CN113322283B - 一种制备后纵韧带骨化患者永生化后纵韧带细胞方法 - Google Patents
一种制备后纵韧带骨化患者永生化后纵韧带细胞方法 Download PDFInfo
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Abstract
本发明提供一种制备后纵韧带骨化患者永生化后纵韧带细胞方法,涉及基因工程技术领域。该制备后纵韧带骨化患者永生化后纵韧带细胞方法,包括以下步骤:S1、永生化后纵韧带细胞的构建;1)SV40Tant i gen基因及Hygromyc i nB抗性基因的确定;2)克隆过表达SV40Tant i ge慢病毒质粒,并携带Hygromyc i nB抗性;3)慢病毒的包装。本发明,永生化后纵韧带细胞系建立可简化研究过程,为研究提供足量而性状稳定的研究细胞,并被广泛用于人类疾病的研究,通过SV40Tant i gen基因及Hygromyc i nB抗性基因一同构建过表达慢病毒质粒成功高效的建立了永生化后纵韧带细胞,用于后纵韧带骨化疾病发病机制的研究。
Description
技术领域
本发明涉及基因工程技术领域,具体为一种制备后纵韧带骨化患者永生化后纵韧带细胞方法。
背景技术
后纵韧带骨化是一种常见的脊柱韧带异位骨化疾病,其患病率在1.9%–4.3%。流行病学调查发现,OPLL主要发生在颈椎和胸椎,该部位所对应的脊髓功能一旦受损,所造成的神经功能障碍往往是广泛的,且对患者生活质量甚至生存时间造成严重的影响。相关的病因学研究显示,年龄、肥胖、饮食、糖尿病、运动或脊柱的异常活动所造成的机械刺激、某些内分泌疾病等都可以增加OPLL的患病风险,说明OPLL存在多种可能的致病因素。因此,寻找OPLL中骨化物生成及生长的机制一直是该领域的研究热点之一。
OPLL的机制研究主要依靠体外细胞实验,由于后纵韧带骨化手术中,直接摘除骨化部位手术风险高,操作难度大,使得OPLL后纵韧带细胞获取困难,许多学者采用临近阶段黄韧带细胞或前纵韧带细胞替代进行研究。且后纵韧带细胞经过10-15代细胞培养后增殖能力基本丧失,细胞活力下降导致研究过程中可用细胞数量受限。
发明内容
(一)解决的技术问题
针对现有技术的不足,本发明提供了一种制备后纵韧带骨化患者永生化后纵韧带细胞方法,解决了现有技术中存在的缺陷与不足。
(二)技术方案
为实现以上目的,本发明通过以下技术方案予以实现:一种制备后纵韧带骨化患者永生化后纵韧带细胞方法,所述方法包括包括以下步骤:
S1、永生化后纵韧带细胞的构建;
1)SV40Tantigen基因及HygromycinB抗性基因的确定;
2)克隆过表达SV40Tantige慢病毒质粒,并携带HygromycinB抗性;
3)慢病毒的包装;
通过Lipo3000将穿梭质粒和包装质粒转染293T细胞,分别在转染后48h和72h收集上清,过滤后超速离心,弃上清,用无菌PBS过夜溶解病毒颗粒,再次过滤后分装冻存;
4)病毒感染后纵韧带细胞并进行抗药性筛选;
将后纵韧带细胞按1.5×105个/孔接种至24孔板中,将制备的慢病毒感染原代后纵韧带细胞,培养一周后开始加入2.5μg/mLHygromycinB进行药物筛选,期间每天观察细胞存活情况及培养液颜色变化,一到两天更换含有2.5μg/mLHygromycinB的培养液进行培养,三周后仍存活的细胞进行进一步扩增培养;
5)单克隆细胞株建立;
将经过药物筛选后的细胞进行单细胞培养,挑选增殖良好的细胞株进行传代培养,经过连续20代细胞传代培养后细胞依然保持良好增殖能力;
S2、永生化人纵韧带细胞的构成;
