CN117165677A - 检测生物标志物在制备神经管畸形疾病的产品的应用 - Google Patents
检测生物标志物在制备神经管畸形疾病的产品的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,公开了一种检测生物标志物在制备神经管畸形疾病的产品的应用,生物标志物为纤毛相关异常基因;生物标志物为Ins2、Mapk1、Ptpn5或HSP90ab1;生物标志物的产品为纤毛芯片生物芯片;制备得到的产品包括辅助诊断或诊断神经管畸形疾病产品,包括试剂盒、试纸、试剂或探针中的一种或多种;本发明利用纤毛相关异常基因,生物标志物Ins2、 Mapk1、Ptpn5或HSP90ab1应用在制备辅助诊断或诊断神经管畸形疾病的产品中,用于NTDs的临床诊断。
Description
技术领域
本发明涉及生物医药技术领域,具体为一种检测生物标志物在制备神经管畸形疾病的产品的应用。
背景技术
神经管是中枢神经细胞(大脑和脊髓)的原基,神经管形成包括初级神经管形成和次级神经管形成两个关键阶段。初级神经管形成始于脊索中胚层诱导胚胎外胚层形成神经板,接着神经板闭合,形成神经管。次级神经管是指初级神经管形成后进行细胞进行自我更新,这一过程是神经管早期发育的细胞学基础,初级神经管的闭合从沿着胚胎轴的特定位置开始,以拉链状的方式分段闭合。在小鼠中,闭合点1发生在胚胎后脑/颈部的边界,分别在孕8.5,闭合失败分别会导致颅脊柱裂的发生;大约12小时后,出现在前脑/中脑的边界和前脑喙区的闭合点 2和闭合3开始进行闭合,其闭合异常会出现无脑畸形/脑膨出畸形。在人类胚胎发育中,神经管的关闭开始于受精后的第17到18天。类似于小鼠胚胎,是不连续的过程,以上过程为初级神经形成,之后在小鼠和人类中,都会通过次级神经形成骶骨下部和尾骨区域的神经管,由胚胎末端尾芽中一种自我更新的干细胞群,沿着初级神经管增殖分化为一个中空的次级神经管。神经管不完全闭合会导致中枢神经系统的严重异常,暴露于羊水环境从而导致大脑和/或脊髓的严重损伤。神经管发育过程依赖多种信号通路,包括音猬因子Shh信号通路、Wnt-β-catenin 信号通路、平面细胞极性PCP信号通路、肌醇代谢信号通路、转化生长因子-β(TGF-β)、哺乳动物雷帕霉素靶蛋白(mTOR)、Notch、血小板源性的生长因子受体(PDGFRs)以及G蛋白偶联受体(GPCRs)相关信号等。这些信号通路的异常会引起神经管的闭合不全,进而导致神经管畸形(neural tube defects,NTDs)的发生。目前,NTDs是出生缺陷中发病率和死亡率最高的类型,也是影响人口素质最常见和最严重的出生缺陷性疾病。NTDs通常发生于怀孕的早期,由于神经管不闭合或闭合不全而造成的头部至脊柱部位畸形,从无脑到轻度脊柱裂不同程度的畸形:包括无脑、露脑、小头畸形、大头畸形、脑积水、脑脊膜膨出、颅脊柱裂和脊柱裂。
纤毛是突出于细胞膜表面的一种高度特化的细胞结构, 广泛存在于从单细胞真核生物到脊椎动物的多种生物体内。纤毛包括有动力的(呼吸纤毛)和无动力的(初级纤毛),有动力的纤毛和无动力纤毛由其基于微管的轴突体结构来区分,其轴突体由九个周边的双重微管组成,在许多有动力的纤毛中,还可能包含一对中央的单微管,这些被称为9+0或9+2的轴突体。与无动力纤毛相比,有动力纤毛还包含有助于运动的额外结构,包括内部和外部的dynein臂、径向辐条和nexin链。目前,已经鉴定出四种主要类型的纤毛:9+2有动力(呼吸纤毛),9+0有动力(结节纤毛),9+2无动力(毛细胞的触毛)和9+0无动力(初级纤毛和光感受细胞)。