CN111961689A - 一种过表达pd-l1基因的间充质干细胞株及其构建方法和应用 - Google Patents
一种过表达pd-l1基因的间充质干细胞株及其构建方法和应用 Download PDFInfo
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Abstract
本发明实施例公开了一种过表达PD‑L1基因的间充质干细胞株及其构建方法和应用,属于生物医学技术领域。所述方法包括以下步骤:1)构建携带有PD‑L1基因的重组慢病毒;2)将所述携带有PD‑L1基因的重组慢病毒转染间充质干细胞,经嘌呤霉素筛选,得到过表达PD‑L1基因的间充质干细胞株。本发明的过表达PD‑L1基因的间充质干细胞株静脉注射类风湿性关节炎小鼠模型后,能够显著减轻类风湿性关节炎症状,降低关节损伤程度,并调节炎性因子的表达,抑制炎症反应,在治疗类风湿性关节炎疾病中有显著性疗效。
Description
技术领域
本发明涉及生物医学技术领域,尤其涉及一种基因工程改造的过表达细胞程序性死亡配体1(PD-L1)基因的间充质干细胞株及其构建方法、及该过表达PD-L1基因的间充质干细胞株在治疗类风湿性关节炎中的应用。
背景技术
类风湿性关节炎(Rheumatoid arthritis,RA)是一种以关节软骨病变、滑膜细胞增生为特征的***性、炎性自身免疫性疾病。患病关节及其周围组织呈现进行性破坏,并致使受损关节发生功能障碍。类风湿性关节炎是一种常见而折磨人的免疫性疾病,发生原因是机体的免疫***紊乱,把自身组织当成了敌人进行攻击,导致关节疼痛、肿胀和破坏,并损伤皮肤、眼、肺等器官。类风湿性关节炎被称之为不死的癌症,当前治疗手段还不能根治该疾病。随着生物技术的发展和完善,干细胞治疗已经为治疗类风湿性关节炎提供了有效的治疗手段。
间充质干细胞(MSCs)是一类具有自我复制能力的多潜能细胞。在一定条件下,它可以分化成多种功能细胞,医学界称为“万用细胞”。同时也表现出抗炎和免疫抑制的特性,从而对炎症性和自身免疫性疾病有潜在的治疗作用。间充质干细胞这些独特的性质赋予它们治疗风湿性疾病的潜能。
干细胞治疗就是提取人体组织中特定的干细胞,在一定的诱导条件下,让这些细胞分化成我们所需的细胞,植入人体,干细胞就会在人体受损部位工作起来,治疗疾病。目前,干细胞治疗在免疫性相关疾病中的作用已经受到了人们的广泛关注。在利用干细胞治疗类风湿性关节炎研究中,已有研究证明间充质干细胞可以抑制T细胞增殖活化,调节相关免疫细胞及因子,缓解炎症反应。同时,间充质干细胞在体外可诱导分化为骨、软骨、脂肪、腱、肌肉等细胞,可聚集至关节病变部位,对关节组织进行损伤修复。
细胞程序性死亡配体1(PD-L1)是大小为40kDa的I型跨膜蛋白。正常情形下免疫***会对聚集在***或脾脏的外来抗原产生反应,诱导具有抗原特异性的T细胞产生。而表达在细胞上的PD-L1可以通过与细胞程序化死亡受体-1(PD-1)结合,阻断T细胞受体信号来抑制其免疫效应,对维持外周免疫耐受和调节T细胞活化具有重要作用。
目前,国内外尚无将MSC细胞与PD-L1结合来治疗关节炎的报道。而现阶段传统治疗关节炎相关疾病的手段及传统药物,虽然其能缓解类风湿性关节炎的症状,但治疗效果不明显,且病情复发率较高,副作用较大。因此,将MSC细胞与PD-L1有效结合,使MSC细胞更大程度的抑制T细胞增殖和活化,进而调节免疫效应与炎症反应。此治疗方法有望成为治疗类风湿性关节炎的新手段,能够有效改善类风湿关节炎的症状,降低不良反应率,安全性好,并有助于患者回归正常生活,改善患者的生活质量。
发明内容
为此,为了克服现有技术的上述不足,本发明的目的在于提供一种过表达PD-L1基因的间充质干细胞株及其构建方法和应用,包括对间充质干细胞进行基因工程改造,建立稳定过表达PD-L1基因的间充质干细胞株;利用慢病毒质粒对干细胞基因转导和基因修饰来制备上述细胞株的方法;以及该细胞株在治疗类风湿性关节炎中的应用。
为了实现上述目的,本发明实施例提供如下技术方案:
根据本发明实施例的第一方面,本发明实施例提供一种过表达PD-L1基因的间充质干细胞株的构建方法,包括以下步骤:
