CN112695049A - 修饰间充质干细胞的融合基因、质粒、修饰得到干细胞及制备方法 - Google Patents
修饰间充质干细胞的融合基因、质粒、修饰得到干细胞及制备方法 Download PDFInfo
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Abstract
本发明公开了修饰间充质干细胞的融合基因、质粒、修饰得到干细胞及制备方法,其中修饰间充质干细胞的融合基因包括Klotho核酸人工序列、自剪切多肽T2A核酸人工序列和FGF23核酸人工序列,且Klotho的核酸人工序列、自剪切多肽T2A核酸人工序列和FGF23核酸人工序列依序串联。本发明所制备的利用融合基因修饰的间充质干细胞,其明显高于正常的干细胞Klotho、FGF23表达量,且能够实现对Klotho、FGF23的持续表达效果。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及修饰间充质干细胞的融合基因、 质粒、修饰得到干细胞及制备方法。
背景技术
慢性肾病(CKD)是一个日益严重的国际问题,2016年慢性肾病影响全球 7.53亿人,其中女性4.17亿,男性3.36亿。CKD患者肾脏Klotho RNA减少, 这一临床观察在许多临床前模型中得到证实。而且单侧肾切除术和对侧缺血再 灌注损伤可下调肾Klotho蛋白和mRNA的表达。在慢性肾小球肾炎模型中, Klotho表达同样减少。Klotho过表达改善肾功能,改善肾脏组织学。在CKD 患者中,冠状动脉钙化非常普遍,增加了心血管疾病的发病率和死亡率。 Klotho-FGF23在血管矿化中起重要作用。因此,CKD是心血管疾病的主要危险 因素,导致发病率增加和寿命缩短。CKD患者肾脏Klotho表达明显降低。
心血管疾病(CVD)是一种普遍存在于人群中的疾病,是导致死亡的首要原 因。Klotho被认为是CVD发展的关键调节器。在一些临床研究中,已经观察 到可溶性Klotho水平低与心血管疾病的发生和严重程度以及在Klotho水平高 时心血管风险降低之间的关系。一些实验研究表明,该蛋白在维持血管稳态中 起作用。Klotho通过促进一氧化氮(NO)的产生改善内皮细胞功能障碍,介导 抑制粘附分子表达、核因子-kappa B的衰减、抑制Wnt信号等抗炎、抗衰老 作用。Klotho调节内皮NO合成酶(eNOs)的表达水平。此外,该蛋白与血管钙 化的减轻和心肌肥厚的预防有关。因此,Klotho蛋白与CVD相关,并在维持 血管功能完整性方面发挥作用。
痴呆是一个宽范畴的大脑疾病,长期引起思考和记忆能力的逐渐降低,足 以影响一个人的日常生活。最常见的痴呆症是阿尔茨海默氏症,占50%到70%。 其他常见类型包括血管性痴呆(25%)、路易体痴呆(15%)和额颞叶痴呆。2015 年全球老年痴呆症影响大约4600万人。老年人变得越来越普遍。大约3%的人 在75年和84年之间,有将近一半的85岁以上的人群。随着老龄化的发展, 痴呆症在整个人群中变得越来越普遍。这是老年人致残最常见的一种原因,它 导致每年6040亿美元的经济损失。神经退行性疾病,特别是阿尔茨海默病 (AD),在西方国家呈上升趋势。阿尔茨海默症协会估计,在美国阿尔茨海默病 是第六大死因,每67秒就有一人被诊断出患有阿尔茨海默病。到2050年,这 些患者的医疗和护理费用将超过1.1万亿美元。AD的特点不仅有神经元和突 触的损失,而且还有β淀粉样蛋白(Aβ斑块)生成的神经毒素及其沉积和神经 原纤维缠结形成。越来越多的证据表明,淀粉样蛋白沉积是该病的主要特征。 最近有研究表明,AD患者脑脊液中抗衰老蛋白Klotho的浓度明显低于年轻患 者或无AD的老年患者。
Klotho基因包含5个外显子,编码I型单通道跨膜蛋白,1012aa。胞内 区很短,只有10个aa。胞外区由2个弱同源性的结构域组成,命名为KL1和 KL2。每个结构域都与I型糖苷酶具有同源性,例如哺乳动物的乳糖-根皮苷、 水解酶,细菌和植物的β葡萄糖苷酶。Klotho基因只在有限的组织和细胞类 型中表达,在肾脏远端小管和大脑的脉络丛中高表达。Klotho蛋白与多种FGFR 结合形成复合共受体,与FGF23结合,参与调节磷酸盐平衡。Klotho可治疗 多种疾病,例如老年性疾病、认知的缺失、肾功能缺失、糖尿病、肿瘤等。
分泌型Klotho蛋白具有公认的唾液酸酶活性,可修饰细胞表面的聚糖, 使分泌型Klotho蛋白具有调控胰岛素、IGF-1、Wnt等多种离子通道和生长因 子活性的能力。分泌型Klotho蛋白还通过一种尚未确定的机制保护细胞和组 织免受氧化应激。
FGF23蛋白是由FGF23基因编码的。FGF23是FGF家族的一员,该家族负 责磷酸盐和维生素D的代谢。FGF23的主要功能似乎是调节血浆中的磷酸盐浓 度。FGF23在应答钙三醇浓度提高时由骨细胞分泌的。FGF23降低肾脏近端小 管磷酸钠共转运体NPT2的表达。因此,FGF23降低了磷酸盐的再吸收,同时 增加了磷酸盐的排泄。FGF23还可以抑制1α-羟化酶,降低其激活维生素D 的能力,进而影响钙的吸收。
FGF23以Klotho依赖性的方式作为磷酸盐尿素和维生素D的反调节激素。 高磷血症导致慢性肾病(CKD)患者血管狭窄、心肌梗死、中风和预期寿命的大 幅缩短。肾脏中缺乏FGF信号导致血清磷酸盐水平升高。
