CN117471099A - APMAP基因及其RNAi干扰系统的应用 - Google Patents
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Abstract
本发明公开了APMAP基因及其RNAi慢病毒干扰系统的应用,涉及生物医药技术领域。本发明的APMAP基因和APMAP基因的RNAi慢病毒干扰系统制备用于治疗结直肠癌的药物,该RNAi慢病毒干扰系统可高效敲低结直肠癌细胞中APMAP基因/蛋白,抑制癌细胞的增殖、侵袭与迁移,操作简单,效率高;本发明还公开了将APMAP基因/蛋白作为精准治疗的靶点,制备用于结直肠癌等高表达APMAP基因/蛋白的疾病诊断或预后判断的试剂盒,也用于制备靶向药物。
Description
技术领域
本发明属于生物医药技术领域,更具体地说,涉及APMAP基因(基因ID:57136,蛋白ID:Q9HDC9)及其RNAi干扰系统在制备用于治疗结直肠癌的药物中的应用。
背景技术
虽然免疫治疗在一定程度上降低了恶化的风险,但仅有伴有卫星不稳定性特征的患者可从免疫治疗中获益。尽管已有大量结直肠癌演进的机制,以及预后相关的科研成果,包括以转录组数据为主的生物信息学数据库,但临床亟需诊疗便捷的分泌性蛋白标记物。
分泌型蛋白是一类合成且修饰后经胞吐方式分泌至胞外的蛋白,由于在血液中半衰期长和特异性高等特点,分泌型蛋白可作为潜在的肿瘤标志物及药物靶标,如游离前列腺的特异性抗原FPSA可用于诊断及观察前列腺癌,人附睾上皮分泌蛋白4可用于随访追踪妇科恶性肿瘤。那么,结直肠癌患者的外周血和组织中高含量的分泌性蛋白是否可作为生物标志物,用于结直肠癌的液体活检,或可成为诊疗靶点。
由于结直肠癌易复发,活检癌组织难以获得,因此利用患者外周血检测结直肠癌标志物更具现实意义。鉴于此,项目组前期从本院生物样本库所存的临床样本中,随机调取6例结直肠癌患者术前和术后外周血血浆,另取6例同龄健康人的外周血血浆为对照,经新一代全扫描的数据独立采集质谱(DIA-MS)检测,鉴定出1035个蛋白质和4825个肽段,从质控后的数据中筛选得到9个差异表达的分泌型蛋白。联合蛋白组学生物信息学分析和ELISA的实验结果,脂肪细胞膜蛋白(Adipocyte Plasma Membrane Associated Protein,APMAP)被证实在结直肠癌患者术前外周血中显著高于术后和健康人;而在其他消化系统肿瘤中并未见显著变化,如胃癌、肝癌等。相应地,APMAP蛋白在结直肠癌组织的表达显著高于良性对照样本。
APMAP是一种整合膜蛋白,可驱动前脂肪细胞分化为成熟脂肪细胞,进而调控脂肪细胞的正常代谢功能。目前的研究表明APMAP可调控炎症反应、肿瘤转移及肿瘤免疫逃逸。APMAP可通过抑制表皮生长因子受体降解或激活β-连环蛋白从而促使前列腺癌或宫颈癌发生转移。除此之外,APMAP的敲除可以促进巨噬细胞的抗体依赖性细胞吞噬作用。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供APMAP基因及其RNAi慢病毒干扰系统的应用,用于制备治疗结直肠癌的药物。
为了解决上述技术问题,本发明所采用的技术方案如下:
APMAP基因在制备用于治疗结直肠癌的药物中的应用。
进一步地,所述的治疗结直肠癌为抑制细胞的增殖。
进一步地,所述的治疗结直肠癌为抑制细胞的迁移。
进一步地,所述的治疗结直肠癌为抑制细胞的侵袭。
用于检测患者外周血或患者癌组织中APMAP蛋白表达量的生物标志物为APMAP基因。
用于检测患者外周血或患者癌组织中APMAP蛋白表达量的试剂盒含有APMAP基因。