1)构建携带有PD-L1基因的重组慢病毒;
2)将所述携带有PD-L1基因的重组慢病毒转染间充质干细胞,经嘌呤霉素筛选,得到过表达PD-L1基因的的间充质干细胞株。
进一步地,所述PD-L1基因的核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQID NO.8所示。
进一步地,所述间充质干细胞为骨髓间充质干细胞。
进一步地,所述间充质干细胞的细胞表型是CD44、CD29和HLA-1。
根据本发明实施例的第二方面,本发明实施例提供了由上述方法构建得到的过表达PD-L1基因的间充质干细胞株。
根据本发明实施例的第三方面,本发明实施例提供了上述的过表达PD-L1基因的间充质干细胞株在制备治疗类风湿关节炎药物中的应用。
本发明实施例具有如下优点:
本发明提供的间充质干细胞经PD-L1基因修饰,通过嘌呤霉素筛选,得到稳定过表达PD-L1基因的间充质干细胞株。过表达PD-L1基因的间充质干细胞株静脉注射类风湿性关节炎动物模型后,能够显著减轻类风湿性关节炎症状,降低关节损伤程度,并调节炎性因子的表达,抑制炎症反应,在治疗类风湿性关节炎疾病中有显著性疗效。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案,下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图仅仅是示例性的,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图引伸获得其它的实施附图。
图1为骨髓间充质干细胞的细胞表型鉴定图。
图2为骨髓间充质干细胞三系分化能力检测。
图3为pCDH-PDL1慢病毒载体图谱。
图4为GFP标记基因检测pCDH-PD-L1转染骨髓干细胞效率。
图5为RT-PCR法分析PD-L1基因的表达。
图6为流式细胞术验证PD-L1转染骨髓干细胞情况。
图7为注射间充质干细胞治疗后小鼠爪子红肿程度。
图8为小鼠肢关节CIA评分结果。
图9为小鼠肢关节HE染色病理检测结果。
图10为小鼠模型血清中炎症因子水平的变化,A:PBS组,B:注射干细胞组,C:注射PD-L1干细胞组。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效,显然,所描述的实施例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。
以下实施例中的实验方法,如无特殊说明,均为本领域常规方法。下述实施例中所用的实验材料,如无特殊说明,均为自常规生化试剂商店购买得到。
实施例1
间充质干细胞的制备与鉴定
1、间充质干细胞培养
(1)抽取骨髓10-15ml(或正常人手术肋骨取骨髓3-5ml),每毫升骨髓加100u肝素抗凝,充分混匀后,保存于4℃冰箱,24小时内分离骨髓单核细胞(MNC)。骨髓先加入等量PBS或Hanks液(或培养液)混匀后,以1份淋巴细胞分离液加2份稀释骨髓的比例,将稀释骨髓缓慢加入ficoll液面上(密度1.077),水平离心机20℃条件下500g(2000r/min)离心25~30分钟。从界面上取出的MNC用PBS液洗涤2~3次后,加适量培养液计数。细胞数量达到106~107/ml后即进行原代培养,按5×106/ml浓度接种于50ml培养瓶中(1×106/cm2培养瓶),每瓶含5ml L-DMEM完全培养液。在体积分数为5%的CO2和37℃条件下静止培养,3d后换液去除非贴壁细胞,以后每3~4d半量换液1次,10~12d细胞达90%融合时传代。
(2)传代培养:首先全部吸出培养液,用无Ca2+、Mg2+PBS液洗涤二次,加入37℃预热的0.25%(2.5g/L,0.25%)胰蛋白酶(含1mmol/L EDTA,即用1mmol/L-EDTA-Na2配制)消化1~2分钟,吸去胰酶,以残留的胰酶继续消化,倒置显微镜下观察,细胞开始皱缩时(细胞开始变圆)加入完全培养液(3ml左右)终止胰酶作用。吸管反复吹打后将细胞收集于15ml离心管中,1500r/min离心10分钟后弃上清,再用PBS液洗涤二次彻底洗去混有的胰蛋白酶。
