CN117551619B - 一种具备高破骨细胞分化能力的thp-1细胞、破骨细胞及制备与应用 - Google Patents
一种具备高破骨细胞分化能力的thp-1细胞、破骨细胞及制备与应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,特别涉及一种具备高破骨细胞分化能力的THP‑1细胞、破骨细胞及制备与应用。本发明提供的具备高破骨细胞分化能力的THP‑1细胞,与正常THP‑1细胞相比,可表达CSF1R和RANK受体;该细胞可以进一步在体外经过分化刺激快速产生具备典型破骨细胞特征的成熟破骨细胞。本发明提供的具备高破骨细胞分化能力的THP‑1细胞、破骨细胞在研究破骨细胞功能、筛选破骨细胞调控药物上具有重要应用价值。
Description
技术领域
本发明涉及细胞生物学技术领域,特别涉及一种具备高破骨细胞分化能力的THP-1细胞、破骨细胞及制备与应用。
背景技术
骨的新陈代谢由成骨细胞介导的骨形成和破骨细胞介导的骨吸收两个过程构成,骨形成过程和骨吸收过程相互协调,共同维持骨的正常新陈代谢。但随着年龄的增长或由于疾病因素的诱导,会导致骨形成和骨吸收的动态平衡被打破。如随着年龄的增加骨吸收活性逐渐强于骨形成活性,进而导致骨量逐渐的减少,当骨量减少到一定程度后即发展为骨质疏松,导致骨脆性增加,骨强度降低,骨折风险增加。尤其是在女性绝经后,由于体内雌激素水平的变化,导致骨吸收被显著激活,出现骨量的快速丢失现象。此外骨关节炎、类风湿性关节炎等骨疾病均有骨吸收的异常激活。大量文献报道包括乳腺癌、前列腺癌在内的多种肿瘤在骨转移灶的部位均出现了骨吸收的激活现象。此外多种药物如糖皮质激素也会引起骨吸收的显著激活,导致骨丢失。因而骨吸收的异常激活与多种疾病的发生发展均有直接的关系,抑制骨吸收是治疗骨吸收激活相关疾病的有效策略。
破骨细胞是体内负责骨吸收过程的唯一功能细胞,而目前直接从体内分离破骨细胞难度极大,运用动物在体内直接干预破骨细胞的分化及功能则费用昂贵,时间成本也很高,不适合运用动物进行体内破骨细胞的大规模研究。因此要研究破骨细胞的功能、揭示破骨细胞分化调控的机制、筛选抑制破骨细胞功能或分化的药物等均需要依赖于合适的体外破骨细胞模型。
目前体外破骨细胞的模型包括:(1)从动物骨髓中分离骨髓来源的巨噬细胞,再经过M-CSF和RANKL细胞因子的刺激分化获得破骨细胞。(2)从小鼠单核细胞系RAW264.7经过RANKL细胞因子的刺激分化获得破骨细胞。(3)分离人的外周血或脐带血的单核PBMCs细胞,再经过M-CSF和RANKL细胞因子的刺激分化获得破骨细胞。(4)从人单核细胞系THP-1,经过PMA和RANKL细胞因子的刺激分化获得破骨细胞。前面第一和第二两种方法虽然来源不受限制,但是因为是动物的细胞无法最真实准确的反应人破骨细胞的分化过程和功能,因此存在一定的缺陷。而第三种方法需要采集人的血液,导致实验细胞的获取受到极大的限制,也不适合推广应用。而第四种方法,从目前文献报道的分化数据来看分化效率极低,而且分化获得的并不是多核的破骨细胞,因此不是一种成熟稳定的人体外破骨细胞分化模型。
综合以上研究现状分析,开发一种能够在体外高效获取人破骨细胞的方法是领域内的迫切需求,该方法对于破骨细胞的功能研究及药物开发,均具有重要的价值。
发明内容
为了克服现有技术的不足和缺点,本发明的首要目的在于提供一种具备高破骨细胞分化能力的THP-1细胞。
本发明的另一目的在于提供上述具备高破骨细胞分化能力的THP-1细胞的制备方法。
本发明的再一目的在于提供上述具备高破骨细胞分化能力的THP-1细胞的应用。
本发明的第四个目的在于提供一种破骨细胞,该破骨细胞由上述具备高破骨细胞分化能力的THP-1细胞分化得到。
本发明的第五个目的在于提供上述破骨细胞的制备方法。
本发明的第六个目的在于提供上述破骨细胞的应用。
本发明的目的通过下述技术方案实现:
一种具备高破骨细胞分化能力的THP-1细胞,该细胞表达或过表达CSF1R和RANK受体;
所述的具备高破骨细胞分化能力的THP-1细胞,优选通过腺病毒包装、腺相关病毒包装、CRISPR/CAS9、慢病毒包装等基因编辑方式实现细胞内CSF1R和RANK受体的表达或过表达;
所述的具备高破骨细胞分化能力的THP-1细胞的制备方法,优选包含如下步骤:
(1)将人CSF1R基因和人RANK基因分别构建到慢病毒载体中,得到含人CSF1R基因片段的重组慢病毒载体和含人RANK基因片段的重组慢病毒载体;
(2)将步骤(1)制得的重组慢病毒载体分别和辅助质粒共转染293T细胞,进行慢病毒包装,得到人CSF1R慢病毒上清液和人RANK慢病毒上清液;
(3)将人CSF1R慢病毒上清液、人RANK慢病毒上清液共感染THP-1细胞,得到具备高破骨细胞分化能力的THP-1细胞,该细胞可同时表达人CSF1R受体和RANK受体;
步骤(1)中所述的慢病毒载体优选为GV717或CV084;
步骤(2)中所述的辅助质粒优选为pHelper1.0和pHelper2.0;
步骤(2)中所述的慢病毒包装的具体操作优选为:
将含相应目的基因片段的重组慢病毒载体和辅助质粒共转染293T细胞;共转染48-72h后,离心收集细胞上清液;对细胞上清液进行浓缩和纯化;
步骤(3)中所述的共感染的具体操作优选为:
①将人CSF1R慢病毒上清液、人RANK慢病毒上清液和感染增强剂HitransG A加入到THP-1细胞,培养2天;
②病毒感染2天后,离心收集THP-1细胞,加入含Puromycin和G418的新鲜生长培养基重悬细胞进行培养;
