重组慢病毒介导STAT3过表达质粒转染促进显性脊柱裂胎鼠骨髓间充质干细胞向神经细胞分化

doi:10.3969/j.issn.1006-6233.2021.03.007

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摘要目的观察重组慢病毒介导STAT3过表达质粒转染骨髓间充质干细胞(BMSCs)移植后,对显性脊柱裂胎鼠脊髓组织神经细胞转分化的影响。方法给予维甲酸致畸建立显性脊柱裂胎鼠模型,利用显微外科注射技术对胎鼠进行干细胞移植治疗。分为空白对照组(12例),阴性对照组(10例)和实验组(11例),采用CCK-8,RT-PCR和Western-blot检测手段,测定干细胞增殖活性的OD值及每组STAT3,pSTAT3,神经标记物(GFAP,NSE,NF和Nestin)和凋亡相关因子(Caspase-8和Bcl-2)的表达情况。结果在干细胞移植后通过CCK-8检测实验组在第8小时OD值达到峰值,之后逐渐下降,与空白对照组和阴性对照组相比有统计学差异(P<0.05),而空白对照组和阴性对照组检测后的OD值高峰时间和峰值未见统计学差异。经过基因和蛋白双验证,实验组pSTAT3,GFAP,NSE,NF和Nestin表达水平显著高于空白对照组和阴性对照组(P<0.05),而STAT3 mRNA在实验组中的表达水平显著降低(P<0.05)。实验组中Caspase-8和Bcl-2在基因和蛋白两个层面的表达水平均明显低于空白对照组和阴性对照组(P<0.05)。结论构建STAT3过表达质粒通过慢病毒介导的方法转染骨髓间充质干细胞,可以提高其向神经细胞转化的效率,为显性脊柱裂的治疗提供新的种子细胞。

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关键词 ：骨髓间充质干细胞, 显性脊柱裂, 转录激活子3, 慢病毒转染

Abstract：Objective: To observe the action of recombinant lentivirus mediated STAT3 overexpression plasmid transfected into bone marrow mesenchymal stem cells on neural cell trans-differentiation in spinal cord tissue of fetal rats with spina bifida aperta. Methods: The fetal rat model of spina bifida aperta was established by retinoic acid and treated by microsurgical injection of stem cells. They were divided into blank control group (12 cases),negative control group (10 cases) and experimental group (11 cases). OD value of BMSCs viability was determined by CCK-8 kit. The expression of STAT3,pSTAT3,nerve markers (GFAP,NSE,NF and nestin) and apoptosis related factors (Caspase-8 and Bcl-2) were tested by the method of real-time PCR and Western blot. Results: OD value in experimental group was the highest at 8 hours after transplantation by CCK-8 kit,and then gradually decreased,which was statistically significant compared with blank control group and negative control group (P<0.05). There was no statistical difference in OD peak time and value between blank control group and negative control group. The expression levels of pSTAT3,GFAP,NSE,NF and nestin in experimental group were remarkably higher than those in blank control group and negative control group (P<0.05),but STAT3 expression in experimental group were statistically decreased (P<0.05). The relative expression levels of Caspase-8 and Bcl-2 in experimental group were tremendously lower than those in blank control group and negative control group (P<0.05). Conclusion: Transfection of recombinant lentivirus mediated STAT3 overexpression plasmid with BMSCs can improve the efficiency of transforming into neural cells and provide new seed cells for the treatment of congenital spina bifida aperta.

Key words： Bone marrow mesenchymal stem cells Spina bifida aperta Signal transducer and activator of transcription 3 Lentivirus transfection

基金资助:2019年度黑龙江省省属高等学校基本科研业务费科研项目,(编号:2019-KYYWF-0332)

引用本文:

姜明宇, 任明永, 高莹, 吴楠, 于忠莹. 重组慢病毒介导STAT3过表达质粒转染促进显性脊柱裂胎鼠骨髓间充质干细胞向神经细胞分化[J]. 河北医学, 2021, 27(3): 384-389.
JIANG Mingyu, REN Mingyong, GAO Ying, et al. Transfection of STAT3 Overexpression Plasmid Mediated by Recombinant Lentivirus Promotes Differentiation of Bone Marrow Mesenchymal Stem Cells into Neural Cells in Fetal Rats with Spina Bifida Aperta. HeBei Med, 2021, 27(3): 384-389.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2021.03.007 或 http://www.hbyxzzs.cn/CN/Y2021/V27/I3/384

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