已经发现分泌型和跨膜型的Klotho与FGF受体共同形成复合物,从而增 加FGF23依赖的信号通路。Klotho作为FGF23信号的共受体,在血清磷酸盐 水平的调节中起着重要作用。Klotho通过调节包括胰岛素样生长因子(IGF-1) 在内的多种信号通路发挥作用。Klotho的一个作用是增加细胞对氧化应激的 抵抗力,涉及到许多不同的病理过程。研究表明,通过调节细胞对氧化应激的 反应,Klotho在阿尔茨海默病和糖尿病等神经退行性疾病中也具有保护作用。 Klotho也被认为是胶原合成的抑制因子,因此在纤维化中可能是有益的。已 有研究表明,Klotho的表达在多种癌细胞中是沉默的,这与细胞生长增强和 肿瘤转移的形成有关。而在癌细胞中过表达Klotho则可以抑制癌细胞生长, 促进癌细胞凋亡。
由于Klotho在多个器官系统中的不同功能作用及其在各种疾病中的潜在 有益作用,将Klotho与FGF23联合应用的新疗法是有前途的治疗方法。到目 前为止,Klotho治疗用药在很大程度上仍未探索。有效的治疗方案,特别是 使局部给药或局部表达Klotho在疾病中对生理环境的影响,或提供持续的 Klotho蛋白在体内表达的方法,仍需探索。
已有报道间充质干细胞回输到病人后表现出免疫逃避特性。在间充质干细 胞移植病例中,间充质干细胞显示出有益的免疫调节作用,从而减少潜在的异 源致病性反应和排斥反应。此外,有报道称间充质干细胞具有抗肿瘤作用。同 时它在创面愈合中也发挥这治疗作用。利用间充质干细胞可通过静脉回输到体 内,是由于它会自动归巢和移植到损伤处。很明显,间充质干细胞在损伤组织 中具有再生作用,其作为治疗蛋白传递载体的作用尚未得到充分的探索。
因此,开发一种可持续表达Klotho和FGF23蛋白的间充质干细胞,不但 具有迫切的研究价值,也具有良好的经济效益和大规模医疗应用潜力,这正是 本发明得以完成的动力所在和基础。
发明内容
为了克服上述所指出的现有技术的缺陷,本发明人对此进行了深入研究, 在付出了大量创造性劳动后,从而完成了本发明。
具体而言,本发明所要解决的技术问题是:提供修饰间充质干细胞的融合 基因、质粒、修饰得到干细胞及制备方法,使间充质干细胞能够持续表达 Klotho和FGF23蛋白。
第一方面,本发明提供了修饰间充质干细胞的融合基因,包括Klotho核 酸人工序列、自剪切多肽T2A核酸人工序列和FGF23核酸人工序列,且Klotho 的核酸人工序列、自剪切多肽T2A核酸人工序列和FGF23核酸人工序列依序串 联。
本发明中,作为一种优选的技术方案,所述Klotho核酸人工序列如SEQ ID NO.2所述。
本发明中,作为一种优选的技术方案,所述自剪切多肽T2A核酸人工序列 如SEQID NO.3所述。
本发明中,作为一种优选的技术方案,所述FGF23核酸人工序列如SEQ ID NO.3所述。
本发明中,作为一种优选的技术方案,所述修饰间充质干细胞的融合基因 其核酸序列如SEQ ID NO.1所述。
第二方面,本发明提供了质粒,其中含有修饰间充质干细胞的融合基因。
本发明中,作为一种优选的技术方案,所述质粒采用如下方法制备得到: 合成修饰间充质干细胞的融合基因,插入pAV-EF1α-GFP载体中,转化到 E.coli(TOP10),经测序正确后,使用质粒提取试剂盒提取并纯化质粒,获得 重组表达载体质粒。
第三方面,本发明提供了间充质干细胞,所述间充质干细胞采用融合基因 修饰得到。
本发明中,作为一种优选的技术方案,所述间充质干细胞采用脐带间充质 干细胞,且采用如下方法制备得到:取新生儿脐带,消毒两次,放在培养皿中, 剥离出其中的华氏胶组织,剪碎后将华氏胶组织转移到培养瓶中,加入含有血 浆替代物的UltraCULTURE培养基培养,干细胞铺满瓶底的80%时,进行传代, 传至P4代,即得到所用的间充质干细胞。
第四方面,本发明提供了间充质干细胞的修饰方法,包括如下步骤:
复苏293T细胞,利用上述得到的重组表达载体质粒转染293T细胞包装成 腺病毒,利用腺病毒感染上述得到的脐带间充质干细胞,制备得到利用融合基 因修饰的间充质干细胞。
由于采用以上技术方案,本发明具有如下有益效果:
本发明中采用腺病毒进行质粒的包装,腺病毒具有安全性高、免疫原性低、 宿主范围广、表达稳定和物理性质稳定等优点。
本发明中采用间充质干细胞作为将Klotho治疗药物传递的工具。由于间 充质干细胞能够迁移到疾病区域,特别是炎症区域,所以间充质干细胞具有抗 炎特性,还可以自动迁移到病变组织。在身体病变区域持续表达Klotho蛋白。
本发明中将Klotho与FGF23融合至同一基因片段,并利用其修饰间充质 干细胞,并使得间充质干细胞稳定的表达Klotho与FGF23是发明人经过大量 反复试验得出的。由于Klotho-FGF23-MSCs在治疗与非预期炎症或免疫反应相 关的疾病方面具有惊人的效果。Klotho-FGF23-mscs由于持续产生Klotho,给 予Klotho-FGF23-MSCs后,修饰后的MSCs可作为生物泵向回输者持续提供 Klotho蛋白。基因修饰的MSCs(间充质干细胞)的归巢能力会使Klotho定位 在病变区域表达。然而,表达的Klotho也可以被血管系统运输到回输者全身, 无论MSCs在体内的任何位置,都可以通过系统运输的方式发挥作用。
本发明中采用的是分泌型Klotho蛋白和FGF23蛋白,其能够保护细胞避 免氧自由基的损害,具有抗衰老功能。因此可用于抗衰美容,使皮肤紧致美白。 也可用于治疗肿瘤、器官纤维化、肾衰竭、老年性疾病、动脉硬化、痴呆、糖 尿病、自身免疫疾病、肺病、各种炎症、慢病纤维化等。
本发明中经修饰的间充质干细胞可以正常静脉注射,也可用于肌肉注射、 皮下注射、鞘内注射或局部注射。因病情不同,需选择合适的注射方式。细胞 使用量也因受试者个体情况和病情严重而不同,通常为1x104~1x107细胞/kg 体重,每次间隔2周以上,可以连续使用3次。