③每隔2天,重复操作步骤②一次(即进行一次细胞换液),一共筛选2周;得到具备高破骨细胞分化能力的THP-1细胞,该细胞可稳定表达CSF1R和RANK受体;
所述的人RANK慢病毒上清液的感染复数MOI优选为50;
所述的人CSF1R慢病毒上清液的感染复数MOI优选为50;
所述的Puromycin在培养基中的终浓度优选为2μg/ml;
所述的G418在培养基中的终浓度优选为200μg/ml;
所述的具备高破骨细胞分化能力的THP-1细胞在制备破骨细胞产品中的应用;
一种破骨细胞,该破骨细胞由上述具备高破骨细胞分化能力的THP-1细胞分化得到;
所述的破骨细胞的制备方法,包含如下步骤:
(1)在具备高破骨细胞分化能力的THP-1细胞中加入含佛波酯(PMA)的细胞培养液进行诱导,使THP-1细胞贴壁分化为巨噬细胞;
(2)当THP-1细胞贴壁分化为巨噬细胞后,开始进行破骨细胞分化诱导,将细胞培养液更换为破骨细胞分化诱导液进行破骨细胞分化诱导,破骨细胞分化诱导液包含如下组分:10-200ng/ml PMA、5-500ng/ml M-CSF、5-500ng/ml RANKL、1-200nM 1a,25(OH)2D3,每2天更换一次破骨细胞分化诱导液;
步骤(1)中所述的细胞培养液中佛波酯(PMA)的浓度优选为100ng/mL;
步骤(1)中所述的诱导时间优选为3天;
步骤(2)中所述的破骨细胞分化诱导液优选包含如下组分:100ng/ml PMA、100ng/ml M-CSF、100ng/ml RANKL、10nM 1a,25(OH)2D3;
步骤(2)所述的破骨细胞分化诱导液还包含:1640培养基、10%FBS和双抗;
步骤(2)中所述的破骨细胞分化诱导的时间优选为5-15天;
所述的破骨细胞在研究破骨细胞功能、筛选调控破骨细胞分化或功能的药物中的应用;
本发明的原理:
(1)本发明首先分析了THP-1细胞分化效率低下的原因。破骨细胞分化需要依赖M-CSF和RANKL两个细胞因子的刺激,M-CSF结合细胞表面的CSF1R受体,RANKL结合细胞表面的RANK受体。而我们通过Q-PCR定量分析发现THP-1细胞不表达CSF1R和RANK受体,因此导致THP-1细胞无法响应M-CSF和RANKL两个细胞因子的刺激,进而无法有效地向破骨细胞分化。
(2)在明确了THP-1分化效率低下的原因后,我们选择采用慢病毒介导THP-1过表达CSF1R和RANK基因的方法来恢复THP-1细胞CSF1R和RANK受体的表达,通过构建过表达CSF1R和RANK基因的慢病毒,并感染THP-1细胞,病毒感染后的细胞经过抗性筛选杀死未被感染的细胞,从而构建高表达CSF1R和RANK破骨分化关键受体的新细胞,命名为THP-1(CSF1Rhi+RANKhi)细胞。
(3)更进一步本发明优化了THP-1细胞的分化条件,通过对佛波酯(PMA)、M-CSF、RANKL、1a,25(OH)2D3四种刺激剂的浓度摸索,最终确定如下分化方案具有最高的促进破骨细胞分化的效率:PMA(100ng/ml)、M-CSF(100ng/ml)、RANKL(100ng/ml)、1a,25(OH)2D3(10nM)。破骨细胞分化过程为:将THP-1(CSF1Rhi+RANKhi)细胞以1×106细胞/mL的密度铺板,加入100ng/mL PMA诱导3天,随后将细胞培养液更换为破骨细胞分化诱导液,配方为:1640培养基+10%FBS+双抗+100ng/ml PMA+100ng/ml M-CSF+100ng/ml RANKL+10nM 1a,25(OH)2D3。此后每2天更换一次破骨细胞分化诱导液,一般THP-1(CSF1Rhi+RANKhi)细胞在经过破骨细胞分化诱导后第5天开始出现破骨细胞,分化第10天可以形成大量的成熟破骨细胞。
(4)本发明将正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞经过如上分化方案进行分化诱导后通过分化第0、5、10天的明场显微镜拍摄,发现在第5天时THP-1(CSF1Rhi+RANKhi)细胞组即可以出现破骨细胞,在第10天时形成大量成熟破骨细胞。而正常THP-1细胞在分化10天后也仅出现少量破骨细胞。
(5)本发明将正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞经过如上分化方案进行分化诱导在第10天进行细胞骨架和细胞核的染色分析,发现THP-1(CSF1Rhi+RANKhi)细胞可以分化产生大量多核的破骨细胞。而正常THP-1细胞在分化10天后仅出现少量多核破骨细胞。
(6)本发明将正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞经过如上分化方案进行分化诱导在第10天进行TRAP染色分析,发现THP-1(CSF1Rhi+RANKhi)细胞可以分化产生大量TRAP+的破骨细胞。而正常THP-1细胞在分化10天后仅出现少量TRAP+的破骨细胞,且TRAP着色非常浅说明分化后的细胞活性很低。
(7)本发明将正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞经过如上分化方案进行分化诱导在第10天进行Q-PCR分析,发现THP-1(CSF1Rhi+RANKhi)细胞分化产生的破骨细胞表达更高水平的破骨标志基因:NFATC1、CTSK、TRAP、MMP9。说明本发明构建的细胞具备更高的破骨细胞特征。
