CN110684803B - 一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法 - Google Patents
一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法 Download PDFInfo
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Abstract
本发明提供了一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法,涉及生物技术领域,所述制备方法包括以下步骤:(1)Drp1‑shRNA慢病毒载体的构建;(2)骨髓细胞的慢病毒体外感染;(3)感染细胞植入小鼠建立模型。本发明率先采用内眦静脉注射方法构建骨髓细胞Drp1基因特异性敲减小鼠模型,并对移植的骨髓细胞数目和病毒感染条件进行了优化,具有快速建模、成功率高、特异性好和经济成本低的优势。不仅为研究Drp1基因免疫学功能提供了动物模型,也为其他骨髓特异性基因模式动物的制备提供了新的依据。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法。
背景技术
近年来,免疫与疾病的研究成为国际热点,尤其在肿瘤、二型糖尿病和心脑血管疾病等领域,有关免疫细胞功能的研究逐年增多。骨髓是机体内的造血组织,主要分布于长骨骨腔和骨松质内,可生成各种干细胞,对于维持造血和免疫功能有至关重要的作用。骨髓来源的免疫细胞主要包括,T细胞、B细胞、NK细胞、单核细胞和巨噬细胞等。其中,T淋巴细胞是来源于骨髓的多能干细胞,可在胸腺中发育成熟,进而具有对抗感染和击杀肿瘤细胞的功能。B细胞也是由骨髓造血干细胞分化而来,可通过分泌抗体对抗病毒感染。NK细胞属于自然杀伤细胞,发育成熟后可发挥免疫调节和击杀癌细胞的作用。单核细胞来源于骨髓造血干细胞,在血液循环中仍属于未发育完全细胞。而单核细胞一旦进入组织器官,便会进一步分化为巨噬细胞,是机体最重要的炎症效应细胞。这些骨髓来源的免疫细胞,不仅对于维持机体正常免疫功能意义重大,也在多数疾病进程中发挥关键作用。对于多数研究者来说,缺少骨髓组织基因敲减或过表达动物模型,是制约相关研究的重要因素。
线粒体是真核细胞内能量合成的关键场所,广泛参与细胞的增殖、分化、凋亡和衰老等调控。线粒体稳态与骨髓来源免疫细胞的分化密切相关,线粒体结构功能异常可引起机体免疫系统的紊乱。而作为线粒体分裂最核心的调控因子,线粒体分裂动力蛋白1(Dynamin-related protein 1,Drp1)在维持线粒体稳态中发挥重要作用。研究发现,Drp1表达和活性变化,可影响线粒体分裂融合动态平衡,进而调控巨噬细胞和T细胞的分化,参与机体免疫调控。而要想在体内研究Drp1对机体免疫功能和相关疾病的调控作用,就必须构建骨髓细胞Drp1特异性敲减的动物模型。目前,骨髓特异性基因模式动物主要依赖于Cre-LoxP基因重组技术,该技术利用骨髓特异性表达Cre重组酶小鼠和Loxp转基因小鼠杂交方式获得,其主要缺点是实验周期长、经济成本高、工作量大,且组织特异性不高。
发明内容
有鉴于此,本发明的目的在于提供一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法。本发明提供的方法具有快速建模、成功率高、特异性好和经济成本低的优势。不仅为研究Drp1基因免疫学功能提供了动物模型,也为其他骨髓特异性基因模式动物的制备提供了新的依据。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法,包括以下步骤:
(1)Drp1-shRNA慢病毒载体的构建
特异性敲减Drp1基因的shRNA的引物如下:
Drp1正向引物如SEQ ID No.1所示,
Drp1反向引物如SEQ ID No.2所示,
通过退火,产生双链shRNA,扩增后回收,并与特异性酶切载体连接获得重组质粒,包装所述重组质粒,获得Drp1-shRNA慢病毒;
(2)骨髓细胞的慢病毒体外感染
取分离的小鼠骨髓细胞,加入Drp1-shRNA慢病毒瞬时转染,并加入10ug/ml聚凝胺和10mmol/L的4-羟乙基哌嗪乙磺酸,静置10min后,800g离心30min,弃上清,无菌PBS重悬细胞,得到Drp1-shRNA病毒感染的骨髓细胞;
(3)感染细胞植入小鼠建立模型
取小鼠,用CO 60进行8Gay辐照量的处理,辐照后的小鼠经内眦静脉注射Drp1-shRNA病毒感染的骨髓细胞。
优选地,所述步骤(2)中骨髓细胞的供体小鼠为C57BL/6-GFP小鼠。
优选地,所述步骤(2)中Drp1-shRNA慢病毒瞬时转染的感染复数为15。
优选地,所述步骤(3)中Drp1-shRNA慢病毒感染的骨髓细胞的移植量为2×106个。
优选地,所述步骤(3)中的小鼠为8周龄C57BL/6小鼠。
本发明提供了一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法。本发明通过慢病毒体外感染和骨髓移植(Bone marrow transplantation,BMT)方式构建骨髓细胞Drp1基因特异性敲减小鼠。本发明中动物骨髓移植主要原理是从供体小鼠中分离骨髓细胞,通过静脉注射回输给经辐照清除自身骨髓的小鼠,受体动物可在2周左右重建骨髓系统和造血功能,较传统骨髓移植多使用尾静脉注射法,小鼠尾静脉较细,穿刺和注射困难较大,成功率不高的方法,本发明采用内眦静脉注射,容易进行静脉注射操作,成功率高。慢病毒(Lentivirus)感染是当前基础医学研究中常用的基因干预手段,可通过感染靶细胞将目的基因整合到宿主染色体上,进而实现对目的基因的稳定敲减或过表达,但由于体内和体外环境存在差异,长时间体外培养可能增加骨髓细胞分化或死亡的风险,为避免上述问题,采用本发明的离心转速和时间,能够促使细胞和病毒沉淀后充分接触,以提高转染效率,缩短感染所需时间。
而且,利用本发明的离心方法提高了感染效率。通过本发明对模型构建中使用的病毒感染复数(MOI)和回输细胞量,在保证骨髓替换率的前提下,既保证了Drp1基因的干扰效率,也减少了病毒用量,节省模型构建成本。最终将感染后的骨髓细胞通过内眦静脉注射回输给经辐照清除自身骨髓的小鼠。模型小鼠骨髓及其分化的免疫细胞均可稳定表达绿色荧光蛋白(GFP),可通过流式细胞术和荧光显微镜进行示踪和定量分析。
附图说明
图1为骨髓移植细胞数量和感染病毒MOI的细胞的GFP阳性率图;其中,A为不同数量骨髓细胞(由C57BL/6-GFP小鼠分离,未感染病毒)移植2周后,检测外周血单个核细胞GFP阳性率;B为Lenti-GFP病毒载体,以不同MOI的病毒浓度感染骨髓细胞,检测GFP阳性细胞率;
图2为以MOI=15浓度的病毒感染骨髓细胞,体外鉴定病毒感染后对Drp1基因的干扰效果图;其中,图左侧为Real-time PCR检测感染细胞Drp1基因mRNA表达的变化;图右侧为Western-blot检测感染细胞Drp1蛋白表达的变化;
图3为2×106个C57BL/6-GFP小鼠骨髓细胞(病毒感染后)回输2周后,EmptyVector组和Drp1-shRNA组小鼠外周血以及各组织脏器中GFP阳性细胞测定图;A为流式细胞术检测外周血单个核细胞、骨髓和脾脏细胞中GFP阳性比率;B为荧光显微镜观察Drp1-shRNA组小鼠的肝脏、肺脏、心脏和脂肪组织中GFP阳性细胞,蓝色DAPI标记细胞核、绿色为GFP阳性细胞、红色自发荧光用来指示组织形态;
