Construction of miR-146b overexpression lentiviral vector and the effect on the proliferation of hippocampal neural stem cells

doi:10.3969/j.issn.2095-4344.3539

Abstract

Abstract: BACKGROUND: More and more studies have confirmed that miR-146b plays an important role in the nervous system, which provides a new therapeutic direction for treatment of nervous system diseases.
OBJECTIVE: To construct rat miR-146b overexpression lentiviral vector and observe the effect of miR-146b overexpression on the proliferation of primary hippocampal neural stem cells.
METHODS: (1) PCR extended to obtain the full-length sequence of rat miR-146b, which was ligated into the lentiviral vector pLVX-Luciferase-Puro. After double digestion and DNA sequencing identification, lentivirus was packed in 293T cells and virus supernatant was collected. (2) Primary hippocampal neural stem cells of newborn SD rats were isolated and cultured, and pLVX-Luciferase-rno-miR-146b-Puro lentivirus was transfected into hippocampal neural stem cells. After 24 and 48 hours of transfection, RT-PCR was applied to detect the expression level of miR-146b, while the expression of Nestin was observed by immunofluorescence staining. MTS method and Edu method were used to measure cell proliferation. After 48 hours of transfection, cell cycle was detected by flow cytometry.
RESULTS AND CONCLUSION: (1) The results of sequencing identification showed that rat miR-146b was successfully cloned into pLVX-Luciferase-Puro overexpression vector. (2) After 24 and 48 hours of transfection, expression level of miR-146b in hippocampal neural stem cells increased significantly. (3) The number of Nestin immunofluorescence-positive cells was significantly reduced after miR-146 overexpression, and the proliferation ratio was decreased, and the cell cycle mainly remained in G1 phase. (4) The results confirm that the rat miR-146b overexpression lentivirus vector was successfully packaged and it could inhibit the proliferation of primary hippocampal neural stem cells after transfection, but the mechanism of its regulation of target genes needs further study.

Key words: neural stem cells, hippocampus, miR-146b, lentivirus, transfection, cell proliferation, rats

CLC Number:

Dai Yaling, Chen Lewen, He Xiaojun, Lin Huawei, Jia Weiwei, Chen Lidian, Tao Jing, Liu Weilin. Construction of miR-146b overexpression lentiviral vector and the effect on the proliferation of hippocampal neural stem cells[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(19): 3024-3030.

Figures/Tables 8

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