CN116536270A - 人卵巢癌细胞系SKOV3-sh-MUC1及其构建方法和应用 - Google Patents
人卵巢癌细胞系SKOV3-sh-MUC1及其构建方法和应用 Download PDFInfo
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Abstract
人卵巢癌细胞系SKOV3‑sh‑MUC1及其构建方法和应用,属于生物医学技术领域。本发明一方面提供了人卵巢癌细胞系SKOV3‑sh‑MUC1,另一方面提供了该人卵巢癌细胞系的构建方法和应用。本发明获得的shRNA分子序列能够干扰MUC1的表达,通过慢病组稳转的方法,将带有可敲低MUC1的shRNA序列的慢病毒载体导入卵巢癌SKOV3细胞中,经过puromycin抗生素筛选稳转株后验证,从而建立了MUC1稳定敲低的细胞株SKOV3‑sh‑MUC1,抑制了卵巢癌细胞的增殖、转移和侵袭能力,可用于以MUC1为靶点的药物作用机制研究,或与MUC1相关的信号通路研究,为卵巢癌的靶向治疗提供新的治疗方案。
Description
技术领域
本发明属于生物医学技术领域,具体涉及人卵巢癌细胞系SKOV3-sh-MUC1及其构建方法和应用。
背景技术
卵巢癌是女性生殖系统的致命妇科恶性肿瘤,由于早期卵巢癌的临床症状不明显,通常诊断出为晚期,且多预后不良。目前对于卵巢癌的治疗方法主要是化疗,紫杉醇类化疗是卵巢癌患者的标准治疗方法,然而癌症易复发,使临床疗效不大,预后仍然较差。除化疗外,针对卵巢癌中某种特殊靶点的靶向药治疗也逐渐应用于临床,研究不同表达基因在各类癌症中的作用,预测药物的疗效和预后显得尤为重要。
粘蛋白1(mucin1,MUC1)是一种Ⅰ型跨膜蛋白,参与多种生理机制,包括粘附、发育和分化。在许多腺癌中,MUC1的异常表达可引起异常的糖基化和细胞增殖。MUC1与许多类型的癌症密切相关,在肿瘤形成和进展中均起致癌作用。MUC1在大多数人类癌中异常表达,包括胰腺癌、卵巢癌、肺癌和卵巢癌。然而,MUC1在分子靶向治疗中的功能在很大程度上是未知的。基础研究中多通过干预目标靶点的表达,对其进行敲低或过表达以观察该靶点改变影响的分子机制、细胞表型变化或靶点对药物敏感性研究。针对研究靶点的敲低较为常见的选择是进行瞬时转染小干扰RNA,一般具有短效性,在研究中需要多次转染,这无疑增加了人力投入及研究成本。本发明构建该稳转细胞株可以直接用于检测实验研究中的指标,可避免多次重复转染,简化实验操作,节约实验时间和成本。该稳转细胞株与对照组细胞相比细胞表型发生变化,敲低MUC1后可使卵巢癌细胞增殖能力显著降低,诱导细胞发生周期阻滞,并且显著抑制了其迁移和侵袭能力,证实MUC1确实在卵巢癌发生发展中起重要的作用。该细胞株可直接用于后续研究靶向药物作用机制,也为进一步深入探索MUC1在卵巢癌进展中发挥何种分子机制作用提供应用基础。
发明内容
针对现有技术存在的问题,本发明的目的在于设计提供人卵巢癌细胞系SKOV3-sh-MUC1及其构建方法和应用的技术方案。
为实现上述目的,本发明采用如下技术方案:
本发明第一方面提供了人卵巢癌细胞系SKOV3-sh-MUC1,保藏单位:中国典型培养物保藏中心,地址:中国武汉武汉大学,保藏日期:2023年01月04日,保藏号为:CCTCC NO:C202308。
本发明第二方面提供了上述的人卵巢癌细胞系的子代细胞系。
本发明第三方面提供了上述的人卵巢癌细胞系的构建方法,其包括以下步骤:
1)设计能够有效敲低MUC1的shRNA分子,shRNA的正义链核苷酸序列如SEQ IDNO.2所示,具体为:
5’-CCGGCCGGGATACCTACCATCCTATCTCGAGATAGGATGGTAGGTATCCC GGTTTTTG-3’;
shRNA的反义链核苷酸序列如SEQ ID NO.3所示,具体为:5’-AATTCAAAAACCGGGATACCTACCATCCTATCTCGAGATAGGATGGTAGG TATCCCGG-3’;
2)合成能够有效敲低MUC1的慢病毒;
3)感染卵巢癌SKOV3细胞,通过抗生素筛选获得稳转细胞。
进一步,所述步骤1)中沉默人MUC1表达的shRNA分子靶标基因的核苷酸序列如SEQID NO.1所示,具体为:CCGGGATACCTACCATCCTAT。
进一步,所述步骤2)中慢病毒载体选用GV493载体,元件顺序为:hU6-MCS-CBh-gcGFP-IRES-puromycin。
