CN111437286A - 基因工程改造外泌体递送miRNA-146a在糖尿病足溃疡治疗方面的应用 - Google Patents
基因工程改造外泌体递送miRNA-146a在糖尿病足溃疡治疗方面的应用 Download PDFInfo
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Abstract
本发明涉及一种基因工程改造外泌体递送miRNA‑146a在糖尿病足溃疡治疗方面的应用，包括：⑴构建质粒：以pCDH‑CMV‑MCS‑EF1‑copGFP慢病毒表达载体为基础，整合miRNA‑146a基因，得到miRNA‑146a过表达慢病毒载体pCDH‑CMV‑MCS‑EF1‑copGFP‑146a；⑵包装获得慢病毒，转染293T：使用慢病毒包装质粒pSPAX2和pMD2.G对pCDH‑CMV‑MCS‑EF1‑copGFP‑146a进行包装，转染293T细胞，富集高滴度的病毒液；⑶侵染脐带充间质干细胞：用包装好的病毒侵染脐带间充质干细胞，得到过表达miRNA‑146a的脐带间充质干细胞hucMSC‑146a；⑷培养hucMSC‑146a，从培养基中分离过表达miRNA‑146a的外泌体Exso‑146a。
Description
技术领域
本发明属于基因工程领域，涉及外泌体表达技术，尤其是一种基因工程改造外泌体递送miRNA-146a在糖尿病足溃疡治疗方面的应用。
背景技术
外泌体是细胞之间的信号传递因子，它广泛存在并分布于各种体液中，携带多种蛋白质、mRNA、miRNA和脂质类物质等。生物体的外泌体形成了一种全新的细胞-细胞间信息传递网络，可参与细胞通讯、细胞迁移、血管新生和肿瘤细胞生长等过程。
脐带间充质干细胞(hucMSC)能够加速损伤皮肤的创面愈合。近年来的研究成果表明，来源于hucMSC的外泌体hucMSC-ex也可以通过介导WNT4信号通路，参与皮肤创伤修复。除此之外，HucMSC-ex还能够显著促进皮肤细胞增殖，抑制H2O2诱导的细胞凋亡。
人类miRNA-146a定位于第五号染色体loc285628基因的第二个外显子区。不同物种间miR-146a的序列高度保守，提示其在生物体的生理、病理过程中可能发挥着重要作用。到目前为止，越来越多的证据表明miR-146a参与了免疫、炎症、造血和肿瘤等多种生物学过程。尤其对于糖尿病来说，患者的外周血单核细胞中的miR-146a表达显著下调，并且随着并发症的增多，miR-146a进一步减少。
研究表明，下调的miR-146a通过NF-kB促进下游促炎性因子的释放增加，提示miR-146a与炎症的关系。另一方面，在创面修复阶段，miR-146a可以促进血管内皮生长因子(VEGF)的表达，进一步促进溃疡创面的愈合。
综上，利用基因工程的手段，构建miR-146a过表达的脐带间充质干细胞外泌体，用于治疗糖尿病足溃疡具备充足的理论依据。
发明内容
本发明的目的在于克服现有技术的不足之处，提供一种利用基因工程的手段，构建miR-146a过表达的脐带间充质干细胞外泌体，用于治疗糖尿病足溃疡的基因工程改造外泌体递送miRNA-146a在糖尿病足溃疡治疗方面的应用。
本发明解决其技术问题是采取以下技术方案实现的：
一种基因工程改造外泌体递送miRNA-146a在糖尿病足溃疡治疗方面的应用，具体方法步骤如下：
步骤一：构建质粒：以基因工程手段构建miRNA-146a过表达的质粒；
步骤二：借用慢病毒等载体，包装后，转染293T，并富集高滴度的病毒液；
步骤三：侵染脐带充间质干细胞：用包装好的病毒侵染脐带间充质干细胞，得到过表达miRNA-146a的脐带间充质干细胞hucMSC-146a；
步骤四：培养hucMSC-146a，从培养基中分离过表达miRNA-146a的外泌体Exso-146a；
步骤五：通过外敷或肌注的方式给药，用Exso-146a治疗糖尿病足溃疡。
而且，所述miRNA-146a的序列为：
GCTCTAGACCGATGTGTATCCTCAGCTTTGAGAACTGAATTCCATGGGTTGTGTCAGTGTCAGACCTCTGAAATTCAGTTCTTCAGCTGGGATATCTCTGTCATCGTGGATCCCG。
本发明的优点和积极效果是：本发明涉及一种基因工程改造外泌体递送miRNA-146a在糖尿病足溃疡治疗方面的应用，包括：⑴构建质粒：以pCDH-CMV-MCS-EF1-copGFP慢病毒表达载体为基础，整合miRNA-146a基因，得到miRNA-146a过表达慢病毒载体pCDH-CMV-MCS-EF1-copGFP-146a；⑵包装获得慢病毒，转染293T：使用慢病毒包装质粒pSPAX2和pMD2.G对pCDH-CMV-MCS-EF1-copGFP-146a进行包装，转染293T细胞，富集高滴度的病毒液；⑶侵染脐带充间质干细胞：用包装好的病毒侵染脐带间充质干细胞，得到过表达miRNA-146a的脐带间充质干细胞hucMSC-146a；⑷培养hucMSC-146a，从培养基中分离过表达miRNA-146a的外泌体Exso-146a。
