CN108096580B - 一种抑制神经母细胞瘤sh-sy5y的靶点及其应用 - Google Patents
一种抑制神经母细胞瘤sh-sy5y的靶点及其应用 Download PDFInfo
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Abstract
本发明属生物医药技术领域,提供一种抑制神经母细胞瘤SH‑SY5Y的靶点,ARL8A基因和/或其参与的自噬途径。ARL8A基因和/或其参与的自噬途径的抑制剂包括:抗ARL8A的抗体、针对ARL8A编码序列的RNA干扰分子或反义寡核苷酸、ARL8A介导的自噬途径抑制剂。RNA干扰分子为定向干扰SH‑SY5Y细胞中ARL8A基因表达的siRNA分子,为ARL8A‑homo‑698。特异性的降解ARL8A基因的mRNA,干扰转录后翻译过程,诱导神经母细胞瘤细胞凋亡,达到抑制肿瘤的作用。对开发新的抗神经胶质瘤药物具有重要指导作用,用于开发制备抗肿瘤药物,对神经胶质瘤的治疗有重大应用前景。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种抑制神经母细胞瘤SH-SY5Y的靶点及其应用。
背景技术
Gie2又称 ARL8A (ADP-ribosylation factor like protein 8A)或ARL10B(ADP-ribosylation factor like protein 10B)是与溶酶体结合来调控细胞自噬的小G蛋白,和另一个小G蛋白Gie1 又称ARL8B (ADP-ribosylation factor like protein 8B) 或ARL10C (ADP-ribosylation factor like protein 10C)的结构高度相似。以下采用ARL8A和ARL10B分别命名这两个蛋白质。正常生理条件下,在全身各器官内这两个蛋白都有表达,在已知的大多数肿瘤细胞内这两个蛋白质也都有表达。2011年《Nature Cell Biology》杂志报道了Hela细胞内ARL8A和ARL8B的功能,当细胞处于营养充足的环境中,二者呈现一定的表达水平,对应的细胞状态为低自噬状态,与自噬相关的溶酶体处于细胞外周。当Hela细胞处于营养缺乏的培养环境中,二者的表达量显著降低,细胞处于高自噬水平,此时溶酶体处于细胞中央发挥自噬。这篇文献证明了ARL8A和ARL8B是Hela细胞处于抵抗饥饿诱导自噬过程中调控溶酶体迁移的关键酶,二者的功能相同。2016年《Oncotarget》杂志报道在前列腺癌细胞内ARLA几乎无表达,可认为是ARL8B单独表达型,降低ARLB表达量会促进大鼠前列腺癌细胞凋亡,证明是一个抗肿瘤药物靶点。从已知的ARLA和ARLB的研究成果来看,ARL8A也可能抗肿瘤药物作用靶点。依据ARL8A和ARL8B的表达量差异,肿瘤细胞可以分为ARL8B单独(或优势)表达型、ARL8A单独(或优势)表达型、二者共有型等三种。迄今为止尚无ARL8A单独表达(或优势)表达型的细胞报导。
神经母细胞瘤是人体神经系统常见的恶性肿瘤,尤以在婴幼儿中多发,发病率和病死率较高,预后不良,目前对于神经母细胞瘤的病因和发病机制尚未明确。人神经母细胞瘤株SH-SY5Y是来源于1970年建立的一神经母细胞瘤株患者的转移骨瘤灶细胞SK-N-SH经三次克隆后的亚系(SK-N-SH→SH-SY→SH-SY5→SH-SY5Y)。该细胞显示中等水平的多巴胺-β-羟基酶活性,与正常多巴胺能神经细胞在功能上相似,因此广泛应用于神经系统退行性病变(如PD)的发病机制和防治措施的研究。SH-SY5Y细胞中含有凋亡相关酪氨酸激酶,在调节神经系统发育过程中发挥重要作用,能够诱导细胞分化,并且能增强其他诱导分化物的作用。如果降低SH-SY5Y细胞内基因的表达量,能导致SH-SY5Y凋亡,所采用的相关原材料和实验方法对抗神经母细胞瘤的新药研发将有很好启迪或促进作用。
RNA干扰(RNAi)是指在进化过程中高度保守的、由双链RNA诱发的、同源mRNA高效特异性降解的现象。由于使用RNAi技术可以特异性干扰特定基因的表达,所以该技术被广泛用于探索基因功能及肿瘤基因的治疗领域。
发明内容