综上所述,本发明提供的修饰间充质干细胞的融合基因,能够对间充质干 细胞进行修饰,使间充质干细胞能够持续表达Klotho和FGF23蛋白。
附图说明
图1为本发明所述的融合基因结构示意图;
图2为本发明脐带间充质干细胞显微镜下视野图;
图3为本发明荧光显微镜下观察收集的病毒颗粒进行的病毒滴度检测,左 图为荧光,右图为明场;
图4为本发明流式检测收集的腺病毒感染293T的效率为18.7%。
图5为流式检测GFP在干细胞中的表达率。
图6为ELISA检测干细胞培养液中Klotho蛋白的含量。
图7为经双氧水处理后细胞的死亡率。
图8为测定的Klotho-FGF23-MCS中NO的含量。
具体实施方式
下面结合具体的实施例对本发明进一步说明。但这些例举性实施方式的用 途和目的仅用来例举本发明,并非对本发明的实际保护范围构成任何形式的任 何限定,更非将本发明的保护范围局限于此。
实施例1
修饰间充质干细胞的融合基因(Klotho-FGF23),包括Klotho核酸人工序 列、自剪切多肽T2A核酸人工序列和FGF23核酸人工序列,且如图1所示,Klotho的核酸人工序列、自剪切多肽T2A核酸人工序列和FGF23核酸人工序 列依序串联,其核酸序列如SEQ ID NO.1。其中,所述Klotho的核酸人工序 列为SEQ ID NO.2;所述自剪切多肽T2A核酸人工序列为SEQ ID NO.3;所述 FGF23核酸人工序列为SEQ ID NO.3。
实施例2
质粒,其中含有修饰间充质干细胞的融合基因。所述质粒采用如下方法制 备得到:合成修饰间充质干细胞的融合基因,插入pAV-EF1α-GFP载体中,转 化到E.coli(TOP10),经测序正确后,使用质粒提取试剂盒提取并纯化质粒, 获得重组表达载体质粒。
更详细的说,本实施例提供的制备方法包括如下步骤:
将融合基因Klotho-FGF23的核酸人工序列,委托生工生物工程(上海)有 限公司合成其整个表达框并插入标准载体pUC上,因此命名为 pUC-Klotho-FGF23,同时将pUC-Klotho-FGF23和pAV-EF1α-GFP载体进行 Fast Digest BamHI(购自ThermoFisher公司)和Fast Digest HindⅢ(购 自ThermoFisher公司)双酶切,37℃,酶切1小时。100μl酶切体系为:10 ×buffer:10μl;DNA 6μg;BamHI酶:3μl;HindⅢ酶:3μl;去离子水补足 体积。利用琼胶电泳将分别把含有Klotho-FGF23的DNA片段和线性化的 pAV-EF1α-GFP DNA片段的琼胶部位切下,放在两个离心管中。
采用DNA extraction kit(购自ThermoFisher公司)将DNA从琼胶中溶 出并浓缩,首先往上述离心管加入500ml DF buffer,55℃作用10分钟,每 2-3分钟摇晃一次,直至琼胶完全溶解。再将琼胶溶液全部吸入DF Column, 并套上Collection Tube(收集过滤液)。8000rpm离心1分钟,将过滤液倒掉。 再加入500ml Wash Buffer,8000rpm离心1分钟,过滤液倒掉。12000rpm离 心2分钟确保乙醇被去除。最后将DF Column转移至上另一干净的微量离心管, 加入25μl Elution Buffer,室温静置2分钟后,12000rpm离心2分钟,微量 离心管内的液体即为纯化的Klotho-FGF23 DNA片段和线性化的pAV-EF1α-GFP DNA片段。
将上述两种DNA片段在16℃进行过夜连接形成pAV-EF1α-Klotho-FGF23 质粒。连接体系为:10×buffer:1μl;T4连接酶:1μl;Klotho-FGF23 DNA: 4μl;线性化的pAV-EF1α-GFP DNA:4μl。
将上述pAV-EF1α-Klotho-FGF23转化到E.coli(DH5α)。筛选鉴定出阳 性克隆,选取阳性克隆37℃、250rpm摇菌(12h-16h),按照质粒提取纯化试 剂盒(购自Invitrogen公司)提取pAV-EF1α-Klotho-FGF23质粒,具体步骤 见说明书。将上述的pAV-EF1α-Klotho-FGF23质粒委托生工生物工程(上海) 有限公司进行测序。经测序正确后备用。
实施例3
脐带间充质干细胞的制备
取医院捐献的新生儿脐带,在超净工作台中先用75wt%酒精消毒两次,放 在培养皿中,用镊子剥离出其中的华氏胶组织,用剪刀剪碎至0.5mm2大小的 小块。将剪碎的华氏胶组织转移到培养瓶中,加入含有血浆替代物的 UltraCULTURE培养基培养,每天用显微镜观察。爬出的干细胞铺满瓶底的80% 时,进行传代,传代后细胞生长速度加快,每2-3天传一代,传至P4代用于 实验(见图2)。
实施例4
(一)293T细胞的复苏
1、从液氮罐中取出冻存的293T细胞,迅速丢入37℃水浴锅中并快速晃 动,直至细胞溶液完全溶解。
2、将细胞溶液转移到50mL离心管中,并在其中加上10mL新鲜的完全培 养基,混匀后离心,1500rpm,5min。
3、去掉上清,加入3mL新鲜的DMEM培养基进行重悬细胞,平均转入六 孔板中,每个孔中补足到3mL培养基。
4、将六孔板平稳放入37℃、5%CO2和95%相对湿度的培养箱中培养。
5、第二天观察细胞存活率,并更换新鲜培养基。以后每天观察细胞生长 情况,细胞铺满孔底时,进行传代,待长满70%瓶底时用于实验。
(二)质粒转染293T细胞包装成病毒
1、取3×105个293T细胞在六孔板中培养,为第二天的转染做准备。
2、转染前将六孔板换成新鲜DMEM培养液(购自Gibco公司),37℃温箱 中培养1h。
3、将LipoFiterTM脂质体转染试剂(购自汉恒生物)回复至室温使用, 使用前摇匀。
