Effect of SHP2 knockdown on the proliferation and osteogenic differentiation of human periodontal ligament stem cells under inflammatory environment
10.12016/j.issn.2096-1456.2022.11.002
- Author:
ZHANG Yuan
1
;
ZHAO Qing
1
;
LV Haodong
1
;
WANG Tiancong
1
;
DOU Zhaojing
1
;
JIN Yuqin
1
;
JI Jun
1
Author Information
1. Department of Orthodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University
- Publication Type:Journal Article
- Keywords:
periodontal ligament stem cell;
proliferation;
periodontitis;
tumor necrosis factor-α;
interleukin-1β;
inflammatory environment;
Src homology-2 domain containing protein tyrosine phosphatase-2(SHP2);
gene sliencing;
osteogenesis;
alkaline phosphatase;
COL-1;
MAPK/NF-κB signaling pathway
- From:
Journal of Prevention and Treatment for Stomatological Diseases
2022;30(11):769-778
- CountryChina
- Language:Chinese
-
Abstract:
Objective : The purpose of this study was to clarify the regulatory effect and mechanism of Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) on human periodontal ligament stem cell (hPDLSC) proliferation and osteogenic differentiation under inflammatory environment and to provide a new target for the treatment of periodontitis.
Methods:SHP2 was knocked down in hPDLSCs, and the transfection efficiency of SHP2 was detected by RT-qPCR and Western blot. An in vitro inflammatory environment was created using tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The effect of SHP2 knockdown on hPDLSC viability under normal and inflammatory conditions was detected by CCK-8, and the osteogenic capacity of hPDLSCs under normal and inflammatory conditions was detected by ALP staining, ALP activity, ARS staining, RT-qPCR and Western blot. The mechanism by which SHP2 knockdown affected the MAPK pathway and its downstream NF-κB pathway under inflammatory conditions was assessed by Western blot.
Results: Green fluorescence was observed after transfection for 72 h, and the titer of SHP2 shRNA recombinant lentivirus was 2.9×108 TU/mL. SHP2 expression was significantly downregulated in lentivirus-transfected cells, as demonstrated by Western blot and RT-qPCR (P<0.001). SHP2 knockdown inhibited hPDLSC proliferation to a certain extent and increased the expression of early osteogenic markers under normal conditions, including increased ALP activity and increased ALP and COL-1 expression (P<0.05). However, SHP2 knockdown exerted no effect on mineralized nodule formation. In the TNF-α- and IL-1β-induced inflammatory environment, SHP2 knockdown exerted no effect on hPDLSC proliferation (P>0.05). Osteogenic markers were upregulated (P<0.05), and mineralized nodules were significantly increased (P<0.05) after SHP2 knockdown. Western blot analysis showed that p65 phosphorylation and IκB-α degradation were reduced in SHP2-knockdown hPDLSCs in the inflammatory environment. Moreover, SHP2 knockdown significantly inhibited the expression of p-p38 and p-JNK MAPK, which represent pathways upstream of the NF-κB pathway (P<0.05).
Conclusion : SHP2 knockdown did not affect cell viability but promoted the osteogenic potential of hPDLSCs by inhibiting the MAPK/NF-κB-mediated signaling pathway under inflammatory environment.
- Full text:炎症环境下敲低SHP2对人牙周膜干细胞增殖和成骨分化的影响.pdf
