CN107502594A - 一种稳定表达外源性ex-4基因的骨髓源间充质干细胞株 - Google Patents
一种稳定表达外源性ex-4基因的骨髓源间充质干细胞株 Download PDFInfo
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Abstract
本发明提供了一种能稳定表达EX‑4的MSCs细胞株。通过慢病毒感染获得稳定表达EX‑4的MSCs细胞株。本发明所用的慢病毒是由PLVTH‑pol‑IRES‑puro慢病毒过表达系统转染293T细胞获得,慢病毒过表达系统包括PLVTH‑EX‑4表达质粒、包膜质粒pMD2.G、包装质粒p8.91。PLVTH‑EX‑4由慢病毒过表达载体质粒PLVTH‑pol‑IRES‑puro和EX‑4目的基因经过双酶切再经T4连接酶连接获得。本发明获得的细胞株一方面能够增强MSCs自身抗凋亡能力、增殖能力、迁移能力;另一方面过表达EX‑4的MSCs组较MSCs组对胰岛β细胞有更好抑制凋亡,促增殖的作用。通过这两方面的作用使胰岛β细胞功能恢复到正常水平,改善MSCs在治疗糖尿病中存在的不足。
Description
技术领域
本发明涉及一种稳定表达外源性Exendin-4(EX-4)基因的骨髓源间充质干细胞株及其制备方法,并且这种细胞株能够有效的抑制胰岛β细胞凋亡,促进增殖的作用。
背景技术
间充质干细胞(Mesenchymal stem cells,MSCs)是一种具有多向分化潜能的干细胞,其能通过直接或间接的多种方式发挥治疗糖尿病的作用。最近研究发现MSCs在高糖、高脂、低氧等恶劣条件下生存能力、增殖能力、分化能力降低,且MSCs经过体外培养后,其表面CCR2等炎症相关分子表达降低,使MSCs向炎症部位的迁移能力降低,大大限制了MSCs的治疗作用。
近来发现MSCs表面表达GLP-1R,且有研究表明在体外用GLP-1处理MSCs后,MSCs的生存能力、增殖能力、分化能力、迁移能力都增强。GLP-1类似物毒蜥外泌肽EX-4是从生长在美国西南部和墨西哥沙漠的希拉毒蜥唾液中发现的一种激素物质,其也是GLP-1R激动剂,而且与GLP-1R的亲和力比GLP-1更强,但其体内半衰期只有3-4h,治疗效果不佳。因此本发明在MSCs中过表达EX-4,使MSCs持续表达EX-4,发现其对胰岛β细胞有较好的保护作用。
发明内容
本发明涉及一种稳定表达外源性EX-4基因的骨髓源间充质干细胞株,解决目前EX-4半衰期短和MSCs不稳定的问题。
为解决上述问题,本发明采用的技术方案是:
通过慢病毒感染获得稳定表达EX-4的MSCs细胞株。本发明所用的慢病毒是由PLVTH-pol-IRES-puro慢病毒过表达系统转染293T细胞获得,慢病毒过表达系统包括PLVTH-EX-4质粒、包膜质粒pMD2.G、包装质粒p8.91。PLVTH-EX-4由慢病毒过表达载体质粒PLVTH-pol-IRES-puro和EX-4目的基因经过双酶切再经T4连接酶连接获得。
稳定表达EX-4的MSCs细胞株能够促进胰岛β细胞增殖,抑制凋亡,对胰岛β细胞有很好的保护作用。其特征在在于它能够在上抑制Bax基因转录,Cleaved-Caspase-9、Beclin-1蛋白的表达。
附图说明
图1 慢病毒过表达系统中过表达载体的具体结构
图2 转染荧光图
图3 感染荧光图
图4 RT-PCR检测EX-4的表达
图5 MTT检测MSCs凋亡情况
图6 RT-PCR检测MSCs的Bax表达
图7 Western Blot检测MSCs中Cleaved-Caspase-9表达
图8 Western Blot检测MSCs中Beclin-1和p-ULK1的表达
图9 划痕实验检测MSCs、MSCs-HA、MSCs-EX-4三种细胞的迁移能力
