CN115216493A - 一种bdnf基因修饰的脐带间充质干细胞及其制备方法 - Google Patents
一种bdnf基因修饰的脐带间充质干细胞及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种BDNF基因修饰的脐带间充质干细胞及其制备方法，属于细胞治疗领域。本发明通过以下步骤制得：首先将人的BDNF基因克隆到慢病毒载体上，与脐带间充质干细胞混合培养得到BDNF基因修饰的脐带间充质干细胞，并通过嘌呤霉素进行抗性筛选。经修饰的间充质干细胞能显著改善动物模型的疾病相关指标，且未引发免疫原性反应，对于治疗视网膜色素变性及其他光感受器退行性病变具有重大的临床治疗价值。
Description
技术领域
本发明属于细胞治疗领域，特别是涉及一种BDNF基因修饰的脐带间充质干细胞及其制备方法。
背景技术
视网膜色素变性（Retinitis Pigmentosa，RP），是一种遗传性眼科疾病，患者出现夜盲、视野狭隘等症状，最后失明。其主要发病机制是眼底视锥细胞及视杆细胞退变。这二者都属于神经上皮细胞，其中视杆细胞主要分布在稍微偏离视网膜中心的部位，与暗视条件下的物体辨别以及视野的宽狭等有关。视锥细胞大多分布在视网膜中心的黄斑部位，主要与中心视力以及色觉等有关。此外，视网膜色素上皮细胞（Retinal PigmentEpithelial, RPE）发生异常，也会引起RP。
目前，尚无视网膜色素变性的有效治疗方法，仅仅是采用对症疗法，但是基因治疗，视网膜移植，人工视网膜、细胞移植等治疗方法的研究正在开展之中。
BDNF(脑源神经营养因子)主要由神经元产生，通过细胞表面受体TrkB，激活ERK、mTOR、AKT等通路，促进神经元细胞的存活、生长。在动物模型上的应用表明，它能够防止神经元萎缩、促进轴突生长及促进神经元间新突触形成和神经通路的再生并抑制神经元凋亡,在中枢神经系统受损伤后可以通过发挥神经保护作用、增强神经可塑性、促进神经再生来修复神经组织。眼底注射BDNF对视网膜神经节细胞(RGCs)、视网膜感光细胞(RPCs)和视网膜色素上皮(RPE)细胞均有保护、营养及抗凋亡作用，具有特别重要的意义。然而BDNF经外周血输入，半衰期短，且无法跨越血-视网膜屏障，而反复经玻璃体注射给药，也存在感染、眼压升高、视网膜损伤等风险。需要寻找新的安全、稳定、持续的作用方式。
间充质干细胞(Mesenchymal Stem Cell, MSC)是能够分化成间充质组织系统的多能前体细胞。MSC能分泌大量的生长因子、免疫调节因子，对周围环境进行调节，抵抗氧化损伤、抑制炎症、促进周围细胞生长、帮助抵抗凋亡，具有极高的临床应用价值。实验证明，MSC移植到视网膜后可以存活至少2个月，并减少视锥细胞视杆细胞的凋亡，增厚视网膜。
发明内容
本发明的目的是提供一种BDNF基因修饰的脐带间充质干细胞及其制备方法，并通过该干细胞实现在治疗视网膜色素变性中的应用。以解决目前针对视网膜色素变性类疾病尚无特效治疗方法，以及近些年开发的基因治疗尚不成熟的现状。
本发明解决上述技术问题的技术方案如下： 首先构建过表达BDNF基因的慢病毒载体，再通过慢病毒载体，在间充质干细胞中，过表达BDNF，形成“BDNF过表达的间充质干细胞”，将BDNF修饰的间充质干细胞用于临床视网膜色素变性的注射治疗。
其中过表达BDNF基因的慢病毒载体构建如下：
将人BDNF基因(NM_170735)克隆到慢病毒载体GV416上，形成如下结构： EF1a-BDNF-3FLAG-CMV-EGFP-T2A-Puromycin
间充质干细胞的修饰如下：