本发明的目的之一是提供了一种抑制神经母细胞瘤SH-SY5Y的靶点,所述靶点为ARL8A基因和/或其参与的自噬途径。
本发明的另一目的是提供了一种ARL8A基因和/或其参与的自噬途径的抑制剂在降低和/或消除神经母细胞瘤SH-SY5Y药物中的应用。
作为优选方案,所述ARL8A基因和/或其参与的自噬途径的抑制剂包括:抗ARL8A的抗体、针对ARL8A编码序列的RNA干扰分子或反义寡核苷酸、ARL8A介导的自噬途径抑制剂。
作为另一优选方案,所述的针对ARL8A编码序列的RNA干扰分子为定向干扰SH-SY5Y细胞中ARL8A基因表达的siRNA分子。
所述siRNA分子为ARL8A-homo-698,其正义链序列为SEQ ID NO.1,反义链序列为:SEQ ID NO.2所示,为:
正义链5'-GGCUUAUUCAACACUCGAATT-3',
反义链5'-UUCGAGUGUUGAAUAAGCCTT-3'。
所述siRNA分子定向干扰SH-SY5Y细胞中ARL8A基因表达的方法包括以下步骤:
(1)将SH-SY5Y细胞接种到细胞培养板中,37℃,5%的CO2培养箱培养至细胞的混浊度达到60%-70%时备用;
(2)用Opti-MEM I稀释所述的siRNA分子和LipofectamineTM 2000,室温下静置5分钟后,将两者混合,混匀并在室温下孵育20分钟;
(3)将步骤(1)中培养细胞的DMEM完全培养基去掉,换成DMEM基本培养基,在37℃,5%的CO2培养箱孵育20min;
(4)将步骤(2)所得的混合物加入步骤(3)的培养细胞中,在37℃,5%的CO2培养箱孵育5h后,去掉培养基,换成DMEM完全培养基继续培养24h-48h;
(5)将步骤(4)所得细胞进行RNA提取并反转录合成cDNA,然后进行荧光定量PCR检测。
本发明在我们利用荧光定量PCR法检测肿瘤细胞内ARL8A和ARL8B表达量差异,从中发现了ARL8B低于检测限,可认为是ARL8A单独表达的SH-SY5Y细胞,而后设计合成具有靶向专一性的siRNA作用于SH-SY5Y细胞后特异性降解ARL8A的mRNA,研究结果显示我们合成的siRNA能专一性抑制ARL8A的表达导致SH-SY5Y细胞凋亡,该结果对临床治疗神经提供良好的指导意义。
有效的靶向治疗是目前国内外不断寻找的辅助治疗方法,在实施靶向ARL8A抗肿瘤研究中,我们利用荧光定量PCR法测定肿瘤细胞内ARL8A和ARL8B的表达量差异,发现了ARL8B几乎不表达的肿瘤细胞株:人神经母细胞瘤株(SH-SY5Y),可认为是ARL8A单独表达的SH-SY5Y细胞,对ARL8A单独表达的肿瘤细胞降低ARL8A能导致明显的细胞凋亡,这个结论在靶向抗肿瘤药物研究方面有重大应用前景,因此将ARL8A基因和/或其参与的自噬途径作为抑制SH-SY5Y的靶点是本专利要保护的内容之一。
ARL8A基因和/或其参与的自噬途径的抑制剂在降低和/或消除神经母细胞瘤SH-SY5Y药物中的应用。该抑制剂包括:抗ARL8A的抗体,针对ARL8A编码序列的RNA干扰分子如:siRNA定向干扰,shRNA定向干扰等,这些方法如果应用在降低诸如神经瘤细胞ARL8A的表达,以此为药物作用机制实施抗肿瘤药物开发,均属于本发明的保护范围。
根据该靶点设计合成具有靶向专一性的siRNA作用于SH-SY5Y细胞后特异性降解ARL8A的mRNA,本发明提供的定向干扰SH-SY5Y细胞中ARL8A基因表达的siRNA分子转染SH-SY5Y细胞后,发现siRNA能特异性的干扰ARL8A基因的mRNA和ARL8A蛋白的表达量,促进神经母细胞瘤株SH-SY5Y的自噬水平,达到抑制肿瘤的作用。该siRNA可以被纳米材料包埋成siRNA抗肿瘤新药候选品来进行抗神经瘤的治疗研究,这个siRNA用于抗肿瘤的用途同样也是本发明所要保护的范围。
本发明所述的siRNA采用人工化学合成方法制备,对于开发新的抗肿瘤基因药物和肿瘤药物的治疗效果具有重要指导作用,可用于开发抗神经母细胞瘤的临床用药,因此该siRNA及其用于研发抗神经母细胞瘤的用途是本项发明要求保护的利益。
附图说明