以上实验数据充分地说明本发明制备的THP-1(CSF1Rhi+RANKhi)细胞可以在体外诱导产生具备典型破骨细胞特征的成熟破骨细胞。分化效率远远高于目前存在的技术方案。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明新构建的THP-1(CSF1Rhi+RANKhi)细胞可以在体外经过分化刺激快速产生具备典型破骨细胞特征的成熟破骨细胞,分化效率远远高于目前存在的技术方案。
(2)THP-1(CSF1Rhi+RANKhi)细胞作为细胞系可以稳定的传代冻存,无需繁琐的原代细胞提取过程,也无需获得人体的血液样本,从而体现出高效、经济、安全、稳定等特点。
(3)本发明揭示了THP-1细胞无法成功向破骨细胞分化的根本原因是其无法表达CSF1R和RANK这两个关键受体。并基于此提出采用慢病毒或其他策略使THP-1细胞高表达CSF1R和RANK基因,从而赋予THP-1高破骨细胞分化潜能的科学思想和实验方法。
(4)本发明筛选获得的破骨细胞分化诱导液配方和细胞分化换液方法可以保证破骨细胞分化的效率和成功率,操作简便。
(5)本发明公布的技术方法可以被具备领域内专业知识的人员重复,具备很高的技术可行性。
附图说明
图1是正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞中RANK和CSF1R基因表达水平的Q-PCR分析结果图。
图2是正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞不同分化时期的显微镜图,其中,星号指示的是分化完成的破骨细胞。
图3是正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞分化为巨噬细胞后进行破骨细胞分化诱导10天后的细胞骨架和细胞核染色显微镜图。
图4是正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞分化为巨噬细胞后进行破骨细胞分化诱导10天后的TRAP染色显微镜图。
图5是正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞分化为巨噬细胞后进行破骨细胞分化诱导10天后的破骨细胞标志基因Q-PCR分析图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的材料和试剂。
实施例1 THP-1细胞的培养
将人THP-1细胞(购买自中国科学院细胞库,货号SCSP-567)饲养于含10%胎牛血清(FBS,依科赛公司,货号FSP500)和1%的双抗(青霉素和链霉素)的1640培养液(Gibco)中;具体培养条件为:细胞生长于37℃、5% CO2的细胞培养箱中,每2-3天换液传代一次;细胞换液时以300g离心5min收集细胞,然后加入新鲜培养基重悬细胞进行传代。
实施例2 CSF1R和RANK基因过表达慢病毒的构建
委托上海吉凯基因科技有限公司构建表达人CSF1R和RANK的慢病毒;其中,人CSF1R基因(GENE ID:1436,转录本编号:NM_005211,核苷酸序列和氨基酸序列如下所示)的CDS表达框长度2919bp,慢病毒载体货号GV717(上海吉凯基因),载体元件顺序CMV-enhancer-CSF1R-SV40-Puromycin,筛选标记嘌呤霉素Puromycin;人RANK基因(GENE ID:8792,转录本编号:NM_003839,核苷酸序列和氨基酸序列如下所示)的CDS表达框长度1851p,慢病毒载体货号CV084(上海吉凯基因),载体元件顺序Ubi-RANK-SV40-Neomycin,筛选标记新霉素Neomycin;具体方法及如下:
(1)将目的基因片段(人CSF1R基因或人RANK基因)构建到慢病毒载体(GV717或CV084)中,得到含相应目的基因片段的重组慢病毒载体CMV-enhancer-CSF1R-SV40-Puromycin和Ubi-RANK-SV40-Neomycin;
(2)将步骤(1)制得的含相应目的基因片段的重组慢病毒载体分别和辅助质粒(pHelper1.0和pHelper2.0)共转染293T细胞;
(3)病毒转染48-72h后,离心收集细胞上清液;
(4)对上述细胞上清液进行0.45μm过滤,超速离心等处理;
(5)质量检测:经物理状态检测、无菌检测和滴度检测后,分别得到CSF1R基因过表达的慢病毒上清液和RANK基因过表达的慢病毒上清液,其中,慢病毒包装滴度1E+8TU/ml,50 μL/tube进行分装后-80度冰箱保存。
人CSF1R基因的核苷酸序列:
ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTGGCATGGTCAGGGAATCCCAGTGATAGAGCCCAGTGTCCCTGAGCTGGTCGTGAAGCCAGGAGCAACGGTGACCTTGCGATGTGTGGGCAATGGCAGCGTGGAATGGGATGGCCCCCCATCACCTCACTGGACCCTGTACTCTGATGGCTCCAGCAGCATCCTCAGCACCAACAACGCTACCTTCCAAAACACGGGGACCTATCGCTGCACTGAGCCTGGAGACCCCCTGGGAGGCAGCGCCGCCATCCACCTCTATGTCAAAGACCCTGCCCGGCCCTGGAACGTGCTAGCACAGGAGGTGGTCGTGTTCGAGGACCAGGACGCACTACTGCCCTGTCTGCTCACAGACCCGGTGCTGGAAGCAGGCGTCTCGCTGGTGCGTGTGCGTGGCCGGCCCCTCATGCGCCACACCAACTACTCCTTCTCGCCCTGGCATGGCTTCACCATCCACAGGGCCAAGTTCATTCAGAGCCAGGACTATCAATGCAGTGCCCTGATGGGTGGCAGGAAGGTGATGTCCATCAGCATCCGGCTGAAAGTGCAGAAAGTCATCCCAGGGCCCCCAGCCTTGACACTGGTGCCTGCAGAGCTGGTGCGGATTCGAGGGGAGGCTGCCCAGATCGTGTGCTCAGCCAGCAGCGTTGATGTTAACTTTGATGTCTTCCTCCAACACAACAACACCAAGCTCGCAATCCCTCAACAATCTGACTTTCATAATAACCGTTACCAAAAAGTCCTGACCCTCAACCTCGATCAAGTAGATTTCCAACATGCCGGCAACTACTCCTGCGTGGCCAGCAACGTGCAGGGCAAGCACTCCACCTCCATGTTCTTCCGGGTGGTAGAGAGTGCCTACTTGAACTTGAGCTCTGAGCAGAACCTCATCCAGGAGGTGACCGTGGGGGAGGGGCTCAACCTCAAAGTCATGGTGGAGGCCTACCCAGGCCTGCAAGGTTTTAACTGGACCTACCTGGGACCCTTTTCTGACCACCAGCCTGAGCCCAAGCTTGCTAATGCTACCACCAAGGACACATACAGGCACACCTTCACCCTCTCTCTGCCCCGCCTGAAGCCCTCTGAGGCTGGCCGCTACTCCTTCCTGGCCAGAAACCCAGGAGGCTGGAGAGCTCTGACGTTTGAGCTCACCCTTCGATACCCCCCAGAGGTAAGCGTCATATGGACATTCATCAACGGCTCTGGCACCCTTTTGTGTGCTGCCTCTGGGTACCCCCAGCCCAACGTGACATGGCTGCAGTGCAGTGGCCACACTGATAGGTGTGATGAGGCCCAAGTGCTGCAGGTCTGGGATGACCCATACCCTGAGGTCCTGAGCCAGGAGCCCTTCCACAAGGTGACGGTGCAGAGCCTGCTGACTGTTGAGACCTTAGAGCACAACCAAACCTACGAGTGCAGGGCCCACAACAGCGTGGGGAGTGGCTCCTGGGCCTTCATACCCATCTCTGCAGGAGCCCACACGCATCCCCCGGATGAGTTCCTCTTCACACCAGTGGTGGTCGCCTGCATGTCCATCATGGCCTTGCTGCTGCTGCTGCTCCTGCTGCTATTGTACAAGTATAAGCAGAAGCCCAAGTACCAGGTCCGCTGGAAGATCATCGAGAGCTATGAGGGCAACAGTTATACTTTCATCGACCCCACGCAGCTGCCTTACAACGAGAAGTGGGAGTTCCCCCGGAACAACCTGCAGTTTGGTAAGACCCTCGGAGCTGGAGCCTTTGGGAAGGTGGTGGAGGCCACGGCCTTTGGTCTGGGCAAGGAGGATGCTGTCCTGAAGGTGGCTGTGAAGATGCTGAAGTCCACGGCCCATGCTGATGAGAAGGAGGCCCTCATGTCCGAGCTGAAGATCATGAGCCACCTGGGCCAGCACGAGAACATCGTCAACCTTCTGGGAGCCTGTACCCATGGAGGCCCTGTACTGGTCATCACGGAGTACTGTTGCTATGGCGACCTGCTCAACTTTCTGCGAAGGAAGGCTGAGGCCATGCTGGGACCCAGCCTGAGCCCCGGCCAGGACCCCGAGGGAGGCGTCGACTATAAGAACATCCACCTCGAGAAGAAATATGTCCGCAGGGACAGTGGCTTCTCCAGCCAGGGTGTGGACACCTATGTGGAGATGAGGCCTGTCTCCACTTCTTCAAATGACTCCTTCTCTGAGCAAGACCTGGACAAGGAGGATGGACGGCCCCTGGAGCTCCGGGACCTGCTTCACTTCTCCAGCCAAGTAGCCCAGGGCATGGCCTTCCTCGCTTCCAAGAATTGCATCCACCGGGACGTGGCAGCGCGTAACGTGCTGTTGACCAATGGTCATGTGGCCAAGATTGGGGACTTCGGGCTGGCTAGGGACATCATGAATGACTCCAACTACATTGTCAAGGGCAATGCCCGCCTGCCTGTGAAGTGGATGGCCCCAGAGAGCATCTTTGACTGTGTCTACACGGTTCAGAGCGACGTCTGGTCCTATGGCATCCTCCTCTGGGAGATCTTCTCACTTGGGCTGAATCCCTACCCTGGCATCCTGGTGAACAGCAAGTTCTATAAACTGGTGAAGGATGGATACCAAATGGCCCAGCCTGCATTTGCCCCAAAGAATATATACAGCATCATGCAGGCCTGCTGGGCCTTGGAGCCCACCCACAGACCCACCTTCCAGCAGATCTGCTCCTTCCTTCAGGAGCAGGCCCAAGAGGACAGGAGAGAGCGGGACTATACCAATCTGCCGAGCAGCAGCAGAAGCGGTGGCAGCGGCAGCAGCAGCAGTGAGCTGGAGGAGGAGAGCTCTAGTGAGCACCTGACCTGCTGCGAGCAAGGGGATATCGCCCAGCCCTTGCTGCAGCCCAACAACTATCAGTTCTGCTGA
