CN106929475A - 一种基于3d温敏型生物凝胶悬滴培养法体外计数肝癌循环肿瘤细胞的方法 - Google Patents
一种基于3d温敏型生物凝胶悬滴培养法体外计数肝癌循环肿瘤细胞的方法 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域，具体是一种基于3D温敏型生物凝胶悬滴培养法体外计数肝癌循环肿瘤细胞的方法，可用于检测肝癌患者外周血中肿瘤细胞数目、并由此判断预后。本发明还提供一种用于悬滴培养法体外计数肝癌循环肿瘤细胞的温敏型生物凝胶及其应用。本发明经实验证明，肝癌循环肿瘤细胞在本发明的温敏型生物凝胶体系中，可以进行扩增生长并形成显微镜下可见的克隆环，从而实现计数的可操作性。本发明为肝癌循环肿瘤细胞的诊断提供了新的临床手段。
Description
技术领域
本发明涉及生物医药技术领域，具体地说，是一种基于3D温敏型生物凝胶悬滴培养法体外计数肝癌循环肿瘤细胞的方法。
背景技术
肝细胞肝癌(hepatocellular carcinoma,HCC)是一种起源于肝细胞的恶性肿瘤，素有“癌中之王”的称号，其发病较其他肿瘤而言相对隐匿，病程短、治疗困难，严重威胁着人类的生命健康。据2016年《A Cancer Journal for Clinicians》杂志的统计数据显示：2015年中国境内共计281.4万人死于各种癌症，其中42.2万人死于肝癌，仅次于肺癌(61.0万)与胃癌(49.8万)，病死率居全国第3位。由于在肝癌的早期，患者多没有典型临床症状和体征，临床上难以发现，而一旦出现典型症状，多已是中、晚期肝癌，此时往往伴随着肝癌的肝内外转移，最终患者多因此死亡。肝癌患者较差的预后主要是由于肝内及肝外转移。而研究表明那些直径仅有0.5cm-1cm的肝癌瘤体(目前的影像学检查的极限)就已经开始向血道散播循环肿瘤细胞(circulating tumor cell,CTC)，甚至在远端器官中形成微小的转移灶，成为肝癌恶性患者出现术后复发和远端转移的高危因子。因此检测肝癌患者体内是否具有CTC显得尤为迫切。
CTC在外周血中数量稀少，其检测诊断存在一定的技术难度。目前检测方法分为两大类：1、不富集CTC，直接根据肿瘤标志物通过流式细胞术、PCR等手段进行检测；2、通过CTC表面标志物磁珠富集后，荧光染色后进行计数。目前这些检测手段都取得了一定的临床数据，其代表为强生公司的CellSearch系统，2004年美国食品和药品管理局(food and drugsadmistration,FDA)批准了该系统用于检测转移性乳腺癌患者外周血CTC，成为第一个获准进入临床运用的CTC检测方法。
遗憾的是，目前现有的检测系统并不适用于肝癌CTC检测。以CellSearch系统为例，仪器分选所用的EpCAM靶点仅适用于上皮来源的CTC。但进一步研究表明，即使用于在上皮来源CTC检测，该系统所获得的数据也极不完善：美国MD Anderson和Baylor医学院的研究人员发现，在肿瘤CTC转移中会发生上皮间质转化(epithelial-mesenchymaltransition,EMT)，从而EpCAM及CK蛋白在CTC中发生降解从而无法被系统捕捉；过高的假阳性也限制了该系统应用于肿瘤筛查，有研究发现克罗恩病人中有11％-19％的患者会出现CK+/EpCAM+阳性信号，这些细胞实际是是肠上皮细胞而非肿瘤细胞；本发明对GEO数据库中样本量最大的一例肝癌数据芯片集进行分析(样本涵盖220例癌旁与225例癌组织)，结果显示EpCAM在肝癌中的阳性率低于30％。这些结果均提示现有的技术手段依然存在技术上的巨大漏洞，并不适用于肝癌CTC检测。
发明内容
本发明的目的在于针对目前肝癌CTC检测面临的临床问题，提供一种新型的肝癌循环肿瘤细胞的计数方案，具体是指一种基于3D温敏型生物凝胶悬滴培养法体外计数肝癌循环肿瘤细胞的方法，该方法在检测肝癌患者外周血中肿瘤细胞数目、并由此判断预后中的应用；本发明还提供了温敏型生物凝胶及其培养上清的制备方案与配方。