图1为荧光定量PCR检测ARL8A和ARL8B基因在肿瘤细胞中的表达情况图,以β-actin为内参,横坐标表示不同肿瘤细胞,纵坐标表示在同一种细胞内的ARL8A和ARL8B相对于该细胞GAPDH的表达量;图2为荧光定量PCR检测GAPDH、ARL8A和ARL8B基因在SH-SY5Y细胞中的表达情况图;图3为不同剂量siRNA转染神经母细胞瘤株SH-SY5Y细胞后的荧光强度图,图中:图中转染试剂配比:1为5ul LipofectamineTM2000、5ul荧光标记siRNA(20uM);2为10ul LipofectamineTM2000、5ul荧光标记siRNA(20uM);3为15ul LipofectamineTM2000、5ul荧光标记siRNA(20uM);4为5ul LipofectamineTM2000、10ul荧光标记siRNA(20uM);5为5ul LipofectamineTM2000、15ul荧光标记siRNA(20uM);6为15ul LipofectamineTM2000、15ul荧光标记siRNA(20uM);图4为siRNA转染SH-SY5Y细胞后实时定量PCR检测ARL8A mRNA表达量柱形图,纵坐标表示ARL8A相对于β-actin的mRNA表达水平;横坐标表示四个处理实验组,control为未处理的正常细胞,NC为无义siRNA干扰的阴性对照组,ARL8A-siRNA1和ARL8A-siRNA2为有义干扰siRNA,其中ARL8A-siRNA2为本发明的siRNA转染实验组;图5A为siRNA转染SH-SY5Y细胞48h后的细胞状态图,其中control为未转染的正常细胞,ARL8A-siRNA2为干扰实验组,图5B为siRNA转染SH-SY5Y细胞48h后的细胞活力柱状图;图6A为Western Blot 检测siRNA转染SH-SY5Y细胞后的ARL8A蛋白表达量及LC3蛋白表达量,图6B为对照组和干扰组中ARL8A、LC3I和LC3II蛋白想对表达量的统计柱状图,图6C为对照组和干扰组中LC3II/LC3I的柱状图。
具体实施方式
为使本发明更容易理解,下面结合具体实施例,进一步阐述本发明。这些实施例只用于说明本发明而不用于限制本发明的范围。
若未特别指明,实施例中所用的化学试剂均为常规市售试剂, 实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
本发明中提供了一种新的抑制神经母细胞瘤的靶点:ARL8A在神经母细胞瘤SH-SY5Y中单独表达,将该蛋白或基因作为靶点,可设计出针对该蛋白及其相关分子的预测、诊断神经母细胞瘤的试剂盒、筛选针对该蛋白及其相关分子的治疗神经母细胞瘤的药物。
实施例1:ARL8A基因单独表达型(或优势表达型)肿瘤细胞的筛选
1.仪器:细胞培养箱(Thermo)、灭菌锅、干式恒温器、低温离心机、微量紫外分光光度计(Thermo)、CFX96 Real-Time System(Bio-Rad)等。
2.试剂和材料:细胞株SH-SY5Y、Hela、HepG2、IMR-32、MDA-MB-15、MGC-803、AGS、SGC7901、MKN-45、GBC-823、HCCC-9810和PC-3,均来自国家实验细胞资源共享平台。MEM 培养基(HyClone)、DMEM高糖培养基(HyClone)、1640培养基(HyClone)、F12培养基(HyClone)、0.25%胰蛋白酶(1×)溶液(HyClone)、胎牛血清(北京博奥龙免疫技术有限公司)、TRIGene总RNA提取试剂(GenStar)、PBS、氯仿、异丙醇、无水乙醇、DEPC、EasyScript First-StrandcDNA Synthesis SuperMix(TRANS)、TransStart Top Green qPCR SuperMix(TRANS)、PCRSTRIP CAPS(Corning)、25cm2 Cell Culture Flash Canted Neck(NEST)、6孔细胞培养板(NEST)等。
3.PCR引物(北京擎科新业生物技术有限公司合成):
ARL8A forward 5’-GATCGCTTTGTTCAACAAGC -3’
ARL8A reverse 5’-GTCCCAGAGCTTGATAGTCACA -3’