人CSF1R蛋白序列:
MGPGVLLLLLVATAWHGQGIPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRVVESAYLNLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWLQCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSVGSGSWAFIPISAGAHTHPPDEFLFTPVVVACMSIMALLLLLLLLLLYKYKQKPKYQVRWKIIESYEGNSYTFIDPTQLPYNEKWEFPRNNLQFGKTLGAGAFGKVVEATAFGLGKEDAVLKVAVKMLKSTAHADEKEALMSELKIMSHLGQHENIVNLLGACTHGGPVLVITEYCCYGDLLNFLRRKAEAMLGPSLSPGQDPEGGVDYKNIHLEKKYVRRDSGFSSQGVDTYVEMRPVSTSSNDSFSEQDLDKEDGRPLELRDLLHFSSQVAQGMAFLASKNCIHRDVAARNVLLTNGHVAKIGDFGLARDIMNDSNYIVKGNARLPVKWMAPESIFDCVYTVQSDVWSYGILLWEIFSLGLNPYPGILVNSKFYKLVKDGYQMAQPAFAPKNIYSIMQACWALEPTHRPTFQQICSFLQEQAQEDRRERDYTNLPSSSRSGGSGSSSSELEEESSSEHLTCCEQGDIAQPLLQPNNYQFC
人RANK基因序列:
ATGGCCCCGCGCGCCCGGCGGCGCCGCCCGCTGTTCGCGCTGCTGCTGCTCTGCGCGCTGCTCGCCCGGCTGCAGGTGGCTTTGCAGATCGCTCCTCCATGTACCAGTGAGAAGCATTATGAGCATCTGGGACGGTGCTGTAACAAATGTGAACCAGGAAAGTACATGTCTTCTAAATGCACTACTACCTCTGACAGTGTATGTCTGCCCTGTGGCCCGGATGAATACTTGGATAGCTGGAATGAAGAAGATAAATGCTTGCTGCATAAAGTTTGTGATACAGGCAAGGCCCTGGTGGCCGTGGTCGCCGGCAACAGCACGACCCCCCGGCGCTGCGCGTGCACGGCTGGGTACCACTGGAGCCAGGACTGCGAGTGCTGCCGCCGCAACACCGAGTGCGCGCCGGGCCTGGGCGCCCAGCACCCGTTGCAGCTCAACAAGGACACAGTGTGCAAACCTTGCCTTGCAGGCTACTTCTCTGATGCCTTTTCCTCCACGGACAAATGCAGACCCTGGACCAACTGTACCTTCCTTGGAAAGAGAGTAGAACATCATGGGACAGAGAAATCCGATGCGGTTTGCAGTTCTTCTCTGCCAGCTAGAAAACCACCAAATGAACCCCATGTTTACTTGCCCGGTTTAATAATTCTGCTTCTCTTCGCGTCTGTGGCCCTGGTGGCTGCCATCATCTTTGGCGTTTGCTATAGGAAAAAAGGGAAAGCACTCACAGCTAATTTGTGGCACTGGATCAATGAGGCTTGTGGCCGCCTAAGTGGAGATAAGGAGTCCTCAGGTGACAGTTGTGTCAGTACACACACGGCAAACTTTGGTCAGCAGGGAGCATGTGAAGGTGTCTTACTGCTGACTCTGGAGGAGAAGACATTTCCAGAAGATATGTGCTACCCAGATCAAGGTGGTGTCTGTCAGGGCACATGTGTAGGAGGTGGTCCCTACGCACAAGGCGAAGATGCCAGGATGCTCTCATTGGTCAGCAAGACCGAGATAGAGGAAGACAGCTTCAGACAGATGCCCACAGAAGATGAATACATGGACAGGCCCTCCCAGCCCACAGACCAGTTACTGTTCCTCACTGAGCCTGGAAGCAAATCCACACCTCCTTTCTCTGAACCCCTGGAGGTGGGGGAGAATGACAGTTTAAGCCAGTGCTTCACGGGGACACAGAGCACAGTGGGTTCAGAAAGCTGCAACTGCACTGAGCCCCTGTGCAGGACTGATTGGACTCCCATGTCCTCTGAAAACTACTTGCAAAAAGAGGTGGACAGTGGCCATTGCCCGCACTGGGCAGCCAGCCCCAGCCCCAACTGGGCAGATGTCTGCACAGGCTGCCGGAACCCTCCTGGGGAGGACTGTGAACCCCTCGTGGGTTCCCCAAAACGTGGACCCTTGCCCCAGTGCGCCTATGGCATGGGCCTTCCCCCTGAAGAAGAAGCCAGCAGGACGGAGGCCAGAGACCAGCCCGAGGATGGGGCTGATGGGAGGCTCCCAAGCTCAGCGAGGGCAGGTGCCGGGTCTGGAAGCTCCCCTGGTGGCCAGTCCCCTGCATCTGGAAATGTGACTGGAAACAGTAACTCCACGTTCATCTCCAGCGGGCAGGTGATGAACTTCAAGGGCGACATCATCGTGGTCTACGTCAGCCAGACCTCGCAGGAGGGCGCGGCGGCGGCTGCGGAGCCCATGGGCCGCCCGGTGCAGGAGGAGACCCTGGCGCGCCGAGACTCCTTCGCGGGGAACGGCCCGCGCTTCCCGGACCCGTGCGGCGGCCCCGAGGGGCTGCGGGAGCCGGAGAAGGCCTCGAGGCCGGTGCAGGAGCAAGGCGGGGCCAAGGCTTGA
