CN105861442B - 高转移肝癌细胞株及其构建方法和应用 - Google Patents
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Abstract
本发明公开了一种高转移肝癌细胞株及其构建方法和应用。肝癌细胞株，命名为人肝癌细胞株Huh‑7/M8，保藏号为：CCTCC NO：C2014247。本发明通过将肝癌细胞株Huh‑7反复8次进行Transwell穿膜实验，筛选得到了Huh‑7/M8细胞株，该细胞株具有高转移性和侵袭性，能够被用于制备哺乳动物肝癌模型，研究肝癌细胞转移机制，提取肝癌转移和复发的标志物，筛选和评估治疗肝癌药物，开发肝癌复发检测试剂盒等用途，具有较高的科研和生产应用的价值，预期能产生良好的科研、经济和社会效益。
Description
技术领域
本发明涉及生物学和肿瘤学领域，特别是涉及一种高转移肝癌细胞株及其构建方法和应用。
背景技术
癌症是严重威胁我国人民生命健康的第一大“杀手”，已成为亟待解决的公共健康问题！最新研究提示我国癌症新发病例和死亡病例逐年增加，其中2015年约有429.2万新发浸润性癌症病例，平均每天新发约12000例；有281.4万癌症死亡病例，平均每天约7500人死于癌症。
原发性肝癌是我国最常见的恶性肿瘤之一，每年新发病例和死亡病例占全球50％以上。据估计2015年我国肝癌新发病例46.6万，死亡病例高达42.2万。尤其是在60岁以下男性人群中，肝癌是发病率和死亡率最高的癌症！
在我国，引起原发性肝癌的原因主要是以下四个因素：1、病毒性肝炎：主要为乙型肝炎病毒(HBV)与丙型肝炎病毒(HCV)引起。我国肝癌病人HBV标记阳性的病人达到85％～90％。2、黄曲霉毒素。3、饮水污染。4、饮酒与吸烟。其中病毒性肝炎是最主要的原因。
由于肝癌缺乏典型的临床表现，早期无明显症状，多数患者确诊时往往已处于晚期，并出现肿瘤远处转移，失去手术根治的机会。而有机会施根治性切除术的肝癌患者，其术后复发率也相当高。大量研究证实肝癌转移和术后复发给临床治疗带来极大困难，严重影响患者的疗效和生存质量，是导致肝癌患者死亡的主要原因。
而且肝癌容易发生转移和复发，如大肝癌切除术后5年复发、转移率为60％～80％，小肝癌也有40％～50％。肝癌最早在肝内转移，很容易侵犯门静脉及分支并形成瘤栓，脱落后在肝内引起多发性转移灶；或者通过血行转移，形成肺转移和淋巴转移。因此，原发性肝癌病人总的预后不佳，迫切需要寻求更加有效的治疗方法。
但目前缺少理想的体外研究细胞模型，制约科研工作者进一步深入研究肝癌转移。建立和鉴定合适的高转移肝癌细胞模型，有助于筛选肝癌复发转移的关键靶点，为成功开发抗复发转移的临床治疗手段提供新策略，有着重要的科学意义和临床价值！
发明内容
本发明的目的是提供一种具有高转移性和侵袭性的肝癌细胞株。
一种肝癌细胞株，命名为人肝癌细胞株Huh-7/M8，保藏于位于中国武汉武汉大学的中国典型培养物保藏中心，保藏日期为2015年3月6日，保藏号为：CCTCC NO：C2014247。
本发明又提供了所述肝癌细胞株的子代细胞。所述子代细胞基本或全部保留了亲代肝癌细胞株Huh-7/M8的特性，也是具有高转移性和侵袭性的。
本发明还提供了一种所述肝癌细胞株的构建方法，所述构建方法为Transwell迁移试验法，起始细胞株为肝癌细胞株Huh-7，试验重复次数为8次。
将Transwell小室放入培养板中，小室内称上室，培养板内称下室，上室内盛装上层培养液，下室内盛装下层培养液，上下层培养液以聚碳酸酯膜相隔，将细胞种在上室内，由于聚碳酸酯膜有通透性，下层培养液中的成分可以影响到上室内的细胞，从而可以研究下层培养液中的成分对细胞生长、运动等的影响。通过将亲代肝癌细胞多次重复Transwell迁移试验，能够筛选驯化得到高转移的肝癌细胞。
Huh-7是由Naka-bayashi等人建立的人肝癌细胞系，细胞源自一个日本男性高分化肝细胞肝癌，HBV阴性，是肝癌研究中常用的细胞模型。STR(短串联重复序列)是广泛分布在真核生物基因组中的简单重复序列，通常采用PCR扩增，扩增产物通过电泳分析并根据大小分离等位基因进行检测。通过STR检测分析发现，Huh-7/M8与亲代Huh-7完全同源，不存在其他细胞的交叉污染，细胞形态和增殖速度没有显著区别。但是Huh-7/M8的迁移和侵袭能力显著强于Huh-7细胞。
本发明还提供了所述的肝癌细胞株在制备哺乳动物肝癌模型中的应用。所述的哺乳动物为裸小鼠或裸大鼠。通过将一定细胞数量的所述人肝癌细胞株Huh-7/M8接种于裸小鼠或裸大鼠的皮下、肝脏、腹腔或者尾静脉等部位，获得高转移性肝癌的动物模型。
