CN114657144A - 一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备及其应用 - Google Patents
一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备及其应用 Download PDFInfo
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Abstract
本发明涉及医学治疗技术领域,尤其涉及一种同时分泌人IL‑10和TGF‑β的可示踪脐带间充质干细胞的制备及其应用,包括以下步骤:步骤S01:构建pLV‑IL10(P2A)TGFb‑EGFP重组慢病毒质粒。本发明在经过测定之后,得出充质干细胞可分泌多种免疫抑制性的细胞因子、生长因子和趋化因子,可以广泛的应用在骨关节炎的治疗,且能够表达荧光报告基因,可通过活体成像技术实时追踪其在关节腔中的位置,存活时间,迁移,从而更方便准确地检测细胞在关节腔的药代动力学相关参数。
Description
技术领域
本发明涉及医学治疗技术领域,特别是涉及一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备及其应用。
背景技术
骨关节炎是一种常见的慢性退行性关节疾病,临床表现为关节疼痛、变形,行走障碍。骨关节炎患者关节软骨损伤后,形成局灶性病变,关节周围组织如滑膜逐渐被侵蚀,导致长期的滑膜慢性炎症。滑膜炎症虽然不是骨关节炎发病的直接诱导因素,但滑膜炎能够加剧软骨破坏,加快骨关节炎的发展。骨关节炎的早期到晚期,均伴有滑膜炎的发生与发展,关节腔镜和组织学检查表明滑膜是骨关节炎中发生炎性反应的主要部位。A型巨噬细胞是滑膜组织的主要组成细胞成分,在滑膜炎症中起到关键的作用。巨噬细胞可分为M1和M2,M1可简述为促炎性细胞,而M2是抑制免疫应答的调节性细胞,在不同的细胞因子微环境中二者可以相互极化。体外培养的环境下M2型巨噬细胞可通过IFN-g或LPS刺激极化为促炎性的巨噬细胞M1,在IL-10、IL-4、TGF-β等细胞因子的诱导下M1巨噬细胞可极化为免疫抑制性的M2巨噬细胞。M2型巨噬细胞不仅可以抑制炎性细胞免疫反应,还可以促进组织修复。通过治疗手段将滑膜组织中的巨噬细胞向M2极化将是有效治疗骨关节炎的重要手段之一。
间充质干细胞可分泌多种免疫抑制性的细胞因子、生长因子和趋化因子等可以调节免疫、促进组织损伤修复等功效。人脐带中间充质干细胞含量、增殖能力优于骨髓间充质干细胞,免疫原性比骨髓间充质干细胞低,并且具有取材方便,无伦理学争议等优点,使其成为理想的再生医学研究和应用的功能细胞。近年来,脐带间充质干细胞在骨关节炎的治疗方面进行了大量的尝试,包括全身性的回输和局部的注射等治疗方式,均取得了良好的治疗效果。虽然间充质干细胞通过分泌多种免疫调节因子来抑制关节炎症性微环境,但是往往关键的免疫调节细胞因子分泌水平较低,导致了骨关节炎症状缓解周期较长,治疗效果往往需要依赖于多次的治疗。因而治疗成本较高,患者也会感到不便。另外,局部注射间充质干细胞的药代动力学参数仍然不是非常明确,需要通过更直观的手段去记录和检测间充质干细胞在局部的存活周期、聚集密度、迁移特点等药代动力学参数,为干细胞临床应用提供更加科学和严谨的数据支撑。
本发明构建了一种能够同时分泌IL-10和TGF-β细胞因子的脐带间充质干细胞,同时该细胞表达绿色荧光报告蛋白。文献报道,IL-10和TGF-β可以有效诱导M2型巨噬细胞,是巨噬细胞向M2极化的重要调节细胞因子,并且IL-10是抑制处于活化的T细胞、B细胞功能最强的调节性细胞因子。TGF-β能够诱导CD4+CD25-T细胞分化为Foxp3+调节性T细胞(Treg)细胞,并且维持Foxp3+Treg细胞的免疫抑制功能的重要细胞因子。因此,分泌高水平IL-10和TGF-β的脐带间充质干细胞用于骨关节炎的治疗,它将发挥以下几方面的作用:
(1)释放抑制性细胞因子刺激滑膜组织巨噬细胞向M2抑制性巨噬细胞极化,抑制免疫应答,降低炎性细胞因子水平,促进组织修复;
(2)诱导和维持Treg细胞在关节腔的形成和聚集,抑制促炎性免疫细胞反应,调节炎性免疫微环境;
(3)通过干细胞自身的的归巢和释放的生长因子等对损伤组织进行修复。以上三点作用机制将从根本上治愈骨关节炎临床。同时,本发明中构建的同时分泌IL-10和TGF-β脐带间充质干细胞能够表达荧光报告基因(绿色荧光蛋白EGFP),可通过活体成像技术实时追踪其在关节腔中的位置、存活时间,迁移等等,从而更方便、准确地检测细胞在关节腔的药代动力学相关参数。