4、pAV-EF1α-Klotho-FGF23和包装质粒、辅助质粒三种质粒以1:1:1的 比例将3.0μg的DNA溶解于100μL的DMEM培养基中,同时12μL的 LipoFiterTM溶于88μL的DMEM培养基中,各自室温静置20分钟。
5、将以上的DNA和LipoFiterTM混匀,室温孵育20分钟。
6、将LipoFiterTM-DNA混合物加入六孔板的一个孔中,培养6小时后, 去除LipoFiterTM-DNA培养液,加入新鲜培养基继续培养。
7、48小时后,显微镜下观察293T细胞转染后的形态变化。
8、将含有病毒的细胞培养上清吸入EP管中,4℃,2000g离心10min,转 移至新的EP管中,4.5μm滤器过滤后-80℃保存。
(三)病毒滴度测定
1、将生长状态良好的293T细胞消化计数后稀释至1×105/mL,加入96 孔板,100μL/孔,为每个病毒稀释度准备6个孔。放入37℃,5%CO2培养 箱中培养。
2、第二天,准备6个1.5mL EP管,第一个EP管中加入10μL病毒液, 然后做10倍梯度稀释,连续6个稀释度。吸取96孔板中原有的培养基,加入 稀释好的病毒液,并做好标记。
3、第三天,在每个孔中再加入100μL完全培养基,利于细胞的生长。
4、第五天,在荧光显微镜下观察结果,如图3所示。荧光百分比在10-30% 的孔计算病毒滴度,用流式细胞仪检测GFP荧光百分比(如图4)。根据公式: 滴度(TU/mL)=细胞数×荧光百分比×MOI(1)×病毒稀释倍数×103计算病毒 滴度,本发明中获得的病毒滴度为3.7×107TU/mL。
实施例5
腺病毒感染脐带间充质干细胞
1、将生长状态良好的间充质干细胞消化计数后稀释至1×105/mL,加入六 孔板中,补加培养基至3mL。放入37℃,5%CO2培养箱中培养24h。
2、从-80℃拿出2mL病毒液,加入终浓度为8μg/mL的聚凝胺(购自Sigma 公司)。
3、从培养箱中拿出六孔板,去掉六孔板中的培养基,加入准备好的病毒 液,放入培养箱中继续培养24h。
4、24h后,去掉六孔板中的病毒液,添加完全培养基进行培养,同时加 入3ug/mL的嘌呤霉素进行筛选,放在37℃,5%CO2培养箱中培养,每两天 换液一次,培养5天,成活的细胞即为腺病毒感染的干细胞。
实施例6
(一)流式细胞仪检测干细胞中GFP的表达率
分别取感染后的第一代细胞、第三代细胞、第五代细胞、第七代细胞、第 九代细胞作为样本,未感染的间充质干细胞作为对照组。
1、样本处理:用血细胞计数仪计数或人工计数,细胞数要求范围0.5× 105-1×107/mL。
2、在1#管中加100μL对照组细胞悬液,在2#、3#、4#、5#、6#管中加 100μl实验组细胞悬液。
3、涡旋混合器混匀。
4、加300uLPBS缓冲液混匀放入MCL架等待上机。
5、将仪器内事先设置好的荧光检测程序拖到工作区,输入架子号、位置 号、标本号,点击检测进行电压以及荧光的调整。当调整完毕按上述调整好的 条件进行GFP的检测。
6、利用分析软件分析检测结果,并记录分析结果。
本发明中第一代干细胞GFP表达率为99.2%,第三代干细胞中GFP表达率 为60%,第五代干细胞中GFP表达率为57%,第七代干细胞中GFP表达率为55%, 第九代干细胞中GFP表达率为54%(如图5)。由于细胞生长过程中会导致插入 的基因缺失,所以随着细胞的生长,GFP表达率会有所降低,降低到一定程度 后,表达率基本不变。
(二)ELISA检测干细胞中Klotho蛋白的表达量
1、样本处理:分别取感染后的第一代细胞、第三代细胞、第五代细胞、 第七代细胞、第九代细胞作为实验组,未感染的间充质干细胞作为对照组。
2、实验前的准备
使用前将试剂盒内所有试剂自冰箱内取出放置室温平衡30分钟(试剂 购自武汉华美生物工程有限公司)。试剂解冻后,实验前进行离心。配制洗涤 液,按照试剂说明书配制,混匀。在进行抗-Klotho检测前,必须仔细检查设 备以保证其运转正常。依据抗-Klotho试剂的说明书,将判读程序编入航天 ZS-3板式酶标仪中。
3、编号:将标准品和样本对应微板孔按序编号,每次设空白对照1孔(双 波长可不设空白对照孔),阴性对照1孔。
4、加样:分别在相应的孔中加入待测样品和标准品100ul,阴性对照孔 中加入空白培养液,轻轻振荡混匀。
5、温育:用封板膜封板后,置于37℃温育2小时。
6、加二抗:小心揭掉封板膜,吸掉孔里的液体,加入100μL的1×的生 物素抗体,37℃温育1小时。
7、洗板:小心揭掉封板膜,用洗板机洗涤3遍,最后一遍尽量用吸水纸 拍干。
8、加酶:每孔加入1×的辣根过氧化酶100ul,空白孔除外,轻轻振荡混 匀。
9、温育:用封板膜封板后,置于37℃温育1小时。
10、洗板:小心揭掉封板膜,用洗板机洗涤5遍,最后一遍尽量用吸水纸 拍干。
11、显色:每孔加入显色剂TMB 90ul,轻轻振荡混匀,37℃避光显色30 分钟。
12、测定:每孔加入终止液50ul,轻轻振荡混匀,10分钟内测定结果。 设定酶标仪450nm为测量波长,540nm为参考波长测定各孔A值。
结果如图6所示,转入Klotho的干细胞其表达率明显高于正常的干细胞 Klotho表达量,随着细胞的生长,整合后不稳定的Klotho基因会缺失,稳定 的Klotho基因就会持续表达,所以表达量会有突然降低再慢慢平稳的变化。
(三)Klotho和FGF23保护细胞免受活性氧的影响
1、分别取5×104的Klotho-FGF23转导的第一代、第三代、、第五代、第 七代、第九代干细胞和未转导的干细胞,铺在六孔板里,放入37℃,5%CO2培 养箱中培养16h。
