生成检测。图中显示培养板(图6A)、镜下(图6B)及统计学(图6C)检测结果。图示代表了三次相似的独立检测结果。
具体实施方式
材料和方法
1、质粒构建和定点突变
携带HA标签的NOK基因(Genebank检索号：KP729000)全长序列通过PCR经XbaI和NotI酶切位点构建到了pCDH-CMV-MCS-EF1-CD511B载体中，同时通过HindIII和XbaI酶切位点构建到了pCDNA3.0载体中，得到了质粒pCDH-CMV-MCS-EF1-CD511B-NOK-HA和pCDNA3.0-NOK。利用Takara Mutant BEST Kit构建了NOK的突变质粒和相应的载体pCDH-CMV-MCS-EF1-CD511B-STYK1-HA和pCDH-CMV-MCS-EF1-CD511B-NOK(V395I)-HA，以及载体pCDNA3.0-STYK1-HA和pCDNA3.0-NOK(V395I)-HA。定点突变上游所用引物包括：5’-GGGGACCTGCTCAGCTTTCTC-3’和5’-GTGTTACAAATACCAGAGTTGGTGG-3’，下游引物包括：5’-CTGGGCCACATCCTCCAACAC-3’和5’-AGCCTCGTCATCTGCAGTTTTA-3’。PCR反应条件为：94℃4min、94℃50s、55℃50s、72℃120s、循环数为30个循环。所有质粒和载体的构建均按照标准PCR和克隆方法，先通过亚克隆到PMD-18T-Simple载体，最后再克隆至目的载体。得到的所有质粒均通过双酶切鉴定和测序鉴定，鉴定合格。
2、细胞培养，瞬时转染及抗体
人胚肾细胞系HEK293T，人宫颈癌细胞系HeLa，小鼠髓单核白血病细胞系WEHI-3B，小鼠前B细胞BaF3均购自中国医学科学院基础医学研究所细胞中心。其中HEK293T细胞系和HeLa细胞系培养于含有10％胎牛血清的DMEM培养基中，WEHI-3B细胞培养于含有10％胎牛血清的RPMI1640培养基中，BaF3细胞培养于含有10％胎牛血清和10％IL-3的RPMI1640培养基中。所有细胞于5％CO2，37℃恒温培养箱中培养。当HEK293T细胞或HeLa细胞汇合度达到约80％时，利用VigoFect转染试剂按照标准操作步骤瞬时转染质粒DNA。本实验所有用到的抗体为：anti-HA(1/5000,Proteintech),anti-Erk和anti-p-Erk(1/1000,Santa CruzBiotechnology),anti-Akt和anti-p-Akt(1/1000,Santa Cruz Biotechnology),anti-STAT1和anti-p-STAT1(1/1000,Bioworld Biotechnology),anti-STAT 3和anti-p-STAT3(1/1000,Santa Cruz Biotechnology),anti-STAT5和anti-p-STAT5(1/1000,BioworldBiotechnology)和anti-β-actin(1/1000,中杉金桥)。
3稳定细胞系构建
利用慢病毒包装系统和VigoFect转染试剂，将辅助质粒pCMV-VSV-G、质粒pMD2.G和质粒RSV-Rev及携带目的基因的骨架质粒pCDH-CMV-MCS-EF1-CD511B瞬时转染HEK293T/N细胞。转然后48小时收获包含有病毒颗粒的培养基，通过离心、过滤等方法纯化病毒颗粒，用无血清培养基重悬病毒颗粒制备的病毒感染液感染目的细胞BaF3，同时加入适量促感染试剂Polybrene(终浓度约为8mg ml-1)。感染约12小时后，更换培养基为新鲜的完全培养基。稳定细胞系感染效率通过流式细胞仪进行分析(GFP表达情况)，阳性克隆子通过RT-PCR和Western Blot进行鉴定，通过有限稀释法获取单克隆。
4、蛋白质印迹
收获转染好的细胞或者稳定细胞系，用预冷的1xPBS洗涤细胞两次，弃去洗液后用含有20mM Tris-HCl(pH7.4),150mM NaCl,1mM EDTA,1％Triton-X100,1mM Na3VO4,2.5mMsodium pyrophosphate,1mMβ-glycerolphosphate,1mM phenylmethylsulfonylfluoride,5μg/ml
