Establishment of stable Schwann cell lines with genetic intervention of MST2 and its appliance in the detection of mitochondrial membrane potential
Journal Title: Chinese Journal of Nervous and Mental Diseases - Year 2024, Vol 50, Issue 2
Abstract
[Objective] To construct rat Schwann cell RSC96 cell lines that stably knock down or overexpress mammalian ste20-like kinase 2 (MST2), and preliminarily explore its regulatory effect on mitochondrial membrane potential. [Methods] The rat Schwann cell lines (RSC96) were divided into normal control group, oxygen and glucose deprivation (OGD) model group, MST2 knockdown control group, MST2 knockdown group, MST2 overexpression control group, MST2 overexpression group. The full-length sequence of rat Mst2 gene was amplified by reverse transcription PCR, and then a recombinant plasmid of overexpressing Mst2 lentiviral expression vector was constructed and identified. The second-generation lentiviral packaging system was used to obtain Mst2 knockdown or overexpression lentiviral venom and then infected the RSC96 cell lines to establish stably transfected cell lines with genetic intervention of MST2. Real-time fluorescence quantitative PCR combined with western blot was used to detect the efficiency of gene intervention. Light microscopy combined with S100 fluorescent staining was used to examine the morphological changes of the cell lines. Cells were exposed to the OGD, and the mitochondrial membrane potential (MMP) level of the cells was detected using a mitochondrial membrane potential detection kit (JC-1). [Results] The Mst2 lentiviral expression vector recombinant plasmid was successfully constructed by using plasmid sequencing. The stably transfected Schwann cell lines had the morphological characteristics of Schwann cells and expressed the Schwann cell-specific marker S100. In lentivirus-infected Schwann cells, MST2 expression was stably and significantly different at both the mRNA and protein levels compared with the control group. The knockdown efficiency exceeded 90% and overexpression was approximately 40 times that of the control. In the OGD model, the genetic intervention of MST2 remained stable as well. OGD could significantly increase MST2 and reduce Schwann cell MMP, indicating the impairment of mitochondrial function while knocking down MST2 significantly improved MMP. [Conclusion] Rat Schwann cell lines that stably knock down and overexpress MST2 are successfully established. In the OGD model, knocking down MST2 can improve the MMP, suggesting a protective effect on mitochondria.
Authors and Affiliations
Beixu HUANG, Chang LIU, Nen LIANG, Songjie LIAO
Keywords
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- EP ID EP750428
- DOI 10.3969/j.issn.1002-0152.2024.02.006
- Views 1
- Downloads 0
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