2、使用双氧水同时加入转染和未转染的干细胞,双氧水的终浓度为50μM, 放在培养箱中培养4h。
3、用消化液处理六孔板中的细胞,并全部收集起来,同时取未经双氧水 处理的干细胞作为对照。
4、将台盼蓝分别加入到实验组和对照组中,显微镜下计数死细胞数量比 例。如图7显示,未经双氧水处理的干细胞死亡率为0.1%,双氧水处理的 Klotho-FGF23转染的第一代干细胞死亡率为21%,第三代干细胞死亡率为50%, 第五代干细胞死亡率为47%,第七代干细胞死亡率为45%,第九代干细胞死亡 率为44%,未转染的干细胞死亡率为95%。
(四)试剂盒测定Klotho和FGF23转染的干细胞中NO含量
1、分别取5×104的Klotho-FGF23转染的第一代、第三代、第五代、第 七代、第九代干细胞和未转染的干细胞,铺在六孔板里,放入37℃,5%CO2培 养箱中培养48h。
2、分别从每个孔中取100μL上清液加试剂一200μL混匀,加试剂二100μL, 漩涡充分混匀后静置10分钟,3500~4000转/分,离心15分钟,取上清液 160μL加入96孔板中。同时加入160μL的双蒸水作为空白对照,以20μmol/L 亚硝酸钠标准液作为标准孔。均加入80μL的显色剂混匀,静置15min,550nm 比色,酶标仪比色,测各孔OD值。
3、计算NO含量。
如图8所示,Klotho-FGF23转染的干细胞产生的NO的含量明显高于正常 的干细胞,随着细胞的生长,整合后不稳定的Klotho-FGF23融合基因会缺失, 稳定的Klotho-FGF23融合基因就会持续表达,所以第一代Klotho-FGF23-MCS 的NO含量最高,传代后会出现突然降低再慢慢平稳的变化。
以上结果综合说明本发明所制备的利用融合基因修饰的间充质干细胞,其 明显高于正常的干细胞Klotho、FGF23表达量,且能够实现对Klotho、FGF23 的持续表达效果。
应当理解,这些实施例的用途仅用于说明本发明而非意欲限制本发明的保 护范围。此外,也应理解,在阅读了本发明的技术内容之后,本领域技术人员 可以对本发明作各种改动、修改和/或变型,所有的这些等价形式同样落于本 申请所附权利要求书所限定的保护范围之内。
序列表
<110> 山东兴瑞生物科技有限公司
<120>修饰间充质干细胞的融合基因、质粒、修饰得到干细胞及制备方法
<130> 2019
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2409
<212> DNA
<213> 人种(Homo sapiens)
<400> 1
atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagccaga 60
cccgagccgg gcgacggcgc gcagacctgg gcccgtttct cgcggcctcc tgcccccgag 120
gccgcgggcc tcttccaggg caccttcccc gacggcttcc tctgggccgt gggcagcgcc 180
gcctaccaga ccgagggcgg ctggcagcag cacggcaagg gtgcgtccat ctgggatacg 240
ttcacccacc accccctggc acccccggga gactcccgga acgccagtct gccgttgggc 300
gccccgtcgc cgctgcagcc cgccaccggg gacgtagcca gcgacagcta caacaacgtc 360
ttccgcgaca cggaggcgct gcgcgagctc ggggtcactc actaccgctt ctccatctcg 420
tgggcgcgag tgctccccaa tggcagcgcg ggcgtcccca accgcgaggg gctgcgctac 480
taccggcgcc tgctggagcg gctgcgggag ctgggcgtgc agcccgtggt caccctgtac 540
cactgggacc tgccccagcg cctgcaggac gcctacggcg gctgggccaa ccgcgccctg 600
gccgaccact tcagggatta cgcggagctc tgcttccgcc acttcggcgg tcaggtcaag 660
tactggatca ccatcgacaa cccctacgtg gtggcctggc acggctacgc caccgggcgc 720
ctggcccccg gcatccgggg cagcccgcgg ctcgggtacc tggtggcgca caacctcctc 780
ctggctcatg ccaaagtctg gcatctctac aatacttctt tccgtcccac tcagggaggt 840
caggtgtcca ttgccctaag ctctcactgg atcaatcctc gaagaatgac cgaccacagc 900
atcaaagaat gtcaaaaatc tctggacttt gtactaggtt ggtttgccaa acccgtattt 960
attgatggtg actatcccga gagcatgaag aataaccttt catctattct gcctgatttt 1020
actgaatctg agaaaaagtt catcaaagga actgctgact tttttgctct ttgctttgga 1080
cccaccttga gttttcaact tttggaccct cacatgaagt tccgccaatt ggaatctccc 1140