发明内容
本发明的目的是提供一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备及其应用,解决了现有技术中缺少一种可同时分泌IL-10和TGF-β,其便于追踪的脐带间充质干细胞的问题。
为了实现上述目的,本发明采用了如下技术方案:
一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备,包括以下步骤:
步骤S01:构建pLV-IL10(P2A)TGFb-EGFP重组慢病毒质粒:通过获取5'端含有EcoRI酶切位点和克扎克序列,3'端包含BamH I酶切位点的IL-10-P2A-TGF-β1多顺反子,来获取IL-10-P2A-TGF-β1序列;将合成基因IL10(P2A)TGFb连接至pLV-EF1a-EGFP(2A)Puro质粒的EcoRI和BamH I之间,得到重组质粒pLV-IL10(P2A)TGFb-EGFP;
步骤S02:重组包装慢病毒:通过将293T细胞以5*105个细胞/孔的密度接种于含有2mL慢病毒包装培养基的6孔培养板中,置于37℃,5%,CO2条件下孵育细胞过夜,保证转染时细胞密度应达到60-70%汇合,完成细胞接种;然后第二天将所有质粒用Opti-MEM稀释至1ug/ul,进行转染制得重组慢病毒Lenti-IL10(P2A)TGF-β-EGFP,最后进行滴定测定,验证细胞体积;
步骤S03:制备IL-10/TGF-β分泌型脐带间充质干细胞:
(1)将人脐带间充质干细胞(hUC-MSC)按照1*105/孔接种至含2mL无血清干细胞培养基的6孔板中,将细胞培养板置37℃,5%CO2培养箱孵育;
(2)待细胞密度达到60-70%,按照感染复数MOI=5加入上述步骤S02中包装的慢病毒Lenti-IL10(P2A)TGF-β-EGFP;
(3)重组慢病毒感染hUC-MSC细胞24小时后,通过荧光显微镜观察绿色荧光蛋白表达情况,判断感染是否成功,并且通过流式细胞检测EGFP的阳性细胞比例;
(4)若结果显示,细胞均表达绿色荧光,阳性率达到93%,则说明成功制备IL-10/TGF-β分泌型脐带间充质干细胞hUC-MSC-IL-10/TGF-β,可通过荧光示踪。
优选的,所述步骤S01中,5'端含有EcoR I酶切位点和克扎克序列,3'端包含BamHI酶切位点的IL-10-P2A-TGF-β1多顺反子的获取,具体包括以下步骤:
(1)从NCBI获取IL-10(Homo sapiens)的CDS序列;
(2)从NCBI获取TGF-β1(Homo sapiens)的CDS序列;
(3)获取P2A序列;
(4)根据上述三个序列,设计出5'端含有EcoR I酶切位点和克扎克序列,3'端包含BamH I酶切位点的IL-10-P2A-TGF-β1多顺反子序列。
优选的,重组质粒pLV-IL10(P2A)TGFb-EGFP的具体操作方法如下:
(1)目的基因酶切;
(2)目的基因和线性化质粒回收;
(3)获得含重组慢病毒质粒pLV-IL10(P2A)TGFb-EGFP的连接产物;
(4)转化后进行质粒抽提,再经过双酶切鉴定和测序进行验证。
优选的,转染制得重组包装慢病毒后,将收集上清转移至无菌的离心管,按照Lenti-X Concentrator:上清液1:3比例加入Lenti-X Concentrator,4℃孵育过夜,完成病毒浓缩;还需加入DMEM基础培养基重悬病毒沉淀,50ul/管分装,-80℃冰箱保存备用,限制分装病毒冻融次数。
优选的,所述重组包装慢病毒滴度测定的具体操作方法为:
(1)293T细胞按照2*105/孔铺板24孔板;
(2)按0.01ul、0.1ul、1ul三个梯度病毒浓缩液加入293T细胞;
(3)48h后流式细胞术检测EGFP阳性的细胞比例;
(4)按公式计算滴度:Titer=(2*105*EGFP阳性率)/加入病毒体积*1000TU/ml。
优选的,所述IL-10和TGF-β分泌水平检测,包括以下步骤:
(1)将野生型hUC-MSC和hUC-MSC-IL-10/TGF-β细胞分别按照5*104/孔接种至24孔板中,每种细胞接种3个重复孔;
(2)将细胞培养板置37℃,5%CO2培养箱孵育72小时后,收集培养上清,5000rpm,离心10分钟,去除细胞碎片;