于10cm2培养皿中培养MSC，培养基为α-MEM+10%血清。生长至90%汇合度，加入3mltrypLE消化5min，加入3ml完全培养基吹打瓶底，转移至15ml离心管，300g离心5min，重悬，计数，取1*106细胞，按MOI 5:1加入慢病毒，加入10ug/ml polybrene，混匀，37度培养箱过夜。24小时后弃上清，换液，感染72小时后，或等细胞生长至90%汇合度时，加入3ml trypLE消化5min，加入3ml完全培养基吹打瓶底，转移至15ml离心管，300g离心5min，1.5ml PBS重悬细胞，取100ul用流式细胞仪对EGFP+细胞占比进行检测。其余细胞以1：3传代，1.并在培养基中加入2ug/ml 嘌呤霉素，在汇合度达90%左右时按需传代或冻存，在细胞收获前的常规培养中，均需加入嘌呤霉素。Puromycin加药传代2次后，可再用流式检测阳性率，一般可达到90%以上阳性细胞再以嘌呤霉素(2ug/ml)进行抗性筛选5天，得到BDNF过表达的间充质干细胞（BDNF-MSC）。BDNF-MSC 以1：3传代至T25 培养瓶中，加培养基5ml进行培养，汇合度至90%时，按前文消化，以PBS 1ml重悬为细胞悬液，并计数，取2ul（含1×106 MSC）用于视网膜下注射。
BDNF-MSC对视网膜色素变性小鼠模型的治疗作用如下所示：
皇室外科学院大鼠模型(Royal College of Surgeons，RCS)：3周的RCS大鼠，予12h/12h明暗交替光照饲养。将1×106 BDNF-MSC（2 μL）网膜下注射。细胞移植后1周，2周，4周分别检测全视野视网膜电图(Electroretinogram,ERG) 。刺激器为Ganzfeld全视野刺激器，双眼同时记录a波和b波的潜伏期与振幅。将大鼠置于无声暗室中6-8小时，腹腔注射10%水合氯醛麻醉，固定，记录电极置于双眼角膜，羟甲基纤维素钠递烟保持角膜湿润。参考电极刺入双侧颊部皮下，接地电极置于尾部。采用0.025 cd.s.m2刺激光强度进行刺激，刺激频率为0.06 Hz，带宽为0.2-300 Hz，每次刺激间隔30s。
结果显示，相比于未处理组，与注射生理盐水组相比，MSC移植组（Ctrl-MSC）的b波振幅增长（89.6±13.7 μV vs 43.4±9.34 μV，p=0.0003），表明MSC移植缓解了RCS大鼠视网膜退变。与MSC移植组相比，BDNF过表达的MSC（BDNF-MSC）的b波振幅增长（123.5±11.7 μV vs 89.6±13.7μV，p=0.003），表明BDNF-MSC进一步改善了视网膜损伤，减轻了视网膜的症状。
本发明的有益效果是：本发明将BDNF基因修饰的间充质干细胞用于治疗治疗视网膜色素变性。与现有技术相比，本发明的经修饰的间充质干细胞具有显著的有利方面。特别地，本发明的经修饰的间充质干细胞展现出显著改善视觉指标的效应，且可安全地施用给动物，而不引发免疫原性反应。因此，本发明的经修饰的间充质干细胞能够用于治疗视网膜色素变性，具有重大的临床价值。
附图说明
通过结合以下附图所作的详细描述，本发明的上述和/或其他方面的优点将变得更清楚和更容易理解，这些附图只是示意性的，并不限制本发明，其中：
图1为实施例1. BDNF-MSC对视网膜色素变性小鼠模型的治疗效果。
具体实施方式
在此记载的实施例为本发明的特定的具体实施方式，用于说明本发明的构思，均是解释性和示例性的，不应解释为对本发明实施方式及本发明范围的限制。除在此记载的实施例外，本领域技术人员还能够基于本申请权利要求书和说明书所公开的内容采用显而易见的其它技术方案，这些技术方案包括采用对在此记载的实施例的做出任何显而易见的替换和修改的技术方案。
实施例1
3周的RCS大鼠，予12h/12h明暗交替光照饲养。将1×106 BDNF-MSC（2 μL）网膜下注射，细胞移植后1周检测全视网膜电(Electroretinogram,CERG) 。刺激器为Ganzfeld全视野刺激器，双眼同时记录a波和b波的潜伏期与振幅。将大鼠置于无声暗室中6小时，腹腔注射10%水合氯醛麻醉，固定，记录电极置于双眼角膜，羟甲基 纤维素钠递烟保持角膜湿润。参考电极刺入双侧颊部皮下，接地电极置于尾部。采用0.025 cd.s.m2刺激光强度进行刺激，刺激频率为0.06 Hz，带宽为0.2-300 Hz，每次刺激间隔30s。