ARL8B forward 5’-GCTGGCGCTCATCTCC -3’
ARL8B reverse 5’-GCTTCGAAATCGGGGTT -3’
GAPDH forward 5’-GTCCACTGGCGTCTTCAC-3’
GAPDH reverse 5’-AGGCATTGCTGATGATCTTGA-3’
β-actin forward 5’-GGACTTCGAGCAAGAGATGG-3’
β-actin reverse 5’-AGCACTGTGTTGGCGTACAG-3’
细胞培养:SH-SY5Y、Hela和HepG2在含10%FBS的DMEM培养基中,IMR-32在含10%FBS的MEM培养基中,MDA-MB-15、MGC-803、AGS、SGC7901、MKN-45、GBC-823、HCCC-9810在含10%FBS的1640培养基中,PC-3在含10%FBS的F12培养基中,37℃、5%CO2培养箱培养。
RNA提取:
(1)细胞样本处理:贴壁细胞数量达90%时,吸尽培养基,每10cm2培养面积(6孔板或35mm平皿)加入1ml TRIGene,用加样器吹打数次,以确保细胞完全裂解,然后转移至离心管中。
(2)裂解产物于室温(15~30℃)放置5min,使核酸-蛋白复合物完全分离。
(3)每1ml TRIGene加入0.2ml氯仿,盖紧管盖,剧烈振荡15秒钟,室温放置2~3min。
(4)4℃≤12000g离心15min,样品会分成三层:橘黄色的下层有机相,中间层和无色的上层水相。
(5)吸取含总RNA的上层水相至一新的离心管中,水相的体积约为所用TRIGene试剂的60%。
(6)每1ml TRIGene的最初使用量加入0.5ml异丙醇,颠倒数次混匀,室温放置10min。
(7)4℃≤12000g离心10min,弃除上清,可见胶状的RNA沉淀。
(8)每1ml TRIGene的最初使用量加入1ml 75%乙醇,颠倒数次混匀,洗涤沉淀。
(9)4℃≤7500g离心5min,弃除上清。
(10)室温倒置5~10min晾干,加入适量DEPC-ddH2O,用加样器吹打数次溶解RNA。
(11)通过紫外分光光度计检测,确定RNA的浓度、纯度。
4.反转录获得第一链cDNA:采用全式金公司的反转录试剂盒(EasyScript®First-Strand cDNA Synthesis Supermix)在逆转录酶EasyScript®RT/RI的作用下高效合成得到第一链cDNA。
对于第一链cDNA合成反应,将以下溶液于冰上化冻,并按相应的体积加入到在无菌的RNase-free 0.5ml的EP管中:Total RNA 1µg;Anchored Oligo(dT)18(0.5ug/ µl)1ul;补RNase-Free Water 至9µl,混匀后65℃孵育5min,打开RNA的二级结构,再冰浴2min,避免RNA的二级结构重新形成。
在冰上加入10ul 2×ES Reaction Mix和1ul EasyScript®RT/RI Enzyme Mix,总体积20ul。轻轻混匀42℃反应15min,随后85℃加热5s失活EasyScript®RT/RI。
5. 荧光定量PCR:引物设计:根据NCBI上公布的相关基因的序列,进行设计引物。设计好的引物均由北京擎科新业生物技术有限公司合成,具体序列如下:
Primer Sequence 5’-3’ Tm
ARL8A forward GATCGCTTTGTTCAACAAGC 56.98℃
ARL8A reverse GTCCCAGAGCTTGATAGTCACA 57.85℃
ARL8B forward GCTGGCGCTCATCTCC 57.29℃
ARL8B reverse GCTTCGAAATCGGGGTT 56.99℃
GAPDH forward GTCCACTGGCGTCTTCAC 57.9˚C
GAPDH reverse AGGCATTGCTGATGATCTTGA 55.4˚C
β-actin forward GGACTTCGAGCAAGAGATGG 51.9˚C
β-actin reverse AGCACTGTGTTGGCGTACAG 51.9˚C