人后纵韧带细胞中随机插入SV40Tantigen基因及HygromycinB抗性基因。
优选的,所述永生化人后纵韧带细胞保留了后纵韧带细胞此前的特性,成骨诱导因子可诱导永生化后纵韧带细胞成骨,且OPLL患者来源的永生化后纵韧带细胞具有更强的成骨表型。
优选的,还包括有后纵韧带原代细胞的获取与培养,具体如下:
1)将手术中取得后纵韧带标本经生理盐水简单清洗去除血迹后迅速放入离心管中低温保存,4-6小时内带回实验室中处理;
2)按以下步骤在细胞房超净台内进行:将标本移入100mm培养皿中,PBS清洗两遍去除血迹及破碎组织,挑选合适组织块转入新的培养皿中并加入少量PBS润湿,小心去除骨组织,将剩余韧带组织剪成1-2mm3大小组织块,在T25培养瓶中预先加入1.5ml完全培养基,使培养瓶底部完全被培养基润湿,倒置培养瓶后小心将组织块间隔1cm排列于培养瓶底部,倒置放入培养箱中4-6小时或过夜,待组织块完全贴附于培养瓶底部后将培养瓶翻转回来;
3)之后每3天更换培养基,每次1.5-2ml,10-14天左右可见组织块周围有细胞向外爬出,待组织块周围爬满细胞并向外生长后,可使用0.25%含EDTA胰酶消化3-5分钟;
4)显微镜下观察细胞变圆并出现漂浮后加入等量含10%FBS完全培养基终止消化,吸管吹打并收集细胞悬液,弃去组织块后1000rpm/5min离心后收集细胞,完全培养基重悬细胞后移入T25培养瓶扩增,此次获得细胞视为P1代,按照1:3比例继续扩增细胞至P3-6代后将细胞冻存备用。
(三)有益效果
本发明提供了一种制备后纵韧带骨化患者永生化后纵韧带细胞方法。具备以下有益效果:
本发明,永生化后纵韧带细胞系建立可简化研究过程,为研究提供足量而性状稳定的研究细胞,并被广泛用于人类疾病的研究,通过SV40Tantigen基因及HygromycinB抗性基因一同构建过表达慢病毒质粒成功高效的建立了永生化后纵韧带细胞,用于后纵韧带骨化疾病发病机制的研究。
附图说明
图1为后纵韧带细胞示意图;
图2为永生化后纵韧带细胞(第5代)示意图;
图3为永生化后纵韧带细胞(第20代)示意图;
图4为SV40慢病毒表达质粒图谱;
图5为酶切鉴定结果示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例:
如图1-5所示,本发明实施例提供一种制备后纵韧带骨化患者永生化后纵韧带细胞方法,该方法包括包括以下步骤:
S1、永生化后纵韧带细胞的构建;
1)SV40Tantigen基因及HygromycinB抗性基因的确定;
2)克隆过表达SV40Tantige慢病毒质粒,并携带HygromycinB抗性;
SV40过表达慢病毒载体的构建;
通过PCR分别克隆出SV40和HygromycinB基因,通过T2A将HygromycinB连接到SV40后,并构建至慢病毒表达载体;PCR过程中所使用的引物分别为:
SV40-F(TTTTCCTACAGATCCTTAATTAAgatggataaagttttaaacagagaggaa);
SV40-R(CTCGACGTCACCGCATGTTAGCAGACTTCCTCTGCCCTCtgtttcaggttcagggggagg);
HygromycinB-F(TGCGGTGACGTCGAGGAGAATCCTGGCCCAatgaaaaagcctgaactcaccgc);
HygromycinB-R(GTAATCCAGAGGTTGATTGttagacttctacacagccatcggt);
通过Gibson组装的方法进行重组,转染大肠杆菌DH5a,涂板后挑取单克隆进行酶切和测序鉴定;
