CN112626118B - 细胞株及其应用 - Google Patents
细胞株及其应用 Download PDFInfo
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- CN112626118B CN112626118B CN202011262960.7A CN202011262960A CN112626118B CN 112626118 B CN112626118 B CN 112626118B CN 202011262960 A CN202011262960 A CN 202011262960A CN 112626118 B CN112626118 B CN 112626118B
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Abstract
本发明公开了细胞株及其应用。本发明的第一方面,提供重组质粒,该重组质粒包含荧光蛋白基因和荧光素酶基因。根据本发明实施例的重组质粒,至少具有如下有益效果:本发明实施例所构建的重组质粒在转染细胞后,可以使细胞同时表达荧光蛋白和荧光素酶,从而使细胞具有体内、体外双重标记的特性,既能够对活体组织生长进行连续监测并进行定量分析,又能在离体的组织和体外共培养体系中标识靶细胞以便于准确的定性和定量。从而实现更加方便快捷、全面精确的监测。
Description
技术领域
本发明涉及生物医学技术领域,尤其是涉及细胞株及其应用。
背景技术
肺癌是目前发病率和死亡率最高的恶性肿瘤,非小细胞肺癌(Non-Small CellLung Cancer,NSCLC)是肺癌的常见类型,约占肺癌的80~85%。然而,NSCLC患者中超过半数在诊断时即为晚期,而且晚期NSCLC生存时间短,无法承受手术治疗。传统的化疗和放疗由于缺乏特异性,在取得疗效的同时也往往给患者带来较大的毒副作用。因此,针对不同分子遗传学背景的群体或/和个体,根据其对药物敏感性相关基因或基因群表达谱的差异,进行有选择性的靶向治疗,达到疗效最优化、毒性最低化是突破NSCLC化疗“瓶颈”的希望所在。
在对NSCLC靶向治疗方法和靶向药进行研究时,研究人员需要对肺癌发生发展机制、肿瘤微环境与肿瘤细胞间的关系及抗癌药物疗效等相关内容有深入的了解,而构建NSCLC动物模型是了解这些内容的重要手段。目前,常用的肺癌动物模型建模方法主要分为诱导法和移植法。诱导法包括鼻滴注致癌物诱导、肺穿刺致癌剂诱导、皮下注射诱导等。诱导法虽然在一定程度上更能反映肺癌在人体内的实际发生情况,但诱导成功率、致癌器官的特异性不太理想。移植法相对而言能够克服这类问题,该方法将具备可移植性的肺癌细胞株直接植入动物体内,从而完成肺癌动物模型的构建。
为了更全面准确地对肺癌进行监测,对于构建得到的肺癌动物模型,研究人员需要在对活体肺癌组织生长进行连续监测和定量分析的同时,在离体的肿瘤组织及体外共培养体系中标识癌细胞以进行准确的定性和定量。然而,以往研究中构建模型所使用的一部分肺癌细胞株虽然能够实现对体外肿瘤细胞的标记,却难以对其进行活体示踪;而另一部分肺癌细胞株尽管解决了在体监测问题,但需要特定底物才能检测,且反应的时效性使其难以对离体的癌细胞作持续稳定的标记。因此,有必要提供一种具备持续稳定的活体示踪和体外标记效果的细胞株。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此,本发明提出一种具备持续稳定的活体示踪和体外标记效果的细胞株及其应用。
本发明的第一方面,提供重组质粒,该重组质粒包含荧光蛋白基因和荧光素酶基因。
根据本发明实施例的重组质粒,至少具有如下有益效果:
本发明实施例所构建的重组质粒在质粒载体上插入有荧光蛋白基因和荧光素酶基因,该重组质粒在转染细胞后,可以使细胞同时表达荧光蛋白和荧光素酶,从而使细胞具有体内、体外双重标记的特性,既能够对活体组织生长进行连续监测并进行定量分析,又能在离体的组织和体外共培养体系中标识靶细胞以便于准确的定性和定量。从而实现更加方便快捷、全面精确的监测。
其中,荧光蛋白基因可以是任选的荧光蛋白的基因,例如可以是绿色荧光蛋白(Green Fluorescent Protein,GFP)、红色荧光蛋白(Red Fluorescent Protein,RFP)、黄色荧光蛋白(Yellow Fluorescent Protein,YFP)的基因,另外也可以是增强绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)以获得更强的荧光效果。荧光素酶基因可以是任选的荧光素酶,例如可以是萤火虫荧光素酶(Firefly Luciferase,编码基因luc)、海肾荧光素酶(Renilla Luciferase,编码基因Rluc)。