本发明的第一方面，提供一种用于悬滴培养法体外计数肝癌循环肿瘤细胞的温敏型生物凝胶，由体积比为8:1:1的A液、B液和C液组成(混合时是将A液、B液和C液三种液体按比例逐一混合，并用无菌铂金棒搅拌均匀，全过程在冰上操作)；
所述的A液，包括生物凝胶的骨架溶液和基质溶液，7mL骨架溶液与1mL基质溶液在冰上混合，并用无菌铂金棒搅拌均匀，4℃保存，即得A液；
所述的生物凝胶的骨架溶液：使用亲水链聚氧乙烯占60％以上的泊洛沙姆，配置体积分数为20-45％的泊洛沙姆(Poloxamer)溶液；配置体积分数为0.2-0.7％的羟丁基壳聚糖溶液；两种液体在冰上冰浴15min后，将两者按体积比1：1充分混合，获得所述的生物凝胶的骨架溶液；
所述的生物凝胶的基质溶液：将0.5-0.8mgIV型胶原、0.01-0.1mg分子量为10000-50000的透明质酸和0.01-0.1mg硫酸氨基葡糖多聚糖于1mL超纯水中4℃下过夜溶解，获得生物凝胶的基质溶液；
所述的B液组分为10倍浓度的DM/F12培养基，每1mL B液中加入1000-5000IU的rh-EGF，作用为调节生物凝胶内的营养成分和离子浓度；
所述的C液组分为pH＝2.5-3.0的乳酸-乳酸钠缓冲液(0.025-0.05mol)，作用为调节生物凝胶内的pH值。
本发明的第二方面，提供上述的温敏型生物凝胶在悬滴培养法体外计数肝癌循环肿瘤细胞中的应用。
本发明的第三方面，提供一种基于上述的3D温敏型生物凝胶悬滴培养法体外计数肝癌循环肿瘤细胞的方法，包括以下步骤：
(a)取肝癌患者10mL外周血，Ficoll密度梯度离心法分离患者外周血有核细胞；
(b)使用StemCell CD45阴选试剂盒去除白细胞，20μL磷酸盐缓冲液(PBS)重悬；
(c)所述的温敏型生物凝胶的A液、B液和C液按比例逐一混合，并用无菌铂金棒搅拌均匀，加入细胞，全过程在冰上操作(具体为800μL A液在冰上先与100μL B液混合，无菌铂金棒搅拌均匀后，加入100μL C液，再次搅拌均匀，加入20μL细胞悬液，全过程在冰上操作)；
(d)24孔培养板在37℃预热30min，细胞凝胶混合液50μL/滴加至预热培养板上，使凝胶迅速转化为果冻状；
(e)37℃、5％CO2细胞培养箱中孵育2h，加入37℃预热的培养上清1mL；
(f)连续培养7天后，4X物镜下进行克隆环计数。
所述的步骤e中的培养上清为：DM/F12培养基中加入8-10％的Gibco 10099-141胎牛血清，每100mL培养基加入L-谷氨酰胺200mM。
本发明的第四方面，提供一种用于肝癌循环肿瘤细胞悬滴培养法的试剂，包括上述的温敏型生物凝胶和悬滴培养法培养上清，所述的悬滴培养法培养上清为：DM/F12培养基中加入8-10％的Gibco 10099-141胎牛血清，每100mL培养基加入L-谷氨酰胺200mM。
本发明的第五方面，提供一种上述的用于肝癌循环肿瘤细胞悬滴培养法的试剂在悬滴培养法体外计数肝癌循环肿瘤细胞中的应用。
本发明优点在于：
1、本发明的温敏型生物凝胶以羟丁基壳聚糖和泊洛沙姆为骨架，添加了肿瘤细胞所在的细胞外基质(ECM)所含有的IV型胶原、透明质酸与硫酸氨基葡糖多聚糖等组分，充分模拟肿瘤在体内的生长环境，使肝癌CTC可以在凝胶中生长，并通过分泌IV型胶原酶溶解部分机制，形成显微镜下可见的克隆环，从而实现计数的可操作性；同时在培养上清中添加重组人表皮生长因子(rh-EGF)，使每一个CTC都可以在凝胶中充分生长，提高计数的敏感性。
2、本发明的温敏型生物凝胶在4℃下呈现溶液状态，在溶液状态下可以加入循环肿瘤细胞，使其在凝胶中充分混匀；37℃下呈现果冻状胶体，肝癌CTC种植后在固定位置生长。
3、本发明的温敏型生物凝胶中的IV型胶原酶和透明质酸等成分在CTC生长过程中被其分泌的胶原酶、透明质酸酶等成分分解，形成折光度不同的克隆环，便于计数。
4、本发明的温敏型生物凝胶与培养上清中均含有rh-EGF，保证所有的肝癌CTC在此体系中可以中充分生长，确保了检测精度。
5、本发明的方法简单，工艺成熟，成本较低，为肝癌循环肿瘤细胞的诊断提供了新的临床手段。
附图说明