人RANK蛋白序列:
MAPRARRRRPLFALLLLCALLARLQVALQIAPPCTSEKHYEHLGRCCNKCEPGKYMSSKCTTTSDSVCLPCGPDEYLDSWNEEDKCLLHKVCDTGKALVAVVAGNSTTPRRCACTAGYHWSQDCECCRRNTECAPGLGAQHPLQLNKDTVCKPCLAGYFSDAFSSTDKCRPWTNCTFLGKRVEHHGTEKSDAVCSSSLPARKPPNEPHVYLPGLIILLLFASVALVAAIIFGVCYRKKGKALTANLWHWINEACGRLSGDKESSGDSCVSTHTANFGQQGACEGVLLLTLEEKTFPEDMCYPDQGGVCQGTCVGGGPYAQGEDARMLSLVSKTEIEEDSFRQMPTEDEYMDRPSQPTDQLLFLTEPGSKSTPPFSEPLEVGENDSLSQCFTGTQSTVGSESCNCTEPLCRTDWTPMSSENYLQKEVDSGHCPHWAASPSPNWADVCTGCRNPPGEDCEPLVGSPKRGPLPQCAYGMGLPPEEEASRTEARDQPEDGADGRLPSSARAGAGSGSSPGGQSPASGNVTGNSNSTFISSGQVMNFKGDIIVVYVSQTSQEGAAAAAEPMGRPVQEETLARRDSFAGNGPRFPDPCGGPEGLREPEKASRPVQEQGGAKA
实施例3 THP-1(CSF1Rhi+RANKhi)细胞的筛选
(1)将THP-1细胞按照1×105细胞/mL的密度铺板于培养板中;然后均按照感染复数MOI=50,加入实施例2制得的CSF1R基因过表达的慢病毒上清液和RANK基因过表达的慢病毒上清液,同时加入感染增强剂HitransG A(100倍稀释,每ml培养基加入10μl稀释后的感染增强剂HitransG A)(上海吉凯基因公司);
(2)在病毒感染2天后,离心收集THP-1细胞,并加入新鲜的生长培养基重悬细胞,在培养基中加入终浓度为2μg/ml的Puromycin和终浓度为200μg/ml的G418进行细胞的筛选;此后每隔2天进行一次细胞换液,未感染慢病毒的细胞将被逐渐杀死,一共筛选2周;得到稳定表达RANK和CSF1R的THP-1细胞,命名为THP-1(CSF1Rhi+RANKhi);
(3)为了检测细胞过表达的效率,我们对正常THP-1细胞和THP-1(CSF1Rhi+RANKhi)细胞的RANK和CSF1R基因的表达进行了实时荧光定量PCR(Q-PCR)分析(具体实验方法按照实施例8-10进行)。
实验结果如图1所示,相比正常THP-1细胞,THP-1(CSF1Rhi+RANKhi)细胞中RANK基因的表达上调了400倍,CSF1R基因上调了200倍,说明细胞构建成功。而正常THP-1细胞不表达CSF1R和RANK受体,进而使得THP-1细胞无法响应M-CSF和RANKL两个细胞因子的刺激,这可能是正常THP-1细胞无法有效地向破骨细胞分化的原因。
实施例4 THP-1细胞向破骨细胞分化条件筛选
(1)将实施例3获得的THP-1(CSF1Rhi+RANKhi)细胞以1×106细胞/mL的密度铺板于12孔板中,1ml/孔;加入佛波酯(PMA或TPA,碧云天公司)(终浓度为100ng/ml)诱导3天,细胞将贴壁分化为巨噬细胞;
(2)当THP-1(CSF1Rhi+RANKhi)细胞贴壁分化为巨噬细胞后,开始进行破骨细胞分化诱导,将细胞培养液更换为破骨细胞分化诱导液(1640培养基+10%FBS+双抗+诱导因子),诱导因子包含如表1所示的组合,诱导因子各成分的浓度范围为:10-200ng/ml PMA、5-500ng/ml M-CSF、5-500ng/ml RANKL、1-200nM 1a,25(OH)2D3;每2天更换一次破骨细胞分化诱导液;
(3)分化第10天在显微镜下观察计数12孔板每个孔中形成的破骨细胞数,其中,计数统计如下:(-)无破骨细胞生成;(+)破骨细胞数小于10个;(++)破骨细胞数介于10-20之间;(+++)破骨细胞数介于20-30之间;(++++)破骨细胞数大于30。
结果显示THP-1(CSF1Rhi+RANKhi)细胞在100ng/ml PMA、100ng/ml M-CSF(Peprotech公司)、100ng/ml RANKL(Peprotech公司)、10nM 1a,25(OH)2D3(Sigma公司)的诱导条件下可最好地刺激破骨细胞的分化,同时药物的用量少,可节省实验成本。
表1 THP-1细胞向破骨细胞分化条件筛选
实施例5 THP-1(CSF1Rhi+RANKhi)细胞向破骨细胞分化诱导
(1)将正常THP-1细胞和THP-1(CSF1Rhi+RANKhi)细胞以1×106细胞/mL的密度铺板于12孔板中,1ml/孔;加入100ng/ml佛波酯(PMA或TPA,碧云天公司)诱导3天,细胞将贴壁分化为巨噬细胞;