图4为2×106个C57BL/6-GFP小鼠骨髓细胞(EmptyVector和Drp1-shRNA慢病毒感染,MOI=15)回输2周后,受体小鼠骨髓细胞、脾脏细胞和腹腔巨噬细胞中Drp1基因敲减效果的鉴定图;其中,图上方从左至右依次为Real-time PCR检测骨髓细胞、脾脏细胞和腹腔巨噬细胞中Drp1的mRNA表达变化;图下方从左至右依次为Western-blot检测骨髓细胞、脾脏细胞和腹腔巨噬细胞中Drp1蛋白表达变化。
具体实施方式
本发明提供了一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法,包括以下步骤:
(1)Drp1-shRNA慢病毒载体的构建
特异性敲减Drp1基因的shRNA的引物如下:
Drp1正向引物如SEQ ID No.1所示,
Drp1反向引物如SEQ ID No.2所示,
通过退火,产生双链shRNA,扩增后回收,并与特异性酶切载体连接获得重组质粒,包装所述重组质粒,获得Drp1-shRNA慢病毒;
(2)骨髓细胞的慢病毒体外感染
取分离的小鼠骨髓细胞,加入Drp1-shRNA慢病毒瞬时转染,并加入10ug/ml聚凝胺和10mmol/L的4-羟乙基哌嗪乙磺酸,静置10min后,800g离心30min,弃上清,无菌PBS重悬细胞,得到Drp1-shRNA病毒感染的骨髓细胞;
(3)感染细胞植入小鼠建立模型
取小鼠,用CO 60进行8Gay辐照量的处理,辐照后的小鼠经内眦静脉注射Drp1-shRNA病毒感染的骨髓细胞。
在本发明中,所述骨髓细胞的慢病毒体外感染中采用离心的方法是为了促使细胞和病毒沉淀后充分接触,以提高转染效率,缩短感染所需时间。
在本发明中,所述特异性敲减Drp1基因的shRNA序列是通过PCR合成,所述扩增shRNA序列的引物分别为:
Drp1正向引物如SEQ ID No.1所示。在本发明中,所述SEQ ID No.1的序列如下所示:GATCCGGCAATTGAGCTAGCGTATATCTTCCTGTCAGAATATACGCTAGCTCAATTGCCTTTTTG;
Drp1反向引物如SEQ ID No.2所示。在本发明中,所述SEQ ID No.2的序列如下所示:AATTCAAAAAGGCAATTGAGCTAGCGTATATTCTGACAGGAAGATATACGCTAGCTCAATTGCCG。
在本发明中,所述转移载体优选为Lenti-H1-shRNA-puro vector载体,酶切载体的位点优选为EcoRI和BamHI。本发明对所述包装质粒的种类没有特殊限定,采用常规包装慢病毒的质粒即可。
在本发明中,所述的骨髓移植的小鼠经钴-60辐照的小鼠,所述的照射剂量优选为8Gy。在本发明中,所述小鼠经钴-60辐照的目的是清除小鼠受体体内自身骨髓细胞,然后将慢病毒体外感染的骨髓细胞移植给小鼠受体,,经过重建后,嵌合体小鼠骨髓及其分化的免疫细胞会稳定表达Drp1-shRNA慢病毒感染。
在本发明中,所述骨髓移植采用的方式为内眦静脉注射。
下面将结合本发明中的实施例,对本发明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1
1.Drp1基因敲减慢病毒载体制备
Drp1引物设计如表1:
表1 Drp1引物
通过退火,产生双链shRNA。酶切载体Lenti-H1-shRNA-puro vector(酶切位点EcoRI,BamHI),使之线性化。双链shRNA连接线性化载体。转化感受态细胞后涂板子,挑克隆测序。测序正确的载体进行扩增,提取质粒包装慢病毒,获得Drp1-shRNA慢病毒载体,空载体(EmptyVector)作为对照。
2.骨髓移植细胞数量和感染病毒MOI的筛选
2.1骨髓移植细胞的数量筛选
(1)骨髓细胞分离方法:C57BL/6-GFP小鼠脱颈处死后,剥离四肢胫骨和股骨,剪断骨头两端,1ml注射器吸取无菌PBS反复冲洗骨腔,70目滤网过滤后,离心收集细胞。1ml红细胞裂解液30s,再次离心后即可得到骨髓原代细胞。