本发明还提供了所述的肝癌细胞株在提取肝癌转移和复发的标志物中的应用。通过与低转移性肝癌细胞进行比较研究，可以发现高转移肝癌的分子标记，针对该分子标记可以进行后续的药物开发等应用。
本发明还提供了所述的肝癌细胞株在筛选和评估治疗肝癌药物中的应用。首先，通过向所述人肝癌细胞株Huh-7/M8培养基中添加不同药物，观察细胞状态变化，获得初步有效的候选药物。然后，将候选药物施药于上述高转移性肝癌动物模型，观察与未施药组动物的存活期、肿瘤大小、转移情况等，筛选获得潜在治疗肝癌的药物。
本发明还提供了所述的肝癌细胞株在开发肝癌复发检测试剂盒中的应用。通过microRNA芯片检测技术比较分析Huh-7和Huh-7/M8细胞中microRNA的表达差异，获得有显著性差异的microRNA共15条。通过实时定量PCR对其中表达差异最大的miR-181a-5p、miR-210、miR-483-5p和miR-483-3p进行验证。相比于亲代细胞Huh-7，Huh-7/M8细胞miR-181a-5p的表达显著降低，与microRNA芯片检测结果一致。检索文献发现miR-181a参与调控肿瘤的转移，因此，miR-181a可以作为肝癌转移的分子标志物。开发出检测miR-181a表达情况的检测试剂盒，该试剂盒可用于检测肝癌复发情况。
本发明通过将肝癌细胞株Huh-7反复8次进行Transwell穿膜实验，筛选得到了Huh-7/M8细胞株，该细胞株具有高转移性和侵袭性，能够被用于制备哺乳动物肝癌模型，研究肝癌细胞转移机制，提取肝癌转移和复发的标志物，筛选和评估治疗肝癌药物，开发肝癌复发检测试剂盒等用途，具有较高的科研和生产应用的价值，预期能产生良好的科研、经济和社会效益。
附图说明
图1为实施例2中Huh-7和Huh-7/M8细胞形态检测图；
图2为实施例4中Huh-7和Huh-7/M8细胞的生长曲线检测结果图；
图3为实施例5中Huh-7和Huh-7/M8细胞的划痕实验结果比较图；
图4为实施例6中Huh-7和Huh-7/M8细胞的迁移实验结果比较图；
图5为实施例7中Huh-7和Huh-7/M8细胞的侵袭实验结果比较图；
图6为实施例8中Huh-7和Huh-7/M8细胞中microRNA表达差异比较图，其中A为microRNA芯片结果，B为实时定量PCR结果。
具体实施方式
实验材料：
细胞株：肝癌细胞株Huh-7，人肝癌细胞株Huh-7/M8。
试剂耗材：MEM培养液(杭州吉诺生物医药技术有限公司)、0.25％胰酶(杭州吉诺生物医药技术有限公司)、胎牛血清(Gibco公司)、Giemsa染色试剂盒(南京建成生物科技有限公司)、60mm和100mm培养皿(Corning公司)、6孔和24孔Transwell小室(孔径8μm，corning公司)、基质胶(matrigel)。
基础培养液：含10％胎牛血清的MEM培养液。
无血清培养液：MEM培养液。
仪器：正置显微镜、倒置显微镜、细胞培养箱、超净工作台、血球细胞计数器。
实施例1 Huh-7/M8细胞株建立
将肝癌细胞株Huh-7用MEM培养液(无血清培养基)制备1×106/mL单细胞悬液，然后在孔径为8μm的6孔板Transwell小室的上室加1mL所述单细胞悬液，下室加2mL基础培养液，培养48h后，收集穿过Transwell小室隔膜的细胞，扩增培养。重复上述操作8次后，获得Huh-7/M8细胞。
实施例2细胞形态学检测
将10万个Huh-7和Huh-7/M8细胞接种于60mm培养皿，培养基为基础培养液，培养72小时，至60％～70％贴壁率，去培养液，PBS洗涤2次，Giemsa染色(完全按照试剂盒操作说明)，正置显微镜下观察，拍照。
如图1所示，与亲代细胞Huh-7比较，Huh-7/M8的细胞大小、细胞轮廓、核质比等与Huh-7差异不大。
实施例3Huh-7/M8细胞STR检测分析
委托中国典型培养物保藏中心对Huh-7/M8细胞进行STR检测分析，检测方法如下：提取待检细胞的DNA，采用GoldeneyeTM 20A STR复合扩增试剂盒进行扩增，在ABI 3100型遗传分析仪上连续对20个已知的STR位点和性别基因Amelogenin进行检测分析，以确定待检测细胞的种属来源，判断其细胞是否存在交叉污染，检测结果如表1所示。
表1
检测报告的结论为：
1、Huh-7/M8细胞系在各基因座均未出现三等位基因现象，细胞中没有发现人类细胞交叉污染。
2、Huh-7/M8细胞系STR数据与美国ATCC、德国和日本JCRB的STR数据库中数据相对比，在JCRB、DSMZ细胞库中均找到与其细胞分型100％相匹配的细胞，细胞名称为Huh-7。