(3)将细胞再悬浮于培养液中,按2~5×105/ml再次接种传代于新的培养瓶中。当这些细胞生长接近融合层时,即得到骨髓间充质干细胞。
2、流式标记法检测骨髓间充质干细胞表面标志
上述培养的第五代骨髓间充质干细胞加入CD44、CD29、CD34、CD38、HLA-1和HLA-DR抗体染色。流式细胞仪检测结果显示已经分离培养获得纯度>95%的骨髓间充质干细胞(MSC),该细胞的表型为CD44、CD29和HLA-1阳性,CD34、CD38、HLA-DR阴性(图1)。
3、骨髓间充质干细胞的成骨、成软骨和成脂三系分化能力
取上述培养的第三代骨髓间充质干细胞消化传代后,以约105/孔接种到6孔板中,成脂、成骨和成软骨分化及对照各2孔,待细胞贴壁后,向分化组加入相应的分化培养液。成脂分化培养基中含1μmol/L***、0.5mmol/L IBMX、60μmol/L吲哚美辛和5U/mL胰岛素,成骨分化培养基中含0.1μmol/L***、10mmol/Lβ-甘油磷酸盐和50μmol/L抗坏血酸,成软骨分化微小细胞团在TGF-β1、***(Dex)及维生素C(Vit C)等诱导下分化,每3~4d换液1次,分化2周后,用4%低聚甲醛将细胞固定,分别用油红O染色、碱性磷酸酶和甲苯胺蓝染色,于显微镜下观察,结果如图2所示,成脂诱导分化1周后,显微镜下可见胞质内有少量脂滴形成,2周后,油红O染色可见胞质内有较多油滴空泡;成骨分化14d,碱性磷酸酶染色后可见胞浆内大量的棕黑色钙结节;成软骨分化,诱导后细胞呈软骨细胞样形态,甲苯胺蓝染色后课件软骨细胞内呈***异染颗粒。
通过体外形态学、流式细胞仪细胞表型检测以及三系分化能力的的鉴定,均表明我们获得了骨髓间充质干细胞。
实施例2
过表达PD-L1基因的骨髓间充质干细胞株PD-L1-MSC的构建
1、PD-L1基因PCR引物的设计与扩增
在NCBI中查询到小鼠PD-L1基因序列,核苷酸序列信息如SEQ ID NO.1所示,氨基酸序列信息如SEQ ID NO.8所示。利用Primer5.0软件设计PD-L1引物。
上游引物PD-L1-F:tctagaATGAGGATATTTGCTGGCATT(SEQ ID NO:2)
下游引物PD-L1-R:gaattcTTACGTCTCCTCGAATTGTG(SEQ ID NO:3)
以小鼠肺cDNA为模板,采用上述设计好的引物,对小鼠PD-L1基因进行PCR扩增。
2、慢病毒包装pCDH-PD-L1质粒
将PD-L1序列构建到慢病毒载体pCDH-CMV-MCS-EF1-copGFP-T2A-Puro上,得到表达pCDH-PD-L1的慢病毒质粒。
构建方法:将上述PCR产物连接到T载体上进行测序,测序正确的质粒命名为T-PD-L1。用XbaI和EcoRI对慢病毒载体质粒和T-PD-L1质粒分别进行双酶切,然后用胶回收试剂盒对目的条带进行胶回收。将胶回收的慢病毒载体pCDH-CMV-MCS-EF1-copGFP-T2A-Puro与T-PD-L1按照摩尔比例为1:7混合,加入T4连接酶与T4连接酶缓冲液(共10μl体系),在4℃连接过夜。连接得到的质粒转化到大肠杆菌stbl3中,然后放置在37℃培养过夜。从转化有pCDH-PD-L1的平板中随机挑取2个单克隆,分别加入含有氨苄青霉素(50μg/ml)的5ml LB培养基中进行扩培,12-16小时后进行质粒提取,并用XbaI和EcoRI进行酶切鉴定。酶切正确的质粒送上海生工进行测序进一步确定。测序正确的即为pCDH-PD-L1表达质粒,表达质粒图谱如图3所示。需要进一步包装成慢病毒。
3、将表达PD-L1的慢病毒感染骨髓间充质干细胞,慢病毒感染完成之后,通过嘌呤霉素筛选稳定表达PD-L1基因的骨髓间充质干细胞株。
4、将筛选后稳定表达PD-L1基因的骨髓间充质干细胞株大规模体外培养,培养到足够数量之后,对细胞进行消化即加入适量胰蛋白酶消化,最后用磷酸盐缓冲液清洗三遍之后,注射进入类风湿性关节炎小鼠模型治疗疾病。
实施例3
骨髓间充质干细胞株转染PD-L1基因的表达鉴定
1、荧光显微镜观察慢病毒pCDH-PD-L1转染骨髓干细胞效率