aacctgaggc aactgctttc ctggattgac cttgaattta accatcctca aatatttatt 1200
gtggaaaatg gctggtttgt ctcagggacc accaagagag atgatgccaa atatatgtat 1260
tacctcaaaa agttcatcat ggaaacctta aaagccatca agctggatgg ggtggatgtc 1320
atcgggtata ccgcatggtc cctcatggat ggtttcgagt ggcacagagg ttacagcatc 1380
aggcgtggac tcttctatgt tgactttcta agccaggaca agatgttgtt gccaaagtct 1440
tcagccttgt tctaccaaaa gctgatagag aaaaatggct tccctccttt acctgaaaat 1500
cagcccctag aagggacatt tccctgtgac tttgcttggg gagttgttga caactacatt 1560
caagtatctc aacttactaa gcctatttct tctcttacta agccttatca tgaaggccga 1620
gggagcctgc tgacatgtgg cgatgtggag gaaaacccag gaccaatggc actgccagtg 1680
accgccctgc tgctgcctct ggccctgctg ctgcacgcag ccagacccta tcccaatgcc 1740
tccccactgc tcggctccag ctggggtggc ctgatccacc tgtacacagc cacagccagg 1800
aacagctacc acctgcagat ccacaagaat ggccatgtgg atggcgcacc ccatcagacc 1860
atctacagtg ccctgatgat cagatcagag gatgctggct ttgtggtgat tacaggtgtg 1920
atgagcagaa gatacctctg catggatttc agaggcaaca tttttggatc acactatttc 1980
gacccggaga actgcaggtt ccaacaccag acgctggaaa acgggtacga cgtctaccac 2040
tctcctcagt atcacttcct ggtcagtctg ggccgggcga agagagcctt cctgccaggc 2100
atgaacccac ccccgtactc ccagttcctg tcccggagga acgagatccc cctaattcac 2160
ttcaacaccc ccataccacg gcggcacacc cggagcgccg aggacgactc ggagcgggac 2220
cccctgaacg tgctgaagcc ccgggcccgg atgaccccgg ccccggcctc ctgttcacag 2280
gagctcccga gcgccgagga caacagcccg atggccagtg acccattagg ggtggtcagg 2340
ggcggtcgag tgaacacgca cgctggggga acgggcccgg aaggctgccg ccccttcgcc 2400
aagttcatc 2409
<210> 2
<211> 1611
<212> DNA
<213> 人种(Homo sapiens)
<400> 2
atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagccaga 60
cccgagccgg gcgacggcgc gcagacctgg gcccgtttct cgcggcctcc tgcccccgag 120
gccgcgggcc tcttccaggg caccttcccc gacggcttcc tctgggccgt gggcagcgcc 180
gcctaccaga ccgagggcgg ctggcagcag cacggcaagg gtgcgtccat ctgggatacg 240
ttcacccacc accccctggc acccccggga gactcccgga acgccagtct gccgttgggc 300
gccccgtcgc cgctgcagcc cgccaccggg gacgtagcca gcgacagcta caacaacgtc 360
ttccgcgaca cggaggcgct gcgcgagctc ggggtcactc actaccgctt ctccatctcg 420
tgggcgcgag tgctccccaa tggcagcgcg ggcgtcccca accgcgaggg gctgcgctac 480
taccggcgcc tgctggagcg gctgcgggag ctgggcgtgc agcccgtggt caccctgtac 540
cactgggacc tgccccagcg cctgcaggac gcctacggcg gctgggccaa ccgcgccctg 600
gccgaccact tcagggatta cgcggagctc tgcttccgcc acttcggcgg tcaggtcaag 660
tactggatca ccatcgacaa cccctacgtg gtggcctggc acggctacgc caccgggcgc 720