本发明的第二方面,提供宿主细胞,该宿主细胞转入有上述的重组质粒。
根据本发明实施例的宿主细胞,至少具有如下有益效果:
该宿主细胞能够同时表达荧光蛋白和荧光素酶而具有体内、体外双重标记的特性,既能够对活体组织生长进行连续监测并进行定量分析,又能在离体的组织和体外共培养体系中标识靶细胞以便于准确的定性和定量。
根据本发明的一些实施例,宿主细胞可以是任选的原核细胞或真核细胞,根据宿主细胞对象的不同,可以采用本领域熟知的各类能够导入包含荧光蛋白基因和荧光素酶基因这两类基因的质粒的方法。
根据本发明的一些实施例,宿主细胞为肿瘤细胞株。在需要对肿瘤细胞进行体内、体外双重标记时,采用上述重组质粒转染得到的宿主细胞进行实验,方便对肿瘤细胞的定性和定量观察。肿瘤细胞株可以是任选的肿瘤细胞,诸如妇科肿瘤细胞、泌尿系统肿瘤细胞、神经系统肿瘤细胞、头颈部肿瘤细胞、肺癌细胞、消化系统肿瘤细胞等。
根据本发明的一些实施例,宿主细胞为肺癌细胞株。
根据本发明的一些实施例,肺癌细胞株为PC9细胞。PC9细胞为酪氨酸酶抑制剂吉非替尼(Gefitinib)高度敏感的肺腺癌细胞,其EGFR基因天然性的外显子19的缺失(delE746-A750)。
根据本发明的一些实施例,PC9细胞经吉非替尼耐药筛选。吉非替尼通过结合于EGFR激酶域ATP口袋(ATP-binding poket,ABP)阻止ATP进入而阻断EGFR激酶活性,从而阻断EGFR下游信号通路,包括MAPK通路以及PI3K通路等,进而抑制细胞增殖分化,并诱导凋亡。经吉非替尼耐药筛选过的PC9细胞更贴近于临床患者产生耐药的过程,从而更好地模拟实际情况下的肺癌细胞。耐药株的具体筛选方法可以是本领域熟知的任选能够达到耐药标准的方法。
本发明的第三方面,提供肺癌细胞株,该肺癌细胞株命名为人肺癌细胞株EGFP-Luc-PC9R,保藏在中国典型培养物保藏中心,保藏日期:2020年9月25日,保藏号为CCTCCNO:C2020179。
本发明的第四方面,提供动物模型的构建方法,该构建方法包括以下步骤:向非人动物施用上述的宿主细胞或肺癌细胞株。
本发明的第五方面,提供上述的宿主细胞或肺癌细胞株在制备或筛选用于治疗或预防肺癌的药物中的应用。
根据本发明的一些实施例,肺癌为非小细胞肺癌。
本发明的第六方面,提供药效评价方法,该药效评价方法包括以下步骤:
S1:向非人动物施用上述的宿主细胞,或上述的肺癌细胞株,构建得到动物模型;
S2:向动物模型施用药物;
S3:根据动物模型体内源自宿主细胞或肺癌细胞株的癌细胞的形成和/或生长情况评价药物的药效。
附图说明
下面结合附图和实施例对本发明做进一步的说明,其中:
图1为本发明实施例的重组质粒制备的技术路线图;
图2为本发明实施例的PC9细胞和PC9R的生长曲线;
图3为本发明实施例的PC9细胞和PC9R细胞在对应浓度吉非替尼处理14天后的照片;
图4为本发明实施例的EGFP-Luc-PC9和EGFP-Luc-PC9R细胞的倒置荧光显微镜观察结果;
图5为本发明实施例的Lumina成像系统检测生物发光活性与细胞数的关系结果。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
下面详细描述本发明的实施例,所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
在本发明的描述中,若干的含义是一个以上,多个的含义是两个以上,大于、小于、超过等理解为不包括本数,以上、以下、以内等理解为包括本数。如果有描述到第一、第二只是用于区分技术特征为目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量或者隐含指明所指示的技术特征的先后关系。
本发明的描述中,除非另有明确的限定,设置、安装、连接等词语应做广义理解,所属技术领域技术人员可以结合技术方案的具体内容合理确定上述词语在本发明中的具体含义。
本发明的描述中,参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
实施例1