无动力的初级纤毛是一个感觉器官,其用于转导许多外部信号,如生长因子、激素和形态发生物,一个完整的初级纤毛是由Hh、WNT、钙、G蛋白偶联受体和受体酪氨酸激酶等介导的信号通路所需的。不同于通常出现在上皮细胞上的有动力纤毛,初级纤毛是一个无动力的感觉器官,出现在大多数静止细胞的顶部表面。纤毛的生物生成涉及锚定近质膜的基底体,一个源于中心体的细胞器,然后聚合基于微管的轴突体并延伸质膜;尽管纤毛膜与质膜连续,纤毛和纤毛膜的蛋白质和脂质内容与细胞质和质膜的主要内容不同。这个特殊的隔室在纤毛生物生成过程中通过形成一个纤毛转换区来建立和维持,这是一个蛋白质结构,它与转换纤维一起,将基底体锚定到质膜,并作为一个纤毛孔来限制从细胞质到纤毛的自由扩散。纤毛组件从分泌系统被定向到纤毛基部,然后通过一个被称为鞭毛内部运输的马达驱动过程被运输到纤毛尖端,其中轴突体的延伸发生。纤毛在信号传导和细胞生物学中的重要性,相关的基因变异,导致了一系列的缺陷和疾病,其中部分会引起NTDs。
有研究发现,初级纤毛在 NTDs 发生过程中发挥着很重要的作用。初级纤毛在正常细胞发育和稳态过程的信号传导中发挥重要作用,因此初级纤毛被看作“细胞天线”。这些信号功能是通过初级纤毛上的无数信号分子来实现的,附着在纤毛膜上的跨膜受体允许细胞对外部的各种刺激做出反应,而位于基体、过渡区和初级纤毛尖端的调节蛋白控制信号级联。由于初级纤毛协调这些信号转导进而影响机体的生长发育及各种器官的正常生理功能,当结构及功能异常会导致多种发育和细胞信号缺陷,从而引起纤毛病,一些初级纤毛相关疾病常常伴发NTDs表征,麦克尔综合征和茹贝尔综合征都有脑膨出的临床表型。其中,有研究提到,在非洲爪蟾胚胎中敲除Ins2基因影响了神经板边缘区域导致眼睛和神经发育缺陷,提示着Ins2基因和神经发育之间可能存在某种联系,而且Ins2基因又是一种编码胰岛素的前体蛋白的基因,对于胰腺中β细胞的正常功能至关重要,参与调节血糖水平和能量代谢。Mapk1基因在多个细胞信号传导通路中起着重要作用,它参与了细胞的增殖、分化和存活等生物学过程。其与神经系统疾病、心血管疾病和免疫系统疾病等多种疾病有关;PTPN5在神经发育和神经系统功能中可能发挥重要作用,其通过调控Src家族激酶的活性,影响与神经功能相关的信号级联;HSP90主要是用于促进特定目标蛋白的成熟、结构维护和适当调控,这些目标蛋白涉及细胞周期控制和信号传导等过程。HSP90蛋白在其ATP酶活性的调控下经历一个功能循环,这个循环可能引发蛋白的构象变化,从而导致它们的激活。但是尚未有关于Ins2、Mapk1、Ptpn5或HSP90ab1基因和神经管畸形相关;更没有通过获得NTDs的分子标志物用于制备神经管畸形疾病的产品以用于NTDs的临床诊断和检测。
发明内容
本发明意在提供一种检测生物标志物在制备神经管畸形疾病的产品的应用,利用纤毛相关异常基因,生物标志物Ins2、Mapk1、Ptpn5或HSP90ab1应用在制备辅助诊断或诊断神经管畸形疾病的产品中,以解决上述问题。
为了实现上述目的,本发明提供如下技术方案:
一种生物标志物在制备辅助诊断或诊断神经管畸形疾病的产品中的应用,所述生物标志物为纤毛相关异常基因。
进一步地,所述生物标志物为Ins2、Mapk1、Ptpn5或HSP90ab1。
检测上述的生物标志物的产品在制备辅助诊断或诊断神经管畸形疾病的产品中的应用。
进一步地,所述检测生物标志物的产品为纤毛芯片。
进一步地,所述辅助诊断或诊断神经管畸形疾病的产品包括试剂盒、试纸、试剂或探针中的一种或多种。
技术方案的优益效果是:
1、本发明提供的检测生物标志物的产品在制备辅助诊断或诊断神经管畸形疾病的产品中的应用,利用纤毛相关异常基因作为生物标志物,在神经管畸形疾病的辅助诊断或诊断中具有重要作用;通过检测Ins2、Mapk1、Ptpn5或HSP90ab1等纤毛相关异常基因,可以提高诊断的准确性;
