CN116536364A - 一种长链非编码rna loc102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法 - Google Patents
一种长链非编码rna loc102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法 Download PDFInfo
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Abstract
本发明涉及细胞基因技术领域,且公开了一种长链非编码RNA LOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法,分离SD大鼠源滑膜间充质干细胞;以人端粒酶催化亚单位重组载体转染的方法,制备永生化的滑膜间充质干细胞系;构建靶向LOC102546541的干扰siRNA,根据siRNA序列合成shRNA模板后构建重组质粒pLKO.1‑shRNA‑puro;将重组质粒与包装质粒psPAX2载体共同转染293T细胞,离心后获得包装shRNA的慢病毒;慢病毒转染细胞系,得到一种沉默LOC102546541永生化的大鼠源滑膜间充质干细胞。
Description
技术领域
本发明涉及细胞基因技术领域,具体为一种长链非编码RNA LOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法。
背景技术
间充质干细胞是一类具有自我更新和具有多向分化潜能的成体干细胞,在体外可以分化为骨、脂肪、神经等多种细胞。目前常用的分离培养方法主要有组织块贴壁法和酶消化法,方法非常繁琐,需要每次从活体组织中提取原代细胞。基因沉默是指生物体中特定基因由于种种原因不表达或是表达减少的现象。长链非编码RNA的基因沉默技术,常有四种方法,即RNA干扰法,反义寡核苷酸法,转录抑制法和基因敲除法。经初步验证,LOC102546541是一种同时存在与滑膜间充质干细胞和软骨细胞的致病基因,为了进一步研究该基因功能,常需要构建具有LOC102546541敲低的干细胞。构建时间长,金钱成本太大,是阻碍实验进展的重要因素。科研人员需要在每次原代提取干细胞的同时另外构建质粒或病毒载体转染细胞,而LncRNA本身为超过2000bp的非编码RNA,其载体构建较一般基因而言难度大,故载体构建成本较为昂贵。
间充质干细胞的原代提取技术和目标基因敲减技术均为成熟的生物科学研究手段,然而过程均非常繁琐,在这两者的基础上结合细胞永生化技术,可以构建致病基因沉默的、能够无限传代的干细胞。如论文《RhoA基因沉默联合脐带间充质干细胞移植脑损伤大鼠功能的恢复》报道了一种脐带间充质干细胞移植同时联合RNAi介导的RhoA基因沉默,观察两者对脑损伤大鼠恢复的影响,脐带间充质干细胞移植可明显改善重型颅脑损伤后大鼠的神经学功能,联合应用RhoA基因沉默有协同效果。本发明构建LOC102546541基因沉默永生化的大鼠滑膜间充质干细胞,可以在体外培养至少40-50代,在保持间充质干细胞干性的同时,沉默了长链非编码RNA基因,可供科研人员长期关注并研究该LncRNA的功能。
发明内容
(一)解决的技术问题
针对现有技术的不足,本发明提供了一种长链非编码RNA LOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法,通过构建沉默该基因的永生化滑膜间充质干细胞,可以构建致病基因沉默的、能够无限传代的干细胞。
该干细胞不仅方便科学家长期追踪LncRNA功能,并且能够用于组织工程技术治疗关节软骨缺损。
(二)技术方案
1.一种长链非编码RNA LOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法:
分离SD大鼠源滑膜间充质干细胞;以人端粒酶催化亚单位(hTERT)重组载体转染的方法,制备永生化的滑膜间充质干细胞系;构建靶向LOC102546541的干扰siRNA,合成shRNA模板后构建重组质粒pLKO.1-shRNA-puro;将重组质粒与包装质粒psPAX2载体共同转染293T细胞,获得慢病毒;慢病毒转染永生化细胞系,得到一种沉默LOC102546541永生化的大鼠源滑膜间充质干细胞。