APMAP基因的RNAi慢病毒干扰系统在制备用于治疗结直肠癌的药物中的应用,所述的APMAP基因的RNAi慢病毒干扰系统的序列如下:
APMAP-shRNA1:5′-GGTCGTCACAGACGATGATGG-3′,
APMAP-shRNA2:5′-CACCGATTCTAGCAGCAAATG-3′。
进一步地,所述的APMAP基因的RNAi慢病毒干扰系统为敲低结直肠癌细胞中APMAP基因。
进一步地,所述的APMAP基因的RNAi慢病毒干扰系统为抑制结直肠癌细胞中APMAP蛋白表达。
相比于现有技术,本发明的有益效果为:
1)本发明RNAi慢病毒干扰可高效敲低结直肠癌细胞中APMAP基因/蛋白,抑制结直肠癌细胞的增殖、侵袭与迁移,该系统操作简单,效率高。
2)APMAP基因/蛋白是精准治疗的一个靶点,制备用于结直肠癌等高表达APMAP基因/蛋白的诊断或预后判断的试剂盒,也用于制备靶向药物。
附图说明
图1为APMAP蛋白在结直肠癌外周血及组织中的表达图(A为DIA-MS检测6例正常人血浆和6例结直肠癌患者血浆中的蛋白含量差异情况图;B为DIA-MS检测的APMAP在6例结直肠癌患者术前血浆中的蛋白含量差异情况图;C为结直肠癌患者血清中高含量肽段与结直肠癌患者术后低含量肽段的交集及其表达热图;D为ELISA检测15例正常人血清、15例结直肠癌、胃癌和肝癌患者术前术后血清中APMAP蛋白的表达情况图:正常人血清APMAP含量5.714±0.572(ng/mL)、结直肠癌患者术前血清APMAP含量8.836±0.551(ng/mL)、结直肠癌患者术后血清APMAP含量6.873±0.667(ng/mL));
图2为免疫荧光检测APMAP蛋白在结直肠癌及良性肠上皮组织中的表达结果图(A为APMAP在结直肠癌组织和良性肠上皮组织中的荧光染色;B为荧光强度的统计图;C为APMAP蛋白表达高低两组与结直肠癌患者预后的关系图);
图3为APMAP蛋白在NCM460细胞、HCT116细胞、LoVo细胞、SW480细胞、DLD-1细胞、SW1116细胞、HCT15细胞和SW620细胞的表达图;
图4为APMAP蛋白在HCT116细胞、SW1116细胞敲低和过表达该基因后的表达图;
图5为慢病毒敲低载体质粒图(A)和慢病毒过表达载体质粒图(B);
图6为APMAP蛋白在DLD-1细胞和SW620细胞敲低和过表达该基因后的表达图;
图7为敲低APMAP后,HCT116细胞的增殖活力图与过表达APMAP后的DLD-1细胞增殖活力图;
图8为敲低APMAP后HCT116细胞和SW1116细胞的迁移能力图与过表达APMAP后DLD-1细胞和SW620细胞的迁移能力图;
图9为敲低APMAP后HCT116细胞和SW1116细胞的侵袭能力图与过表达APMAP后DLD-1细胞和SW620细胞的侵袭能力图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面结合具体实施例对本发明进一步进行描述。以下实施例中如无特殊说明,所用的技术手段均为本领域技术人员所熟知的常规手段。
南通大学附属医院生物样本库结直肠癌石蜡组织芯片(2004年-2009年间手术,患者术前没有接受过免疫治疗、化疗或放疗,临床病例资料完整),其中结直肠癌组织120例,良性组织40例。
以下实施例中使用的主要试剂、耗材,动物模型为:
Opal 7色免疫组化试剂盒(美国Perkin Elmer公司)。
抗人APMAP抗体(25953-1-AP,中国武汉Proteintech公司)。
抗CK抗体。
结直肠癌细胞株(HCT116,LoVo,SW480,DLD-1,SW1116,HCT15,SW620)和肠上皮细胞系(NCM460)购自南京科佰生物科技有限公司。
RPMI-1640培养基、DMEM高糖培养基和胎牛血清(美国Gibco公司)。
以下实施例中使用的主要仪器如下:
多光谱病理扫描系统:美国Perkin Elmer公司。
倒置荧光显微镜:德国蔡司公司。
凝胶成像系统:中国天能公司。
多功能酶标仪:美国Thermo公司。
实施例1
直接法酶联免疫吸附(ELISA):
将所用抗原用包被稀释液稀释空白对照,阴性对照,并设置,每孔抗原加入100μL,置于4℃过夜后弃去孔中液体;5%小牛血清置37℃封闭1h,封闭结束后用洗涤液满孔洗涤3遍,每次3min,倾去液体后在吸水纸上拍干;将稀释好的样品加入酶标反应孔中,每样品至少3个复孔,每孔100μL,置于37℃孵育1h,用满孔洗涤3遍,每次3min;酶标抗体根据酶结合物提供商提供的参考工作稀释度进行,37℃,30-60min之间(短于30min往往结果不稳定),每孔加100μL,洗涤同前;加入TMB-过氧化氢尿素溶液,每孔100μL,置37℃避光放置3-5分钟;每孔加入终止液50μL终止反应,于15min内测定实验结果,检测450nm波长处吸光度值,进行读数,并计算。
结果如图1所示,结直肠癌患者术前外周血APMAP蛋白含量,显著高于其在术后和健康人外周血中的表达,且与质谱检测结直肠癌血浆中APMAP蛋白变化趋势一致。
实施例2
组织芯片置烘片仪上烤片后,浸于二甲苯中脱蜡后进行梯度酒精脱水;蒸馏水冲洗后,在耐高温切片架上置于pH为6.0的AR9修复液中,高温抗原修复;自然冷却至室温后用PBS冲洗,滴加一抗封闭液封闭10min;滴加200μL的稀释比例为1∶100的抗人APMAP抗体工作液(25953-1-AP)于组织芯片上,4℃过夜;取出组织芯片复温0.5h后用PBS冲洗;在组织芯片上滴加200μL的二抗工作液,室温10min后PBS冲洗;向组织芯片上滴加配好的荧光染料,避光室温孵育10min,接着用PBS冲洗;如要接着孵第二个抗体,则进行高温抗原修复,方法如上述;如不再孵抗体,则在干燥透明后,用DAPI封片。
荧光显微镜下观察染色结果,细胞相应部位出现染色作为阳性表现。使用Vectra3自动成像软件在20倍放大倍率下捕获每个样本。使用inForm4.1.0(Perkin Elmer)对图像进行分析和评分,并为每个细胞设置阳性或阴性细胞的阈值。计算每个区域的细胞百分比并进行评分(0-100),APMAP蛋白表达分数的分界点由X-tile软件根据生存时间及生存状态得出。评分如下:0-58.73为低表达或无表达,58.74-100为高表达。所有数据均用统计软件SPSSV.22.0处理,用卡方检验进行组间比较,用Cox比例风险回归分析患者的预后因素;用Kaplan-Meier方法和log-rank检验进行单因素分析患者预后,所有检验结果P<0.05为差异有统计学意义。结果如图2所示,APMAP蛋白在结直肠癌组织中表达高于良性肠上皮上皮细胞,且患者组高表达的生存期短,预后差。
实施例3
1、结直肠癌细胞系:DLD-1细胞、SW1116细胞、HCT116细胞、HCT15细胞用RPMI-1640完全培养基培养;LoVo细胞、SW480细胞和SW620细胞DMEM高糖完全培养基培养,肠上皮细胞NCM460细胞用RPMI-1640完全培养基培养,培养箱内保持温度37℃、5%的CO2饱和湿度,培养箱内常规传代培养,选取处于对数生长期的细胞进行实验。
2、细胞总蛋白提取