aprotinin,和5μgml leupeptin(pH7.5)的反应液于冰上裂解细胞30分钟。裂解后于4℃高速离心后取上清裂解液，定量测定裂解液中的蛋白浓度。用相同蛋白总量的细胞裂解液进行10％SDS-PAGE胶电泳。电泳结束后，于300mA恒流条件下将SDS-PAGE胶中蛋白转印到硝酸纤维膜上，转膜约2.5小时。用5％脱脂牛奶溶液于室温封闭蛋白膜1小时，之后于相应一抗溶液中4℃孵育过夜。第二天，用辣根过氧化物酶耦联的相应二抗进行杂交，室温孵育1小时。最后于暗室中显影检测蛋白表达情况。
5、细胞增值实验和锚定非依赖生长实验
针对细胞增殖实验，取约1×104个转染后的HEK293T细胞和HeLa细胞种植于96孔板中的一个孔中，每组至少设置4个平行孔。于细胞培养箱中培养一段时间后，于每孔中加入相应试剂，根据最后培养基颜色的变化确定细胞的增殖，增殖实验按照cell countingkit-8试剂盒标准说明操作。针对锚定非依赖生长实验，首先制备2ml含有0.7％琼脂 的RPMI1640完全培养基，将此培养基倒入细胞培养皿中，待其冷却后作为底层胶。再准备约1×104个各稳定细胞系，分别与2ml含有0.4％琼脂糖的RPMI1640完全培养基混合均匀，将混匀后的细胞悬液倒入底层胶，待细胞悬液凝固，往凝固好的上层胶上加入1ml RPMI1640完全培养基，并置于细胞培养箱中培养约两周。两周后观察各皿中集落形成情况，选取其中直径大于0.2mm的集落作为阳性集落，并拍照、计数。
6、免疫沉淀
利用VigoFect转染试剂将各质粒瞬时转染HEK293T细胞，转染48小时后，收获转染后的细胞并应预冷的1xPBS洗涤细胞两遍。弃去洗液后用加入了磷酸酶抑制剂的细胞裂解液(50mM Tris,at pH7.4.,150mM NaCl,10％glycerol,1mM EDTA,1％Triton X-100)于冰上裂解细胞半小时。离心后收获上清蛋白溶液，并加入适量anti-HA beads，4℃孵育过夜。过夜孵育后的蛋白液于8000×g离心30s后，弃上清。再用Wash Buffer洗涤珠子5遍，每次10分钟。最后弃去洗液并加入SDS-PAGE loading buffer。样品煮沸10分钟后，进行SDS-PAGE电泳。电泳后的蛋白通过电转至硝酸纤维膜上。剪取所需位置的膜块，用5％脱脂牛奶室温封闭1小时，再用相应的一抗4℃杂交孵育过夜，第二天用辣根过氧化物酶标记的二抗孵育膜块，最后于暗室显影。
7、自身磷酸化分析
用VigoFect转染试剂分别将质粒pCDNA3.0、pCDNA3.0-NOK、pCDNA3.0-STYK1和pCDNA3.0-V395I瞬时转染HEK293T细胞。转染48小时后，收获各细胞，并用预冷的1×PBS洗涤细胞两遍。于4000rpm离心5分钟后获取细胞。加入适量的添加了磷酸酶抑制剂的RIPAbuffer于冰上裂解细胞半小时。低温高速离心后，获取裂解上清液。在裂解上清中加入anti-HA beads并于4℃孵育过夜，使目的蛋白与珠子充分结合。第二天，8200×g离心30s收获珠子，并用wash buffer洗涤珠子5次，每次10分钟。弃去洗液后得到结合了目的蛋白的珠子。对结合了目的蛋白的珠子进行SDS-PAGE电泳和western blot分析，一抗使用anti-p-Tyr抗体，最后通过显影检测NOK及突变组中的NOK蛋白的磷酸化水平。
8、体外酪氨酸激酶活性测试
用VigoFect转染试剂分别将质粒pCDNA3.0、pCDNA3.0-NOK、pCDNA3.0-STYK1和pCDNA3.0-V395I瞬时转染HEK293T细胞。转染48小时后，收获各细胞，并用预冷的1×PBS洗涤细胞两遍。于4000rpm离心5分钟后获取细胞。加入适量的添加了磷酸酶抑制剂的RIPAbuffer于冰上裂解细胞半小时。低温高速离心后，获取裂解上清液。采用BCA发定量测定总蛋白量。吸取5μg总蛋白样进行酪氨酸激酶活性测试。简而言之，将5μg总蛋白样与底物肽一起加入至包含有50μl激酶反应缓冲液(100mM Tris-HCl,pH7.4,50mM MgCl2,5mM MnCl2,5mMdithiothreitol,1mM ATP,50mM sodium orthovanadate)的条形小孔中于室温孵育30分钟，其中底物肽可以与条形小孔中的链霉亲和素结合。孵育结束后，用1×TBS/T洗涤条形小孔5次，之后于每个小孔中加入100μl anti-phosphotyrosine recombinant 4G10TM，室温反应20-30分钟。弃去反应液后用1×TBS/T洗涤条形小孔5次。之后加入75μl TMB底物混合液，室温反应最多15分钟。待对照组中出现淡蓝色时，即可于每孔中加入适量2M的硫酸溶液终止反应。于分光光度计上测定450nm波长下的吸光值，并记录读数。