进一步,所述步骤3)中抗生素为puromycin抗生素。
本发明第四方面提供了上述的人卵巢癌细胞系或上述的子代细胞系在构建卵巢癌发生、发展或转移的细胞或动物模型中的应用。
本发明第五方面提供了上述的人卵巢癌细胞系或上述的子代细胞系在构建用于MUC1为靶点药物作用机制研究的细胞或动物模型中的应用。
本发明第六方面提供了上述的人卵巢癌细胞系或上述的子代细胞系在构建与MUC1相关信号通路研究的细胞或动物模型中的应用。
本发明第七方面提供了上述的人卵巢癌细胞系或上述的子代细胞系在构建卵巢癌靶向治疗的细胞或动物模型中的应用。
本发明第八方面提供了抑制MUC1表达的shRNA分子,所述shRNA的正义链核苷酸序列如SEQ ID NO.2所示,shRNA的反义链核苷酸序列如SEQ ID NO.3所示。
本发明的有益效果为:本发明获得的shRNA分子序列能够干扰MUC1的表达,通过慢病毒稳转的方法,将带有可敲低MUC1的shRNA序列的慢病毒载体导入卵巢癌SKOV3细胞中,经过puromycin抗生素筛选稳转株后验证,从而建立了MUC1稳定敲低的细胞株SKOV3-sh-MUC1,抑制了卵巢癌细胞的增殖、转移和侵袭能力,可用于以MUC1为靶点的药物作用机制研究,或与MUC1相关的信号通路研究,为卵巢癌的靶向治疗提供新的治疗方案。
附图说明
图1为慢病毒载体图谱。
图2为实验组、阴性对照组提取RNA,通过Realtime PCR方法检测MUC1基因mRNA水平的表达图。NC表示未经任何处理的SKOV3细胞组,sh-Control表示感染阴性对照病毒的SKOV3细胞组,sh-MUC1为感染MUC1敲低慢病毒的SKOV3细胞株。*表示与sh-Control相比,*P<0.05,**P<0.01。
图3为实验组、阴性对照组提取蛋白质,通过Western Blot方法检测MUC1基因蛋白水平的表达图。
图4为实验组、阴性对照组细胞增殖检测图,图4A:CCK-8检测;图4B:EdU检测。
图5为实验组、阴性对照组细胞周期检测图。
图6为实验组、阴性对照组细胞迁移和侵袭检测图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,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。
实施例1:沉默人MUC1表达的特异性干扰序列shRNA的获得
根据网站:
www.sigmaaldrich.cn/CN/zh/semi-configurators/shrna?activeLink=productSearch设计shRNA序列,获得的靶基因序列:
sh-MUC1:CCGGGATACCTACCATCCTAT;
sh-Control:TTCTCCGAACGTGTCACGT;
shRNA的正义链序列:
5’-CCGGCCGGGATACCTACCATCCTATCTCGAGATAGGATGGTAGGTATCCC GGTTTTTG-3’;
shRNA的反义链序列:
5’-AATTCAAAAACCGGGATACCTACCATCCTATCTCGAGATAGGATGGTAGG TATCCCGG-3’。
实施例2:MUC1的shRNA-慢病毒表达载体的构建
将hU6-MCS-CBh-gcGFP-IRES-puromycin载体,采用EcoR I/Age I进行双酶切线状化,并切胶回收;根据shRNA设计特异性引物完成引物退火;将shRNA和酶切回收的线状载体同源重组形成环状,转入制备好的细菌感受态细胞,挑选单克隆菌落进行测序鉴定,比对正确的克隆即为构建成功的shRNA沉默载体(见图1)。
具体步骤如下:
(1)载体线状化酶切回收,酶切体系如表1所示:
表1酶切体系
37℃(最适温度)反应1h后,对载体酶切产物进行1%琼脂糖凝胶电泳,回收目的片段。
(2)目的片段与载体的重组,通过T4 DNAligase将双酶切线性化的载体和退火双链DNA连接,16℃连接1-3h,或连接过夜。反应体系如表2:
表2链接体系
*根据载体大小做相应调整
(3)转化及阳性克隆的PCR鉴定与测序
将5μl连接产物加入到50μl大肠杆菌感受态细胞中,冰浴30min,42℃热激80s,冰浴3min;加入450μL无抗生素的LB液体培养基,150rpm于37℃摇床震荡培养45min;取菌液均匀涂抹在含有Amp的LB固体培养基上,于37℃培养箱中过夜培养。次日,挑取单克隆,一个离心管中放一个单克隆,管内加8ml含有氨苄青霉素的液体LB培养基,37℃,220rpm,振荡培养14h。培养至菌液混浊,收集菌液,并提质粒进行测序(上海吉凯基因有限公司),进行测序比对后,鉴定阳性克隆即为构建成功的MUC1-shRNA慢病毒表达载体。