Real-time PCR反应:采用Trans基于SYBR Green染料的荧光实时定量PCR技术检测基因表达情况,反应条件按照说明书操作。所有实验都在Bio-RadCFX96系统上进行。每个正向引物和反向引物的浓度为10µmol/L。实验步骤如下:
(1)在冰上按照顺序加入以下的试剂:cDNA (50ng) 1 µl;2 × TransStart TopGreen qPCR SuperMix 10 µl;Forward Primer (10 µM) 0.4 µl;Reverse Primer (10 µM) 0.4µl;ddH2O 8.2 µl,总体积为20 µl。
(2)实时荧光定量PCR:采用三步法进行扩增,具体反应的条件如下:首先,将样本进行预变性,94℃,30s;随后进行PCR反应,94℃,5s,52℃,30s,72℃,10s,进行40个循环。每个样本重复3次;
(3)在PCR扩增反应完成后,根据2-∆∆CT法分析样本间的表达情况,具体分析方法为:每对基因在每个模板中做3个重复管,得到的Ct值取平均值;每个目的基因的Ct平均值减去对应模板内参基因(β-actin)的Ct平均值,得到△Ct。实验组(ARL8A和ARL8B)的△Ct减去对照组(GAPDH)的△Ct,得到△△Ct值,对照组和实验组的待测基因的倍数关系用2-∆∆CT表示。每种细胞做3次生物重复,计算偏差值。
实验结果:用实时定量PCR 2-∆∆CT法分析实验结果,并作出柱状图,如图1和图2所示,横坐标为不同种类细胞,纵坐标为不同种细胞内ARL8A和ARL8B相对该种细胞GAPDH的表达量,β-actin为内参,结果显示,在SH-SY5Y细胞中ARL8A为单独表达的肿瘤细胞。
实施例2:化合成siRNA验证ARL8A沉默效果
仪器:细胞培养箱(Thermo)、灭菌锅、干式恒温器、低温离心机、微量紫外分光光度计(Thermo)、CFX96 Real-Time System(Bio-Rad)、正置荧光显微镜等;
试剂和材料:DMEM高糖培养基(HyClone)、0.25%胰蛋白酶(1×)溶液(HyClone)、胎牛血清(北京博奥龙免疫技术有限公司)、LipofectamineTM2000(Invitrogen)、防荧光淬灭试剂、TRIGene总RNA提取试剂(GenStar)、PBS、氯仿、异丙醇、无水乙醇、DEPC、EasyScriptFirst-Strand cDNA Synthesis SuperMix(TRANS)、TransStart Top Green qPCRSuperMix(TRANS)、PCR STRIP CAPS(Corning)、25cm2 Cell Culture Flash Canted Neck(NEST)、6孔细胞培养板(NEST)、载玻片、盖玻片等。
1.siRNA设计与化学合成(上海吉玛制药技术有限公司)
根据人源ARL8A的mRNA序列(NCBI No: AAH63125.1),利用干扰RNA序列设计软件SiDirect2 (http://sidirect2.rnai.jp/)、生成初步设计结果,再结合RNAi 效率预测公式进行评估,包括分析自由能特性,与靶标位置mRNA的二级空间结构,SNP分析,Blast分析等,最终筛选出2条最佳的RNAi靶标序列,如下:
ARL8A-siRNA1,即ARL8A-homo-504,正义链:5'-CCACAACCUACUGGACAAATT-3'
ARL8A-siRNA1,即ARL8A-homo-504,反义链:5'-UUUGUCCAGUAGGUUGUGGTT-3'
ARL8A-siRNA2,即ARL8A-homo-698,正义链:5'-GGCUUAUUCAACACUCGAATT-3'
ARL8A-siRNA2,即ARL8A-homo-698,反义链:5'-UUCGAGUGUUGAAUAAGCCTT-3'
Negative control,正义链:5'-UUCUCCGAACGUGUCACGUTT-3'
Negative control,正义链:5'-ACGUGACACGUUCGGAGAATT-3'
Negative control FAM,正义链:5'-UUCUCCGAACGUGUCACGUTT-3'
Negative control FAM,正义链:5'-ACGUGACACGUUCGGAGAATT-3'