实施例1：构建含有不同位点突变的突变子STYK1和V395I
利用生物信息学手段，通过与其它受体型酪氨酸激酶成员如FGFR和PDGFR进行序列比对，我们已经找NOK诸多保守的丝/苏/酪氨基酸残基位点和结构域，如VAIL，HRD和DFG结构域。而在本研究中，我们构建了另外的两种不同突变。分别是NOK基因P203L的突变即STYK1，和V395I的突变。STYK1首次克隆和鉴定于人的胚胎cDNA文库中，V395I的突变则发现于人的多形性胶质瘤样本中，而NOK是从患有甲状腺瘤病人的扁桃体样本中克隆和鉴定得到的。和NOK相比，STYK1的突变位点203位位于胞内ICD的sub-domain1中，V395I则位于C末端(图1)。
实施例2：自身磷酸化检测
实验室前期实验表明Y417F的突变不仅能显著性促进NOK介导的有丝分裂信号通路，还能导致NOK通过增强自身磷酸化而活化。鉴 于此研究结果，我们检测了STYK1和V395I是否对自身磷酸化水平有影响。利用HEK293T细胞，通过瞬时转染后IP检测p-Tyr的表达水平发现，在44kD附近NOK组和STYK1组的表达水平相当，但是均高于V395I组的水平。从条带上分析NOK和STYK1中p-Tyr的水平比V395I组高出约3倍(图2A)。除此之外，在75kD附近和120kD附近也有新条带的出现。这些条带可能是NOK的高级结构如多聚体，可能是与NOK相互作用的相关蛋白。针对这两个位置的条带中p-Tyr的水平发现，NOK组均要显著高于STYK1和V395I组的水平，其中在75kD附近NOK组约为V395I组的5倍，STYK1组约为V395I组的2.5倍。尤其在120kD附近，NOK组p-Tyr信号显著增强，而STYK1和V395I组信号极弱。这可能说明突变影响到了NOK与其他未知蛋白之间的相互作用或者自身的磷酸化水平。当然，这些结果和蛋白需要更进一步的实验进行发掘，鉴定和证实。
实施例3：激酶活性测试
既然在突变子中自身磷酸化水平发生了改变，那么突变会不会也影响到激酶活性？所以我们研究了突变对激酶活性的影响。同样利用HEK293T细胞和体外激酶活性测试系统，我们发现NOK和突变子都保留了自身激酶的活性。而且通过对三者激酶活性进行比较分析后发现STYK1和V395I相对于NOK的突变并未对激酶活性产生较大的影响(图2B)，和NOK组相比较，STYK1和V395I对激酶活性的改变在7％和2％之内。这就说明突变没有从根本上对NOK的激酶活性产生正面或者负面的影响。
实施例4：突变对PI3K/AKT,RAS/MAPK以及JAK/STAT信号通路的影响
考虑到受体型酪氨酸激酶可以通过自身磷酸化而活化，在此过程中其自身p-Tyr的水平会显著升高，因此我们推测这样的改变会可能会影响到信号通路的传递。因为NOK与FGFRs存在相类似交叉的信号通路如RAS/MAPK和PI3K/AKT信号通路，于是我们检测了突变与NOK对在这两条经典信号通路的影响。我们发现在HEK293T细胞中，NOK能显著性地提高p-ERK和p-AKT的表达水平，但是STYK1不能激活这些重要信号分子的磷酸化，V395I虽然能在一定程度上激活 p-ERK和p-AKT的水平，但是相对于NOK而言这种能力明显削弱(图3A)。我们也检测了JAK-STAT信号通路，发现突变STYK1和V395I都没有对该信号通路产生明显影响，各信号分子如p-STAT1，p-STAT3和p-STAT5都基本维持在相对稳定的水平(图3A)。同样，我们利用HeLa细胞进行了类似的检测，发现STYK1和V395I均能在一定程度上负性调节ERK和AKT的磷酸化水平，这和HEK293T是一致的。但是针对JAK-STAT信号通路而言，我们发现在HeLa细胞中，STYK1和V395I能显著性抑制p-STAT3的表达，这与HEK293T细胞中的情况不一样，可能因为不同细胞系而导致的结果(图3B)。