(4)慢病毒包装及滴度测定
将提取的慢病毒表达载体质粒分别与包装质粒共转染293T细胞(转染试剂由上海吉凯公司提供),于48h收集病毒上清,观察细胞形态和GFP表达,将提取的病毒纯化和浓缩,采用梯度稀释法测定病毒滴度,将制备完成的病毒浓缩液分装于-80℃保存。
实施例3:MUC1-shRNA慢病毒感染人卵巢癌细胞
将人卵巢癌SKOV3细胞制备以2.5×105个/孔的密度接种于6孔板,待细胞密度达到70%时,分别更换为sh-MUC1、sh-Control两种慢病毒溶解液(MOI=10)与感染试剂至培养基,摇晃混匀孵育8-10h后更换为正常完全培养基继续培养,72h后荧光显微镜观察GFP绿色荧光表达量,约90%以上说明感染成功。
Puromycin抗生素筛选稳转细胞株:预实验设置抗生素浓度梯度选取可杀死未感染病毒SKOV3细胞的最低抗生素浓度(即工作浓度)为2μg/mL,使用该工作浓度对上述感染成功的细胞进行药筛。细胞培养基中加入2μg/mL Puromycin抗生素连续培养48h,换液洗去未感染成功的阴性细胞,带有GFP荧光的存活细胞即为目标阳性细胞,后续使用1μg/mL抗生素浓度(维持浓度)长期培养以维持该稳转株。
将稳转株进行保藏,保藏单位:中国典型培养物保藏中心,地址:中国武汉武汉大学,保藏日期:2023年01月04日,保藏号为:CCTCC NO:C202308,建议的分类命名:人卵巢癌细胞SKOV3-sh-MUC1 Human ovarian cancer cell line SKOV3-sh-MUC1。
实施例4:SKOV3细胞中MUC1 mRNA水平检测
收集抗生素药筛后的两种稳转细胞株,按照Total RNA提取试剂盒(15596026,Invitrogen公司)说明书提取细胞总RNA,用反转录试剂盒(RR037A,Takara公司)反转录合成cDNA。进行Real-Time PCR检测MUC1基因mRNA表达(扩增引物见表3),程序为:95℃10min;(95℃15s,64℃30s,72℃20s)*40个循环;95℃15s;60℃,1min;95℃15s。Real-Time PCR采用2-△△Ct法进行相对定量分析。
表3引物序列
RT-qPCR检测发现sh-MUC1组的MUC1水平显著低于sh-Control组(P<0.01)(见图2)。
实施例5:SKOV3细胞中MUC1蛋白水平检测
分别提取各组细胞的总蛋白,BCA法测定蛋白浓度。加入适量上样缓冲液于100℃煮沸变性10min;每孔20μg,80V恒压SDS-PAGE电泳,在4℃,200mA恒流条件下电转120min,将蛋白转移到PVDF膜上。5%脱脂牛奶室温封闭1.5h,加入一抗鼠源MUC1(1:1000,CST公司)、兔源β-actin单克隆抗体(1:5000,Abcam公司)4℃孵育过夜。分别加入HRP标记的羊抗兔IgG与羊抗鼠IgG(1:5000,Abcam公司),室温孵育2h,TBST漂洗5min×3次,ECL化学发光显影。
Western Blot检测发现,所述shRNA可显著降低SKOV3细胞中MUC1蛋白表达(P<0.01)(见图3)。
实施例6:CCK-8与EdU检测SKOV3-sh-MUC1细胞活力与增殖
CCK-8:取对数生长期的细胞消化离心,调整细胞数为5×104个/mL,取100μL接种于96孔板内,每组5个复孔。避光加入10μL/孔的CCK-8试剂,37℃孵育1h,多功能酶标仪检测450nm处的吸光度值,分别于24h、48h、72h、96h检测细胞活力。以上实验重复至少三次,绘制细胞活力曲线图。细胞活力(%)=[(OD实验组-OD空白组)/(OD对照组-OD空白组)]*100。
EdU检测:6孔板中铺入细胞,培养过夜细胞贴壁后加入2×EdU工作液,继续培养2h进行EdU标记;去除培养基,每孔加入4%多聚甲醛1mL,室温固定细胞15min,去除固定液后每孔,加洗涤液洗涤3次,每次5min;每孔加入1mL通透液,室温孵育15min后使用洗涤液洗涤三次;每孔加入0.5mL Click反应液,室温避光孵育30min;洗涤液洗涤3次;每孔加入1XHoechst 33342溶液1mL进行细胞核染色,洗涤液洗涤3次,使用荧光显微镜进行荧光检测。
所述SKOV3-sh-MUC1与阴性对照细胞相比,其细胞增殖能力显著降低(P<0.01)(见图4)。