图10 MTT检测Min6细胞活力
图11 RT-PCR检测Min6的Bax表达
图12 Western Blot检测Min6中Cleaved-Caspase-9表达
图13 Western Blot检测Min6中Beclin-1和p-ULK1的表达
具体实施方式
1.一种稳定表达外源性EX-4的骨髓源间充质干细胞株的建立方法,它包括以下步骤:
1)骨髓源间充质干细胞的培养:MSCs细胞购于上海通派生物科技有限公司,在MSCs完全培养基,于37℃,5%CO2培养箱中培养。所用的MSCs完全培养基是含10%FBS(澳洲胎牛血清)F12培养基。
2)PLVTH-EX-4过表达载体的构建:EX-4完整的基因序列由生物公司合成,通过T4DNA连接酶将EX-4基因与PLVTH-pol-IRES-puro过表达质粒连接,并转化到DH5a大肠埃希菌中,通过克隆筛选与鉴定获得PLVTH-EX-4过表达载体。
3)转染293T细胞获得慢病毒:293T细胞来自于中国药科大学,用含有10%FBS的DMEM培养基,在37℃,5%CO2培养箱中培养。PLVTH-EX-4、包膜质粒pMD2.G和包装质粒p8.91按3∶2∶1的比例共转染293T细胞,所用的转染试剂为Lipfectamine 6000,处理48-60h观察荧光强度,收集上清2000rpm离心5min,去除细胞沉淀,并用0.45μm滤膜过滤,得到慢病毒。
4)慢病毒感染MSCs细胞株:感染前24h时,将MSCs铺于6孔板中,在细胞融合度在40%-50%左右感染最佳。将病毒上清和预热的新鲜完全培养基按3∶1混合,加入终浓度为8μg/ml的polybrere并混匀,然后加入6孔板,进行感染。感染24h,换液,48h和72h时观察荧光,获得稳定表达EX-4的MSCs细胞株。
2.所述过表达EX-4能增强MSCs自身抗凋亡能力、增殖能力、迁移能力;具体实施方式如下:
1)MTT检测MSCs细胞活力
将MSCs、MSCs-HA、MSCs-EX-4三种细胞铺于96孔板,每孔5×103个细胞,每组6个复孔,用终浓度0.3mM H2O2处理12h。去掉上清后每孔加入5mg/ml的MTT溶液10μl,培养箱孵育4h,然后去掉上清,每孔加入DMSO 150μl,摇床低速震摇10min,使结晶充分溶解,测定OD490。
2)RNA水平检测Bax表达
通过RT-PCR检测不同组别Bax mRNA的表达水平,显示过表达EX-4组较模型组明显下降。
3)Western Blot检测Beclin-1、Cleaved-caspase-9和p-ULK1的表达
Western Blot检测不同组别MSCs中Cleaved-caspase-9、Beclin-1和p-ULK1的表达情况,显示过表达EX-4组较模型组明显下降。
3.MSCs迁移实验
用10μl枪头垂直在孔中央划痕,用1ml PBS洗3遍,然后加入含1%FBS的F12培养基,显微镜下拍照,记为0h,继续培养24h,拍照,并与0h时的比较。
4.所述过表达EX-4的MSCs能增强Min6抗凋亡能力、增殖能力;具体实施方式如下:
1)MTT检测Min6细胞活力
将Min6细胞铺96孔板,每孔5×103个细胞,MSCs细胞培养48h的条件培养基处理6h作为实验组,终浓度5mM STZ处理24h。每孔加入5mg/ml的MTT溶液10μl,培养箱孵育4h,然后去掉上清,每孔加入DMSO 150μl,摇床低速震摇10min,使结晶充分溶解,测定OD490。
2)RNA水平检测Bax表达
通过RT-PCR检测不同组别Min6细胞中Bax mRNA的表达水平,显示过表达EX-4的MSCs能显著抑制Min6中Bax mRNA的表达。
4)Western Blot检测Beclin-1、Cleaved-caspase-9和p-ULK1的表达