各种结直肠癌细胞均用含10%胎牛血清的完全培养基于37℃、5%的CO2条件下培养;收集结直肠癌细胞,弃培养基,用预冷的PBS洗涤细胞2次;根据细胞培养瓶的大小和细胞的生长密度,加入不同的细胞裂解液,后用细胞刮子刮净细胞,转移至干净的EP管中;将刮下的细胞蛋白在冰上充分裂解30min;4℃离心留上清,用BCA方法和多功能酶标仪测细胞蛋白的浓度,保存在-20℃冰箱备用。
3、蛋白免疫印迹(Western blot)
制备聚丙烯酰胺凝胶(5%浓缩胶,10%分离胶);蛋白Marker及提取的蛋白样品上样后,运行电压调至100V,结束后将胶取出进行转膜(PVDF膜);恒流300mA电转1.5h,转膜需在冰盒中进行;转膜结束后,将PVDF膜放入封闭液,室温封闭2h;用封闭液配制一抗稀释液,PVDF膜上均匀滴加稀释的一抗,孵育4℃过夜;洗膜后,用TBST配制二抗稀释液,PVDF膜上均匀滴加稀释的二抗,室温1.5h;洗膜结束后将PVDF膜平铺于显影仪相应位置,ECL发光液用TBST稀释,均匀滴加于PVDF膜上,凝胶成像系统拍照、保存。
结果如图3所示,APMAP在HCT116和SW1116结直肠癌细胞中表达相对较高,在DLD-1和SW620结直肠癌细胞中表达相对较低。
实施例4
1、筛选基因敲除阳性克隆
1)针对APMAP基因序列,特异性靶向APMAP基因的RNAi慢病毒干扰系统的基因序列如下所示,构建慢病毒介导的RNAi干扰体系:
APMAP-shRNA1:5′-GGTCGTCACAGACGATGATGG-3′,
APMAP-shRNA2:5′-CACCGATTCTAGCAGCAAATG-3′。
2)选择合适的病毒感染浓度,进行目的细胞的慢病毒感染,加入助染试剂;感染12-16h后,换液继续培养,同时观察细胞状态是否有异常;感染72-96h后,在倒置荧光显微镜观察荧光,并对感染的细胞进行药物筛选,以收集较多感染成功的细胞。
3)有限稀释法稀释细胞至10块96孔板中;一周后观察单克隆生长情况,约两周后将长起来的单克隆转移至48孔板中扩大培养;依次将长起来的单克隆转移至24孔板,12孔板中扩大培养;当每株单克隆扩大培养至2个12孔时,取出一个孔的细胞,裂解提蛋白,使用Western blot检测基因敲低的单克隆株。
结果如图4所示,与对照组相比,经RNAi慢病毒干扰系统处理后的HCT116细胞和SW1116细胞的APMAP相对蛋白表达量明显降低,说明HCT116细胞和SW1116细胞的APMAP蛋白表达被有效地抑制。
2、过表达体系构建及稳定株筛选
1)利用首尾引物扩增APMAP mRNA的开放阅读框架区域,并利用基因重组技术,将APMAP表达片段重组至pCDH-CMV-MCS-EF1-EGFP-2A-puro载体(图5)。
2)包装成病毒后,选择合适的病毒感染浓度,进行目的细胞的慢病毒感染,加入助染试剂;感染12-16h后,换液继续培养;感染72-96h后,在倒置荧光显微镜观察荧光,并对感染的细胞进行药物筛选,以收集较多感染成功的细胞。
3)单克隆的制备和生长:有限稀释法稀释细胞至10块96孔板中;一周后观察单克隆生长情况,约两周后将长起来的单克隆转移至48孔板中扩大培养;依次将长起来的单克隆转移至24孔板,12孔板中扩大培养,当每株单克隆扩大培养至2个12孔时,取出一个孔的细胞,裂解提蛋白,使用Western blot检测APMAP过表达的单克隆株。
结果如图6所示,与对照组相比,经过表达处理后的细胞的APMAP相对蛋白表达量明显增高,说明DLD-1细胞和SW620细胞的APMAP蛋白表达被有效地上调。
3、细胞增殖(CCK-8)