实施例4细胞生长曲线检测
分别将5万个Huh-7和Huh-7/M8细胞接种于100mm培养皿，培养基为基础培养液，培养72～96小时，至70％～90％贴壁率，更换新鲜培养液，0.25％胰酶消化，收集细胞，细胞计数，配制10000个/mL浓度的单细胞悬液，分别每孔接种5mL单细胞悬液至30个60mm培养皿，每总细胞数为50000个。依据情况更换培养液，保证充足的营养成分，分别在24、48、72、96、120、144、168、196小时，消化3皿细胞计数，取平均值，绘制生长曲线。
结果如图2所示，Huh-7/M8与Huh-7生长曲线大致重合，Huh-7/M8与Huh-7体外增殖速度没有显著性区别。
实施例5划痕实验
在6孔板中接种50万个Huh-7和Huh-7/M8细胞，培养基为基础培养液，长至100％融合度时，换无血清培养液，用无菌的200μL枪头划痕，洗涤2遍，去除悬浮细胞，换无血清培养液培养，选取划痕宽度合适，边界规整的区域标记，每隔24小时显微镜下观察拍照，比较两种细胞水平移动能力的差异。
结果如图3所示，无血清培养48小时后，Huh-7/M8细胞的划痕间隙宽度明显小于Huh-7细胞，说明Huh-7/M8细胞的水平方向的运动能力显著强于Huh-7细胞。
实施例6细胞迁移实验
消化Huh-7和Huh-7/M8细胞，用无血清培养液制取5×105/mL单细胞悬液，在24孔板Transwell(孔径为8μm)上室加200μL单细胞悬液，下室加600μL基础培养液。36小时后，去除Transwell上室细胞，Gimsa染色，割取Transwell隔膜，封片后正置显微镜下观察，并拍照。
结果如图4所示，同样条件下，Huh-7/M8细胞穿过Transwell隔膜的细胞显著多于Huh-7细胞，说明Huh-7/M8细胞的迁移能力显著强于Huh-7细胞。
实施例7细胞侵袭实验
用基质胶(matrigel)和无血清培养液按1∶5混合，每个Transwell小室(24孔板，孔径为8μm)加30μL混合液，均匀覆盖隔膜，37℃孵化包被4小时，备用。消化Huh-7和Huh-7/M8细胞，用MEM培养液制取5×105/mL单细胞悬液，在已经用基质胶(matrigel)包被好的Transwell上室加200μL单细胞悬液，下室加600μL基础培养液。48小时后，去除Transwell上室细胞，Gimsa染色，割取Transwell隔膜，封片后正置显微镜下观察，并拍照。
结果如图5所示，同样条件下，Huh-7/M8细胞穿过Transwell隔膜(基质胶(matrigel)包被)的数目显著多于Huh-7细胞。说明Huh-7/M8细胞的侵袭能力显著强于Huh-7细胞。
实施例8表达差异显著的microRNA筛选
为了进一步研究microRNA调控肝癌转移的分子机制，我们委托上海伯豪生物技术有限公司，应用microRNA芯片检测技术比较分析了Huh-7和Huh-7/M8细胞中microRNA的表达差异。筛选出信号值大于200，表达变化倍数大于1.5倍，p值小于0.01的microRNA共15条，如miR-181a-5p、miR-196a-5p、miR-21a-3p、miR-210、miR-483、miR-130b-3p等(图6A)。如图6B所示，通过实时定量PCR对其中表达差异最大的miR-181a-5p、miR-210、miR-483-5p和miR-483-3p进行验证。相比于亲代细胞Huh-7，Huh-7/M8细胞miR-181a-5p的表达显著降低，与microRNA芯片检测结果一致。检索文献发现miR-181a参与调控肿瘤的转移，因此我们锁定miR-181a为调控肝癌转移的潜在microRNA，是抑制肝癌转移的分子标志物。
Claims (6)
1.肝癌细胞株，其特征在于，命名为人肝癌细胞株Huh-7/M8，保藏号为：CCTCC NO：C2014247，
所述肝癌细胞株的构建方法为Transwell迁移试验法，起始细胞株为肝癌细胞株Huh-7，试验重复次数为8次。
2.如权利要求1所述的肝癌细胞株的子代细胞。
3.如权利要求1所述的肝癌细胞株在建立哺乳动物肝癌模型中的应用。
4.如权利要求3所述的应用，其特征在于，所述的哺乳动物为裸小鼠或裸大鼠。
5.如权利要求1所述的肝癌细胞株在提取肝癌转移和复发的标志物中的应用。
6.如权利要求1所述的肝癌细胞株在开发肝癌复发检测试剂盒中的应用。
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