Western Blot检测不同组别Min6中Cleaved-caspase-9、Beclin-1和p-ULK1的表达情况。显示过表达EX-4的MSCs能显著抑制Min6中Cleaved-caspase-9、Beclin-1和p-ULK1的表达。
Claims (4)
1.高效稳定表达毒蜥外泌肽(Exendin-4,EX-4)的MSCs细胞株,其特征在于它是由慢病毒感染MSCs细胞株获得。
2.如权利要求1所述1.一种稳定表达外源性EX-4的骨髓源间充质干细胞株的建立方法,它包括以下步骤:
1)骨髓源间充质干细胞的培养:MSCs细胞购于上海通派生物科技有限公司,在MSCs完全培养基,于37℃,5%CO2培养箱中培养。所用的MSCs完全培养基是含10%FBS(澳洲胎牛血清)F12培养基。
2)PLVTH-EX-4过表达载体的构建:EX-4完整的基因序交由南京钟鼎生物合成,通过T4DNA连接酶将EX-4基因与PLVTH-pol-IRES-puro过表达质粒相连接,并转化到DH5a大肠埃希菌中,通过克隆筛选与鉴定获得PLVTH-EX-4过表达载体。
3)转染293T细胞获得慢病毒:293T细胞来自于中国药科大学微生物教研室,用含有10%FBS的DMEM培养基,在37℃,5%CO2培养箱中培养。PLVTH-EX-4、包膜质粒pMD2.G和包装质粒p8.91按3∶2∶1的比例共转染293T细胞,所用的转染试剂为Lipfectamine 6000,处理48-60h观察荧光强度,收集上清2000rpm离心5min,去除细胞沉淀,并用0.45μm滤膜过滤,得到慢病毒。
4)慢病毒感染MSCs细胞株:感染前24h时,将MSCs铺与6孔板中,待感染时细胞融合度在40%-50%左右最佳。将病毒上清和预热的无PS的新鲜完全培养基按3∶1混合,加入8μg/ml的polybrere并混匀,然后加入已经吸出培养基的6孔板,进行感染。感染后24h,用预热的无PS的完全培养基换掉含病毒的培养基,6孔板继续培养,此后根据细胞状态换液,在感染后第48h和72h时观察荧光,可获得稳定表达EX-4的MSCs细胞株。
3.如权利要求1或2所述的高效稳定表达EX-4的MSCs细胞株能够促进胰岛β细胞增殖,抑制凋亡。对胰岛β细胞有很好的保护作用。
4.如权利要求3所述高效稳定表达EX-4的MSCs细胞株对胰岛β细胞的保护作用,其特征在在于它能够在mRNA水平上抑制Bax的转录,Cleaved-Caspase-9、Beclin-1蛋白的表达。
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CN114025776A (zh) * | 2019-08-22 | 2022-02-08 | 布瑞克斯奥根株式会社 | 包含源自诱导多能干细胞来源间充质干细胞前体细胞的外泌体的非酒精性脂肪肝炎预防或治疗用组合物 |
CN114025776B (zh) * | 2019-08-22 | 2024-04-12 | 布瑞克斯奥根株式会社 | 包含源自诱导多能干细胞来源间充质干细胞前体细胞的外泌体的非酒精性脂肪肝炎预防或治疗用组合物 |
CN110628723A (zh) * | 2019-09-05 | 2019-12-31 | 清华大学 | 基因修饰MSCs治疗2型糖尿病 |
WO2021042321A1 (zh) * | 2019-09-05 | 2021-03-11 | 清华大学 | 基因修饰MSCs治疗2型糖尿病 |
CN110628723B (zh) * | 2019-09-05 | 2021-05-04 | 清华大学 | 基因修饰MSCs治疗2型糖尿病 |
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