(2)当正常THP-1细胞和THP-1(CSF1Rhi+RANKhi)细胞贴壁分化为巨噬细胞后,开始进行破骨细胞分化诱导,将细胞培养液更换为破骨细胞分化诱导液(1640培养基+10%FBS+双抗+诱导因子),诱导因子包含如下组分:100ng/ml PMA、100ng/ml M-CSF(Peprotech公司)、100ng/ml RANKL(Peprotech公司)、10nM 1a,25(OH)2D3(Sigma公司);此后每2天更换一次破骨细胞分化诱导液。
图2为正常THP-1细胞和THP-1(CSF1Rhi+RANKhi)细胞在分化前、分化第5天和第10天的显微镜图片。如图2所示,THP-1(CSF1Rhi+RANKhi)细胞在经过破骨细胞分化诱导后第5天开始出现破骨细胞,分化第10天可以形成大量的成熟破骨细胞;而正常THP-1细胞经过破骨细胞分化诱导后第10天仅形成少量成熟破骨细胞。
实施例6 细胞骨架染色
正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞分化诱导至第10天后进行细胞骨架和细胞核的染色分析,具体方法如下:
(1)将实施例5中分化诱导至第10天的破骨细胞从培养箱中取出,取出细胞培养基,用PBS溶液清洗细胞1次,每次3min;
(2)在步骤(1)清洗后的细胞中加入质量分数为4%的多聚甲醛(加入量与原细胞培养基体积相同)室温固定细胞10min,固定结束后用PBS溶液清洗细胞3次,每次3min;
(3)在步骤(2)清洗后的细胞中加入鬼笔环肽细胞骨架染色液(加入量为原细胞培养基体积的1/2)(货号40734ES75,翌圣生物科技),室温避光染色30min,染色结束后用PBS溶液清洗细胞3次,每次3min;
(4)在步骤(3)清洗后的细胞中加入DAPI细胞核染色液(加入量为原细胞培养基体积的1/2)(货号C1002,碧云天生物),室温避光染色20min,染色结束后用PBS溶液清洗细胞3次,每次3min;随后进行荧光显微镜的拍摄。
结果如图3所示,THP-1(CSF1Rhi+RANKhi)细胞可以分化产生大量多核的破骨细胞,而正常THP-1细胞在分化10天后仅出现少量多核破骨细胞。
实施例7 破骨细胞TRAP染色分析
正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞分化诱导至第10天后进行TRAP染色分析,按照TRAP染色试剂盒(Sigma-Aldrich,货号387)说明书进行,具体过程如下:
(1)配置染色液:取1.5mL EP管,将试剂Fast garnet GBC Base solution和Sodium Nitrite Solution各取50μL混匀并静置2min,得到Fast garnet GBC Basesolution和Sodium Nitrite Solution的混合液;另取15mL离心管,加入试剂Naphthol AS-BI phosphate solution 50μL,试剂Acetate solution 200μL,试剂Tartrate solution100μL,加入4.5mL 37℃蒸馏水和配好的试剂Fast garnet GBC Base solution和SodiumNitrite Solution的混合液,混合均匀后37℃温浴备用;
(2)细胞固定与染色:取实施例5中分化诱导至第10天的细胞(96孔板为例),吸弃培养基,加入50μl质量分数为4%的多聚甲醛室温固定15min,弃掉多聚甲醛,50μl 37℃蒸馏水洗2次后,加入30μl染色剂37℃染色1h,50μL蒸馏水洗1次,加入20μl蒸馏水,显微镜下观察破骨细胞并计数。注意:加液过程中要缓慢加入,避免细胞脱落。
结果如图4所示,THP-1(CSF1Rhi+RANKhi)细胞可以分化产生大量TRAP+的破骨细胞。而正常THP-1细胞在分化10天后仅出现少量TRAP+的破骨细胞,且TRAP着色非常浅说明分化后的细胞活性很低。
实施例8 细胞总RNA提取
正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞分化诱导至第10天后提取细胞总RNA,其中,细胞总RNA的提取按照天根生化科技(北京)有限公司TRNzol Universal总RNA提取试剂(天根生物科技,货号DP424)的说明书进行,具体过程如下:
(1)6孔板每个孔加入1ml TRNzol裂解细胞并用枪头反复吹打,室温作用5min;
(2)将裂解液移入1.5ml离心管中,加入200μl氯仿,盖紧管盖,涡旋混匀室温静置3min,4℃,12000g/min离心15min;
(3)离心后,将上层水相液体转移至新的1.5ml离心管中,加入500μl异丙醇,涡旋混匀室温静置10min,4℃,12000g/min离心10min;
(4)将离心后获得的上清用移液器吸取,丢弃,保留底部白色沉淀;
(5)加入1ml体积分数为75%的乙醇(DEPC水配制),用手轻轻将白色沉淀弹起,然后在4℃条件下,7500g/min离心5min;
(6)弃上清,室温干燥10-15min,加入30-40μl DEPC水重新溶解,RNA置于-80℃冰箱中保存或直接使用。
实施例9 总RNA的反转录实验
使用反转录试剂盒(TaKaRa,货号RR047A),根据试剂盒的操作流程,将实施例8提取的1000ng总RNA反转录成cDNA,具体过程如下:
(1)RNA测定浓度后,按照10 μl反转录体系加入500ng总RNA的量计算需要加入的RNA和DEPC水的体积;
