CN108251454A - 一种脂氧合酶基因修饰间充质干细胞的获取方法及其应用 - Google Patents

一种脂氧合酶基因修饰间充质干细胞的获取方法及其应用 Download PDF

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CN108251454A
CN108251454A CN201711436333.9A CN201711436333A CN108251454A CN 108251454 A CN108251454 A CN 108251454A CN 201711436333 A CN201711436333 A CN 201711436333A CN 108251454 A CN108251454 A CN 108251454A
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stem cell
lipoxygenase
mescenchymal stem
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周蒙滔
陈必成
余华军
施哲好
叶雯
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First Affiliated Hospital of Wenzhou Medical University
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Abstract

本发明公开了一种脂氧合酶基因修饰间充质干细胞的获取方法，先通过将多能诱导性干细胞放入诱导培养基中进行诱导，诱导周期为10‑20天，形成诱导性间充质干细胞；再从构建好的慢病毒中转染脂氧合酶基因来修饰诱导性间充质干细胞，得到脂氧合酶基因修饰的间充质干细胞。本发明还提供了通过上述方法获得的脂氧合酶基因修饰间充质干细胞作为抗炎药物的应用，通过该方法获取的脂氧合酶基因修饰间充质干细胞不仅具有早期靶向定位功能，而且具有双重抗炎效用。

Description

一种脂氧合酶基因修饰间充质干细胞的获取方法及其应用
技术领域
本发明涉及一种脂氧合酶基因修饰间充质干细胞的获取方法及其应用，属于生物工程技术领域。
背景技术
目前，对于一些重症胰腺炎(SAP)病人，其易发生全身炎症反应综合症（SIRS），而SIRS 极易引发远处器官损伤，导致多脏器功能障碍综合征（MODS），此问题是SAP 治疗的最后障碍。为改善上述病症，国内外相关报告指出可通过脂氧素类药品进行配合治疗，但由于现有的脂氧素类药品均是体外合成，价格昂贵，且进入体内后作用周期短，易失效，从而限制了其临床应用，存在改善空间。同时，国内外相关报告指出间充质干细胞（MesenchymalStem Cells，MSCs）具有炎症趋向性的特性，因此若能获得脂氧合酶基因修饰的间充质干细胞，则不仅具有早期靶向定位功能，而且具有双重抗炎效用，然而现有技术尚未发现将脂氧合酶基因修饰的间充质干细胞作为抗炎药物的应用以及有效获取脂氧合酶基因修饰的间充质干细胞的方法。
发明内容
针对现有技术的不足，本发明提供了一种脂氧合酶基因修饰间充质干细胞的获取方法及其应用，通过该方法获取的脂氧合酶基因修饰间充质干细胞不仅具有早期靶向定位功能，而且具有双重抗炎效用。
为实现上述目的，本发明提供了一种脂氧合酶基因修饰间充质干细胞的获取方法，先通过将多能诱导性干细胞放入诱导培养基中进行诱导，诱导周期为10-20天，形成诱导性间充质干细胞；再从构建好的慢病毒中转染脂氧合酶基因来修饰诱导性间充质干细胞，得到脂氧合酶基因修饰的间充质干细胞。
本发明可进一步设置为所述的脂氧合酶为5-脂氧合酶和15-脂氧合酶中的一种或两种混合。
本发明还提供了通过上述方法获得的脂氧合酶基因修饰间充质干细胞作为抗炎药物的应用。
本发明的有益效果是：通过采用上述方法，可简单安全的获得脂氧合酶基因修饰间充质干细胞，而将获得的脂氧合酶基因修饰间充质干细胞作为抗炎药物应用时，其不仅具有早期靶向定位功能，而且具有双重抗炎效用。
附图说明
图1为诱导前iPS的形态图；
图2为诱导后iMSCs的形态图；
图3为iMSCs的成骨实验分化图；
图4为iMSCs的成脂实验分化图；
图5为iMSCs流式鉴定图表；
图6为荧光镜下5-LO的表达图；
图7为普通光镜下5-LO的表达图；
图8为荧光镜下15-LO的表达图；
图9为普通光镜下15-LO的表达图；
图10为通过PCR鉴定5-LO和15-LO过表达水平的图表；
图11为通过Elisia检测所得图表。
具体实施方式
本发明提供了一种脂氧合酶基因修饰间充质干细胞的获取方法及其应用：
1、通过将多能诱导性干细胞（iPS）放入诱导培养基中进行诱导，诱导周期为10-20天，形成诱导性间充质干细胞（induced Mesenchymal Stem Cells，iMSCs），将获得的iMSCs进行成骨和成脂实验证明其符合MSCs的特性（如图3和4所示），再如图5所示经流式鉴定iMSCsCD73、CD90、CD29阳性，CD45、CD34、Anti-HLA-DR阴性，也符合MSCs的细胞特性。
2、从构建好的慢病毒（ALOX15 载体信息：Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin；ALOX5载体信息：Ubi-MCS-3FLAG-SV40-Cherry）中通过基因转染技术转染脂氧合酶基因来修饰诱导性间充质干细胞，得到脂氧合酶基因修饰的间充质干细胞。所述的脂氧合酶为5-脂氧合酶（5-LO）和15-脂氧合酶（15-LO）中的一种或两种混合。完成后如图6-9所示采用荧光镜和普通光镜技术分别检测细胞内 5-LO、15-LO 的表达。
3、利用聚合酶链式反应（PCR）鉴定5-LO、15-LO过表达的水平，如图10所示发现5-LO和15-LO都有明显的表达增高。其中，5Y- iMSCs和15Y- iMSCs为空载体-诱导性间充质干细胞。
4、将慢病毒转染后的iMSCs与5-LO和/或15-LO培养后，加入低浓度炎症底物（花生四烯酸），并在15-30分钟内取上清液进行酶联免疫吸附（Elisia）检测，如图11所示发现脂氧素（LXA4）的浓度在5-LO和/或15-LO的培养组都有明显表达增高。
因此，综上所述，通过本发明所提供的方法，不仅可简单安全的获得脂氧合酶基因修饰间充质干细胞，而且将获得的脂氧合酶基因修饰间充质干细胞作为抗炎药物应用时，因其具备MSCs炎症趋向性的特性，因此能在炎症早期、趋向性、高浓度地获得脂氧素（LXA4）干预，并将高浓度LXA4靶向到重症胰腺炎病人的炎症部位，同时因MSCs和LXA4都具有抗炎效果，因此获得的脂氧合酶基因修饰间充质干细胞还具有双重抗炎效用。

Claims (3)

1.一种脂氧合酶基因修饰间充质干细胞的获取方法，其特征在于：先通过将多能诱导性干细胞放入诱导培养基中进行诱导，诱导周期为10-20天，形成诱导性间充质干细胞；再从构建好的慢病毒中转染脂氧合酶基因来修饰诱导性间充质干细胞，得到脂氧合酶基因修饰的间充质干细胞。
2.根据权利要求1所述的一种脂氧合酶基因修饰间充质干细胞的获取方法，其特征在于：所述的脂氧合酶为5-脂氧合酶和15-脂氧合酶中的一种或两种混合。
3.权利要求1或2所述方法获得的脂氧合酶基因修饰间充质干细胞作为抗炎药物的应用。
CN201711436333.9A 2017-12-26 2017-12-26 一种脂氧合酶基因修饰间充质干细胞的获取方法及其应用 Pending CN108251454A (zh)

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