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Femtosecond laser assisted photo-transfection and differentiation of mouse embryonic stem cells

机译:Femtosecond激光辅助光转染和小鼠胚胎干细胞的分化

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In tissue engineering research, stem cells have been used as starting material in the synthesis of mammalian cells for the treatment of various cell based diseases. This is done by manipulating the DNA content of the cells to induce a specific effect such as increased proliferation or developing a new cell type through the process of differentiation. Such controlled gene expression of stem cells is achieved by the method of transfection, where exogenous plasmid deoxyribonucleic acid (pDNA) is inserted into a stem cell using chemical, viral or physical methods. In this research, we used femtosecond (fs) laser pulses from a home-build microscope system to perforate the cellular membrane and allow entry of selected pDNA to alter the behaviour of mouse embryonic stem cells (mESCs). In one set of experiments, we induce fluorescence on mESCs using green fluorescence protein plasmid (pGFP) while in other tests; differentiation of mESCs into endoderm cells is performed using Sox-17 plasmid DNA (pSox-17). Primitive endoderm formation was thereafter confirmed using polymerase chain reactions (PCR) and the Sox-17 primer. Cell viability studies using adenosine triphosphate were also conducted. From the data, it was concluded that the photo-transfection method is biocompatible since it was able to induce fluorescence in mESCs. Secondly, it was confirmed that Sox-17 was photo-transfected successfully using 6 μW laser power, 128 fs pulses and 1kHz pulse repetition rate.
机译:在组织工程研究中,干细胞已被用作哺乳动物细胞合成中的起始材料,用于治疗各种细胞基疾病。这是通过操纵细胞的DNA含量来诱导特定效果,例如通过分化过程增加增殖或开发新的细胞类型。通过转染方法实现干细胞的这种受控基因表达,其中使用化学,病毒或物理方法将外源性质粒脱氧核糖核酸(PDNA)插入干细胞中。在本研究中,我们使用来自家庭构建显微镜系统的飞秒(FS)激光脉冲,以穿孔细胞膜并允许选择的PDNA进入以改变小鼠胚胎干细胞的行为(MESCS)。在一组实验中,我们在其他试验中使用绿色荧光蛋白质质粒(PGFP)诱导MESCS上的荧光;使用SOX-17质粒DNA(PSOx-17)进行MESCS进入内胚层细胞的分化。之后使用聚合酶链反应(PCR)和SOX-17引物证实原始内胚层形成。还进行了使用腺苷三磷酸的细胞活力研究。从数据中,得出结论,光转染方法是生物相容的,因为它能够在麦塞斯诱导荧光。其次,证实SOX-17使用6μW激光功率,128 FS脉冲和1KHz脉冲重复率成功进行了光转染。

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