本实施例提供重组质粒,其制备方法的技术路线图参考图1,具体步骤如下:
1.质粒转化、提取及酶切验证
以pEGFP-N2质粒、M50 super 8×TOPFlash两种质粒常规转化大肠杆菌DH5α感受态细胞,将转化的菌液涂布于含氨苄霉素的LB固体平板,培养过夜。挑取单克隆菌落置入含有对应抗性LB培养液的菌种管中,37℃摇菌12h,提取质粒。以EcoRⅠ及BamH I位点对pEGFP-N2及M50 super 8×TOPFlash质粒进行酶切,电泳验证酶切结果。
2.Luciferase基因的PCR扩增及纯化回收
根据Genbank数据库得到Luciferase基因的cDNA序列及其相关信息,通过Primer5软件设计引物,在上游引物的5′端加上酶切位点EcoRⅠ,下游引物的5′端加上酶切位点BamHI对M50 super 8×TOPFlash质粒进行PCR反应,产物纯化回收,得到Luciferase基因小片段,待用。
表1.Luciferase基因引物序列及扩增产物大小
3.pEGFP-N2及Luciferase双酶切,连接重组与鉴定
取步骤2回收的Luciferase小片段、步骤1中pEGFP-N2质粒经酶切纯化回收得到的载体大片段与DNA Ligation Kit Solution I混匀后于4℃孵育过夜。连接产物转化感受态细胞,涂布于LB固体平板,培养过夜。挑取单克隆菌落置入含有对应抗性LB培养液的菌种管中,37℃摇菌12h,提取质粒。将重组质粒通过琼脂糖凝胶电泳及EcoR I和BamH I酶切后电泳鉴定连接产物。酶切鉴定正确的质粒送金唯智工程科技有限公司测序。构建成功的质粒命名为pCMV-EGFP-N2-Luciferase,该重组质粒包含荧光蛋白基因EGFP及荧光素酶基因Luciferase。
实施例2
本实施例提供宿主细胞,其制备方法及验证方法的具体步骤如下:
1.重组质粒的转染
将PC9细胞接种于6孔板,当细胞汇合度达80%时,各孔以实施例1中的重组质粒pCMV-EGFP-N2-Luciferase 2μg、脂质体6μl进行转染,37℃、5%CO2孵育24h。
2.稳定筛选细胞系
在PC9细胞转染72h后吸弃上清液,加入含有10%FCS、800μg/ml G418的RPMI 1640培养基,培养约3周,期间换液以去除死亡的细胞,筛选出稳定表达pCMV-EGFP-N2-Luciferase的细胞,将其命名为EGFP-Luc-PC9。
3.检测生物发光活性
消化计数EGFP-Luc-PC9细胞,以0.25×106个、0.5×106个、1×106个、1.5×106个、2×106个细胞分别接种至6孔板,预留一孔仅添加培养基作为空白对照。细胞贴壁过夜后,6孔板每孔添加15mg/ml的D-luciferin(荧光素酶催化底物),轻轻摇匀置于Lumina成像系统进行检测,验证其生物发光活性。
实施例3
本实施例提供耐药细胞株,其构建方法如下:
PC9细胞用高浓度吉非替尼冲击、逐渐增加低浓度吉非替尼诱导的方法建立耐药株,在人NSCLC Gefitinib敏感细胞株PC9的培养基中,按0.001、0.002、0.005、0.01、0.02、0.05、0.075、0.10、0.25、0.5、1mol/L浓度梯度逐步增加吉非替尼浓度,使用浓度为1μmol/L的吉非替尼维持细胞耐药性,并用10%胎牛血清的RPMI培养基传代培养,成功建立对吉非替尼耐药的PC9细胞株,命名为PC9R。
采用Alarmar Blue法对PC9R和PC9细胞检测不同浓度吉非替尼对两种细胞的影响,吉非替尼的浓度梯度为0.5nM、1nM、5nM、10nM、50nM、100nM、500nM、1000nM、5000nM、10000nM,处理72h,绘制相应的生长曲线,结果见图2,A为PC9细胞的生长曲线,B为PC9R的生长曲线。从图2可以看出,本实施例所提供的耐药细胞株PC9R的IC50为10300nM,为PC9细胞的1000倍以上。图3为PC9细胞和PC9R细胞在对应浓度吉非替尼处理14天后的照片。从图3可以看出,该耐药细胞株PC9R可以在吉非替尼浓度为1μM的环境下稳定生长,而PC9细胞仅能构耐受1nM的吉非替尼。
实施例4
本实施例提供一种肺癌细胞株,制备方法参考实施例2,与实施例2的区别在于,导入质粒所用的宿主细胞采用实施例3所提供的耐药细胞株PC9R,制备得到的肺癌细胞株命名为EGFP-Luc-PC9R。其制备方法参考实施例2。