以上SiRNA由上海吉玛制药技术有限公司合成,合成方法依据化学合成寡聚RNA方法实行,合成产物用HPLC纯化,纯度大于99%。
2.利用Negative control FAM进行转染条件筛选
(1)细胞爬片制作:转染前一天,在6孔板里放入盖玻片,分别将2ml 含SH-SY5Y细胞悬浮液加入到6孔中,混匀后放入到37℃、5%CO2培养箱中培养,当细胞浑浊度达50%-70%时使用。
(2)荧光标记siRNA瞬间转染:将合成的Negative control FAM用DEPC水溶解,得到20uM浓度的溶液。配置6种配比转染混合液:1)5ul LipofectamineTM2000、5ul荧光标记siRNA、500ul opti-MEM,2)10ul LipofectamineTM2000、5ul荧光标记siRNA、500ul opti-MEM,3)15ul LipofectamineTM2000、5ul荧光标记siRNA、500ul opti-MEM,4)5ulLipofectamineTM2000、10ul荧光标记siRNA、500ul opti-MEM,5)5ulLipofectamineTM2000、15ul荧光标记siRNA、500ul opti-MEM,6)15ulLipofectamineTM2000、15ul荧光标记siRNA、500ul opti-MEM,混合静置20min后,分别加入6孔板中对应的编号1、2、3、4、5、6的孔中,5h后换完全培养液,继续培养24h后,制成玻片,于正置显微镜下观察细胞GFP绿色荧光强度,筛选最好的转染条件。结果如图3,荧光强度最高的转染条件为6、15ul LipofectamineTM2000、15ul荧光标记siRNA、500ul Opti-MEM。
3.siRNA干扰序列筛选
1.siRNA瞬时转染
(1)转染的前一天,将SH-SY5Y细胞接种到6孔细胞培养板中,在37℃,5%的CO2培养箱培养,保证在转染时候细胞的混浊度达到50%-70%。
(2)采用筛选到的转染效果最好的转染条件进行转染,即用250ul Opti-MEM I分别稀释15ul siRNA分子和15ul LipofectamineTM 2000,室温下静置5分钟后,将两者混合,轻轻混匀并在室温下孵育20分钟。
(3)将第(1)步培养细胞的DMEM完全培养基去掉,换成DMEM基本培养基,在37℃,5%的CO2培养箱孵育20min。
(4)将第(2)步所获得的混合物加入第(3)步培养细胞中,在37℃,5%的CO2培养箱孵育5h后,去掉培养基,换成DMEM完全培养基继续培养24h。之后进行RNA提取并反转录合成cDNA。
2.荧光定量PCR检测
数据分析方法为:每对基因在每个模板中做3个重复管,得到的Ct值取平均值;每个目的基因的Ct平均值减去对应模板内参基因(β-actin)的Ct平均值,得到△Ct。实验组和阴性对照组的△Ct减去对照组(未转染)的△Ct,得到△△Ct值,对照组和实验组的待测基因的倍数关系用2-∆∆CT表示。实验结果如图4,能够有效干扰ARL8A基因的序列为ARL8A-siRNA2,即ARL8A-homo-698,其正义链:5'-GGCUUAUUCAACACUCGAATT-3',反义链:5'-UUCGAGUGUUGAAUAAGCCTT-3'。
实施例3:ARL8A基因沉默对神经母细胞瘤株SH-SY5Y细胞增殖的影响
材料及仪器:神经母细胞瘤株SH-SY5Y、DMEM高糖培养基、胎牛血清、细胞培养箱(Thermo)、LipofectamineTM2000(Invitrogen)、CCK8试剂盒、蛋白定量试剂盒(北京博奥龙免疫技术有限公司)、PageRuler Prestained Protein Ladder、脱脂奶粉(BD)、SIGMAFASTTM Protein Inhibitor Cocktail Tablets,EDTA-Free(SIGMA)、细胞裂解液(北京博奥龙免疫技术有限公司)、PMSF、PVDF Transfer Membranes(Immobilon-P)、电泳仪(BIO-RAD)、Mini-PROTEAN Tetra System(BIO-RAD)、高速台式冷冻离心机(湘仪)、化学发光成像系统(Chemi)、6孔细胞培养板(NEST)等。
