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, 12 (10), 3710-3718
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The Effects of Sema3A Overexpression on the Proliferation and Differentiation of Rat Gingival Mesenchymal Stem Cells in the LPS-induced Inflammatory Environment

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The Effects of Sema3A Overexpression on the Proliferation and Differentiation of Rat Gingival Mesenchymal Stem Cells in the LPS-induced Inflammatory Environment

Tian Tian et al. Int J Clin Exp Pathol.

Abstract

Semaphorin3A has been identified as a potent osteoprotective factor that simultaneously inhibits bone resorption and promotes bone formation. The present study demonstrates the effect of the overexpression of Sema3A on the proliferation and differentiation of rat gingival mesenchymal stem cells (GMSCs) in the lipopolysaccharide (LPS)-induced inflammatory environment. rGMSCs were transfected with viral stocks of pLenO-GTP-Sema3A (Lv-Sema3A group) or pLenO-GTP (Lv-NC group), with rGMSCs as a control. The transfection efficiency was determined by flow cytometry. Cell proliferation was assessed using a Cell Counting Kit-8 assay. The expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2) were determined at 3, 7 and 14 days after the osteogenic induction culture with or without LPS using real-time PCR and Western blot. Alizarin Red staining was performed at 28 days. A pLenO-GTP-Sema3A-mediated transfection of rGMSC stably overexpressing Sema3A was built up. The overexpression of Sema3A promoted cell proliferation in the LPS-induced inflammatory environment. In addition, osteogenesis-related genes were upregulated in the Lv-Sema3A group compared with the control group. Also, after LPS administration, the overexpression of Sema3A enhanced the expression of the osteogenic genes in the LPS-induced inflammatory environment. Hence, Sema3A gene-modified rGMSCs show better osteogenic differentiation and proliferation capacities compared with rGMSCs in the LPS-induced inflammatory environment.

Keywords: Semaphorin3A; bone tissue engineering; gingival mesenchymal stem cells; lipopolysaccharide; transfection.

Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Transfection efficiency of rGMSCs under fluorescence microscopy. The transfection rate of the Lv-Sema3A group was 60% (B) and it was the same in the Lv-NC group (A) at 72 h after transfection.
Figure 2
Figure 2
Transfection efficiency of rGMSCs by flow cytometry. The transfection rate of the Lv-Sema3A group was 90.6% (A) and that of the Lv-NC group was 90.3% (B) after screening by puromycin for 3 days.
Figure 3
Figure 3
The effect of the Sema3A gene transfection of rGMSC using real-time PCR and Western blot detection. The mRNA and protein levels of Sema3A were significantly higher in the Lv-Sema3A group compared with the control and Lv-NC groups (A-C) (**P < 0.01).
Figure 4
Figure 4
Cell proliferation was measured using a Cell Counting Kit-8. There are statistical differences between the Lv-Sema3A group and the control groups at 7 days culture with DM and 3, 5, 7 days with DM+LPS (*P < 0.05).
Figure 5
Figure 5
The osteogenic gene expression of ALP, Runx2, and OCN were analyzed using Real-time PCR at 3, 7, and 14 days. The mRNA levels of ALP, Runx2, and OCN were up-regulated in the Lv-Sema3A group after being cultured with DM or DM+LPS (*P < 0.05, **P < 0.01).
Figure 6
Figure 6
The protein expressions of ALP, Runx2, and OCN were detected by Western blot at 7 and 14 days. There are statistical differences between the Lv-Sema3A group and the LPS Lv-Sema3A groups cultured at 7 and 14 days with DM or DM+LPS (*P < 0.05, **P < 0.01).
Figure 7
Figure 7
Mineralized nodules by Alizarin Red staining (original magnification 200×, scale bar = 100 µm) (A-F). The relative amount of calcium was calculated by absorbance at 562 nm (G). The mineralized nodules in the Lv-Sema3A group were higher in quantity than those in the control group in the LPS-induced inflammation (**P < 0.01).

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