2、本发明提供的检测生物标志物的产品在制备辅助诊断或诊断神经管畸形疾病的产品中的应用,纤毛芯片作为一种检测生物标志物的工具可以实现高通量的快速检测,可以用于早期筛查神经管畸形疾病的风险;早期诊断和预防对于这类疾病来说尤为重要,可以更早地进行针对性治疗和管理,减少病情进展及并发症的风险。
附图说明
图1为构建神经管畸形小鼠模型畸形的胚胎表型;
图中,A为正常胚胎,B为模型组中正常胚胎,C为模型组中NTDs表型-露脑畸形;
图2为对照例中,电镜观察孕10.5小鼠室管膜细胞纤毛状态;
A为正常胚胎组,B为模型组中正常表型小鼠胚胎,C为模型组中NTDs表型小鼠胚胎,D为统计分析。
具体实施方式
下面结合附图和实施方式对本发明作进一步的详细说明:
诱导构建纤毛异常引起的神经管畸形小鼠模型
选择6~8周,体重18~21g SPF级C57BL/6J小鼠,繁殖饲料喂养,常规饮水。在晚上6点以1:1 雌雄鼠合笼后,于第二天早上检查阴拴者记为孕0.5天,孕鼠随机分为对照组和模型组,在孕7.5天,模型组孕鼠给予360~375mg/kg剂量的碳酸锂(255823,sigma)溶液腹腔注射;正常对照组孕鼠给予等体积的生理盐水。孕10.5天时,断颈处死,剖腹取出妊娠子宫,放入盛有PBS(PH7 .4)的培养皿,在体视显微镜下剥离出胎鼠,观察其畸形情况并拍照,计算致畸率。
取正常胚胎和畸形胚胎固定于4%的中性甲醛进行定,360~375mg/kg剂量组神经管畸形的比例比较多,其中受不同厂家不同批次的碳酸锂药物影响,因此根据购买的实际碳酸锂浓度选择所用药物适宜的剂量为最佳的造模剂量。畸形的表型如图1所示。
实施例1
扫描电镜观察纤毛
取诱导构建的小鼠模型,于孕10.5天取材,在体式显微镜下观察对照组和模型组胎鼠情况,取正常胚胎的胎鼠的全脑和模型组中表型正常的胎鼠全脑,模型组中表型为神经管畸形的胎鼠的全脑,取材时候要快速清洗后马上置于2.5%戊二醛固定液1 mL,室温固定4个小时后放置4 ℃,然后进行扫描电镜处理。
扫描电子显微镜观察在 5 000~20 000 倍扫描电子显微镜下观察胎鼠脑组织中纤毛的长度,通过扫描电镜的测量软件在每个胎鼠胎脑中纤毛的长度,测量每个视野中至少10根完整纤毛的长度并拍照。应用 SPSS 23.0 统计软件,计量资料以x±s表示,多组间比较采用重复测量数据的方差分析,P<0.05 为差异有统计学意义。如图2所示,孕10.5小鼠室管膜细胞纤毛状态,结果显示对照组纤毛长度和模型组中表型正常小鼠胚胎与模型组中NTDs表型小鼠胚胎均有统计学差异(P<0.05)且均高于处理组纤毛长度。而模型组中正常胚胎脑组织纤毛长度和NTDs胚胎脑组织纤毛也有统计学差异(P<0.05),且长度高于NTDs胚胎脑组织纤毛长度。
实施例2
利用纤毛相关异常基因和/或Ins2、Mapk1、Ptpn5或HSP90ab1作为生物标志物制备纤毛芯片对小鼠模型NTDs的检测
一、在已建立的C57BL/6J 小鼠模型样本库中随机选取对照(Control)组、肌醇干预表型正常(Non-NTDs)组及神经管畸形表型(NTDs)组的胚胎神经组织用于纤毛芯片检测。具体步骤如下:
1、组织匀浆
每10~20mg组织样品,加入1ml的TRIZOL试剂,用电动匀浆器进行匀浆;其中,所加样品体积不能超过匀浆此样品所使用的TRIZOL试剂体积的10%。
2、两相分离
匀浆后样品于15~30℃ 孵育5分钟,以便核酸蛋白复合体完全解离。每1ml的TRIZOL试剂匀浆的样品中加入0.2ml的氯仿,盖紧EP管盖。手动剧烈振荡管体15秒后,再在15~30℃ 孵育2~3分钟,接着在4℃ 下12000g离心15分钟;离心后混合液体将分为下层的红色酚氯仿相,中间层和上层的无色的水相,RNA全部被分配于水相中,水相的体积大约是匀浆时加入的TRIZOL试剂的60%。