结果显示，相比于未处理组，与注射生理盐水组相比，MSC移植组（Ctrl-MSC）的b波振幅增长（103.3 μV vs 52.74 μV，p=0.0003），表明MSC移植缓解了RCS大鼠视网膜退变。与MSC移植组相比，BDNF过表达的MSC（BDNF-MSC）的b波振幅增长（135.2 μV vs 103.3μV，p=0.003）
上述披露的各技术特征并不限于已披露的与其它特征的组合，本领域技术人员还可根据发明之目的进行各技术特征之间的其它组合，以实现本发明之目的为准。
Claims (10)
1.一种BDNF基因修饰的脐带间充质干细胞及其制备方法，其特征在于：首先构建过表达BDNF基因的慢病毒载体，再通过慢病毒载体，在间充质干细胞中，过表达BDNF，形成“BDNF基因修饰的间充质干细胞（BDNF-MSC）”，BDNF修饰的间充质干细胞用于视网膜色素变性的注射治疗。
2.根据权利要求1所述的BDNF基因修饰的脐带间充质干细胞及其制备方法，其特征在于，所述过表达BDNF基因的慢病毒载体通过将人BDNF基因(NM_170735)克隆到慢病毒载体GV416上，形成如下结构：EF1a-BDNF-3FLAG-CMV-EGFP-T2A-Puromycin。
3.根据权利要求1所述的BDNF基因修饰的脐带间充质干细胞及其制备方法，其特征在于，间充质干细胞为羊膜、牙髓、骨髓、脂肪等组织来源的间充质干细胞。
4.根据权利要求1所述的BDNF基因修饰的脐带间充质干细胞及其制备方法，其特征在于，所述BDNF过表达的间充质干细胞（BDNF-MSC）通过过表达BDNF基因的慢病毒与间充质干细胞在细胞培养条件下充分感染得到。
5.根据权利要求4所述的BDNF基因修饰的脐带间充质干细胞及其制备方法，其特征在于，感染过程中按MOI 5~10:1加入慢病毒，同时加入10ug/ml polybrene混匀培养。
6.根据权利要求5所述的BDNF基因修饰的脐带间充质干细胞及其制备方法，其特征在于，用流式细胞仪对EGFP+细胞占比进行检测，阳性率高于95%，且汇合度达90%左右时按需传代或冻存。
7.根据权利要求6所述的BDNF基因修饰的脐带间充质干细胞及其制备方法，其特征在于，用2ug/ml的嘌呤霉素对汇合度达标的阳性细胞进行抗性筛选5天，得到BDNF过表达的间充质干细胞（BDNF-MSC）。
8.根据权利要求7所述的BDNF基因修饰的脐带间充质干细胞及其制备方法，其特征在于，所述BDNF过表达的间充质干细胞以1：3传代，培养至90%汇合度后重悬为细胞悬液。
9.根据权利要求7所述的BDNF基因修饰的脐带间充质干细胞及其制备方法，其特征在于，将BDNF修饰的间充质干细胞，通过静脉输注、或者玻璃体腔注射的方式注射进入体内。
10.权利要求1-9所述的干细胞应用在视网膜色素变性类疾病治疗中。
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| CN104593421A (zh) * | 2015-03-02 | 2015-05-06 | 刘爽 | 一种抑制胶质瘤生长的过表达bdnf的间充质干细胞的制备方法及其临床应用 |
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| CN104593421A (zh) * | 2015-03-02 | 2015-05-06 | 刘爽 | 一种抑制胶质瘤生长的过表达bdnf的间充质干细胞的制备方法及其临床应用 |
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