附图说明
图1为本发明pCDH-CMV-MCS-EF1-copGFP-146a的质粒构建图；
图2为本发明分离得到的Exo-146a的western-blot外泌体鉴定结果图；
图3为本发明Exo-146a对角质形成细胞中NF-kB的影响结果图；
图4为本发明Exo-146a对角质形成细胞中IL-4的影响结果图；
图5为本发明外泌体HucMSC-ex及Exo-146a对糖尿病足溃疡动物模型伤口愈合的影响结果图；
图6为本发明外泌体hucMSC-ex和Exo-146a对组织中VEGF水平的影响。
具体实施方式
下面结合附图并通过具体实施例对本发明作进一步详述，以下实施例只是描述性的，不是限定性的，不能以此限定本发明的保护范围。
如图1，一种基因工程改造外泌体递送miRNA-146a在糖尿病足溃疡治疗方面的应用具体方法步骤如下：
步骤一：构建质粒：以pCDH-CMV-MCS-EF1-copGFP慢病毒表达载体为基础，整合miRNA-146a基因，得到miRNA-146a过表达慢病毒载体pCDH-CMV-MCS-EF1-copGFP-146a；
步骤二：包装获得慢病毒，转染293T：使用慢病毒包装质粒pSPAX2和pMD2.G对pCDH-CMV-MCS-EF1-copGFP-146a进行包装，转染293T细胞，富集高滴度的病毒液；
步骤三：侵染脐带充间质干细胞：用包装好的病毒侵染脐带间充质干细胞，得到过表达miRNA-146a的脐带间充质干细胞hucMSC-146a；
步骤四：培养hucMSC-146a，从培养基中分离过表达miRNA-146a的外泌体Exso-146a；
步骤五：通过外敷或肌注的方式给药，用Exso-146a治疗糖尿病足溃疡。
GCTCTAGACCGATGTGTATCCTCAGCTTTGAGAACTGAATTCCATGGGTTGTGTCAGTGTCAGACCTCTGAAATTC AGTTCTTCAGCTGGGATATCTCTGTCATCGTGGATCCCG。
TCTAGA为限制性酶切位点XbaⅠ，GGATCC为BamHⅠ。
本发明通过外敷或肌注的方式给药，用Exso-146a治疗糖尿病足溃疡
本方法实现了基因工程改造外泌体递送miRNA-146a治疗糖尿病足溃疡的方法，能够降低溃疡炎症的发生率，治疗糖尿病足等疾病。
本发明通过对分离得到的Exo-146a的western-blot外泌体鉴定，具体为将超速离心获得组分用western-blot的方法检测，如图2，实验结果显示，Tsg101、CD9、CD63、CD81等特征表面蛋白都能检测到。可知，经过该离心组分确实含有外泌体。
本发明以人皮肤角质形成细胞为研究对象，以Phorbol myristate acetate(PMA)佛波醇酯为刺激剂，诱导刺激促炎性白细胞介素分泌，模拟炎症状态，如图3所示，由图可知，PMA的刺激下，核因子激活的B细胞的κ-轻链增强(NF-kB)分泌增多；而同时添加PMA和Exo-146a培养的角质形成细胞中，NF-kB仍保持正常细胞的水平。
图4所示为以Lipopolysaccharide(LPS)脂多糖为刺激剂，诱导刺激促炎性白细胞介素IL-4分泌，模拟炎症状态。由图4可知，LPS的刺激下，IL-4分泌显著增多；而同时添加LPS和Exo-146a培养的角质形成细胞中，IL-4保持在一个相对低的水平。
由此上述两个实验可见，Exo-146a可以帮助炎症细胞恢复正常状态。
糖尿病动物模型的建立
将6只毕格犬随机分为对照组(n＝2)和实验组(n＝4),实验组在5％戊巴比妥钠麻醉下,经前肢静脉缓慢推注50g/L四氧嘧啶(200mg/kg)；对照组经前肢静脉推注等量生理盐水。
6只毕格犬左侧后肢备皮,5％戊巴比妥钠全麻下右侧卧位消毒铺巾,皮肤切开约5cm,分离皮下组织,钝性分离浅层肌肉,植入金属垫板,分层缝合肌肉、皮下组织以及皮肤全层,然后安装皮肤表垫板和弹簧及螺母,调节螺母增加压力至49牛顿左右,每只犬共植入4枚压迫器。然后将犬仰卧位,消毒左下肢内侧皮肤,在股动脉搏动处切开皮肤,分离皮下组织,游离出股动脉后结扎股动脉主干及主要分支,缝合伤口。4-7d压迫器周围皮肤坏死后,压迫器可自行脱落或手术取下,形成溃疡。
外泌体对糖尿病足溃疡动物模型的创面修复作用分析
