CN113801849B - 一种人乳腺良性叶状肿瘤细胞株bpt-0526及其应用 - Google Patents
一种人乳腺良性叶状肿瘤细胞株bpt-0526及其应用 Download PDFInfo
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Abstract
本发明公开了一种人乳腺良性叶状肿瘤细胞株BPT‑0526,所述细胞系BPT‑0526保藏于中国典型培养物保藏中心,保藏日期为2021年5月26日,保藏地址为:中国,武汉,武汉大学,保藏编号为CCTCC NO:C2021143。本发明还公开了人乳腺良性叶状肿瘤细胞株作为研究肿瘤发生发展机理的细胞模型以及在筛选抗肿瘤药物中的应用。本发明的肿瘤细胞系可作为研究乳腺良性叶状肿瘤的模型,对于了解人乳腺良性叶状肿瘤病人的发病机制有很大的帮助。
Description
技术领域
本发明涉及医学技术领域,具体涉及一种人乳腺良性叶状肿瘤细胞株BPT-0526及其应用。
背景技术
乳腺叶状肿瘤是由乳腺间质和上皮组织两种成分组成的纤维上皮性肿瘤,约占乳腺肿瘤的0.3%~1%。根据叶状肿瘤的临床病理特征,WHO将其分为良性、交界性和恶性叶状肿瘤3类。其中,良性叶状肿瘤较交界性、恶性叶状肿瘤发病年龄小、病程长、生长缓慢,而交界性和恶性叶状肿瘤血供更丰富、肿瘤呈浸润性生长、细胞核异型性明显、核分裂活跃并伴有间质明显过度生长。
目前,在乳腺叶状肿瘤的临床诊断中,空心针穿刺活检阳性率可达83%~93%。然而,化疗、内分泌治疗、靶向治疗、免疫治疗等对乳腺叶状肿瘤的治疗效果均不佳,其主要治疗方式仍为手术治疗。因此,进一步提高乳腺叶状肿瘤术前诊断准确率对后续手术治疗方案的选择显得尤为重要。除此之外,精准化、个体化医疗的不断推广也使得科研工作者不得不探索效率更高、创伤更小、靶点更精准的乳腺叶状肿瘤相关治疗方案。
人乳腺叶状肿瘤的发生、发展机制的研究以及相关临床诊治方案的探索和改进一直都是热点问题,而建立良好的细胞实验模型是开展相关研究的基础之一。提供理想的细胞模型是人乳腺叶状肿瘤相关研究及探索道路中亟需解决的问题
发明内容
基于上述问题,本发明的目的在于克服上述现有技术的不足之处而提供一株人乳腺良性叶状肿瘤细胞株BPT-0526,以填补目前国内外人群来源的叶状肿瘤细胞系的空缺,本发明的人乳腺良性叶状肿瘤细胞株BPT-0526能用于研究乳腺叶状肿瘤发生发展机理及相关药物。
为实现上述目的,本发明采取的技术方案包括以下几个方面:
在第一个方面,本发明提供了一株人乳腺良性叶状肿瘤细胞株BPT-0526,所述细胞系保藏于中国典型培养物保藏中心,保藏日期为2021年5月26日,保藏地址为:中国,武汉,武汉大学,保藏编号为CCTCC NO:C2021143。
在第二个方面,本发明提供了上述所述的人乳腺良性叶状肿瘤细胞株BPT-0526在制备用于研究肿瘤发生发展机理的细胞模型中的应用。
优选地,所述肿瘤为良性肿瘤。
优选地,所述肿瘤为人乳腺良性叶状肿瘤。
在第三个方面,本发明提供了上述所述的人乳腺良性叶状肿瘤细胞株BPT-0526在筛选抗肿瘤药物中的应用。
优选地,所述肿瘤为良性肿瘤。
优选地,所述肿瘤为人乳腺良性叶状肿瘤。
本发明的有益效果为:本发明的人乳腺良性叶状肿瘤细胞系是从中国人来源建立的,建系时间短,生物遗传性稳定,且目前市面上缺乏人乳腺良性叶状肿瘤细胞系,以该人乳腺良性叶状肿瘤细胞系作为研究模型,对于了解乳腺叶状肿瘤病人的发病机制有很大的帮助。
附图说明
图1是本发明人乳腺良性叶状肿瘤细胞株BPT-0526的形态学观察示意图。
图2是本发明人乳腺良性叶状肿瘤细胞株BPT-0526的STR检测结果观察示意图。
图3是本发明人乳腺良性叶状肿瘤细胞株BPT-0526的免疫组化实验结果观察示意图。
具体实施方式
本发明涉及微生物动物细胞系领域,具体涉及一株人乳腺良性叶状肿瘤细胞及其建立方法。本发明中的人乳腺良性叶状肿瘤细胞来源于患有良性叶状肿瘤的30岁的女病人的右乳肿物,命名为人乳腺良性叶状肿瘤细胞BPT-0526,己保藏于中国典型培养中心,保藏日期为2021年5月26日,保藏地址为:中国,武汉,武汉大学,保藏编号为CCTCC NO:C2021143。