(2)骨髓移植方法:取40只C57BL/6小鼠(受体),分为5组,每组8只。照射前一周开始饲喂含庆大霉素(32万U/L)和红霉素(250mg/L)的无菌水。小鼠CO 60照射(8Gay剂量)24小时内,异氟烷吸入麻醉。取新鲜分离的C57BL/6-GFP小鼠骨髓细胞悬液,调整细胞浓度至5×107、2.5×107、1×107、5×106和1.5×106个cells/ml,分别用1ml注射器经内眦静脉丛回输200μl。最终,每只动物回输细胞量分别为1×107、5×106、2×106、1×106和3×105。
(3)骨髓移植2周后检测外周血中GFP阳性细胞比率。综合考虑骨髓置换率和细胞用量,选择2×106作为模型构建中细胞用量。见图1。
2.细胞感染病毒MOI的筛选
DMEM培养基中加入10μg/ml的聚凝胺(增强感染效率)和10mmol/L的4-羟乙基哌嗪乙磺酸(调节酸碱度),配置成病毒稀释液。取新鲜分离的C57BL/6小鼠骨髓细胞,以不同浓度Lenti-GFP阳性病毒瞬时感染,MOI=5,10,15,30,45,60。静置10min后,800g离心30min。流式细胞仪检测,以未感染病毒的细胞作参照,比较各浓度MOI病毒感染后细胞的GFP阳性比率。选择MOI=15作为细胞感染时的病毒MOI。见图1。
3.病毒感染细胞植入小鼠构建嵌合体
3.1骨髓细胞慢病毒感染
取新鲜分离的C57BL/6-GFP小鼠骨髓细胞,分为两份,分别加入Empty Vector和Drp1-shRNA病毒瞬时转染(病毒MOI=15),并加入10μg/ml的聚凝胺和10mmol/L的4-羟乙基哌嗪乙磺酸,静置10min后,800g离心30min。弃上清,1ml无菌PBS重悬细胞,浓度为1×107/ml。感染完成后,分别提取细胞mRNA和蛋白。Real-time PCR结果表明,Drp1基因mRNA的抑制效率达到91.0%。免疫印迹(Western-blot)结果显示,与Empty Vector组相比,Drp1-shRNA组蛋白表达显著下调,见图2。
3.2感染细胞的骨髓移植
取8周龄C57BL/6小鼠10只,分为两组,每组5只,CO 60(8Gay剂量)辐照。24小时内,第一组小鼠经内眦静脉注射200μl细胞悬液(Empty Vector感染细胞,浓度为1×107/ml),第二组小鼠经内眦静脉注射200μl细胞悬液(Drp1-shRNA病毒感染细胞,浓度为1×107/ml)。2周后受体小鼠骨髓重建,可获得骨髓细胞Drp1基因特异性敲减的嵌合体小鼠,EmptyVector组小鼠作为模型组的平行对照。
4.骨髓细胞Drp1基因特异性敲减小鼠的鉴定
4.1小鼠骨髓来源免疫细胞的分离
(1)外周血细胞的分离
取新鲜分离的小鼠尾静脉外周血1滴,加入红细胞裂解液1ml,1500转速离心5min。细胞沉淀再用1ml红细胞裂解液清洗一次。
(2)脾脏细胞的分离
取新鲜分离的小鼠脾脏组织,剥除周围结缔组织,PBS漂洗后,置于70目细胞筛,轻轻挤压冲洗获细胞。800g离心10min,弃去上清,加入红细胞裂解液2ml,离心后获得脾细胞。
(3)骨髓细胞的分离
小鼠脱颈处死后,剥离四肢胫骨和股骨,剪断骨头两端,1ml注射器吸取无菌PBS反复冲洗骨腔,70目滤网过滤后,离心收集细胞。1ml红细胞裂解液30s,再次离心后即可得到骨髓细胞。
(4)腹腔巨噬细胞的分离
小鼠腹腔注射3%的巯基乙酸盐培养液2ml,处理48小时,诱导巨噬细胞生成。麻醉后脱颈处死,消毒灭菌,腹腔注射无菌PBS,注射器反复冲洗腹腔,离心收集并培养细胞,4小时后贴壁的即为巨噬细胞。
4.2外周血、脾脏和骨髓中GFP阳性细胞测定