(2)向PCR管中依次加入DEPC水,总RNA和5×PrimeScriptTM RT Master Mix(TaKaRa,货号RR047A),并混合均匀;
(3)于PCR仪上按照37℃反应15 min,85℃反应5 sec,4℃保存的程序进行反转录扩增;
(4)扩增后的cDNA用灭菌的三蒸水稀释30-50倍后用于荧光定量PCR扩增,或于-20℃长期保存。
实施例10 实时荧光定量PCR扩增(Q-PCR)
参考试剂盒说明书(TaKaRa,货号RR820A)使用实时荧光定量PCR技术以GAPDH为内参基因进行Q-PCR实验检测基因的表达水平。具体过程如下:
(1)实验前准备好检测引物(Q-PCR引物见表2),灭菌三蒸水,96孔PCR板,荧光定量试剂;
(2)根据待检测基因的数目分配反应产物在96孔PCR板中的具体位置;
(3)按照每个待检测基因每个样品三个复孔的比例配制反应液:20μl反应体系中含cDNA模板2μl(实施例9获取),2×SYBR® Premix Ex TaqTM II(TaKaRa,货号RR820A)试剂10μl,上游引物(10 μM)1μl,下游引物(10 μM)1μl,三蒸水 6μl;
(4)将全部反应液加入相应孔后,用专用封口膜封闭96孔PCR板,并2500 rpm离心3min;
(5)离心完成后,于BIO-RAD公司CFX96 TouchTM荧光定量PCR检测系统上机检测,反应程序为:A、预变性95℃ 30 sec;B、PCR反应:95℃ 5 sec,60℃ 30 sec,读板,循环40次;C、拟合曲线并读板;
(6)反应完成后,用系统自带分析软件进行数据分析,排除3个复孔中CT值差异大于0.5的数值,并根据内参基因计算各检测基因的相对表达水平。
表2 Q-PCR引物列表
如图5所示,我们将正常THP-1和THP-1(CSF1Rhi+RANKhi)细胞进行分化诱导在第10天进行Q-PCR分析,发现THP-1(CSF1Rhi+RANKhi)细胞分化产生的破骨细胞表达更高水平的破骨标志基因:NFATC1、CTSK、TRAP、MMP9,进一步说明本发明构建的细胞具备更高的破骨细胞特征。
综上,本发明采用慢病毒介导THP-1细胞CSF1R和RANK基因过表达,并筛选稳定细胞株,从而获得新型具备高破骨细胞分化能力的新THP-1细胞THP-1(CSF1Rhi+RANKhi)的方法。此外,也可以采用腺病毒、腺相关病毒、CRISPR/CAS9等技术策略实现THP-1细胞中CSF1R和RANK基因的表达,只要是领域内存在的介导基因编辑和过表达的策略均可以用来构建THP-1(CSF1Rhi+RANKhi)细胞,因此不应在此方面限制本专利的保护范围。同时,本发明筛选确定THP-1(CSF1Rhi+RANKhi)细胞高效分化为成熟破骨细胞的分化液配方和细胞分化换液方法可以保证破骨细胞分化的效率和成功率,操作简便。本发明提供的THP-1(CSF1Rhi+RANKhi)细胞破骨分化模型在研究破骨细胞功能、筛选破骨细胞调控药物上具有重要应用价值。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (6)
1.一种破骨细胞的制备方法,其特征在于包含如下步骤:
(1)在具备高破骨细胞分化能力的THP-1细胞中加入含佛波酯的细胞培养液进行诱导,使THP-1细胞贴壁分化为巨噬细胞;
(2)当THP-1细胞贴壁分化为巨噬细胞后,开始进行破骨细胞分化诱导,将细胞培养液更换为破骨细胞分化诱导液进行破骨细胞分化诱导,破骨细胞分化诱导液包含如下组分:100ng/ml PMA、100ng/ml M-CSF、100ng/ml RANKL、10 nM 1a,25(OH)2D3;
所述的具备高破骨细胞分化能力的THP-1细胞过表达CSF1R和RANK受体。
2.根据权利要求1所述的破骨细胞的制备方法,其特征在于:
所述的具备高破骨细胞分化能力的THP-1细胞通过基因编辑方式实现细胞内CSF1R和RANK受体的过表达。
3.根据权利要求1所述的破骨细胞的制备方法,其特征在于:
所述的具备高破骨细胞分化能力的THP-1细胞的制备方法,包含如下步骤:
(1)将人CSF1R基因和人RANK基因分别构建到慢病毒载体中,得到含人CSF1R基因片段的重组慢病毒载体和含人RANK基因片段的重组慢病毒载体;
(2)将步骤(1)制得的重组慢病毒载体分别和辅助质粒共转染293T细胞,进行慢病毒包装,得到人CSF1R慢病毒上清液和人RANK慢病毒上清液;
(3)将人CSF1R慢病毒上清液、人RANK慢病毒上清液共感染THP-1细胞,得到具备高破骨细胞分化能力的THP-1细胞,该细胞可同时表达人CSF1R受体和RANK受体。
4.根据权利要求3所述的破骨细胞的制备方法,其特征在于:
步骤(3)中所述的共感染的具体操作为:
①将人CSF1R慢病毒上清液、人RANK慢病毒上清液和感染增强剂HitransG A加入到THP-1细胞,培养2天;
②病毒感染2天后,离心收集THP-1细胞,加入含Puromycin和G418的新鲜生长培养基重悬细胞进行培养;
③每隔2天,重复操作步骤②一次,一共筛选2周;得到具备高破骨细胞分化能力的THP-1细胞。
5.根据权利要求1所述的破骨细胞的制备方法,其特征在于:
步骤(2)中所述的破骨细胞分化诱导的时间为5-15天。
6.权利要求1-5任一项所述的破骨细胞的制备方法在研究破骨细胞功能、筛选调控破骨细胞分化或功能的药物中的应用。
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