ctggcccccg gcatccgggg cagcccgcgg ctcgggtacc tggtggcgca caacctcctc 780
ctggctcatg ccaaagtctg gcatctctac aatacttctt tccgtcccac tcagggaggt 840
caggtgtcca ttgccctaag ctctcactgg atcaatcctc gaagaatgac cgaccacagc 900
atcaaagaat gtcaaaaatc tctggacttt gtactaggtt ggtttgccaa acccgtattt 960
attgatggtg actatcccga gagcatgaag aataaccttt catctattct gcctgatttt 1020
actgaatctg agaaaaagtt catcaaagga actgctgact tttttgctct ttgctttgga 1080
cccaccttga gttttcaact tttggaccct cacatgaagt tccgccaatt ggaatctccc 1140
aacctgaggc aactgctttc ctggattgac cttgaattta accatcctca aatatttatt 1200
gtggaaaatg gctggtttgt ctcagggacc accaagagag atgatgccaa atatatgtat 1260
tacctcaaaa agttcatcat ggaaacctta aaagccatca agctggatgg ggtggatgtc 1320
atcgggtata ccgcatggtc cctcatggat ggtttcgagt ggcacagagg ttacagcatc 1380
aggcgtggac tcttctatgt tgactttcta agccaggaca agatgttgtt gccaaagtct 1440
tcagccttgt tctaccaaaa gctgatagag aaaaatggct tccctccttt acctgaaaat 1500
cagcccctag aagggacatt tccctgtgac tttgcttggg gagttgttga caactacatt 1560
caagtatctc aacttactaa gcctatttct tctcttacta agccttatca t 1611
<210> 3
<211> 54
<212> DNA
<213> 人种(Homo sapiens)
<400> 3
gaaggccgag ggagcctgct gacatgtggc gatgtggagg aaaacccagg acca 54
<210> 4
<211> 744
<212> DNA
<213> 人种(Homo sapiens)
<400> 4
atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagccaga 60
ccctatccca atgcctcccc actgctcggc tccagctggg gtggcctgat ccacctgtac 120
acagccacag ccaggaacag ctaccacctg cagatccaca agaatggcca tgtggatggc 180
gcaccccatc agaccatcta cagtgccctg atgatcagat cagaggatgc tggctttgtg 240
gtgattacag gtgtgatgag cagaagatac ctctgcatgg atttcagagg caacattttt 300
ggatcacact atttcgaccc ggagaactgc aggttccaac accagacgct ggaaaacggg 360
tacgacgtct accactctcc tcagtatcac ttcctggtca gtctgggccg ggcgaagaga 420
gccttcctgc caggcatgaa cccacccccg tactcccagt tcctgtcccg gaggaacgag 480
atccccctaa ttcacttcaa cacccccata ccacggcggc acacccggag cgccgaggac 540
gactcggagc gggaccccct gaacgtgctg aagccccggg cccggatgac cccggccccg 600
gcctcctgtt cacaggagct cccgagcgcc gaggacaaca gcccgatggc cagtgaccca 660
ttaggggtgg tcaggggcgg tcgagtgaac acgcacgctg ggggaacggg cccggaaggc 720
tgccgcccct tcgccaagtt catc 744
Claims (10)
1.修饰间充质干细胞的融合基因,其特征在于:包括Klotho核酸人工序列、自剪切多肽T2A核酸人工序列和FGF23核酸人工序列,且Klotho的核酸人工序列、自剪切多肽T2A核酸人工序列和FGF23核酸人工序列依序串联。
2.如权利要求1所述的修饰间充质干细胞的融合基因,其特征在于:所述Klotho核酸人工序列如SEQ ID NO.2所述。
3.如权利要求2所述的修饰间充质干细胞的融合基因,其特征在于:所述自剪切多肽T2A核酸人工序列如SEQ ID NO.3所述。
4.如权利要求3所述的修饰间充质干细胞的融合基因,其特征在于:所述FGF23核酸人工序列如SEQ ID NO.3所述。