(3)根据IL-10和TGF-β酶联吸附测定试剂盒操作步骤检测细胞上清中IL-10和TGF-β的水平;
(4)若结果显示,hUC-MSC-IL-10/TGF-β细胞上清中分泌高水平的IL-10和TGF-β,而对照组hUC-MSC细胞培养上清中几乎检测不到IL-10和TGF-β的表达,则表明间充质干细胞通过基因修饰可分泌特定的多种免疫抑制性的细胞因子、生长因子和趋化因子。
本发明至少具备以下有益效果:
在经过测定之后,得出充质干细胞通过基因修饰可稳定分泌多种免疫抑制性的细胞因子、生长因子和趋化因子,可以广泛的应用在骨关节炎的治疗,且能够表达荧光报告基因,可通过活体成像技术实时追踪其在关节腔中的位置,存活时间,迁移,从而更方便准确地检测细胞在关节腔的药代动力学相关参数。
附图说明
为了更清楚地说明本发明实施例技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,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,还可以根据这些附图获得其他的附图。
图1为重组质粒pLV-IL10(P2A)TGFb-EGFP示意图;
图2为琼脂糖凝胶电泳示意图;
图3为Lenti-IL10(P2A)TGF-β-EGFP感染的人脐带间充质干细胞示意图;
图4为三个样品流式细胞检测EGFP阳性细胞比例示意图;
图5为细胞因子释放水平检测示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例一
参照图1-5,一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备及其应用,包括以下步骤:
步骤S01:构建pLV-IL10(P2A)TGFb-EGFP重组慢病毒质粒:通过获取5'端含有EcoRI酶切位点和克扎克序列,3'端包含BamH I酶切位点的IL-10-P2A-TGF-β1多顺反子,来获取IL-10-P2A-TGF-β1序列;将合成基因IL10(P2A)TGFb连接至pLV-EF1a-EGFP(2A)Puro质粒的EcoR I和BamH I之间,得到重组质粒pLV-IL10(P2A)TGFb-EGFP;
步骤S02:重组包装慢病毒:通过将293T细胞以5*105个细胞/孔的密度接种于含有2mL慢病毒包装培养基的6孔培养板中,置于37℃,5%,CO2条件下孵育细胞过夜,保证转染时细胞密度应达到60-70%汇合,完成细胞接种;然后第二天将所有质粒用Opti-MEM稀释至1ug/ul,进行转染制得重组慢病毒Lenti-IL10(P2A)TGF-β-EGFP,最后进行滴定测定,验证细胞体积;
步骤S03:制备IL-10/TGF-β分泌型脐带间充质干细胞:
(1)将人脐带间充质干细胞(hUC-MSC)按照1*105/孔接种至含2mL无血清干细胞培养基的6孔板中,将细胞培养板置37℃,5%CO2培养箱孵育;
(2)待细胞密度达到60-70%,按照感染复数MOI=5加入上述步骤S02中包装的慢病毒Lenti-IL10(P2A)TGF-β-EGFP;
(3)重组慢病毒感染hUC-MSC细胞24小时后,通过荧光显微镜观察绿色荧光蛋白表达情况,判断感染是否成功,并且通过流式细胞检测EGFP的阳性细胞比例;
(4)若结果显示,细胞均表达绿色荧光,阳性率达到85%以上,则说明成功制备IL-10/TGF-β分泌型脐带间充质干细胞hUC-MSC-IL-10/TGF-β,可通过荧光示踪;如图3和图4所示;
本实施例中:在经过测定之后,得出充质干细胞通过基因修饰可稳定分泌多种免疫抑制性的细胞因子、生长因子和趋化因子,可以广泛的应用在骨关节炎的治疗,且能够表达荧光报告基因,可通过活体成像技术实时追踪其在关节腔中的位置,存活时间,迁移,从而更方便准确地检测细胞在关节腔的药代动力学相关参数。
实施例二
参照图1-5,构建pLV-IL10(P2A)TGFb-EGFP重组慢病毒质粒具体包括:
步骤A:5'端含有EcoR I酶切位点和克扎克序列,3'端包含BamHI酶切位点的IL-10-P2A-TGF-β1多顺反子的获取,具体包括以下步骤:
(1)从NCBI获取IL-10(Homo sapiens)的CDS序列:
ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA
(2)从NCBI获取TGF-β1(Homo sapiens)的CDS序列:
ATGCCGCCCTCCGGGCTGCGGCTGCTGCCGCTGCTGCTACCGCTGCTGTGGCTACTGGTGCTGACGCCTGGCCGGCCGGCCGCGGGACTATCCACCTGCAAGACTATCGACATGGAGCTGGTGAAGCGGAAGCGCATCGAGGCCATCCGCGGCCAGATCCTGTCCAAGCTGCGGCTCGCCAGCCCCCCGAGCCAGGGGGAGGTGCCGCCCGGCCCGCTGCCCGAGGCCGTGCTCGCCCTGTACAACAGCACCCGCGACCGGGTGGCCGGGGAGAGTGCAGAACCGGAGCCCGAGCCTGAGGCCGACTACTACGCCAAGGAGGTCACCCGCGTGCTAATGGTGGAAACCCACAACGAAATCTATGACAAGTTCAAGCAGAGTACACACAGCATATATATGTTCTTCAACACATCAGAGCTCCGAGAAGCGGTACCTGAACCCGTGTTGCTCTCCCGGGCAGAGCTGCGTCTGCTGAGGCTCAAGTTAAAAGTGGAGCAGCACGTGGAGCTGTACCAGAAATACAGCAACAATTCCTGGCGATACCTCAGCAACCGGCTGCTGGCACCCAGCGACTCGCCAGAGTGGTTATCTTTTGATGTCACCGGAGTTGTGCGGCAGTGGTTGAGCCGTGGAGGGGAAATTGAGGGCTTTCGCCTTAGCGCCCACTGCTCCTGTGACAGCAGGGATAACACACTGCAAGTGGACATCAACGGGTTCACTACCGGCCGCCGAGGTGACCTGGCCACCATTCATGGCATGAACCGGCCTTTCCTGCTTCTCATGGCCACCCCGCTGGAGAGGGCCCAGCATCTGCAAAGCTCCCGGCACCGCCGAGCCCTGGACACCAACTATTGCTTCAGCTCCACGGAGAAGAACTGCTGCGTGCGGCAGCTGTACATTGACTTCCGCAAGGACCTCGGCTGGAAGTGGATCCACGAGCCCAAGGGCTACCATGCCAACTTCTGCCTCGGGCCCTGCCCCTACATTTGGAGCCTGGACACGCAGTACAGCAAGGTCCTGGCCCTGTACAACCAGCATAACCCGGGCGCCTCGGCGGCGCCGTGCTGCGTGCCGCAGGCGCTGGAGCCGCTGCCCATCGTGTACTACGTGGGCCGCAAGCCCAAGGTGGAGCAGCTGTCCAACATGATCGTGCGCTCCTGCAAGTGCAGCTGA
(3)获取P2A序列:
GGA AGC GGA GCT ACT AAC TTC AGC CTG AAG CAG GCT GGA GAC GTG GAG AACCCT GGA CCT
(4)根据上述三个序列,设计出5'端含有EcoR I酶切位点和克扎克序列,3'端包含BamH I酶切位点的IL-10-P2A-TGF-β1多顺反子序列:
GAATTCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAAC GGAAGC GGA GCT ACT AAC TTC AGC CTG AAG CAG GCT GGA GACGTG GAG AAC CCT GGA CCT
ATGCCGCCCTCCGGGCTGCGGCTGCTGCCGCTGCTGCTACCGCTGCTGTGGCTACTGGTGCTGACGCCTGGCCGGCCGGCCGCGGGACTATCCACCTGCAAGACTATCGACATGGAGCTGGTGAAGCGGAAGCGCATCGAGGCCATCCGCGGCCAGATCCTGTCCAAGCTGCGGCTCGCCAGCCCCCCGAGCCAGGGGGAGGTGCCGCCCGGCCCGCTGCCCGAGGCCGTGCTCGCCCTGTACAACAGCACCCGCGACCGGGTGGCCGGGGAGAGTGCAGAACCGGAGCCCGAGCCTGAGGCCGACTACTACGCCAAGGAGGTCACCCGCGTGCTAATGGTGGAAACCCACAACGAAATCTATGACAAGTTCAAGCAGAGTACACACAGCATATATATGTTCTTCAACACATCAGAGCTCCGAGAAGCGGTACCTGAACCCGTGTTGCTCTCCCGGGCAGAGCTGCGTCTGCTGAGGCTCAAGTTAAAAGTGGAGCAGCACGTGGAGCTGTACCAGAAATACAGCAACAATTCCTGGCGATACCTCAGCAACCGGCTGCTGGCACCCAGCGACTCGCCAGAGTGGTTATCTTTTGATGTCACCGGAGTTGTGCGGCAGTGGTTGAGCCGTGGAGGGGAAATTGAGGGCTTTCGCCTTAGCGCCCACTGCTCCTGTGACAGCAGGGATAACACACTGCAAGTGGACATCAACGGGTTCACTACCGGCCGCCGAGGTGACCTGGCCACCATTCATGGCATGAACCGGCCTTTCCTGCTTCTCATGGCCACCCCGCTGGAGAGGGCCCAGCATCTGCAAAGCTCCCGGCACCGCCGAGCCCTGGACACCAACTATTGCTTCAGCTCCACGGAGAAGAACTGCTGCGTGCGGCAGCTGTACATTGACTTCCGCAAGGACCTCGGCTGGAAGTGGATCCACGAGCCCAAGGGCTACCATGCCAACTTCTGCCTCGGGCCCTGCCCCTACATTTGGAGCCTGGACACGCAGTACAGCAAGGTCCTGGCCCTGTACAACCAGCATAACCCGGGCGCCTCGGCGGCGCCGTGCTGCGTGCCGCAGGCGCTGGAGCCGCTGCCCATCGTGTACTACGTGGGCCGCAAGCCCAAGGTGGAGCAGCTGTCCAACATGATCGTGCGCTCCTGCAAGTGCAGCTGAGGATCC
步骤B:重组质粒pLV-IL10(P2A)TGFb-EGFP的具体操作方法如下:
(1)酶切
目的基因酶切:将合成的IL-10-P2A-TGF-β1基因片段1ug,加入1ul限制性内切酶EcoR I(NEB,R0145S)和1ul限制性内切酶BamH I(NEB#R3101),5ulBuffer(NEB),最后加入双蒸水使整个反应体积达到50ul,置于37℃水浴锅孵育1小时,同样的方法双酶切质粒pLV-EF1a-EGFP(2A)Puro得到线性化骨架质粒;
(2)目的基因和线性化质粒回收
所需试剂1*TAE:4.84gTris(solarbio,#T8060-100g)、0.744g Na2EDTA.2H2O(沪试,#10009717)、1.142ml冰醋酸(国药,#10000218)充分溶解到适量ddH2O中,定容到1L后使用;琼脂糖(Sigma-Aldrich)琼脂糖凝胶回收试剂盒:普通琼脂糖凝胶DNA回收试剂盒(天根,#DP209-02);DNA loading:6x DNA loading(全式金,#GH101);
实验步骤如下:
A:1%琼脂糖凝胶制备
称取0.5g琼脂糖,加入50mL 1*TAE溶液后混合均匀,微波炉中煮沸2min后加入凝胶槽中进行冷凝;
B:琼脂糖凝胶电泳
将所有样品加入DNA loading至1*后加入点样孔后,120V电泳30min;
C:目的条带切胶
在紫外切胶仪下将含目的条带的琼脂糖凝胶切下转移到新的1.5mL离心管中;
D:目的条带回收
根据琼脂糖凝胶回收试剂盒步骤操作回收目的片段,测定浓度后保存在-20℃。
(3)连接
所需试剂
连接试剂盒:Ligation highVer.2(TOYOBO,#LGK-201)
实验步骤步骤如下:
A:反应体系
配制如下连接反应体系:组分,体积,Ligation high Ver.210μL,线性化载体*2.5μL,目的基因片段*7.5μL,回收的目的基因片段和线性化载体按照10:1(摩尔比)加入反应体系;
B:连接
混匀后置16℃,反应30min;
C:储存
反应结束后获得含重组慢病毒质粒pLV-IL10(P2A)TGFb-EGFP的连接产物立即使用或者保存在-20℃备用;
(4)转化