3、RNA沉淀
将水相转移到新离心管中,水相与异丙醇混合以沉淀其中的RNA,加入异丙醇的量为每个样品匀浆时加入1ml TRIZOL试剂的此时加0.5ml的异丙醇;混匀后15~30℃ 孵育10分钟后,于4℃ 12000 g离心10分钟。此时,离心前不可见的RNA沉淀将在管底部和侧壁上形成胶状沉淀块。
4、RNA清洗
移去上清液,每1ml TRIZOL试剂匀浆的样品中加入至少1ml的75%乙醇,清洗RNA沉淀,振荡后,4℃ 7,500g离心5分钟。
5、重新溶解RNA沉淀
去除乙醇溶液,空气中干燥RNA沉淀5~10分钟,切勿真空离心干燥。注意RNA沉淀不要完全干燥,否则将大大降低RNA的可溶性。部分溶解的RNA样品A260/280比值将小于1.6。溶解RNA时,先加入无RNA酶的水用枪反复吹打几次,然后55到60℃ 孵育10分钟。获得的RNA溶液保存于-70℃。
6、小量RNA的抽提
从少量的组织(20mg)中抽提RNA:加入800µl的TRIZOL试剂至样品中,样品裂解后,按步骤2加氯仿进行两相分离。在加异丙醇沉淀RNA前,加5~10µg的无RNA酶的糖原作为水相载体。为降低溶液粘度,在加氯仿前先将样品两次过26号针头以剪切基因组DNA。两相分离后糖原留在水相中并和RNA共沉淀。只要糖原浓度不高于4mg/ml,不会影响cDNA的合成,同样也不会对PCR过程产生影响。
7、cDNA合成
1)配制混合物:
500ng/ul oligo (dT) 18 1µl
总RNA 1.5ug
10mM dNTPs Mix 1µl
灭菌水 up to13 μl;
2)65℃水浴5分钟后,冰上放置至少1分钟;
3)短暂离心后,在离心管中依次加入:
5 X First-Strand Buffer 4µl
0.1M DTT 1µl
RNase Inhibitor 1µl
SuperScript III RT 1µl;
4)移液枪轻轻吸打几次混合均匀;
5)50℃ 温育60分钟;
6)70℃ 温育15分钟使酶失活;
7)每20µl cDNA加91µl灭菌水混匀,置冰浴待用或-20℃保存。
二、实时定量PCR
1、样品准备
2X SuperArray PCR master mix 1050µl
已稀释的cDNA 105µl
ddH2O 945µl
总体积 2100µl;
2、加样
1)小心打开PCR Array上的膜;
2)加20ul混合液到PCR Array对应的每个孔中;
3)小心盖上盖子密封PCR Array;
4)在设置PCR程序前将准备好的PCR Array放在冰上;
5)、实时定量PCR程序设置完后,将PCR Array置于实时定量PCR仪进行PCR反应。
所设置的程序为:
聚合酶激活/变性,温度为95℃,时间为10分钟;扩增40个循环,温度为95℃,时间为15秒;收集荧光的温度为60℃,时间为1分钟;
采用ΔΔCt 方法进行数据分析:
1)计算每个处理组中的每个通路相关基因的ΔCt
ΔCt (group 1) = average Ct – average of HK genes’ Ct for group 1array
ΔCt (group 2) = average Ct – average of HK genes’ Ct for group 2array
2)计算2个PCR Array(或两组)中每个基因的ΔΔCt
ΔΔCt = ΔCt (组2) - ΔCt (组1)
其中,组1为对照组,组2为模型组