5.如权利要求4所述的修饰间充质干细胞的融合基因,其特征在于:所述修饰间充质干细胞的融合基因其核酸序列如SEQ ID NO.1所述。
6.质粒,其特征在于:其中含有如权利要求5所述的修饰间充质干细胞的融合基因。
7.如权利要求6所述的质粒,其特征在于:所述质粒采用如下方法制备得到:合成修饰间充质干细胞的融合基因,插入pAV-EF1α-GFP载体中,转化到E.coli(TOP10),经测序正确后,使用质粒提取试剂盒提取并纯化质粒,获得重组表达载体质粒。
8.间充质干细胞,其特征在于:所述间充质干细胞采用如权利要求5所述的融合基因修饰得到。
9.如权利要求8所述的间充质干细胞,其特征在于:所述间充质干细胞采用脐带间充质干细胞,且采用如下方法制备得到:取新生儿脐带,消毒两次,放在培养皿中,剥离出其中的华氏胶组织,剪碎后将华氏胶组织转移到培养瓶中,加入含有血浆替代物的UltraCULTURE培养基培养,干细胞铺满瓶底的80%时,进行传代,传至P4代,即得到所用的间充质干细胞。
10.间充质干细胞的修饰方法,其特征在于:包括如下步骤:复苏293T细胞,利用如权利要求7所述的重组表达载体质粒转染293T细胞包装成腺病毒,利用腺病毒感染如权利要求9所述的脐带间充质干细胞,制备得到利用融合基因修饰的间充质干细胞。
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---|---|---|---|---|
CN113444730A (zh) * | 2021-03-17 | 2021-09-28 | 昆明市延安医院 | 一种原发性肝细胞klotho基因转导干细胞筛选构建方法 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110195077A1 (en) * | 2010-01-29 | 2011-08-11 | Novartis Ag | Methods and compositions using fgf23 fusion ppolypeptides |
US20150079065A1 (en) * | 2012-04-16 | 2015-03-19 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Klotho variant polypeptides and uses thereof in therapy |
US20170233446A1 (en) * | 2008-01-28 | 2017-08-17 | Novartis Ag | Methods and compositions using klotho-fgf fusion polypeptides |
CN107810201A (zh) * | 2014-12-04 | 2018-03-16 | 诺华股份有限公司 | 使用klotho变体多肽的方法和组合物 |
CN110229835A (zh) * | 2019-06-12 | 2019-09-13 | 山东兴瑞生物科技有限公司 | 修饰间充质干细胞的融合基因、质粒、修饰得到干细胞及制备方法 |
CN111154791A (zh) * | 2019-06-25 | 2020-05-15 | 山东兴瑞生物科技有限公司 | 重组cd6基因、及利用其修饰的t细胞、制备方法和应用 |
-
2020
- 2020-08-20 CN CN202010844629.XA patent/CN112695049A/zh active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170233446A1 (en) * | 2008-01-28 | 2017-08-17 | Novartis Ag | Methods and compositions using klotho-fgf fusion polypeptides |
US20110195077A1 (en) * | 2010-01-29 | 2011-08-11 | Novartis Ag | Methods and compositions using fgf23 fusion ppolypeptides |
US20150079065A1 (en) * | 2012-04-16 | 2015-03-19 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Klotho variant polypeptides and uses thereof in therapy |
CN107810201A (zh) * | 2014-12-04 | 2018-03-16 | 诺华股份有限公司 | 使用klotho变体多肽的方法和组合物 |
CN110229835A (zh) * | 2019-06-12 | 2019-09-13 | 山东兴瑞生物科技有限公司 | 修饰间充质干细胞的融合基因、质粒、修饰得到干细胞及制备方法 |
CN111154791A (zh) * | 2019-06-25 | 2020-05-15 | 山东兴瑞生物科技有限公司 | 重组cd6基因、及利用其修饰的t细胞、制备方法和应用 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113444730A (zh) * | 2021-03-17 | 2021-09-28 | 昆明市延安医院 | 一种原发性肝细胞klotho基因转导干细胞筛选构建方法 |
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