3)慢病毒的包装;
正确构建质粒后,通过Lipo3000将穿梭质粒和包装质粒转染293T细胞,分别在转染后48h和72h收集上清,过滤后超速离心,弃上清,用无菌PBS过夜溶解病毒颗粒,再次过滤后分装冻存,将获得的慢病毒命名为SV40-Hygro,利用慢病毒滴度检测试剂盒(ABM公司)进行病毒滴度测定,所得SV40-Hygro病毒的滴度3.2×109IU/mL;
4)病毒感染后纵韧带细胞并进行抗药性筛选;
将后纵韧带细胞按1.5×105个/孔接种至24孔板中,将制备的慢病毒感染原代后纵韧带细胞,培养一周后开始加入2.5μg/mLHygromycinB进行药物筛选,期间每天观察细胞存活情况及培养液颜色变化,一到两天更换含有2.5μg/mLHygromycinB的培养液进行培养,三周后仍存活的细胞进行进一步扩增培养;
5)单克隆细胞株建立;
将经过药物筛选后的细胞进行单细胞培养,挑选增殖良好的细胞株进行传代培养,经过连续20代细胞传代培养后细胞依然保持良好增殖能力;
后纵韧带原代细胞的获取与培养,具体如下:
1)将手术中取得后纵韧带标本经生理盐水简单清洗去除血迹后迅速放入离心管中低温保存,4-6小时内带回实验室中处理;
2)按以下步骤在细胞房超净台内进行:将标本移入100mm培养皿中,PBS清洗两遍去除血迹及破碎组织,挑选合适组织块转入新的培养皿中并加入少量PBS润湿,小心去除骨组织,将剩余韧带组织剪成1-2mm3大小组织块,在T25培养瓶中预先加入1.5ml完全培养基,使培养瓶底部完全被培养基润湿,倒置培养瓶后小心将组织块间隔1cm排列于培养瓶底部,倒置放入培养箱中4-6小时或过夜,待组织块完全贴附于培养瓶底部后将培养瓶翻转回来;
3)之后每3天更换培养基,每次1.5-2ml,10-14天左右可见组织块周围有细胞向外爬出,待组织块周围爬满细胞并向外生长后,可使用0.25%含EDTA胰酶消化3-5分钟;
4)显微镜下观察细胞变圆并出现漂浮后加入等量含10%FBS完全培养基终止消化,吸管吹打并收集细胞悬液,弃去组织块后1000rpm/5min离心后收集细胞,完全培养基重悬细胞后移入T25培养瓶扩增,此次获得细胞视为P1代,按照1:3比例继续扩增细胞至P3-6代后将细胞冻存备用;
S2、永生化人纵韧带细胞的构成;
人后纵韧带细胞中随机插入SV40Tantigen基因及HygromycinB抗性基因。
本发明,还包括HygromycinB药筛浓度测试;
1)Day0:将OPLL细胞按1.5×105个/孔接种至24孔板中;
2)Day1:按2000、1000、800、400、200、100、50、25、0μg/mL终浓度加入HygromycinB,每个浓度设两个副孔。后续每天观察细胞状态,每2天用含相应浓度HygromycinB换液一次;
3)Day2:加入HygromycinB浓度大于400μg/mL孔细胞出现漂浮,死亡明显;
4)Day6:加入HygromycinB浓度大于200μg/mL孔细胞出现漂浮,死亡明显;
5)Day8:加入HygromycinB浓度等于100μg/mL孔细胞出死亡不明显;
6)Day12:加入HygromycinB浓度小于50μg/mL孔未观察到明显细胞死亡,状态与未加药孔无异;
结论:200μg/mLHygromycinB终浓度是能够有效杀死OPLL细胞的最低浓度,选择该浓度作为后续细胞筛选浓度。
永生化OPLL细胞构建;
1)Day0:将OPLL细胞按1×106个/孔接种至6孔板中;
2)Day1:按10MOI值向细胞中加入慢病毒;
3)Day3-Day10:每2-3天用常规完全培养基细胞换液,培养细胞至80%左右密度;