消化收集转染后48h的各组细胞,离心待用;用完全培养基重悬细胞,将细胞密度调整至20000个/mL;每孔加入100μL细胞悬液,每组设5个复孔,轻轻拍打96孔板,使细胞分布均匀;待细胞贴壁后(约6-8h),分别在0、24、48、72、96h加入CCK-8试剂(每孔10μL),轻轻拍打96孔板,放入培养箱2h后取出,在酶标仪上检测450m下的吸光度值,注意酶标仪的线性范围;用Graphpad prism统计处理所测数据,绘制折线图。
结果如图7所示,经RNAi慢病毒干扰系统敲低APMAP后,HCT116细胞细胞的增殖活力下降(A),而过表达APMAP后的DLD-1细胞增殖活力明显升高(B)。
4、细胞迁移(Transwell小室法)
消化收集稳定转染的各组细胞,离心待用;用基培重悬细胞,调整细胞密度至3×104/mL;在24孔板中加入800μL的完全培养基,放入小室,充分浸润,取100μL的细胞悬液加入transwell小室的上室;常规培养24-48h后取出,1×PBS洗2次,4%的多聚甲醛固定20min,1×PBS洗2次;在24孔板中加入500μL的结晶紫染液,放入小室,10min后取出,1×PBS洗2次,倒置小室,用棉签轻轻擦去上室内未穿过去的细胞;
结果如图8所示,用倒置显微镜观察结果,结果显示经RNAi慢病毒干扰体系敲低APMAP后HCT116细胞和SW1116细胞的迁移能力下降,而过表达APMAP后DLD-1细胞和SW620细胞的迁移能力提高。
5、细胞侵袭(Transwell小室法)
先配置水凝胶(50μL水凝胶混合于350μL稀释液中,再加入50μL基培混合),加入transwell小室的上室,每个100μL,避免产生气泡;消化收集稳定转染各组细胞,离心待用;用基培重悬细胞,调整细胞密度至5×104/mL;在24孔板中加入800μL的完全培养基,放入小室,充分浸润,取100μL的细胞悬液加入transwell小室的上室;常规培养24-48h后取出,1×PBS洗2次,4%的多聚甲醛固定20min,1×PBS洗2次;在24孔板中加入500μL的结晶紫染液,放入小室,10min后取出,1×PBS洗2次,倒置小室,用棉签轻轻擦去上室内未穿过去的细胞;
结果如图9所示,用倒置显微镜观察结果,结果显示经RNAi慢病毒干扰体系敲低APMAP后HCT116细胞和SW1116细胞的侵袭能力下降,而过表达APMAP后DLD-1细胞和SW620细胞的侵袭能力提高。
Claims (9)
1.APMAP基因在制备用于治疗结直肠癌的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述的治疗结直肠癌为抑制细胞的增殖。
3.根据权利要求1所述的应用,其特征在于,所述的治疗结直肠癌为抑制细胞的迁移。
4.根据权利要求1所述的应用,其特征在于,所述的治疗结直肠癌为抑制细胞的侵袭。
5.用于检测患者外周血或患者癌组织中APMAP蛋白表达量的生物标志物,其特征在于,为APMAP基因。
6.用于检测患者外周血或患者癌组织中APMAP蛋白表达量的试剂盒,其特征在于,含有APMAP基因。
7.APMAP基因的RNAi慢病毒干扰系统在制备用于治疗结直肠癌的药物中的应用,其特征在于,所述的APMAP基因的RNAi慢病毒干扰系统的序列如下:
APMAP-shRNA1:5′-GGTCGTCACAGACGATGATGG-3′,
APMAP-shRNA2:5′-CACCGATTCTAGCAGCAAATG-3′。
8.根据权利要求7所述的应用,其特征在于,所述的APMAP基因的RNAi慢病毒干扰系统为敲低结直肠癌细胞中APMAP基因。
9.根据权利要求7所述的应用,其特征在于,所述的APMAP基因的RNAi慢病毒干扰系统为抑制结直肠癌细胞中APMAP蛋白表达。
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