所需试剂:DH5α感受态细胞(康体生命,#KTSM101L);LB液体培养基:10g胰蛋白胨(Sigma-Aldrich,#T9410-250G)、5g酵母提取物(Sigma-Aldrich,#T9410-250G)、10g NaCl(沪试,#10019308)加入ddH2O溶解后调节pH7.0定容到1L,121℃、15min高压灭菌后冷却至室温使用;LB固体培养基:10g胰蛋白胨(Sigma-Aldrich,#T9410-250G)、5g酵母提取物(Sigma-Aldrich,#T9410-250G)、10gNaCl(沪试,#10019308)后调节pH 7.0,然后加入10g琼脂粉,定容到1L,121℃、15min高压灭菌后冷却至室温使用;抗生素:称取1g对应的抗生素粉末(sigma),充分溶解到20mL ddH2O,0.22μm除菌过滤洗头除菌后分装保存于-20℃;
实验步骤如下:
A:取一支DH5α感受态(100μl)置于冰上解冻,待感受态细胞解冻后枪头吸取10μL步骤(3)的连接产物加入感受态细胞中,轻轻吹打几次混匀后置于冰上冰浴20min;
B:冰浴结束,置于水浴锅中42℃热激90s后立刻放于冰上2min;
C:混合物中加入200μl无抗LB液体培养基,37℃,200rpm复苏1h;
D:吸取适量复苏后菌液涂布到含有100μg/mL含有对应抗生素的LB固体培养基上;
E:37℃培养箱中倒置过夜培养,可见LB固体培养基上出现单克隆菌落;
(5)质粒抽提
所需试剂:LB液体培养基:10g胰蛋白胨(Sigma-Aldrich,#T9410-250G)、5g酵母提取物(Sigma-Aldrich,#70161-500G)、10gNaCl(沪试,#10019308)加入ddH2O溶解后调节pH7.0定容到1L,121℃、15min高压灭菌后冷却至室温使用;质粒小提试剂盒:Omega质粒小提试剂盒(Omega,#D6943);抗生素:称取1g对应的抗生素粉末(sigma),充分溶解到20mLddH2O,0.22μm除菌过滤洗头除菌后分装保存于-20℃。
实验步骤如下:
A:挑取步骤(4)LB固体培养基生长的单克隆,加入5mL含有100μg/mL含有对应抗生素的LB液体培养基,在37℃细菌培养摇床200rpm过夜培养(12-16h);
B:取1.5~5ml菌液室温10000*g离心1min;
C:去上清,加250μl溶液Ⅰ(含RNase A),涡漩振荡器震荡至菌体完全悬浮;
D:加入250μl溶液Ⅱ,温和颠倒离心管4~6次,获得澄清的裂解液;最好室温孵育2min,剧烈混合会使剪切染色体DNA,降低质粒纯度(储存溶液Ⅱ应拧紧瓶盖);
E:加350μl溶液Ⅲ,温和颠倒数次混合,至出现白色絮状沉淀,室温10000*g离心10min;
F:特别小心吸取上清,移至洁净的装配好容积2ml离心管的吸收柱中,要保证没有吸入沉淀和细胞碎片,室温10000*g离心1min,至裂解物完全通过吸收;
G:弃滤过液,加500μl Buffer HB,10000*g离心1min,清洗吸收柱,除去残余蛋白质保证DNA的纯度;
H:如果接下来的步骤对质粒纯度要求不高,如酶消化法等其它筛选方法,此步可省略;
J:弃滤过液,再用100%乙醇稀释的750μl Wash Buffer清洗吸收柱,10000*g离心1min注意:Wash Buffer浓缩液用前必须用纯乙醇稀释,方法见标签如果经过冷冻,用前必须恢复室温;
K:此步可选:再加750μl WashBuffer清洗吸收柱;
L:必须将吸收柱10000*g离心1min确保乙醇被去除,乙醇会影响下面的步骤;
M:将吸收柱放入干净1.5ml离心管,加50-100μl(取决于需要的终浓度)无菌去离子水或TE缓冲液在滤膜上,10000*g离心5min;
N:测浓度后,于-20℃冰箱保存备用;
(6)双酶切鉴定
取步骤(5)抽提的重组质粒1ug,加入1ul限制性内切酶EcoRI(NEB,R0145S)和1ul限制性内切酶BamH I(NEB#R3101),5ulBuffer(NEB),最后加入双蒸水使整个反应体积达到50ul,置于37℃水浴锅孵育1小时,随后通过1%琼脂糖凝胶电泳进行鉴定,显示切下一条约1700bp的条带则证明目的条带连接上去;如图2所示;
(7)测序验证
将步骤(5)抽提的质粒送泓迅生物科技股份有限公司进行Sanger测序验证,测序结果与我们设计的IL-10-P2A-TGF-β1序列一致,则说明重组慢病度质粒pLV-IL10(P2A)TGFb-EGFP构建成功。
实施例三
参照图1-5,慢病毒包装,具体实施过程如下:
(1)第1天(下午):细胞接种
将293T细胞以5*105个细胞/孔的密度接种于含有2mL慢病毒包装培养基的6孔培养板中,置于37℃,5%CO2条件下孵育细胞过夜,保证转染时细胞密度应达到60-70%汇合;