CCK8法检测siRNA对SH-SY5Y细胞增殖的抑制结果
(1)在96孔板中接种细胞悬液(100ml/孔)。转染ARL8A-siRNA2后,将培养板放在37℃、5%的CO2的培养箱培养,24h、36h、48h后分别取样检测;检测时,向每孔加入10ul的CCK-8溶液(注意不要在孔中生成气泡,它们会影响O.D值的读数)。
(2)将培养板在培养箱内孵育 30min,用酶标仪测定在450 nm处的吸光度。
实验结果见图5和表1。图5为光镜下观察到的细胞生长状况,cell为未处理组,siRNA表示干扰实验组,表2为siRNA不同作用时间对SH-SY5Y细胞生长的抑制率,由图知,随siRNA干扰ARL8A时间的延长,细胞的生长率下降,抑制率上升。
表1: CCK8法检测的siRNA转染SH-SY5Y细胞后的不同转染时间的细胞活力结果
Western Blot检测siRNA对ARL8A表达的抑制情况
将神经母细胞瘤株细胞SH-SY5Y接种到6孔板中,在37℃、5%的CO2的培养箱培养一天至细胞的混浊度达到50%-70%。按上述转染操作转染细胞,37℃孵育24h后,吸除培养基,用PBS洗两遍,加入100ul细胞裂解液,在冰浴裂解10min,用细胞刮棒将细胞刮下,转移至1.5ml离心管中,15000rpm离心15min后,取上清液测定蛋白浓度。每组取40ug蛋白进行SDS-PAGE,电泳完成后转移至PVDF膜上,封闭后进行ARL8A、LC3、β-actin一抗及羊抗兔二抗处理,然后进行发光反应,显影。结果见图6,加入siRNA后细胞的ARL8A的表达量明显低于未处理的正常细胞组。
在SH-SY5Y细胞中,ARL8A-siRNA2能敲低ARL8A的表达量,并且ARL8A表达量下降时,LC3II/LC3I比值增大,细胞自噬水平升高,细胞生长受到抑制,所以本发明所述的siRNA对于神经胶质瘤的治疗具有重大应用前景。该siRNA可以被纳米材料包埋成siRNA抗肿瘤新药候选品来进行抗神经瘤的治疗研究。
序列表
<110> 中国医学科学院药用植物研究所
<120> 一种抑制神经母细胞瘤SH-SY5Y的靶点及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggcuuauuca acacucgaa 19
<210> 2
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
uucgaguguu gaauaagcc 19
Claims (2)
1.抑制ARL8A基因表达的siRNA分子在制备用于降低和/或消除神经母细胞瘤SH-SY5Y药物制剂中的应用,所述siRNA分子为ARL8A-homo-698,其正义链序列为SEQ ID NO.1,反义链序列为:SEQ ID NO.2所示,为:正义链5'-GGCUUAUUCAACACUCGAATT-3',反义链5'-UUCGAGUGUUGAAUAAGCCTT-3'。
2.根据权利要求1所述的抑制ARL8A基因表达的siRNA分子在制备用于降低和/或消除神经母细胞瘤SH-SY5Y药物制剂中的应用,其特征在于:所述siRNA分子定向干扰SH-SY5Y细胞中ARL8A基因表达的方法包括以下步骤:
(1)将SH-SY5Y细胞接种到细胞培养板中,37℃,5%的CO2培养箱培养至细胞的混浊度达到60%-70%时备用;
(2)用Opti-MEM I稀释所述的siRNA分子和LipofectamineTM 2000,室温下静置5分钟后,将两者混合,混匀并在室温下孵育20分钟;
(3)将步骤(1)中培养细胞的DMEM完全培养基去掉,换成DMEM基本培养基,在37℃,5%的CO2培养箱孵育20min;
(4)将步骤(2)所得的混合物加入步骤(3)的培养细胞中,在37℃,5%的CO2培养箱孵育5h后,去掉培养基,换成DMEM完全培养基继续培养24h-48h;
(5)将步骤(4)所得细胞进行RNA提取并反转录合成cDNA,然后进行荧光定量PCR检测。
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