优选的,所述从100-180g、6-8周龄的SD大鼠双侧膝关节周围的滑膜组织分离出滑膜间充质干细胞。
优选的,所述人端粒酶催化亚单位重组载体的构建用EcoRⅠ和NotⅠ分别对pCDH-CMVMCS-EF1-copGFP和pCI-neo-hTERT进行双酶切,DNA回收试剂盒回收纯化人端粒酶催化亚单位目的基因片段和pCDH-CMV-MCS-EF1-copGFP载体片段,T4-DNA连接酶对这两个片段进行定向连接。
优选的,所述干细胞的筛选与鉴定包括细胞形态梭形,成栅栏状生长,且在含有10%FBS的DMEM血清的培养基中能够稳定增殖;采用免疫荧光技术和流氏细胞仪检测干细胞表面分子,用试剂盒鉴定成骨分化、成脂分化、成软骨分化的能力。
优选的,所述重组质粒pLKO.1-shRNA-puro的制备包括通过构建LOC102546541对应的靶向干扰siRNA,根据其合成shRNA模板,连接到慢病毒载体pLKO.1-puro,构建重组质粒pLKO.1-shRNA-puro。
优选的,所述得到一种沉默LOC102546541永生化的大鼠源滑膜间充质干细胞包括将重组质粒与包装质粒psPAX2载体共同转染293T细胞,48-72h后收集上清,离心去除细胞碎片,继续离心上清浓缩病毒,用无血清培养基重悬沉淀物得到包装携带shRNA的慢病毒,将慢病毒转染永生化的滑膜间充质干细胞系后制得。
(三)有益的技术效果
分离SD大鼠源滑膜间充质干细胞;以人端粒酶催化亚单位重组载体转染的方法,制备永生化的滑膜间充质干细胞系;构建靶向干扰siRNA,合成shRNA模板后构建重组质粒pLKO.1-shRNA-puro;将重组质粒与包装质粒psPAX2载体共同转染293T细胞,获得慢病毒;慢病毒转染永生化细胞系,得到一种沉默LOC102546541永生化的大鼠源滑膜间充质干细胞。经动物实验表明,将该干细胞用于膝关节腔内原位注射剂,可以有效治疗早期关节软骨缺损,表现为关节软骨细胞的增殖,增加COL2的分泌以及减少MMP13的表达,从而达到治疗效果。
经过对LOC102546541基因功能初步验证,我们确信该基因在人体中参与到骨关节炎疾病发展中,且扮演重要角色。因此,对该基因的追踪性研究显得尤为重要。通过构建沉默该基因的永生化滑膜间充质干细胞将有利于该基因的机制研究。一旦对该基因致病的作用机制探索完成,后续可结合组织工程技术,制备用于治疗骨关节炎的生物制剂,因此本发明具有很强的临床转化意义。目前,实现对目标基因在某种细胞中的特殊功能的研究,至少需要用到干细胞原代提取技术:程序过于繁琐;LncRNA基因沉默技术:时间周期长,金钱成本太大。虽然这两种技术都有各自明显的缺点,但是在这两者的基础上结合细胞永生化技术,可以构建致病基因沉默的、能够无限传代的干细胞,将一劳永逸地为科学家提供便利。
附图说明
图1基因沉默的永生化滑膜间充质干细胞。
图2RT-qPCR检测结果。
图3micro-CT实验结果。
图4病理染色与免疫组化。
具体实施方式
1.滑膜间充质干细胞的采集
从100g、8周龄的SD大鼠双侧膝关节周围的滑膜组织分离出滑膜间充质干细胞。
将修剪过的组织块加入到10mL DMEM培养基,在37℃、5%CO2条件下消化14h,200目不锈钢网过滤小组织块,用100mL磷酸缓冲液清洗,然后将滤液加到离心管中,1000r/min离心10min,去除上清液,20mL磷酸缓冲液重悬,1000r/min离心10min,去除上清液,反复3次,收集细胞转入添加了10%的小牛血清的DMEM培养基,2天更换一次培养基,布满细胞时用0.05%胰蛋白酶处理,分离培养。
2.永生化细胞系的构建及鉴定
2.1构建人端粒酶催化亚单位重组载体
用EcoRⅠ和NotⅠ分别对pCDH-CMVMCS-EF1-copGFP和pCI-neo-hTERT进行双酶切,DNA回收试剂盒回收纯化3.4kb的人端粒酶催化亚单位目的基因片段和7.5kb的pCDH-CMV-MCS-EF1-copGFP载体片段,T4-DNA连接酶对这两个片段进行定向连接后,转化DH5α感受态细胞,挑取阳性克隆并提取质粒。
2.2人端粒酶催化亚单位重组载体转染
将待转染的细胞配制成8×105个细胞浓度接种,5%CO2和37℃培养,24h后用含人端粒酶催化亚单位重组载体的重组逆转录病毒感染,1周后用2μg/mL嘌呤霉素筛选,转染成功可获得细胞克隆。
