Expression of recombinant Nkx2.5 and its effect during human bone marrow mesenchymal stem cells reprogramming into cardiomyocytes

doi:10.3969/j.issn.2095-4344.1559

Abstract

Abstract:

BACKGROUND: Most studies mainly focus on using small molecules or viruses to obtain cardiomyocytes, but reports about the use of recombinant proteins to reprogram cells to obtain cardiomyocytes are rare.
OBJECTIVE: To explore the effect of recombinant Nkx2.5 protein on the transdifferentiation of human bone marrow mesenchymal stem cells into cardiomyocytes.
METHODS: The prokaryotic expression vector of Nkx2.5 was constructed. Nkx2.5 protein was induced to express at different time points, and then was isolated and purified. Nkx2.5 was encapsulated with nano-transducers to form nano-protein complexes, which were transfected into human bone marrow mesenchymal stem cells. The protein transfection efficiency was then detected. After successful protein transduction, the expression of myosin heavy chain, a specific marker of cardiomyocytes, was detected and the transformation of human bone marrow mesenchymal stem cells into cardiomyocytes was observed.
RESULTS AND CONCLUSION: The protein expression was higher when the cells were induced with isopropyl-β-d-thiogalactoside at 28 oC for 4 hours. To conclude, the nano-Nkx2.5 complexes can be efficiently transfected into human bone marrow mesenchymal stem cells and promote the reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes, but future studies are needed to improve the efficiency of the transdifferentiation.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Transcription Factors, Myocytes, Cardiac, Tissue Engineering

CLC Number:

Mai Liping, Yang Xiangyu, Wu Yueheng, He Guodong, Li Xiaohong. Expression of recombinant Nkx2.5 and its effect during human bone marrow mesenchymal stem cells reprogramming into cardiomyocytes[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 685-690.

Figures/Tables 2

2.1 Nkx2.5片段的获取重组pReceiver-B01-Nkx2.5表达载体经限制性内切酶XmnⅠ和XhoⅠ双酶切后琼脂糖凝胶电泳结果，见图1。从图中可以看出pReceiver-B01-Nkx2.5经酶切后有目标片段出现，该片段长度约在1 kb，与Nkx2.5片段的理论大小一致。 2.2 Nkx2.5重组蛋白诱导表达情况与破菌检测结果重组表达质粒pReceiver-B01-Nkx2.5经转化鉴定后进行表达，结果显示：加入IPTG诱导剂，28 ℃振荡培养4 h获取的蛋白表达较高，在35-40 kD之间有明显的目标片段Nkx2.5蛋白出现，见图2。裂解细菌包涵体后离心分别检测上清和沉淀，检测到沉淀中有蛋白表达，上清则没有，见图3。 2.3 Nkx2.5重组蛋白纯化上述所获得的沉淀进行纯化和复性后，取蛋白溶液进行SDS-PAGE检测，结果可见：包涵体蛋白样在35-45 kD之间出现明显的目标片段，见图4。 2.4 Nkx2.5重组蛋白的鉴定采用Dot blotting方法检测各次分离纯化后的蛋白，结果可见在硝酸纤维素膜上目的蛋白出现明显黑色斑点，而阴性对照则不显影，见图5。 2.5 纳米-Nkx2.5复合体转染骨髓间充质干细胞根据实验室前期的研究[18]，纳米转导材料能够以复合体的形式携载蛋白，随后穿膜并在细胞内释放和表达；前期研究结果显示：纳米粒子和蛋白质的最适摩尔比为4∶1。纳米-Nkx2.5复合体转染至骨髓间充质干细胞后可见转染率高达95%，对照组仅用纳米粒子转染骨髓间质干细胞则未见表达，见图6。Western Blot检测转染后的细胞，结果显示Nkx2.5蛋白表达明显上调，见图7。 2.6 Nkx2.5重组蛋白能促进骨髓间质干细胞向心肌样细胞转化实验组培养基中加入Nkx2.5重组蛋白，对照组培养基中未加入Nkx2.5重组蛋白，细胞培养至第8天时，采用免疫荧光法检测心肌的特异性标志物MHC表达，显微镜下计数每1 000个细胞含有绿色荧光标记的MHC数目，阳性细胞占(5.67±1.53)%，而对照组未见MHC阳性表达，见图8。同时采用Western Blot检测MHC蛋白表达，结果可见加入Nkx2.5重组蛋白的细胞与对照组比较，MHC蛋白表达水平上调，见图9。"

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