慢病毒pCDH-PD-L1转染骨髓干细胞的24小时后,用新鲜完全培养基替换原培养基,继续培养,48小时后于荧光倒置显微镜观察。结果如图4所示,发现PD-L1的转染效率大于90%。
2、RT-PCR法分析PD-L1基因的表达
Trizol法提取转染pCDH-PD-L1的骨髓干细胞的RNA,反转录成cDNA。同时提取转染pCDH-CMV-MCS-EF1-copGFP-T2A-Puro空载体的干细胞的RNA,反转录成cDNA,做对照试验。设计引物,同时以这两种cDNA为模板,扩增PD-L1基因片段,同时内参基因GAPDH。引物信息如下表所示。
RT-PCR结果如图5所示,分别以转染pCDH-PD-L1病毒的第1、3、7天骨髓干细胞的cDNA为模板进行PCR扩增,均可以检测到PD-L1的基因片段,而转染空载体的骨髓干细胞中没有出现PD-L1的基因片段,说明PD-L1基因已经成功***到干细胞基因组并表达。
3、流式细胞术验证PD-L1是否转染成功
上述转染PD-L1和转染空载体的骨髓间充质干细胞中加入PD-L1抗体处理检测。流式细胞仪检测结果显示PD-L1成功转染骨髓间充质干细胞(结果如图6所示)。
实施例4
过表达PD-L1的骨髓间充质干细胞株治疗类风湿性关节炎的疗效
1、构建小鼠类风湿性关节炎模型
(1)雄性Balb/c小鼠,6周龄;
(2)首次免疫,利用2mg/ml牛Ⅱ型胶原蛋白溶液与等体积的完全弗式佐剂冰上乳化,得到1mg/ml最终胶原,注射剂量为100μl,于小鼠尾巴根部皮下注射;
(3)在首次免疫后第21天,进行二次免疫,取2mg/ml牛Ⅱ型胶原蛋白溶液与等体积的不完全弗式佐剂冰上乳化,得到1mg/ml的最终胶原,注射剂量为50μl,于小鼠尾巴根部皮下注射;
(4)正常条件饲养小鼠,并每天观察小鼠爪子的关节肿胀和红斑的出现情况,根据Wood法对关节炎的严重程度进行分级评估。
2、过表达PD-L1的骨髓间充质干细胞株治疗类风湿性关节炎
(1)成功构建类风湿性关节炎小鼠模型后,将小鼠模型随机地分为三组,分别为注射PBS组、注射未转染PD-L1正常干细胞组、注射过表达PD-L1干细胞组,每组6只小鼠;
(2)小鼠在二次免疫后开始进行治疗处理,注射PBS组、注射未转染PD-L1正常干细胞组、注射过表达PD-L1干细胞组,三组小鼠分别注射200μl PBS注射液,200μl(2×106个)骨髓间充质干细胞,200μl(2×106个)过表达PD-L1的骨髓间充质干细胞。注射时间点为二次免疫后开始第一次注射,每周注射一次,连续两周;
(3)在第二次免疫后第28天后,小鼠注射戊巴比妥钠进行麻醉,处死,取小鼠外周血、爪子及踝关节等组织进行下一步实验检测。
3、过表达PD-L1基因的骨髓间充质干细胞株在类风湿性关节炎小鼠模型中的治疗效果
(1)小鼠模型建立后,每天给小鼠拍照,观察小鼠爪子红肿程度,及使用游标卡尺测量小鼠爪子厚度,并记录分析。结果显示,对比注射PBS组的小鼠,注射干细胞组的小鼠爪子红肿程度,明显减轻,注射过表达PD-L1间充质干细胞组的小鼠爪子红肿程度基本消失(图7)。在第36天PBS对照组的CIA评分达到最高12.3分,而MSC组的评分为6.8分,过表达PD-L1 MSC组的评分为3.5分,具体评分曲线如图8所示。结果表明应用过表达PD-L1基因的间充质干细胞株可以有效改善关节炎小鼠的关节肿胀畸形。
(2)过表达PD-L1基因的骨髓间充质干细胞株对关节病理学变化的影响
HE(Haematoxylin and eosin)染色,检测关节腔狭窄程度,观察关节炎性细胞浸润程度及关节破坏程度。具体步骤为:将小鼠关节组织包埋切片,脱蜡至水,苏木素伊红染色,使用不同浓度酒精进行脱水,二甲苯进行透明,滴加半滴中性树胶封片。于显微镜下400×镜观察染色效果及拍照。结果如图9所示,注射PBS组的小鼠关节关节破坏程度严重;注射干细胞组的小鼠关节损伤程度较PBS组稍轻;而注射过表达PD-L1干细胞组的小鼠关节损伤最小,关节炎性细胞浸润程度及关节破坏程度最低。可见过表达PD-L1间充质干细胞可以有效改善关节炎小鼠的关节肿胀,减轻关节软骨损伤。
实施例5
过表达PD-L1的骨髓间充质干细胞株对血清中炎症因子水平的影响