将EGFP-Luc-PC9和EGFP-Luc-PC9R细胞在倒置荧光显微镜下观察荧光蛋白的表达情况,结果见图4。其中,左列为正常视野下的细胞,右列为激发后的荧光图像。从图中可以看出,两种肺癌细胞株均能够较好地表达出荧光蛋白EGFP。
消化计数EGFP-Luc-PC9R细胞,以0.4×106个、0.8×106个、1.2×106个、1.6×106个、2×106个细胞分别接种至6孔板,预留一孔仅添加培养基作为空白对照。细胞贴壁过夜后,于6孔板每孔15mg/ml Dluciferin,轻轻摇匀置于Lumina成像系统进行检测,Lumina成像系统检测生物发光活性与细胞数关系见图5。从图中可以看出,通过细胞株中的荧光素酶基因Luciferase可以进行有效的成像检测。
实施例5
本实施例提供一种动物模型,该动物模型的构建方法包括以下步骤:
以0.6%戊巴比妥钠对小鼠腹腔注射进行麻醉,取右侧卧位固定于鼠板,以75%乙醇常规消毒,在左腋下行1cm左右切口,钝性分离皮下及肌层至肋骨表面层次,能清晰看见粉红色左肺上下运动,在肋骨上缘以微量进样器垂直刺入3~4mm,缓慢注射含有2×106个EGFP-Luc-PC9R细胞的Matrigel胶悬液100μL(对照组注射不含该细胞的Matrigel胶悬液100μL)于肺内,停止数秒后,缓慢拔出微量进样器,医用胶封闭针孔。逐层缝合关腹,再次乙醇消毒。
建模后第5天、第10天、第15天、第20天、第25天每天处死3只小鼠,解剖留取小鼠双肺、胸膜结节、心包、肝脏、肾脏、脊柱、肋骨、大脑行病检,观察肿瘤生长和转移情况。结果表明,第15天解剖时,左肺叶肉眼可见癌结节,第25天解剖时,发现胸腔结构不清晰,可见血性胸腔积液,胸膜、心包、纵隔及侧肺均肉眼可见转移性癌结节。上述结果表明,采用实施例4的肺癌细胞株建模成功。
以15mg/ml的D-luciferin对小鼠进行腹腔注射,以异氟烷氧气实施吸入麻醉,以仰卧位摆放于Lumina成像系统暗箱中进行活体成像。结果显示,实验组小鼠于第15天可见较为明显的生物发光信号,并且随着时间的推移而不断增强,而对照组小鼠均未见明显阳性信号。
第25天处死的小鼠的双肺制备冰冻切片,室温干燥,添加1%BSA抗体稀释液100μl封闭30min后,100μl的Hoechst稀释液染细胞核10min。1×PBS清洗3次,50%丙三醇封片,荧光显微镜下观察。结果显示,肺癌细胞表达绿色荧光蛋白,而非肿瘤细胞不发出荧光,两者可以明显区分。
综合上述结果可以看到,本实施例提供的小鼠肺癌模型既可以对活体肺癌组织生长进行在体监测,又可以对离体的肿瘤组织进行准确的定性和定量。
实施例6
本实施例提供一种肺癌药物筛选方法,该方法包括以下步骤:
根据实施例5所提供的方法构建小鼠肺癌模型,分为空白组和给药组,第1天、第3天、第5天、第7天、第9天分别给药,给药前后活体成像,检测肺癌细胞的生长情况;第10天处死小鼠,制备组织冰冻切片,荧光显微镜观察各个部位癌细胞的形成和生长情况。根据检测结果,定性或定量对药物的药效进行评价,根据在体和离体的评价结果综合筛选,从而由药物库中得到合适的肺癌药物。
相对于相较于一般细胞株移植后的触诊、肿瘤体积测量等传统方法,利用上述实施例提供的动物模型进行药物评价,由于其中的肿瘤细胞可以同时稳定表达荧光蛋白及萤光素酶,从而可以更灵敏的发现残余病灶点或尽早发现肿瘤的复发,更准确地对药物治疗效果进行判定。
上面结合附图对本发明实施例作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
SEQUENCE LISTING
<110> 清华-伯克利深圳学院筹备办公室
<120> 细胞株及其应用
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<160> 2
<170> PatentIn version 3.5
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<213> 人工序列
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Claims (1)
1. 肺癌细胞株,其特征在于,命名为人肺癌细胞株EGFP-Luc-PC9R,保藏在中国典型培养物保藏中心,保藏编号为CCTCC No:C2020179。
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