3)通过2-ΔΔCt计算组2与组1对应基因的表达差异。
实施例3
选择Actb、B2m、Gapdh作为作为内参基因,结果如下表1至表3所示,
表1 模型组中NTDs表型胚胎脑组织和对照组正常表型胚胎脑组织相比
由表1可知,NTDs表型胚胎脑组织与对照组之间的差异基因,具有统计学意义(p值小于0.05)的上调基因包括Ift172、Map2k1、Ptch1、Sufu和Hsp90ab1,它们的差异倍数分别为1.235344857、1.456549625、1.254389198、1.314878277、1.385570352。同样,下调的基因中Igf1、Ins2、Mapk1和Nphp1显示了明显的差异,其倍数为1.291869574、3.049319768、2.454261068、1.755088503。基于p值和差异倍数,筛选下调大于2倍的基因,Ins2(3.049319768倍)和Mapk1(2.454261068倍)。
表2 模型组中正常表型胚胎脑组织和对照组正常表型胚胎脑组织相比
由表2可知,模型组中正常表型胚胎脑组织和对照组相比的差异基因,具有统计学意义(p值小于0.05)的上调基因包括Map2k1、Sufu和Hsp90ab1,它们的差异倍数分别为1.3875075376、1.2990527870、1.4104656461。同样,下调的基因中Arl6、Igf1和Ptpn5显示了明显的差异,其倍数为1.2084982633、1.5059811918、2.2946362921。基于p值和差异倍数,筛选下调大于2倍的基因,Ptpn5 (2.2946362921倍);另外,Hsp90ab1基因在表1中统计上调1.385570352倍,在表2中统计上调1.4104656461倍。
表3 模型组中NTDs表型胚胎脑组织和模型组中正常表型胚胎脑组织相比
由表3可知,模型组中NTDs表型胚胎脑组织和模型组中正常表型胚胎脑组织相比的差异基因,只有2个基因(Cdc42,Mapk1)具有统计学意义(p值小于0.05),其基因表达水平下调,差异倍数分别为1.191462940、2.877628401。为进一步筛选,选择下调倍数大于2倍的基因,Mapk1 (2.877628401倍),值得注意的是,Mapk1基因在分别在表1和表2中都呈现上调。
综上可知,生物标志物Ins2、Mapk1、Ptpn5或HSP90ab1在统计学上具有显著意义,而且它们的差异倍数也很明显。因此,可以将其用于NTDs的诊断和检测。
以上所述的仅是本发明的实施例,方案中公知的具体技术方案或特性等常识在此未作过多描述。应当指出,对于本领域的技术人员来说,在不脱离本发明技术方案的前提下,还可以作出若干变形和改进,这些也应该视为本发明的保护范围,这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准,说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (5)
1.检测生物标志物在制备神经管畸形疾病的产品的应用,其特征在于,所述生物标志物为纤毛相关异常基因。
2.如权利要求1所述的应用,其特征在于,所述生物标志物为Ins2、Mapk1、Ptpn5或HSP90ab1。
3.检测如权利要求1或2中所述的生物标志物的产品在制备辅助诊断或诊断神经管畸形疾病的产品中的应用。
4.如权利要求3所述的应用,其特征在于,所述生物标志物的产品为纤毛芯片。
5.如权利要求3所述的应用,其特征在于,所述辅助诊断或诊断神经管畸形疾病的产品包括试剂盒、试纸、试剂或探针中的一种或多种。
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