图1为悬滴法培养肝癌CTC的技术流程示意图：简而言之是通过Ficoll分离患者外周血有核细胞后，使用CD45磁珠阴选去除白细胞，将剩余细胞与温敏型3D生物凝胶(具有4℃呈液体，37℃呈果冻样的特点)混合后，50μL/滴加至预热培养板上凝结，并加入液体培养基作为营养支持。
图2是悬滴法培养肝癌CTC7天后，凝胶中形成的一个典型的CTC克隆。外周一圈折光度不同的圆环命名为“克隆环”，是由肝癌CTC在生长过程中分解基质胶中的ECM成分所形成。
图3是通过单细胞荧光定量PCR法，检测挑取的5个CTC克隆与正常肝细胞之间肿瘤标志物GPC3和AFP的相对表达量。
图4是通过抽取健康志愿者外周静脉血10mL，分别掺入含有100个、250个、500个和1000个Hep3B或SMMC-7721肿瘤细胞系细胞。充分混匀后取1mL作为检测样本，按照实施例1中的分离培养方法，进行CTC分离、培养与计数。其敏感度计算公式按正文中所示。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
除非另有描述，本发明的实施将采用分子生物学、微生物学、重组DNA和免疫学的常规技术，这些均是本领域技术人员所知的。这些技术在下列文献中有完整的描述：例如，Sambrook《分子克隆实验指南》第2版(1989)；《DNA克隆》第I和II卷(D.N.Glover编辑1985)；《寡核苷酸合成》(M.J.Gait编辑，1984)；《核酸杂交》(B.D.Hames和S.J.Higgins编辑.1984)；《蛋白质纯化：原理和实践》第2版(Springer-Verlag，N.Y.)，以及《实验免疫学手册》I-IV卷(D.C.Weir和C.C.Blackwell编辑1986)。或者，可按照试剂生产商所提供的说明书进行。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1：一例肝癌晚期患者术前CTC培养结果
1.实验方法：
(1)抽取该患者10mL术前外周血，与HBSS缓冲液1:1混合，置于50mL离心管中。在另一50mL离心管中加入10mL Ficoll密度梯度液，将稀释后的外周血轻轻加至密度梯度液上，使其形成明显的分层。2000rpm室温离心20min，此时液体分为4层，用3mL巴氏滴管吸取有核细胞层，加入10mLHBSS缓冲液中，1000rpm离心5min收集细胞并充分去除上清，加入1mLHBSS(含有2％血清)重悬细胞，并转移至5mL无菌流式管中。
(2)加入StemCell CD45阴选磁珠20μL，轻柔混匀后室温孵育15min，将其放入磁场中，静置30min，轻柔吸取全部上清，转移至新的5mL无菌流式管中，重复上述步骤一次。1000rpm离心5min收集细胞，充分去除上清，20μL磷酸盐缓冲液(PBS)重悬。
(3)800μL生物凝胶A与100μL B液和100μL C液，按已经阐明的步骤混合后，加入20μL细胞悬液，全过程在冰上操作。
(4)24孔培养板在37℃预热30min，细胞凝胶混合液按50μL/滴加至预热培养板上，使凝胶迅速转化为果冻状。之后培养板在37℃、5％CO2细胞培养箱中孵育2h，使凝胶充分凝固。
(5)2h后，沿培养板孔壁缓慢加入37℃预热的培养上清1mL。
(6)连续培养7天后，4X物镜下进行克隆环计数。
(7)克隆挑取与鉴定：在培养出的CTC克隆中，挑取5个进行单细胞荧光定量PCR检测肝癌标志物GPC3和AFP的表达量，以10-20个人肝细胞作为对照(胶原酶消化法后倍比稀释获取)。
2.实验结果：
整个实验流程简图如图1所示。经两名实验人员独立计数，该患者10mL外周血中一共培养出46个CTC克隆。典型的克隆形态如图2所示。经过单细胞荧光定量PCR鉴定，相比于正常肝细胞，挑取的5个克隆均高表达GPC3和AFP，为肝癌细胞(图3)。
实施例2：肝癌CTC温敏型生物凝胶悬滴培养法敏感度检测
1.实验方法：
(1)抽取健康志愿者外周静脉血10mL，分别掺入含有100个、250个、500个和1000个Hep3B或SMMC-7721肿瘤细胞系细胞。
(2)充分混匀后，取1mL血，按实施例1中的分离培养方法，进行CTC分离、培养与计数。其敏感度按照如下方法计算：