实施例7:流式细胞术检测SKOV3-sh-MUC1细胞周期
细胞于实验前一日更换为无血清培养基使细胞饥饿12h,后更换为完全培养基继续培养24h,取对数生长期细胞消化离心后用预冷PBS重悬,再次离心弃上清,使用70%乙醇重悬细胞,于4℃过夜固定。离心弃去上清,预冷PBS重悬洗一次,弃上清,加入0.5mL PI染液,室温避光孵育20min,流式细胞仪上机检测细胞周期,实验独立重复三次。
所述SKOV3-sh-MUC1与阴性对照细胞相比,其细胞周期发生G0/G1期阻滞,S期缩短,表明MUC1的敲低可显著影响细胞周期进程(P<0.01)(见图5)。
实施例8:MUC1低表达抑制卵巢癌细胞迁移和侵袭
Transwell细胞迁移与侵袭实验:实验前将细胞饥饿12h,胰蛋白酶消化离心,用无血清培养基稀释重悬并进行细胞计数,使用具有8.0μm孔径的Transwell小室,取无血清细胞悬液(2×104个/100μL)接种在上室(侵袭实验在接种细胞前预先铺入100μl1:8稀释的Matrigel基质胶,37℃孵育2h待胶凝固),下室加600μL含10%FBS的完全培养基;培养24h,取出Transwell小室,弃上室培养基,用PBS洗两次,棉签轻轻擦去上室表面细胞;下室加入4%多聚甲醛固定细胞30min,自然风干后用0.1%结晶紫溶液染色30min,随机计数5个视野细胞数量计算平均细胞数,实验独立重复三次。
所述SKOV3-sh-MUC1与阴性对照细胞相比,其迁移和侵袭能力均显著降低(P<0.01)(见图6)。
统计学分析:数据用SPSS 22.0软件进行统计学分析。计量资料以均值±标准差(x±s)表示,组间比较单因素方差分析(One-way ANOVA)和t检验,P<0.05表明差异有显著性。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (10)
1.人卵巢癌细胞系SKOV3-sh-MUC1,保藏编号为CCTCC NO:C202308。
2.如权利要求1所述的人卵巢癌细胞系的子代细胞系。
3.如权利要求1所述的人卵巢癌细胞系的构建方法,其特征在于包括以下步骤:
1)设计能够有效敲低MUC1的shRNA分子,shRNA的正义链核苷酸序列如SEQ ID NO.2所示,shRNA的反义链核苷酸序列如SEQ ID NO.3所示;
2)合成能够有效敲低MUC1的慢病毒;
3)感染卵巢癌SKOV3细胞,通过抗生素筛选获得稳转细胞。
4.如权利要求3所述的构建方法,其特征在于所述步骤2)中慢病毒载体选用GV493载体,元件顺序为:hU6-MCS-CBh-gcGFP-IRES-puromycin。
5.如权利要求3所述的构建方法,其特征在于所述步骤3)中抗生素为puromycin抗生素。
6.如权利要求1所述的人卵巢癌细胞系或权利要求2所述的子代细胞系在构建卵巢癌发生、发展或转移的细胞或动物模型中的应用。
7.如权利要求1所述的人卵巢癌细胞系或权利要求2所述的子代细胞系在构建用于MUC1为靶点药物作用机制研究的细胞或动物模型中的应用。
8.如权利要求1所述的人卵巢癌细胞系或权利要求2所述的子代细胞系在构建与MUC1相关信号通路研究的细胞或动物模型中的应用。
9.如权利要求1所述的人卵巢癌细胞系或权利要求2所述的子代细胞系在构建卵巢癌靶向治疗的细胞或动物模型中的应用。
10.抑制MUC1表达的shRNA分子,其特征在于shRNA的正义链核苷酸序列如SEQ ID NO.2所示,shRNA的反义链核苷酸序列如SEQ ID NO.3所示。
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CN1455680A (zh) * | 2000-09-11 | 2003-11-12 | 达纳-法伯癌症协会有限公司 | Muc1胞外域和癌症治疗组合物及方法 |
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WO2008109377A1 (en) * | 2007-03-02 | 2008-09-12 | Mdrna, Inc. | Nucleic acid compounds for inhibiting vegf family gene expression and uses thereof |
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