4)Day11:加入相应终浓度的HygromycinB进行筛选;
5)Day12-Day20:每2-3天用含HygromycinB完全培养基细胞换液,每天观察细胞状态;
6)Day21:细胞在加入HygromycinB后细胞出现大量死亡,少量存活的细胞在后续培养中扩增起来;
7)Day21-:SV40组细胞扩增至足够数量后冻存;
8)上述获得的细胞经过扩增后挑选单细胞进行培养,获得永生化OPLL单细胞株,并进行反复传代培养,经过观察,细胞传代20代以上仍然保持良好的增殖能力。
永生化人后纵韧带细胞保留了后纵韧带细胞此前的特性,成骨诱导因子可诱导永生化后纵韧带细胞成骨,且OPLL患者来源的永生化后纵韧带细胞具有更强的成骨表型。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (1)
1.一种制备后纵韧带骨化患者永生化后纵韧带细胞方法,其特征在于,所述方法包括以下步骤:
S1、永生化后纵韧带细胞的构建;
1)SV40T抗原基因及潮霉素B抗性基因的确定;
2)克隆过表达SV40T抗原慢病毒质粒,并携带潮霉素B抗性;
3)慢病毒的包装;
通过Lipo3000将穿梭质粒和包装质粒转染293T细胞,分别在转染后48h和72h收集上清,过滤后超速离心,弃上清,用无菌PBS过夜溶解病毒颗粒,再次过滤后分装冻存;
4)病毒感染后纵韧带细胞并进行抗药性筛选;
将后纵韧带细胞按1.5×105个/孔接种至24孔板中,将制备的慢病毒感染原代后纵韧带细胞,培养一周后开始加入2.5µg/mL潮霉素B进行药物筛选,期间每天观察细胞存活情况及培养液颜色变化,一到两天更换含有2.5µg/mL潮霉素B的培养液进行培养,三周后仍存活的细胞进行进一步扩增培养;
5)单克隆细胞株建立;
将经过药物筛选后的细胞进行单细胞培养,挑选增殖良好的细胞株进行传代培养,经过连续20代细胞传代培养后细胞依然保持良好增殖能力;
S2、永生化人后纵韧带细胞的构成;
人后纵韧带细胞中随机插入SV40T抗原基因及潮霉素B抗性基因;
所述方法还包括后纵韧带原代细胞的获取与培养,具体如下:
1)将手术中取得后纵韧带标本经生理盐水简单清洗去除血迹后迅速放入离心管中低温保存,4-6小时内带回实验室中处理;
2)按以下步骤在细胞房超净台内进行:将标本移入100mm培养皿中,PBS清洗两遍去除血迹及破碎组织,挑选合适组织块转入新的培养皿中并加入少量PBS润湿,小心去除骨组织,将剩余韧带组织剪成1-2mm3大小组织块,在T25培养瓶中预先加入1.5ml完全培养基,使培养瓶底部完全被培养基润湿,倒置培养瓶后小心将组织块间隔1cm排列于培养瓶底部,倒置放入培养箱中4-6小时或过夜,待组织块完全贴附于培养瓶底部后将培养瓶翻转回来;
3)之后每3天更换培养基,每次1.5-2ml,10-14天可见组织块周围有细胞向外爬出,待组织块周围爬满细胞并向外生长后,使用0.25%含EDTA胰酶消化3-5分钟;
4)显微镜下观察细胞变圆并出现漂浮后加入等量含10%FBS完全培养基终止消化,吸管吹打并收集细胞悬液,弃去组织块后1000rpm/5min离心后收集细胞,完全培养基重悬细胞后移入T25培养瓶扩增,此次获得细胞视为P1代,按照1∶3比例继续扩增细胞至P3-6代后将细胞冻存备用;
所述永生化人后纵韧带细胞保留了后纵韧带细胞此前的特性,成骨诱导因子可诱导永生化后纵韧带细胞成骨,且OPLL患者来源的永生化后纵韧带细胞具有更强的成骨表型。
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