2.3干细胞筛选与鉴定
细胞形态成梭形,成栅栏状生长,且在含有10%FBS的DMEM血清的培养基中能够稳定增殖;采用免疫荧光技术和流氏细胞仪检测干细胞表面分子,用试剂盒鉴定干细胞具有成骨分化、成脂分化、成软骨分化的能力。
3.永生化干细胞系装配RNA干扰基因
3.1构建靶向干扰siRNA
根据人类基因组数据库、RNA结合的Tm值及特异性的对比结果,设计LOC102546541对应的siRNA,并确保其有高度保守性。
3.2合成shRNA模板
根据siRNA序列,设计并合成2条shRNA模板,退火后将其插入Pgenesil-1质粒,再通过限制性酶切割和鉴定。
3.3重组质粒pLKO.1-shRNA-puro的构建
将shRNA模板,连接到慢病毒载体pLKO.1-puro,构建重组质粒pLKO.1-shRNA-puro。
3.4转染干细胞
将重组质粒与包装质粒psPAX2载体共同转染293T细胞,48h后收集上清,离心去除细胞碎片,继续离心上清浓缩病毒,用无血清培养基重悬沉淀物得到包装携带shRNA的慢病毒,将慢病毒转染永生化的滑膜间充质干细胞系。
4.制成
一种沉默LOC102546541永生化的大鼠源滑膜间充质干细胞。
5.基因沉默永生干细胞检测
5.1基因沉默永生干细胞的准备
取基因沉默永生干细胞接种于10%小牛血清的DMEM完全培养液中进行细胞培养,每隔三天更换培养液一次,5天左右细胞铺满瓶底90%,0.28%胰酶消化3min,800r/min离心5min,按1:2接种于培养瓶中,在37℃、5%CO2恒温培养箱中扩增培养。扩增培养的基因沉默永生化干细胞显微镜下观察结果如图1所示。
5.2实验动物分组
选择15只8周龄,280g-310g左右的SD大鼠,随机分为基因空白对照组(n=3)、假手术组(n=3)、关节软骨缺损组(n=3)、PBS组(n=3)和沉默永生干细胞组(n=3)。
5.3关节软骨缺损动物模型的构建与治疗
将所有15只大鼠的左侧膝关节设置为对照组,右侧膝关节设置为处理组。按照上述实验分组分别做不同处理,具体如下:空白对照组,不做任何处理;假手术组,切开并分离膝关节组织后立即缝合;关节软骨缺损组,用环钻磨损膝关节软骨,构建环形缺损(直径=3mm,深度=1mm);PBS组,在关节软骨缺损的基础上,用PBS注射治疗;沉默永生干细胞组,在关节软骨缺损的基础上,注射沉默永生化干细胞治疗。经过6周后,进行检测。
5.4实验结果的检测
①RT-PCR检测
动物模型构建6周后,每一组随机取3只大鼠的膝关节股骨组织,迅速置于液氮中,并放置过夜。隔日,从液氮中迅速取出组织,用组织剪分离关节软骨,置于1.5ml研磨试管中,加入预冷过的无酶研磨钢珠和800μLTrizol,于研磨机上研磨组织。待研磨完全后,转移至1.5ml无酶试管中,室温放置5min;加入氯仿200μL,室温放置3min;4℃,12000rpm离心15min;取上清液至新的离心管,加入0.5mL异丙醇,冰上静置10min;4℃,12000rpm离心10min;弃上清,加入75%无水乙醇1mL,4℃,7500rpm离心5min;弃上清,漂洗一次,以20μLDEPC的水溶解沉淀。设计并合成目标基因引物(表格1),按试剂盒进行PCR扩增及上机检测。每只大鼠的实验结果,以处理组(右侧膝关节)与对照组(左侧膝关节)的数据比值表示,实验结果如图2所示。
②小动物CT(micro-CT)
动物模型构建6周后,每一组随机取3只大鼠的膝关节股骨组织,将每只大鼠的股骨远端分离并固定在4%多聚甲醛中,然后使用Bruker Micro CT Skyscan 1276系统(Kontich,Belgium)进行扫描。使用以下扫描设置:体素大小为6.533712μm1,中等分辨率,85kV,200uA,1mmAl滤波器,积分时间为384ms。密度测量值校准为制造商提供的羟基磷灰石钙(CaHA)体模。使用制造商的评估软件进行分析。使用CT分析仪软件(版本1.18.8.0)进行二维分析。实验结果如图3所示。
③病理染色和免疫组化
