CN111593026A - 一种荧光标记的人三阴性乳腺癌骨转移细胞株 - Google Patents
一种荧光标记的人三阴性乳腺癌骨转移细胞株 Download PDFInfo
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Abstract
本发明提供了一种荧光标记的人三阴性乳腺癌骨转移细胞株,通过下述方法制备而成:一个培养人源三阴性乳腺癌细胞MDA‑MB‑231的步骤;扩增含有红色荧光蛋白RFP的慢病毒载体,制备含有红色荧光标记的MDA‑MB‑231细胞;将RFP‑MDA‑MB‑231细胞移植到裸鼠第四对乳腺脂肪垫位置,生长成瘤;4‑6周后取后肢胫骨,用缓冲溶液冲取骨髓,再采用胶原酶消化,获得骨髓来源单细胞,体外培养扩增,流式细胞技术分选红色荧光细胞;通过裸鼠体内成瘤筛选,得到红色荧光标记的MDA‑MB‑231人三阴性乳腺癌骨转移细胞株。本发明具有骨转移特征,并且有荧光蛋白标记,实现了细胞在体内和体外的可视化、可示踪。
Description
技术领域
本发明属于生物工程领域,涉及一种细胞株,具体来说是一种荧光标记的人三阴性乳腺癌骨转移细胞株。
背景技术
本世纪以来,全球乳腺癌发病率快速增长,成为城市女性的第一杀手。尽管乳腺癌早期诊断以及手术技术和放化疗水平的提高降低了乳腺癌死亡率,但恶性乳腺癌伴随肺、骨、脑等器官转移的致死率仍居高不下。据统计,未治愈的乳腺癌约70%会出现肺转移,约60%会出现骨转移和肝转移,约15%会出现脑转移。
尤其是三阴性乳腺癌约占乳腺癌的15%,恶性程度极高,易侵袭、易转移。因缺乏雌激素受体(estrogen receptor,ER)、孕激素受体(progesterone receptor,PR)和人表皮生长因子受体-2(human epithelial growth factor receptor,HER2)等治疗靶点,一旦发生扩散和转移,难治愈且预后差。因此,阻止恶性乳腺癌扩散、转移,是从根本上解决恶性乳腺癌的高致死率的关键点。
MDA-MB-231细胞株是经典的、被全球医学科学家广泛认同并使用的三阴性乳腺癌细胞。MDA-MB-231细胞恶性程度高,移植小鼠后易成瘤,肿瘤生长速度快,而且容易转移到肺、骨等器官。
并非所有三阴性乳腺癌细胞都能够发生骨转移。建立具有高骨转移能力的三阴性乳腺癌细胞,必将有助于鉴定骨转移乳腺癌细胞亚群的基因表达特征,更有助于科学家寻找乳腺癌骨转移的治疗靶点和干预措施。这是本发明的初衷。
发明内容
本发明的目的在于提供一种荧光标记的人三阴性乳腺癌骨转移细胞株,所述的这种荧光标记的人三阴性乳腺癌骨转移细胞株要解决现有技术中的人三阴性乳腺癌骨转移细胞株不能可视化和可示踪的技术问题。
本发明提供了一种荧光标记的人三阴性乳腺癌骨转移细胞株,通过下述的方法制备而成:
(1)一个培养人源三阴性乳腺癌细胞MDA-MB-231的步骤;
(2)扩增含有红色荧光蛋白RFP的慢病毒载体,利用慢病毒转染细胞的方法,转染MDA-MB-231细胞,制备含有红色荧光标记的MDA-MB-231细胞;
(3)通过流式细胞仪分选,获得红色荧光RFP-MDA-MB-231细胞;
(4)将RFP-MDA-MB-231细胞移植到裸鼠第四对乳腺脂肪垫位置,然后进行哺育4-6周成瘤;
(5)乳腺肿瘤直径达到0.8-1.2cm时,处死裸鼠,解剖,取后肢胫骨,无菌环境下,用PH7.4的1x PBS缓冲溶液冲取骨髓,再采用胶原酶消化,获得骨髓来源单细胞,体外培养扩增;
(6)利用流式细胞分析仪,检测并分选从乳腺肿瘤转移到骨的红色荧光乳腺癌细胞,体外培养扩增,作为P1代乳腺癌骨转移细胞;
(7)扩增P1代乳腺癌骨转移细胞,移植裸鼠,重复上述第4-第6步,获得P2代乳腺癌骨转移细胞;
(8)扩增P2代乳腺癌骨转移细胞,移植裸鼠,重复上述第4-第6步,获得P3代乳腺癌骨转移细胞;
(9)通过上述三轮的裸鼠体内成瘤筛选,得到红色荧光标记的MDA-MB-231人三阴性乳腺癌骨转移细胞株。
进一步的,上述的一种荧光标记的人三阴性乳腺癌骨转移细胞株,其保藏号为GDMCC No:61073。