在模型犬的溃疡创伤面上，挑三处，通过外敷方式分别敷3种样品：control样品为海藻酸钠湿性敷料，HucMSC-ex样品为天然来源的脐带间充质干细胞外泌体与海藻酸钠湿性敷料的组合，Exo-146a样品为过表达miRNA-146a的脐带间充质干细胞外泌体与海藻酸钠湿性敷料的组合。实验过程中，7天换一次药。由实验结果如图5所示，可知，Exo-146a能在hucMSC-ex的基础上，进一步加速糖尿病足溃疡创口的愈合。
外泌体对糖尿病足溃疡组织的VEGF蛋白的影响分析
有文献报道，糖尿病足溃疡患者VEGF(血管内皮生长因子)蛋白含量低于非糖尿病患者，随着糖尿病足溃疡的病程越长，VEGF的含量越少，糖尿病病程与VEGF含量呈负相关。即，随着糖尿病足溃疡病程时间的增加，患者溃疡局部所含VEGF量减少。神经病变、感染都会显著减少溃疡组织中的VEGF水平。
在正常犬正常部位、糖模犬溃疡部位、实施例中第14天的溃疡组织(敷hucMSC-ex)、第14天的溃疡组织(敷Exo-146a)中取样，分析对比其样品中VEGF的含量，如图6所示，可知，外泌体hucMSC-ex及Exo-146a对溃疡组织中的VEGF有提高的效果，其中Exo-146a的提高程度明显高于hucMSC-ex，证明miRNA-146a对VEGF有调节作用。
尽管为说明目的公开了本发明的实施例和附图，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例和附图所公开的内容。
Claims (3)
1.一种基因工程改造外泌体递送miRNA-146a在糖尿病足溃疡治疗方面的应用，其特征在于：具体方法步骤如下：
步骤一：构建质粒：以基因工程手段构建miRNA-146a过表达的质粒；
步骤二：借用慢病毒等载体，包装后，转染293T，并富集高滴度的病毒液；
步骤三：侵染脐带充间质干细胞：用包装好的病毒侵染脐带间充质干细胞，得到过表达miRNA-146a的脐带间充质干细胞hucMSC-146a；
步骤四：培养hucMSC-146a，从培养基中分离过表达miRNA-146a的外泌体Exso-146a；
步骤五：通过外敷或肌注的方式给药，用Exso-146a治疗糖尿病足溃疡。
2.根据权利要求1所述的一种基因工程改造外泌体递送miRNA-146a在糖尿病足溃疡治疗方面的应用，其特征在于：所述miRNA-146a的序列为：
GCTCTAGACCGATGTGTATCCTCAGCTTTGAGAACTGAATTCCATGGGTTGTGTCAGTGTCAGACCTCTGAAATTC AGTTCTTCAGCTGGGATATCTCTGTCATCGTGGATCCCG
其中，下划线序列为限制性酶切位点XbaⅠ和BamHⅠ。
3.根据权利要求1-2所述的基因工程改造外泌体，其特征在于：能够降低溃疡炎症的发生率，治疗糖尿病足等疾病。
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| CN116042517A (zh) * | 2021-05-17 | 2023-05-02 | 山东大学齐鲁医院 | 间充质干细胞衍生的外泌体在制备治疗2型糖尿病药物中的应用 |
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-
2020
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Non-Patent Citations (1)
| Title |
|---|
| YING WANG ET AL.: "Exosomes Derived From Mesenchymal Stromal Cells Pretreated With Advanced Glycation End Product-Bovine Serum Albumin Inhibit Calcification of Vascular Smooth Muscle Cells" * |
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| CN116042517A (zh) * | 2021-05-17 | 2023-05-02 | 山东大学齐鲁医院 | 间充质干细胞衍生的外泌体在制备治疗2型糖尿病药物中的应用 |
| CN113373117A (zh) * | 2021-06-17 | 2021-09-10 | 江苏大学 | 一种过表达miR-13474工程化外泌体及其制备方法与应用 |
| CN113373117B (zh) * | 2021-06-17 | 2023-02-17 | 江苏大学 | 一种过表达miR-13474工程化外泌体及其制备方法与应用 |
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