Establishment of a lentiviral vector carrying rat miR-21 gene and its effect on apoptosis of bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.01.001

Abstract

Abstract:

BACKGROUND: Apoptosis in bone marrow mesenchymal stem cells (BMSCs) occurs after transplantation, which is mainly responsible for unsatisfactory therapeutic effects on premature ovarian failure
induced by chemotherapy. miR-21 participates in the regulation of cell proliferation and apoptosis, and has important anti-apoptotic effect.
OBJECTIVE: To construct a lentiviral vector for rat miR-21 and observe its effects on apoptosis of BMSCs induced by phosphoramide nitrogen mustard.
METHODS: Rat miR-21 gene was synthesized, amplified, and connected with the lentiviral plasmid pLVX-shRNA2. Both double enzyme digestion and gene sequencing were done to identify the successful construction of the vector pLVX-shRNA2-rno-miR-21-5p. The vector was packaged, and the titer was examined. BMSCs were transfected by the vector with different multiplicities of infection (MOI=20 or 40), and the efficiency was observed. miR-21 expression in the transfected cells was detected using real-time PCR. Phosphoramide nitrogen mustard was added into the cell culture media of BMSCs, then the apoptotic rate of BMSCs was detected by flow cytometry, and apoptotic index was examined by Hoechst33342 staining.
RESULTS AND CONCLUSION: The target gene was successfully connected with the lentiviral vector. The viral titer was 6×10¹¹ CFU/L. The vector transfected BMSCs had a high efficiency above 90%. Real-time PCR results showed the expression levels of miR-21 in miR-21 group 1 (MOI=20) and miR-21 group2 (MOI=40) were higher than that in BMSCs group and blank vector group (P=0.000). Flow cytometry and Hoechst33342 staining results showed the apoptotic rate and apoptotic index of miR-21 transfected groups were lower than those of blank vector group and BMSCs group (P=0.001). Overall, we successfully established rat miR-21 lentivirus vector pLVX-shRNA2-rno-miR-21-5p and transfected it into the BMSCs. Upregulation of miR-21 could reduce BMSCs apoptosis, and enhance cell proliferation.

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: Ovarian Diseases, MicroRNAs, Transfection, Apoptosis, Tissue Engineering

CLC Number:

R394.2

Fu Xia-fei, He Yuan-li, Wang Xue-feng, Chen Xiao-ying. Establishment of a lentiviral vector carrying rat miR-21 gene and its effect on apoptosis of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 1-5.

Figures/Tables 1

2.1 rno-miR-21-5p的扩增和序列测定设计引物按照既定程序进行PCR扩增，所得到的PCR反应液进行琼脂糖凝胶电泳检测扩增反应产物。电泳结果显示，PCR成功特异性扩增出miR-21，得到大小为117 bp的特异性片段(图1)，没有非特异性杂带，且表达量较高，光信号较强，无拖尾及弥散。 2.2 慢病毒载体pLVX-shRNA2-rno-miR-21-5p的构建与鉴定经EcoRⅠ及BamHⅠ双酶切，琼脂糖凝胶电泳鉴定，得到大小分别为7 888 bp和117 bp的片段(图2)。阳性克隆的测序结果显示重组质粒中插入的rno-miR-21-5p序列与目标序列完全一致。因此，可以证明成功构建了miR-21慢病毒载体系统。 2.3 慢病毒载体pLVX-shRNA2-rno-miR-21-5p包装与滴度测定结果将构建的慢病毒载体pLVX-shRNA2-rno- miR-21-5p及包装质粒载体，以磷酸钙共沉淀法转染包装细胞(293FT)，收集培养上清液中的慢病毒液，采用抗性克隆法测得病毒滴度为6×1011 CFU/L。 2.4 慢病毒载体pLVX-shRNA2-rno-miR-21-5p转染骨髓间充质干细胞携带miR-21基因的慢病毒液以MOI为20和40感染骨髓间充质干细胞，荧光显微镜下均可见到绿色荧光蛋白的表达(图3)，转染效率分别为(93.13±1.93)%、(94.57±1.75)%，两组相比差异无显著性意义(t=-1.236，P=0.251)。 2.5 骨髓间充质干细胞转染载体后miR-21的表达实时定量PCR结果显示：骨髓间充质干细胞组、空载组、miR-21组1(MOI为20)、miR-21组2(MOI为40)miR-21的表达量分别为1.07±0.05，1.12±0.05，4.05±0.07，4.06±0.04，经单向方差分析，4组间差异有显著性意义(F=3 014.962，P=0.000)。采用SNK法进行两组间比较，骨髓间充质干细胞组与空载组相比，差异无显著性意义(P > 0.05)，miR-21组1与miR-21组2的表达量高于骨髓间充质干细胞组及空载组(P < 0.05)，但miR-21组1与miR-21组2相比，差异无显著性意义(P > 0.05)，见图4。 2.6 骨髓间充质干细胞的凋亡流式细胞仪结果显示，骨髓间充质干细胞组、空载组、转染组的凋亡率分别为(33.33±3.42)%，(31.74±2.93)%，(29.78±3.06)%。经单向方差分析，3组间差异有显著性意义(F=11.777，P=0.001)。采用SNK法进行两组间比较，骨髓间充质干细胞组与空载组相比，差异无显著性意义，转染组的凋亡率低于骨髓间充质干细胞组及空载组，见图5。 Hoechst33342染色结果显示：骨髓间充质干细胞组、空载组、转染组的凋亡指数分别为：(34.36±4.24)%，(35.73± 3.6)%，(24.48±3.17)%，经单向方差分析，3组间差异有显著性意义(F=13.144，P=0.001)。采用SNK法进行两组间比较，骨髓间充质干细胞组与空载组相比，差异无显著性意义，转染组的凋亡指数低于骨髓间充质干细胞组及空载组，与流式细胞仪凋亡结果一致。表明在加入化疗药物诱导模拟卵巢局部微环境，骨髓间充质干细胞转染miR-21慢病毒载体后，其凋亡受到抑制，从而促进细胞的增殖，提高细胞的存活率，有利于骨髓间充质干细胞功能的持续发挥与作用。"

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