通过上述的方法,本发明建立了可视化、可示踪的人三阴性乳腺癌骨转移细胞系,可用于乳腺癌骨转移的机制研究、药物开发、治疗靶点研究等等。
本发明和已有技术比较,其技术进步是显著的。本发明和现有的MDA-MB-231细胞相比,本发明有荧光蛋白标记,实现了细胞在体内和体外的可视化、可示踪。而且和现有的MDA-MB-231细胞相比,本发明获得的细胞株完成了小鼠体内三代成瘤和筛选,其骨转移能力及骨转移特异性比亲本更强。
附图说明
图1显示了红色荧光标记的MDA-MB-231细胞。
图2荷瘤小鼠骨髓细胞的流式细胞分选,有27.5%细胞带有红色荧光。
图3显示了筛选得到的红色荧光标记的骨转移MDA-MB-231-BM细胞。
图4为通过细胞克隆成球实验,显示了MDA-MB-231-BM细胞比亲本MDA-MB-231细胞有更强的增殖能力。
图5显示了MDA-MB-231-BM细胞比亲本MDA-MB-231细胞有更强的迁移能力。
图6显示了MDA-MB-231-BM细胞比亲本MDA-MB-231细胞有更强的骨转移能力。
图7显示了MDA-MB-231-BM细胞比亲本MDA-MB-231细胞有更强的体内再生肿瘤能力。
具体实施方式
实施例1
本发明提供了一种荧光标记的人三阴性乳腺癌骨转移细胞株,通过下述的方法制备而成:
(1)培养亲本人源三阴性乳腺癌细胞MDA-MB-231(购买自ATCC公司);
(2)扩增含有红色荧光蛋白RFP的慢病毒载体pLemiR(Open biosystems公司产品)。利用慢病毒转染细胞的方法,转染MDA-MB-231细胞,制备含有红色荧光标记的MDA-MB-231细胞;
(3)通过流式细胞仪分选,获得红色荧光RFP-MDA-MB-231细胞(图1A:RFP-MDA-MB-231细胞白光图像,图1B:RFP-MDA-MB-231细胞荧光图像);
(4)将1百万个RFP-MDA-MB-231细胞,移植到裸鼠第四对乳腺脂肪垫位置,一周后开始成瘤,四到六周后肿瘤直径达到0.8-1.2cm;
(5)处死裸鼠,解剖,取后肢胫骨,无菌环境下,用PH=7.4的1x PBS溶液冲取骨髓,再采用胶原酶消化,获得骨髓来源单细胞,体外培养扩增;
(6)利用流式细胞分析仪,检测并分选从乳腺肿瘤转移到骨的红色荧光乳腺癌细胞(图2A是用于流式细胞仪分选的细胞,图2B是流式细胞仪分选后细胞),体外培养扩增,作为P1代乳腺癌骨转移细胞;
(7)扩增P1代乳腺癌骨转移细胞,移植裸鼠,重复上述第4-第6步,获得P2代乳腺癌骨转移细胞;
(8)扩增P2代乳腺癌骨转移细胞,移植裸鼠,重复上述第4-第6步,获得P3代乳腺癌骨转移细胞;
(9)通过上述三轮的裸鼠体内成瘤筛选,得到红色荧光标记的MDA-MB-231人三阴性乳腺癌骨转移细胞株,命名MDA-MB-231-BM((图3A:MDA-MB-231-BM细胞白光图像,图3B:MDA-MB-231-BM细胞荧光图像)。
本发明获得的MDA-MB-231-BM细胞株是经典细胞株MDA-MB-231细胞的一个亚型,具有骨转移特征,带有红色荧光蛋白。
上述的红色荧光标记的MDA-MB-231-BM人三阴性乳腺癌骨转移细胞株,属于三阴性乳腺癌骨转移细胞,其分类学名称为MDA-MB-231,保藏于广东省微生物菌种保藏中心(GDMCC)中,广东省微生物菌种保藏中心的地址为:广东省广州市越秀区先烈中路100号大院实验大楼5楼,保藏日期为2020年7月1日,保藏号为GDMCC No:61073。
实施例2
对本发明得到的MDA-MB-231-BM细胞进行细胞增殖、细胞迁移、骨转移和成瘤能力检测,并对MDA-MB-231-BM细胞进行基因表达特征检测。实验步骤如下:
1:克隆形成实验(colony formation assay)检测细胞增殖能力。
1)处于对数生长期、状态良好的MDA-MB-231和MDA-MB-231-BM消化后,加入完全培养基制成细胞悬液;
2)将细胞悬液进行细胞计数,取2000个细胞接种于12孔板中,12孔板中加入1mL完全培养基,使细胞均匀散布于培养板;
3)12孔板置于细胞培养箱,待细胞贴壁后每隔2-3天换液,避免污染;