上述组织用于小动物CT检测后,用EDTA脱钙1-2个月,然后用多聚甲醛固定,并包埋在石蜡中。将大鼠股骨切片切成4μm的切片,并按照制造商的说明进行番红固绿染色和马松染色。此外,我们根据说明书中的推荐比例,使用稀释的COL2抗体(Proteintech 1:200)和MMP13抗体(Abcam 1:200)对股骨切片进行了免疫组织化学检查。实验结果如图4所示。
表1 RT-qPCR引物
综上所述,本发明构建了LOC102546541沉默的永生化滑膜间充质干细胞系,该细胞系具备稳定传代功能,可供科学家长期研究。同时,经动物实验发现,将该干细胞作为关节腔内注射剂,可以有效治疗早期关节软骨缺损。其治疗效果主要表现为:促进关节软骨细胞分泌二型胶原,减少基质金属蛋白酶13(MMP13)的生成,从而使得软骨基质的合成速率大于降解速率,继而修复软骨组织。据不完全统计,我国目前每年因关节软骨缺损或骨关节炎,接受膝关节置换术的患者数量超过50万;尤其随着我国医保制度的不断完善和人口老龄化,这一数字持续上升,到2023年底约有1亿人患病。如果,这种现象保持不变或继续增长,将对社会造成继肿瘤疾病之后最大的疾病负担。因此,在早期发展新技术治疗关节软骨缺损显得尤为重要。未来,本发明将有实现临床转化的巨大潜力。
本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书界定。
Claims (6)
1.一种长链非编码RNALOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法,其特征在于:
分离SD大鼠源滑膜间充质干细胞;以人端粒酶催化亚单位(hTERT)重组载体转染的方法,制备永生化的滑膜间充质干细胞系;构建靶向干扰siRNA,根据siRNA序列合成shRNA模板后构建重组质粒pLKO.1-shRNA-puro;将重组质粒与包装质粒psPAX2载体共同转染293T细胞,获得慢病毒;慢病毒转染永生化细胞系,得到一种沉默LOC102546541永生化的大鼠源滑膜间充质干细胞。
2.根据权利要求1所述的一种长链非编码RNALOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法,其特征在于:所述从100-180g、6-8周龄的SD大鼠双侧膝关节周围的滑膜组织分离出滑膜间充质干细胞。
3.根据权利要求1所述的一种长链非编码RNALOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法,其特征在于:所述人端粒酶催化亚单位重组载体的构建用EcoRⅠ和NotⅠ分别对pCDH-CMVMCS-EF1-copGFP和pCI-neo-hTERT进行双酶切,DNA回收试剂盒回收纯化人端粒酶催化亚单位目的基因片段和pCDH-CMV-MCS-EF1-copGFP载体片段,T4-DNA连接酶对这两个片段进行定向连接。
4.根据权利要求1所述的一种长链非编码RNALOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法,其特征在于:所述干细胞的筛选与鉴定包括细胞形态梭形,成栅栏状生长,且在含有10%FBS的DMEM培养基中能够稳定增殖;采用免疫荧光技术和流氏细胞仪检测干细胞表面分子,试剂盒鉴定成骨分化、成脂分化、成软骨分化的能力。
5.根据权利要求1所述的一种长链非编码RNALOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法,其特征在于:所述重组质粒pLK O.1-shRNA-puro的制备包括通过构建LOC102546541对应的靶向干扰siRNA,根据其序列合成shRNA模板,连接到慢病毒载体pLKO.1-puro,构建重组质粒pLKO.1-shRNA-puro。
6.根据权利要求1所述的一种长链非编码RNALOC102546541基因沉默永生化鼠源滑膜间充质干细胞的制备方法,其特征在于:所述得到一种沉默LOC102546541永生化的大鼠源滑膜间充质干细胞包括将重组质粒与包装质粒psPAX2载体共同转染293T细胞,48-72h后收集上清,离心去除细胞碎片,继续离心上清浓缩病毒,用无血清培养基重悬沉淀物得到包装携带shRNA的慢病毒,将慢病毒转染永生化的滑膜间充质干细胞系后制得。
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