为了更加简洁明了的展示本发明的技术方案、目的和优点,下面结合具体实施例详细说明本发明的技术方案。
实施例1
本发明的人乳腺良性叶状肿瘤细胞株通过以下方法获得:
一、原代组织的制备与检测
1、病人信息概述
该患者,女,诊断为良性叶状肿瘤,术后病理报告显示乳腺纤维上皮肿瘤,部分导管上皮及间质细胞增生活跃,部分区域界欠清,核分裂象约1个/HPF,诊断为良性叶状肿瘤。
2、获得原代细胞的具体步骤
(1)获得新鲜的上述病例的临床手术切除的良性叶状肿瘤标本,取材严格保证无菌。用组织块放置于10ml玻璃培养皿中,用含10%胎牛血DMEM F12双抗培养液漂洗,去除多余血液及周边结缔组织,用眼科剪将组织剪切成小块(每块约1mm3大小)。
(2)用5mL含10%胎牛血DMEM F12双抗培养液均匀润湿组织小块后,将组织小块用眼科剪剪碎后置于50mL离心管中,加入含1.5mg/mL胶原酶I、1.5mg/mL胶原酶II、6%胰酶的10%胎牛血DMEM F12双抗培养液,置于37℃温箱消化1.5h。
(3)将消化后的细胞悬液用100uM滤网过滤后离心,离心速度1000rpm,8min,升速9,降速9。
(4)离心后弃去上清,用含20%胎牛血DMEM F12双抗培养液重悬后接种于25cm培养瓶中。加入含20%胎牛血DMEM F12双抗培养液后将培养瓶瓶底朝上放置于37℃,5.0%CO2温箱内静置培养。待细胞贴壁生长,密切观察细胞生长状况,换液。
二、人乳腺良性叶状肿瘤细胞株的建立
(1)传代
传代培养的具体步骤如下:
用0.25%胰酶消化原代细胞,待细胞变圆终止消化,1000rpm离心3min后,用含20%胎牛血清重完全培养基悬后放入培养瓶中,放置于37℃,5.0%CO2温箱内静置培养。初期换液采用半换液方法培养,待细胞传代20代之后,采取全换液方法培养,密切观察细胞生长状况。
(2)筛选
待步骤(1)中的细胞生长稳定后,采用反复贴壁法并使用含20%胎牛血DMEM F12双抗培养液进行培养,其目的是为了促进上皮和间质细胞生长,抑制其中纤维细胞生长。之后经反复冻存,以稳定传代80代以上。稳定后接种100000细胞于六孔板中,贴壁12h后进行慢病毒转染,每个孔中分别加入SV40慢病毒300000TU/ml、TERT慢病毒300000TU/ml、5ug/ml聚凝胺(polybrene),转染24h后换液,48h后用3ug/ml的嘌呤霉素进行筛选。筛选后用新鲜含20%胎牛血DMEM F12双抗培养液进行传代培养。得到本发明的人乳腺良性叶状肿瘤细胞株BPT-0526。
SV40病毒转染对于成纤维细胞、间质细胞这一类细胞永生化效果较HPV慢病毒好,其永生化更加稳定。本发明的人乳腺良性叶状肿瘤细胞株BPT-0526在培养条件简单的情况下可以稳定传代,并且生长速度佳。且此细胞株来源的病人肿物大,临床及病理特征非常明显。
实施例2人乳腺良性叶状肿瘤细胞株的鉴定
对实施例1得到的人乳腺良性叶状肿瘤细胞株进行鉴定,具体包括以下几种方法:
1.人乳腺良性叶状肿瘤细胞株细胞形态学鉴定,结果如图1所示。
2.人乳腺良性叶状肿瘤细胞株STR鉴定,结果如表1、2和图2所示,表1、表2中Allele为基因位点,STR为短串联重复序列。图2的结果是将此细胞系与人细胞库进行了比对得到的STR相关信息。
表1 STR鉴定结果
表2 STR鉴定结果
3.人乳腺良性叶状肿瘤组织免疫组化实验,检测标志物包括:CD34、CD56、Ki67、ER、PR、P63。结果如图3所示。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (2)
1.一种人乳腺良性叶状肿瘤细胞株BPT-0526,其特征在于,所述细胞系BPT-0526保藏于中国典型培养物保藏中心,保藏日期为2021年5月26日,保藏地址为:中国,武汉,武汉大学,保藏编号为CCTCC NO:C2021143。
2.如权利要求1所述的人乳腺良性叶状肿瘤细胞株BPT-0526在制备用于研究人乳腺良性叶状肿瘤发生发展机理的细胞模型中的应用。
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