2.实验结果：
经过独立三次独立重复实验，该体系在Hep3B细胞系中的检出率为60％-80％，在SMMC7721细胞系中的检出率为70％-90％。结果如图4所示。
实施例3：6例肝癌患者外周血悬滴培养法计数与CellSearch法计数敏感度对比
1.实验方法：
临床随机挑取6名肝癌确诊患者，每人抽取20mL外周静脉血，10mL由第三方(中国人民解放军第二军医大学附属东方肝胆医院)利用CellSearch系统检测，10mL由我方进行悬滴培养法计数。
2.实验结果：
1号患者CellSearch检出CTC数目为3，悬滴培养法计数CTC为26；
2号患者CellSearch检出CTC数目为6，悬滴培养法计数CTC为15；
3号患者CellSearch检出CTC数目为5，悬滴培养法计数CTC为40；
4号患者CellSearch检出CTC数目为13，悬滴培养法计数CTC为72；
5号患者CellSearch检出CTC数目为1，悬滴培养法计数CTC为29；
6号患者CellSearch检出CTC数目为5，悬滴培养法计数CTC为61。
悬滴培养法计数的敏感度要远高于CellSearch系统。
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 (6)
1.一种用于悬滴培养法体外计数肝癌循环肿瘤细胞的温敏型生物凝胶，其特征在于，由体积比为8:1:1的A液、B液和C液组成；
所述的A液，包括生物凝胶的骨架溶液和基质溶液，7mL骨架溶液与1mL基质溶液在冰上混合，并用无菌铂金棒搅拌均匀，4℃保存，即得A液；
所述的生物凝胶的骨架溶液：使用亲水链聚氧乙烯占60％以上的泊洛沙姆，配置体积分数为20-45％的泊洛沙姆溶液；配置体积分数为0.2-0.7％的羟丁基壳聚糖溶液；两种液体在冰上冰浴15min后，将两者按体积比1:1充分混合，获得所述的生物凝胶的骨架溶液；
所述的生物凝胶的基质溶液：将0.5-0.8mgIV型胶原、0.01-0.1mg分子量为10000-50000的透明质酸和0.01-0.1mg硫酸氨基葡糖多聚糖于1mL超纯水中4℃下过夜溶解，获得所述的生物凝胶的基质溶液；
所述的B液组分为10倍浓度的DM/F12培养基，每1mL B液中加入1000-5000IU的rh-EGF；
所述的C液组分为pH＝2.5-3.0的乳酸-乳酸钠缓冲液0.025-0.05mol。
2.一种如权利要求1所述的温敏型生物凝胶在悬滴培养法体外计数肝癌循环肿瘤细胞中的应用。
3.一种基于3D温敏型生物凝胶悬滴培养法体外计数肝癌循环肿瘤细胞的方法，其特征在于，包括以下步骤：
(a)取肝癌患者10mL外周血，Ficoll密度梯度离心法分离患者外周血有核细胞；
(b)使用StemCell CD45阴选试剂盒去除白细胞，20μL磷酸盐缓冲液重悬；
(c)如权利要求1所述的温敏型生物凝胶的A液、B液和C液三种液体按比例逐一混合，并用无菌铂金棒搅拌均匀，加入细胞，全过程在冰上操作；
(d)24孔培养板在37℃预热30min，细胞凝胶混合液50μL/滴加至预热培养板上，使凝胶迅速转化为果冻状；
(e)37℃、5％CO2细胞培养箱中孵育2h，加入37℃预热的培养上清1mL；
(f)连续培养7天后，4X物镜下进行克隆环计数。
4.根据权利要求3所述的基于3D温敏型生物凝胶悬滴培养法体外计数肝癌循环肿瘤细胞的方法，其特征在于，所述的步骤e中的培养上清为：DM/F12培养基中加入8-10％的Gibco10099-141胎牛血清，每100mL培养基加入L-谷氨酰胺200mM。
5.一种用于肝癌循环肿瘤细胞悬滴培养法的试剂，其特征在于，包括权利要求1所述的温敏型生物凝胶和悬滴培养法培养上清，所述的悬滴培养法培养上清为：DM/F12培养基中加入8-10％的Gibco 10099-141胎牛血清，每100mL培养基加入L-谷氨酰胺200mM。
6.一种如权利要求5所述的用于肝癌循环肿瘤细胞悬滴培养法的试剂在悬滴培养法体外计数肝癌循环肿瘤细胞中的应用。
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