以野生(WT)C57BL/6小鼠相应组织样本为参照,流式细胞术检测Empty Vector组和Drp1-shRNA组小鼠外周血、脾脏和骨髓中GFP阳性细胞比率。根据检测结果可知,EmptyVector组小鼠的外周血细胞、脾脏细胞和骨髓细胞中GFP阳性细胞比率分别为92.6%、87.1%和91.4%;Drp1-shRNA组小鼠的外周血细胞、脾脏细胞和骨髓细胞中GFP阳性细胞比率分别为93.8%、86.0%和90.6%见图3。结果表明,EmptyVector组和Drp1-shRNA组小鼠的骨髓细胞置换率均较高。
4.3肝脏、肺脏、心脏和脂肪组织GFP阳性细胞检测
分离Drp1-shRNA组小鼠的肝脏、脂肪、肺脏和心脏组织,石蜡包埋切片,厚度约5-7μm。脱蜡后,DAPI对细胞核进行染色,自发红色荧光指示组织形态,荧光显微镜观察动物体肝脏、脂肪、肺脏和心脏组织的GFP阳性细胞情况。结果表明,Drp1-shRNA组小鼠的心脏、肺脏、肝脏和脂肪组织中表达了C57BL/6-GFP小鼠(供体)骨髓来源的免疫细胞,且这些细胞经过体外Drp1-shRNA的慢病毒感染,见图3。
4.4Drp1基因mRNA和蛋白水平鉴定
提取EmptyVector组和Drp1-shRNA组小鼠脾脏组织、骨髓细胞和腹腔巨噬细胞中总RNA,反转获得cDNA模板,然后采用Real-time PCR检测mRNA转录水平的变化,其中PCR检测用的Drp1引物序列如表2:
表2 PCR检测用的Drp1引物
结果表明与EmptyVector组相比,Drp1-shRNA组基因表达显著降低,见图4。
提取EmptyVector组和Drp1-shRNA组小鼠脾脏组织、骨髓细胞和腹腔巨噬细胞中的总蛋白,定量后,采用Western-blot检测Drp1蛋白表达变化。结果表明与EmptyVector组相比,Drp1-shRNA组蛋白表达也显著降低,见图4。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国人民解放军第四军医大学
<120> 一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法
<141> 2019-10-14
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<213> 人工序列(Artificial Sequence)
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Claims (3)
1.一种构建骨髓细胞Drp1基因特异性敲减小鼠模型的方法,其特征在于,包括以下步骤:
(1)Drp1-shRNA慢病毒载体的构建
特异性敲减Drp1基因的shRNA的引物设计如下:
Drp1正向引物如SEQ ID No.1所示,
Drp1反向引物如SEQ ID No.2所示,
通过退火,产生双链shRNA,扩增后回收,并与特异性酶切载体连接获得重组质粒,包装所述重组质粒,获得Drp1-shRNA慢病毒;
(2)骨髓细胞的慢病毒体外感染
取分离的小鼠骨髓细胞,加入Drp1-shRNA慢病毒瞬时转染,并加入10ug/ml聚凝胺和10mmol/L的4-羟乙基哌嗪乙磺酸,静置10min后,800g离心30min,弃上清,无菌PBS重悬细胞,得到Drp1-shRNA病毒感染的骨髓细胞;
所述Drp1-shRNA慢病毒瞬时转染的感染复数为15;
(3)感染细胞植入小鼠建立模型
取小鼠,用CO 60进行8Gay辐照量的处理,辐照后的小鼠经内眦静脉注射Drp1-shRNA病毒感染的骨髓细胞;
所述Drp1-shRNA慢病毒感染的骨髓细胞的移植量为2×106个。
2.根据权利要求1所述的方法,其特征在于,所述步骤(2)中骨髓细胞的供体小鼠为C57BL/6-GFP小鼠。
3.根据权利要求1所述的方法,其特征在于,所述步骤(3)中的小鼠为8周龄C57BL/6小鼠。
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