酶联免疫吸附试验(Elisa)检测过表达PD-L1的骨髓间充质干细胞对小鼠血清中炎症因子水平的影响。本实验中我们采用IL-1,TNF Elisa试剂盒检测小鼠血清中炎症因子水平。具体步骤如下:已稀释好的ABC和TMB显色液在加入酶标板孔前都应预先在37℃中平衡至少30min。将浓度为500pg/ml、250pg/ml、125pg/ml、62.5pg/ml、31.3pg/ml、15.6pg/ml的标准品各0.1ml依次加入一排6孔中,1孔只加样品稀释液的作为零孔。样品孔中加入样品100μl。酶标板加上盖,37℃反应90min。甩去酶标板内液体,再对着吸水纸拍几下,不洗。将准备好的抗体工作液按每孔0.1ml依次加入(TMB空白显色孔除外)。37℃反应60min。将准备好的ABC工作液按每孔0.1ml依次加入(TMB空白显色孔除外),37℃反应30min。按每孔90μl依次加入已在37℃平衡30min的TMB显色液,37℃避光反应25min。按每孔0.1ml依次加入TMB终止液,顺序同加TMB,此时蓝色立转黄色。用酶标仪在450nm测定OD值。结果如图10所示,过表达PD-L1间充质干细胞组的IL-1和TNF水平显著低于PBS对照组和MSC组。这些结果提示,经PD-L1修饰的骨髓间充质干细胞可以有效的改善RA的炎性反应。
虽然,上文中已经用一般性说明及具体实施例对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
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Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn
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Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn
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Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
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Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu
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Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp
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Claims (6)
1.一种过表达PD-L1基因的间充质干细胞株的构建方法,其特征在于,包括以下步骤:
1)构建携带有PD-L1基因的重组慢病毒;
2)将所述携带有PD-L1基因的重组慢病毒转染间充质干细胞,经嘌呤霉素筛选,得到过表达PD-L1基因的间充质干细胞株。
2.根据权利要求1所述的过表达PD-L1基因的间充质干细胞株的构建方法,其特征在于,所述PD-L1基因的核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.8所示。
3.根据权利要求1所述的过表达PD-L1基因的间充质干细胞株的构建方法,其特征在于,所述间充质干细胞为骨髓间充质干细胞。
4.根据权利要求1所述的过表达PD-L1基因的间充质干细胞株的构建方法,其特征在于,所述间充质干细胞的细胞表型是CD44、CD29和HLA-1。
5.权利要求1-4任一项所述方法构建得到的过表达PD-L1基因的间充质干细胞株。
6.权利要求5所述的过表达PD-L1基因的间充质干细胞株在制备治疗类风湿关节炎药物中的应用。
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