(2)第2天(早上):转染
将所有质粒用Opti-MEM稀释至1ug/ul;
A:A管:250ul无血清Opti-MEM培养基稀释7ul Lipofectamine3000,涡旋混匀10s;
B:B管:250ul无血清Opti-MEM培养基稀释4ug质粒(pLV-IL10(P2A)TGFb-EGFP:2ul,psPAX2:1.2ul,pMD2G:0.8ul)以及6ul的P3000试剂,涡旋混匀10s;
C:制备脂质体-DNA复合物:将A管内容物转移至B管并充分混匀,室温孵育10min;加入复合物前,每孔去除1mL的培养基,使每孔总体积为1mL;
D:向每孔加入500uL的脂质体DNA复合物,小心地靠着孔壁加入液体,以免破坏细胞,轻轻搅拌培养板,使其分布均匀;
E:将培养板置于37℃,5%CO2条件下孵育8小时;
F:转染8小时后,更换每孔中的平板培养基,从每孔中小心吸去包含脂质体-DNA复合物的培养基,吸出的培养基用10%漂白溶液处理后再处置,替换成2mL预热的慢病毒包装培养基;
G:将培养板放回培养箱,在37℃,5%CO2下继续孵育;
H:转染48小时后,从每孔中收集2mL含重组慢病度的上清液,装入15mL锥形管中置于4℃保存;6孔板中补加2mL预热的慢病毒包装培养基,将培养板置于37℃,5%CO2条件下继续孵育;
J:转染约72小时后,从每孔中再次收集2mL含重组慢病度的上清液,与首次收集的上清液混合,使收集上清液总体积为4mL;
(3)室温下以2000rpm离心10分钟,去除细胞碎片,收集并转移上清液,弃去细胞沉淀,制得重组慢病毒Lenti-IL10(P2A)TGF-β-EGFP;病毒浓缩:将收集上清转移至无菌的离心管,按照Lenti-X Concentrator:上清液1:3比例加入Lenti-X Concentrator,4℃孵育过夜;4℃1500g离心60min,弃上清;加入DMEM基础培养基重悬病毒沉淀,50ul/管分装,-80℃冰箱保存备用,限制分装病毒冻融次数,以维持病毒滴度;
重组包装慢病毒滴度测定的具体操作方法为:
A:293T细胞按照2*105/孔铺板24孔板;
B:按0.01ul、0.1ul、1ul三个梯度病毒浓缩液加入293T细胞;
C:48h后流式细胞术检测EGFP阳性的细胞比例;
D:按公式计算滴度:Titer=(2*105*EGFP阳性率)/加入病毒体积*1000TU/ml。
实施例四
参照图1-5,IL-10和TGF-β分泌水平检测,包括以下步骤:
(1)将野生型hUC-MSC和hUC-MSC-IL-10/TGF-β细胞分别按照5*104/孔接种至24孔板中,每种细胞接种3个重复孔;
(2)将细胞培养板置37℃,5%CO2培养箱孵育72小时后,收集培养上清,5000rpm,离心10分钟,去除细胞碎片;
(3)根据IL-10和TGF-β酶联吸附测定试剂盒操作步骤检测细胞上清中IL-10和TGF-β的水平;
(4)若结果显示,hUC-MSC-IL-10/TGF-β细胞上清中分泌高水平的IL-10和TGF-β,而对照组hUC-MSC细胞培养上清中几乎检测不到IL-10和TGF-β的表达,则表明间充质干细胞通过基因修饰可分泌特定的多种免疫抑制性的细胞因子、生长因子和趋化因子;如图5所示;
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,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的范围内。本发明要求的保护范围由所附的权利要求书及其等同物界定。
Claims (6)
1.一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备,其特征在于,包括以下步骤:
步骤S01:构建pLV-IL10(P2A)TGFb-EGFP重组慢病毒质粒:通过获取5'端含有EcoR I酶切位点和克扎克序列,3'端包含BamH I酶切位点的IL-10-P2A-TGF-β1多顺反子,来获取IL-10-P2A-TGF-β1序列;将合成基因IL10(P2A)TGFb连接至pLV-EF1a-EGFP(2A)Puro质粒的EcoRI和BamH I之间,得到重组质粒pLV-IL10(P2A)TGFb-EGFP;