为了进一步验证实验结果，我们利用BaF3稳定细胞系进行了同样的研究。在稳定细胞系中，因突变带来的影响比瞬时转染要更加明显。在STYK1和V395I中，p-ERK、p-STAT1、p-STAT3和p-STAT5的水平和NOK相比收到了显著抑制(图4)。比如p-STAT5，在NOK组中过表达，但是在STYK1和V395I中，其表达水平几乎接近于对照组；虽然p-AKT的表达水平在各组中的变化不是很明显，但是p-ERK的表达在STYK1和V395I中和NOK组相比都收到了强烈的抑制，表达量几乎下降到了和对照组接近的水平。以上的实验结果更进一步验证和证实了NOK能显著性地激活有丝分裂信号通路，而STYK1和V395I能在一定程度上抑制NOK所介导的PI3K/AKT、RAS/MAPK、和JAK/STAT信号通路中信号的传导。
实施例5：STYK1和V395I能阻断依赖于IL-3的细胞生长
既然STYK1和V395I能对信号通路产生如此明显的影响，这些变化是否会因此改变细胞的增殖能力？实验室前期利用稳定细胞系已证实，当细胞过表达NOK时其增殖能力会显著提升。利用CCK8实验方法我们首先检测了STYK1和V395I对HEK293T细胞增殖能力的影响。在转染48小时后发现CCK8实验结果所反映的细胞增殖能力与信号通路中对PI3K/AKT和MAPK/ERK通路的活化情况相一致，即STYK1和V395I都在一定程度上抑制细胞的增殖(图5A)。同样为了进一步证实实验结果，利用HeLa细胞和稳定细胞系和CCK8系统做了同样的检测，得到了同样的结论(图5B)。尤其在稳定细胞系中，在48小时之前NOK组与STYK1和V395I组之间并不存在明显差异，但是在48小时 后，NOK组细胞依然能持续增殖生长，而STYK1和V395I组中的细胞和对照组一样出现明显的凋亡，细胞数量剧减。其中STYK1组的细胞略高于对照组和V395I组，而V395I组和对照组则处于相似水平(图5C)。这些实验说明突变STYK1和V395I能抑制NOK依赖于IL-3的细胞增殖能力。这也从侧面证实了该突变对NOK所介导的信号通路的负性调控。
实施例6：STYK1和V395I削弱了NOK促进的锚定非依赖生长能力
为了进一步从功能上验证以上实验结果，我们研究了突变对稳定细胞系的克隆形成能力。各稳定细胞系饥饿数小时后进行软琼脂实验，在培养箱培育15天左右后对形成的克隆集落进行分析计数。大于0.2微米的细胞集落被划分为阳性克隆。我们发现NOK组中细胞集落形成数量远远高于其他三组。其中V395I组中细胞的集落约为NOK组的24％，而STYK1组中的集落和对照组相当，约为NOK组的6.7％。这说明V395I和STYK1均抑制了NOK所介导的细胞锚定非依赖生长特性(图6)。
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Claims (1)
1.一种分离的核酸在制备用于治疗肿瘤的药物中的用途，其中所述分离的核酸是NOK基因的P203L或V395I突变体，所述肿瘤选自B淋巴瘤、宫颈癌和肾细胞癌，并且其中所述NOK基因的P203L或V395I突变体分别如SEQ ID NO:1或SEQ ID NO:2所示。
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| CN1570108A (zh) * | 2004-05-13 | 2005-01-26 | 清华大学 | 鼠源epor胞外区与人源nok胞内区嵌合受体及编码基因与应用 |
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| CN1570108A (zh) * | 2004-05-13 | 2005-01-26 | 清华大学 | 鼠源epor胞外区与人源nok胞内区嵌合受体及编码基因与应用 |
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