4)显微镜下可以看到的细胞圆形团块时(约第10天),取出12孔板,弃去培养液,用PH=7.4的1x PBS缓冲液清洗2遍;
5)甲醇固定10分钟后,每孔加入1mL 1x结晶紫染色1-2小时,继续用PH=7.4的1xPBS缓冲液清洗2遍;
6)光学成像仪下拍照、观察并量化克隆数量。
通过细胞克隆成球实验,图4显示了MDA-MB-231-BM细胞比亲本MDA-MB-231细胞有更强的增殖能力。
2:伤口愈合(wound healing)实验检测细胞迁移能力。
1)处于对数生长期、状态良好的MDA-MB-231和MDA-MB-231-BM消化后,加入完全培养基制成细胞悬液;
2)将细胞悬液进行细胞计数,6孔板培养,每孔中加入5x10^5个细胞;
3)待细胞完全铺满培养板(细胞密度接近100%)时,用10uL枪头进行划痕;
4)划完后用PH=7.4的1x PBS缓冲液清洗3遍,去除脱落的细胞;
5)加入含0.1%FBS的DMEM培养基,放入37℃、5%CO2的细胞培养箱中;
6)于0h、24h时间点拍照,并用imageJ软件计算划痕两边的距离。图5显示了MDA-MB-231-BM细胞比亲本MDA-MB-231细胞有更强的迁移能力。
3:动物实验检测细胞骨转移及成瘤能力。
1)培养MDA-MB-231和MDA-MB-231-BM细胞。
2)将1百万个MDA-MB-231和MDA-MB-231-BM细胞分别种植于裸鼠第四对乳腺脂肪垫,构建乳腺癌荷瘤裸鼠;
3)4-6周后处死裸鼠,取裸鼠胫骨、股骨于解剖荧光显微镜下观察骨组织转移情况;图6显示了MDA-MB-231-BM细胞比亲本MDA-MB-231细胞有更强的骨转移能力。
4)取乳腺原位瘤,测量肿瘤大小及重量,观察MDA-MB-231和MDA-MB-231-BM成瘤情况。图7显示了MDA-MB-231-BM细胞比亲本MDA-MB-231细胞有更强的体内再生肿瘤能力。
4:MDA-MB-231-BM细胞基因表达特征检测。
1)培养MDA-MB-231和MDA-MB-231-BM细胞,抽提细胞总RNA;
2)将足量总RNA样本送至华大基因测序有限公司进行深度RNA测序并分析测序结果(表1:和亲本MDA-MB-231细胞相比,MDA-MB-231-BM细胞有66个基因下调,表2:和亲本MDA-MB-231细胞相比,MDA-MB-231-BM细胞有61个基因下调),61个基因上调(表2)。这些上调及下调的基因,决定了该细胞株具有骨转移的特异性。
表1:MDA-MB-231-BM细胞中66个表达下调的基因
表2:MDA-MB-231-BM细胞中61个表达上调的基因
Claims (2)
1.一种荧光标记的人三阴性乳腺癌骨转移细胞株,其特征在于通过下述的方法制备而成:
1)一个培养人源三阴性乳腺癌细胞MDA-MB-231的步骤;
2)扩增含有红色荧光蛋白RFP的慢病毒载体,利用慢病毒转染细胞的方法,转染MDA-MB-231细胞,制备含有红色荧光标记的MDA-MB-231细胞;
3)通过流式细胞仪分选,获得红色荧光RFP-MDA-MB-231细胞;
4)将RFP-MDA-MB-231细胞移植到裸鼠第四对乳腺脂肪垫位置,然后进行哺育4-6周成瘤;
5)肿瘤直径达到0.8-1.2cm时,处死裸鼠,解剖,取后肢胫骨,无菌环境下,用PH7.4的1x PBS缓冲溶液冲取骨髓,再采用胶原酶消化,获得骨髓来源单细胞,体外培养扩增;
6)利用流式细胞分析仪,检测并分选从乳腺肿瘤转移到骨的红色荧光乳腺癌细胞,体外培养扩增,作为P1代乳腺癌骨转移细胞;
7)扩增P1代乳腺癌骨转移细胞,移植裸鼠,重复上述第4-第6步,获得P2代乳腺癌骨转移细胞;
8)扩增P2代乳腺癌骨转移细胞,移植裸鼠,重复上述第4-第6步,获得P3代乳腺癌骨转移细胞;
9)通过上述三轮的裸鼠体内成瘤筛选,得到红色荧光标记的MDA-MB-231人三阴性乳腺癌骨转移细胞株。
2.一种荧光标记的人三阴性乳腺癌骨转移细胞株,其特征在于:其保藏号为GDMCC No:61073。
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