步骤S02:重组包装慢病毒:通过将293T细胞以5*105个细胞/孔的密度接种于含有2mL慢病毒包装培养基的6孔培养板中,置于37℃,5%,CO2条件下孵育细胞过夜,保证转染时细胞密度应达到60-70%汇合,完成细胞接种;然后第二天将所有质粒用Opti-MEM稀释至1ug/ul,进行转染制得重组慢病毒Lenti-IL10(P2A)TGF-β-EGFP,最后进行滴定测定,验证细胞体积;
步骤S03:制备IL-10/TGF-β分泌型脐带间充质干细胞:
(1)将人脐带间充质干细胞(hUC-MSC)按照1*105/孔接种至含2mL无血清干细胞培养基的6孔板中,将细胞培养板置37℃,5%CO2培养箱孵育;
(2)待细胞密度达到60-70%,按照感染复数MOI=5加入上述步骤S02中包装的慢病毒Lenti-IL10(P2A)TGF-β-EGFP;
(3)重组慢病毒感染hUC-MSC细胞24小时后,通过荧光显微镜观察绿色荧光蛋白表达情况,判断感染是否成功,并且通过流式细胞检测EGFP的阳性细胞比例;
(4)若结果显示,细胞均表达绿色荧光,阳性率达到93%,则说明成功制备IL-10/TGF-β分泌型脐带间充质干细胞hUC-MSC-IL-10/TGF-β,可通过荧光示踪。
2.根据权利要求1所述的一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备,其特征在于,所述步骤S01中,5'端含有EcoR I酶切位点和克扎克序列,3'端包含BamHI酶切位点的IL-10-P2A-TGF-β1多顺反子的获取,具体包括以下步骤:
(1)从NCBI获取IL-10(Homo sapiens)的CDS序列;
(2)从NCBI获取TGF-β1(Homo sapiens)的CDS序列;
(3)获取P2A序列;
(4)根据上述三个序列,设计出5'端含有EcoRI酶切位点和克扎克序列,3'端包含BamHI酶切位点的IL-10-P2A-TGF-β1多顺反子序列。
3.根据权利要求1所述的一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备,其特征在于,重组质粒pLV-IL10(P2A)TGFb-EGFP的具体操作方法如下:
(1)目的基因酶切;
(2)目的基因和线性化质粒回收;
(3)获得含重组慢病毒质粒pLV-IL10(P2A)TGFb-EGFP的连接产物;
(4)转化后进行质粒抽提,再经过双酶切鉴定和测序进行验证。
4.根据权利要求1所述的一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备,其特征在于,转染制得重组包装慢病毒后,将收集上清转移至无菌的离心管,按照Lenti-X Concentrator:上清液1:3比例加入Lenti-X Concentrator,4℃孵育过夜,完成病毒浓缩;还需加入DMEM基础培养基重悬病毒沉淀,50ul/管分装,-80℃冰箱保存备用,限制分装病毒冻融次数。
5.根据权利要求1所述的一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备,其特征在于,重组包装慢病毒滴度测定的具体操作方法为:
(1)293T细胞按照2*105/孔铺板24孔板;
(2)按0.01ul、0.1ul、1ul三个梯度病毒浓缩液加入293T细胞;
(3)48h后流式细胞术检测EGFP阳性的细胞比例;
(4)按公式计算滴度:Titer=(2*105*EGFP阳性率)/加入病毒体积*1000TU/ml。
6.根据权利要求1所述的一种同时分泌人IL-10和TGF-β的可示踪脐带间充质干细胞的制备,其特征在于,IL-10和TGF-β分泌水平检测,包括以下步骤:
(1)将野生型hUC-MSC和hUC-MSC-IL-10/TGF-β细胞分别按照5*104/孔接种至24孔板中,每种细胞接种3个重复孔;
(2)将细胞培养板置37℃,5%CO2培养箱孵育72小时后,收集培养上清,5000rpm,离心10分钟,去除细胞碎片;
(3)根据IL-10和TGF-β酶联吸附测定试剂盒操作步骤检测细胞上清中IL-10和TGF-β的水平;
(4)若结果显示,hUC-MSC-IL-10/TGF-β细胞上清中分泌高水平的IL-10和TGF-β,而对照组hUC-MSC细胞培养上清中几乎检测不到IL-10和TGF-β的表达,则表明间充质干细胞通过基因修饰可分泌特定的多种免疫抑制性的细胞因子、生长因子和趋化因子。
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